Kidney Transplantation, Bioengineering and Regeneration. Kidney Transplantation in the Regenerative Medicine Era [1st Edition] 9780128018361, 9780128017340

Table of contents :
Content:
Front-matter,Copyright,List of Contributors,Biographies,Kidney Transplantation, Bioengineering, and RegenerationEntitled to full textPart I: Kidney TransplantationSection I: Epidemiology of Kidney Disease and TransplantationChapter 1 - Epidemiology of End-Stage Renal Failure: The Burden of Kidney Diseases to Global Health, Pages 5-11, Matias Trillini, Norberto Perico, Giuseppe Remuzzi
Chapter 2 - Transplant Programs Worldwide and the Spanish Miracle, Pages 13-27, Rafael Matesanz, Beatriz Domínguez-Gil, Elisabeth Coll, Beatriz Mahíllo, Gloria de la Rosa, María O. Valentín
Chapter 3 - The Deceased Kidney Donor, Pages 31-39, Hany El Hennawy, Jeffrey Rogers
Chapter 4 - The Living Donor, Pages 41-50, Andrea Pietrabissa, Luigi Pugliese, Massimo Abelli, Elena Ticozzelli, Teresa Rampino
Chapter 5 - Criteria for Kidney Allocation in the United States, Pages 51-58, Linda Ohler
Chapter 6 - Strategies to Increase the Donor Pool, Pages 59-83, Michael A. Rees, David E. Fumo
Chapter 7 - Kidney Preservation, Pages 87-100, Ina Jochmans, John M. O’Callaghan, Rutger J. Ploeg, Jacques Pirenne
Chapter 8 - Ex-vivo Normothermic Perfusion in Renal Transplantation, Pages 101-109, Sarah A. Hosgood, Michael L. Nicholson
Chapter 9 - Kidney Transplant Recipient Surgery, Pages 111-125, Fabio Vistoli, Vittorio Perrone, Gabriella Amorese, Ugo Boggi, Giuseppe Orlando
Chapter 10 - Robotic-Assisted Kidney Transplantation, Pages 127-133, Ivo Tzvetanov, Pier C. Giulianotti, Giuseppe D’Amico, Raquel G. Roca, Enrico Benedetti
Chapter 11 - Orthotopic Kidney Transplantation, Pages 135-140, Bulang He
Chapter 12 - Kidney Transplantation Combined With Other Organs, Pages 141-157, Junichiro Sageshima, Linda Chen, Gaetano Ciancio, Alberto Pugliese, George W. Burke III
Chapter 13 - Pediatric Renal Transplantation, Pages 159-184, Ashton Chen, Jen-Jar Lin, Andrew M. South
Chapter 14 - Indications for Renal Transplantation: Evaluation of Transplant Candidates, Pages 187-197, Opas Traitanon, Lorenzo Gallon
Chapter 15 - Early Postoperative ICU Care of the Kidney Transplant Recipient, Pages 199-210, Xavier Wittebole, Diego Castanares-Zapatero, Michel Mourad, Virginie Montiel, Christine Collienne, Pierre-François Laterre
Chapter 16 - Approaches to Minimize Delayed Graft Function in Renal Transplantation, Pages 211-219, Maria Francesca Egidi, Domenico Giannese
Chapter 17 - Histocompatibility Testing in the Transplant Setting, Pages 223-234, Michael D. Gautreaux
Chapter 18 - The Immune Response to the Allograft, Pages 235-246, Fiona Carty, Karen English
Chapter 19 - Induction Immunosuppression in Kidney Transplantation, Pages 247-258, Josep M. Grinyó, Oriol Bestard
Chapter 20 - Maintenance Immunosuppression in Kidney Transplantation, Pages 259-276, Monica Cortinovis, Giuseppe Remuzzi, Norberto Perico
Chapter 21 - Novel Drugs in Kidney Transplantation, Pages 277-290, Sindhu Chandran, Flavio Vincenti
Chapter 22 - Hematopoietic Stem Cell Transplantation for Induction of Allograft Tolerance, Pages 291-301, Kiyohiko Hotta, Tetsu Oura, A. Benedict Cosimi, Tatsuo Kawai
Chapter 23 - Regulatory T Cell Therapy in Transplantation, Pages 303-318, Scott McEwen, Qizhi Tang
Chapter 24 - Mesenchymal Stromal Cells to Improve Solid Organ Transplant Outcome: Lessons From the Initial Clinical Trials, Pages 319-331, Marlies E.J. Reinders, Johannes W. De Fijter, Maarten L. Zandvliet, Ton J. Rabelink
Chapter 25 - Renal Transplantation Across HLA and ABO Barriers, Pages 333-354, Shaifali Sandal, Robert A. Montgomery
Chapter 26 - Pathology of the Renal Allograft, Pages 357-372, Loredana Melchiorri, Christian C. Morrill, Gino Coletti, Paul Persad
Chapter 27 - Imaging-Based Monitoring of the Renal Graft, Pages 373-402, Corinne Deurdulian, Hisham Tchelepi
Chapter 28 - Immune Monitoring in Kidney Transplantation, Pages 403-417, Mark Nguyen, Anna Geraedts, Minnie Sarwal
Chapter 29 - Renal Function Measurements, Pages 419-427, Esteban Porrini
Chapter 30 - Pharmacokinetics and Genomics of Immunosuppressive Drugs, Pages 429-443, Marek Drozdzik
Chapter 31 - Gene Expression Technology Applied to Kidney Transplantation, Pages 445-457, Richard Danger, Sophie Brouard
Chapter 32 - Acute Cellular Rejection, Pages 461-474, Madhav C. Menon, Paolo Cravedi, Fadi El Salem
Chapter 33 - Acute Antibody-Mediated Rejection, Pages 475-487, Ivy A. Rosales, Robert B. Colvin
Chapter 34 - Vascular Complications in Renal Transplantation, Pages 491-502, Hany El-Hennawy, Christian C. Morrill, Giuseppe Orlando, Alan C. Farney
Chapter 35 - Infections in Kidney Transplant Recipients, Pages 503-512, Aynaa Alsharidi, Deepali Kumar, Atul Humar
Chapter 36 - Metabolic Disorders Following Kidney Transplantation, Pages 513-523, Quirino Lai, Francesco Pisani
Chapter 37 - Cancer After Kidney Transplantation, Pages 525-542, Renaud Snanoudj, Jacques Dantal, Céleste Lebbé, Christophe Legendre
Chapter 38 - Cardiovascular Disease and Renal Transplantation, Pages 543-554, Robert J. Applegate, P. Matthew Belford, Sanjay K. Gandhi, Michael A. Kutcher, Renato M. Santos, David X. Zhao
Chapter 39 - Graft and Patient Survival, Pages 557-571, Aneesha A. Shetty, Ekamol Tantissattamo, Bing Ho, Anton Skaro, Lihui Zhao, Samantha Montag, Michael Abecassis
Chapter 40 - Urological Complications of the Renal Graft, Pages 573-587, Tristan Keys, Majid Mirzazadeh
Chapter 41 - Impact of De Novo Donor-specific Alloantibody in Primary Renal Allografts, Pages 589-596, Matthew J. Everly
Chapter 42 - De Novo and Recurrence of Renal Disease, Pages 597-608, Quirino Lai, Fabio Melandro, Francesco Pisani
Chapter 43 - Kidney Transplantation in the Elderly, Pages 611-631, Robert J. Stratta
Chapter 44 - Dual Kidney Transplantation, Pages 633-642, Paolo Cravedi, Andrea Angeletti, Piero Ruggenenti
Chapter 45 - Kidney Transplantation in the Diabetic Patient, Pages 643-651, Angelika C. Gruessner, Rainer W.G. Gruessner
Chapter 46 - Kidney Transplantation in the Hepatitis C Infected Recipient, Pages 653-663, Roberta Angelico, Giuseppe Iaria, Mario Angelico
Chapter 47 - Pregnancy After Kidney Transplantation, Pages 665-676, Marialuisa Framarino-dei-Malatesta
Chapter 48 - Nutrition of the Kidney Transplant Recipients, Pages 677-683, Paolo Dionigi, Mario Alessiani
Chapter 49 - Kidney Transplantation in Developing Countries, Pages 687-698, Goce Spasovski, Mirela Busic, Mirjana S. Matovinovic, Francis L. Delmonico
Chapter 50 - Health-Related Quality of Life Outcomes After Kidney Transplantation, Pages 699-708, Aneesha A. Shetty, Jason A. Wertheim, Zeeshan Butt
Chapter 51 - Costs of Kidney Transplantation, Pages 709-718, Charles Strom, Yanik Bababekov, David Axelrod
Chapter 52 - Kidney Transplantation: Have the Promises Been Kept and Needs Met, Pages 721-736, Charles Strom, Eliot Heher, A. Benedict Cosimi
Chapter 53 - Innovations in Kidney Transplant Research, Pages 737-751, Sarwat Ahmad, Eric Siskind, Jonathan S. Bromberg
Chapter 54 - Converging Organ Transplantation Towards Regenerative Medicine, Pages 757-767, Ravi Katari, Riccardo Tamburrini, Lauren Edgar, Giuseppe Orlando
Chapter 55 - A Systems Engineering Approach to Restoring Kidney Structure and Function, Pages 769-784, David F. Williams
Chapter 56 - Kidney Development in the Mammal, Pages 787-799, Melissa H. Little
Chapter 57 - Renal Branching Morphogenesis, Pages 801-814, Joshua Blake, Norman D. Rosenblum
Chapter 58 - Principles of Stem Cell Biology Applied to the Kidney, Pages 817-827, Brooke E. Chambers, Rebecca A. Wingert
Chapter 59 - Extracellular Matrix Biology Applied to the Kidney, Pages 829-841, Rachel Lennon
Chapter 60 - Interplay Between Growth Factor Receptors, Small GTPases, and Mechanical Stress in the Maintenance of Kidney Glomerular Homeostasis, Pages 843-859, Manuel Chiusa, Xiwu Chen, Roy Zent, Ambra Pozzi
Chapter 61 - Bioengineering Approach to Immunomodulation, Pages 861-872, Lauren Brasile, Bart Stubenitsky
Chapter 62 - Principles of Organ Bioengineering, Pages 873-876, Abritee Dhal, Matthew Brovold, Anthony Atala, Shay Soker
Chapter 63 - Recellularization of Kidney Scaffold With Stem Cells, Pages 877-886, Marina Figliuzzi, Giuseppe Remuzzi, Andrea Remuzzi
Chapter 64 - Bioreactors for Cell Culture Systems and Organ Bioengineering, Pages 889-899, Chiara Attanasio, Paolo A. Netti
Chapter 65 - Synthetic Biomaterial for Regenerative Medicine Applications, Pages 901-921, Tiziana Nardo, Irene Carmagnola, Francesca Ruini, Silvia Caddeo, Stefano Calzone, Valeria Chiono, Gianluca Ciardelli
Chapter 66 - Immune Responses to Biomaterials Used in Renal Engineering, Pages 923-933, David F. Williams
Chapter 67 - Regeneration of Kidney From Human Reprogrammed Stem Cells, Pages 937-955, Melissa H. Little, Kenji Osafune
Chapter 68 - Bioprinting Complex 3D Tissue and Organs, Pages 957-971, Carlos Kengla, Amritha Kidiyoor, Sean V. Murphy
Chapter 69 - Principles of Kidney Regeneration, Pages 973-988, Maria L. Angelotti, Francesca Becherucci, Benedetta Mazzinghi, Anna Peired, Paola Romagnani
Chapter 70 - Markers of Repair and Regeneration in the Marginal Kidney, Pages 989-996, Stephen J. Walker, Susan Y. Zhao
Chapter 71 - Nephron Repair in Mammals and Fish, Pages 997-1003, Zhenzhen Peng, Veronika Sander, Alan J. Davidson
Chapter 72 - Imaging of Glomerular Regeneration, Pages 1005-1011, János Peti-Peterdi, Kengo Kidokoro, Anne Riquier-Brison
Chapter 73 - Reversibility of Renal Fibrosis, Pages 1013-1023, Christos E. Chadjichristos, Panagiotis Kavvadas, Jean-Claude Dussaule, Ahmed Abed, Christos Chatziantoniou
Chapter 74 - Pharmacological Induction of Kidney Regeneration, Pages 1025-1037, Elena Gagliardini, Ariela Benigni, Norberto Perico
Chapter 75 - Developmental Approaches to Kidney Regeneration, Pages 1039-1050, Valentina Benedetti, Barbara Imberti, Christodoulos Xinaris, Giuseppe Remuzzi
Chapter 76 - Nephron Progenitors, Pages 1053-1065, Ilaria Santeramo, Bettina Wilm, Patricia Murray
Chapter 77 - Urine Progenitor Cells for Potential Application in Renal Tissue Repair, Pages 1067-1073, Peng Li, Xiongbing Lu, Junhong Deng, Andrea Peloso, Yuanyuan Zhang
Chapter 78 - Endothelial Progenitor Cells in Kidney Disease, Pages 1075-1082, Michael S. Goligorsky
Chapter 79 - Mesenchymal Stromal Cells for Acute Renal Injury, Pages 1085-1095, Cinzia Rota, Serge Cedrick Mbiandjeu Toya, Marina Morigi
Chapter 80 - Amniotic Fluid Cells: Kidney Injury and Regeneration, Pages 1097-1107, Stefano Da Sacco, Astgik Petrosyan, Laura Perin
Chapter 81 - Renal Cells From Spermatogonial Germline Stem Cells for Protection Against Kidney Injury, Pages 1109-1115, Sharmila Fagoonee, Letizia De Chiara, Elvira Smeralda Famulari, Lorenzo Silengo, Fiorella Altruda
Chapter 82 - Kidney-on-a-Chip: Technologies for Studying Pharmacological and Therapeutic Approaches to Kidney Repair, Pages 1119-1133, Rosalinde Masereeuw, Jelle Vriend, Martijn J. Wilmer
Chapter 83 - Renal Replacement Devices, Pages 1135-1149, Christopher J. Pino, H. David Humes
Chapter 84 - Xenotransplantation and Kidney Regenerative Technology, Pages 1151-1161, Kazuhiko Yamada, Masayuki Tasaki, Adam Griesemar, Jigesh Shah
Chapter 85 - Embryonic Organoid Transplantation, Pages 1163-1166, Shinya Yokote, Takashi Yokoo
Chapter 86 - Lineage Reprogramming Toward Kidney Regeneration, Pages 1167-1175, Yun Xia, Nuria Montserrat, Josep M. Campistol, Juan Carlos Izpisua Belmonte
Chapter 87 - Regenerating Kidney Structure and Function: An Industry Perspective, Pages 1177-1187, Joydeep Basu, Timothy A. Bertram, John W. Ludlow
Index, Pages 1189-1225

Citation preview

KIDNEY TRANSPLANTATION, BIOENGINEERING, AND REGENERATION

KIDNEY TRANSPLANTATION, BIOENGINEERING, AND REGENERATION Kidney Transplantation in the Regenerative Medicine Era Edited by

GIUSEPPE ORLANDO Wake Forest University Health Sciences, Winston-Salem, NC, United States

GIUSEPPE REMUZZI IRCCS—Mario Negri Institute for Pharmacological Research, Bergamo, Italy Azienda Socio Sanitaria Territoriale Papa Giovanni XXIII, Bergamo, Italy University of Milan, Milan, Italy

DAVID F. WILLIAMS Wake Forest Institute of Regenerative Medicine, Winston-Salem, NC, United States

Academic Press is an imprint of Elsevier 125 London Wall, London EC2Y 5AS, United Kingdom 525 B Street, Suite 1800, San Diego, CA 92101-4495, United States 50 Hampshire Street, 5th Floor, Cambridge, MA 02139, United States The Boulevard, Langford Lane, Kidlington, Oxford OX5 1GB, United Kingdom Copyright r 2017 Elsevier Inc. All rights reserved. No part of this publication may be reproduced or transmitted in any form or by any means, electronic or mechanical, including photocopying, recording, or any information storage and retrieval system, without permission in writing from the publisher. Details on how to seek permission, further information about the Publisher’s permissions policies and our arrangements with organizations such as the Copyright Clearance Center and the Copyright Licensing Agency, can be found at our website: www.elsevier.com/permissions. This book and the individual contributions contained in it are protected under copyright by the Publisher (other than as may be noted herein). Notices Knowledge and best practice in this field are constantly changing. As new research and experience broaden our understanding, changes in research methods, professional practices, or medical treatment may become necessary. Practitioners and researchers must always rely on their own experience and knowledge in evaluating and using any information, methods, compounds, or experiments described herein. In using such information or methods they should be mindful of their own safety and the safety of others, including parties for whom they have a professional responsibility. To the fullest extent of the law, neither the Publisher nor the authors, contributors, or editors, assume any liability for any injury and/or damage to persons or property as a matter of products liability, negligence or otherwise, or from any use or operation of any methods, products, instructions, or ideas contained in the material herein. British Library Cataloguing-in-Publication Data A catalogue record for this book is available from the British Library Library of Congress Cataloging-in-Publication Data A catalog record for this book is available from the Library of Congress ISBN: 978-0-12-801734-0 For Information on all Academic Press publications visit our website at https://www.elsevier.com/books-and-journals

Publisher: Mica Haley Acquisition Editor: Mica Haley Editorial Project Manager: Fenton Coulthurst Production Project Manager: Sue Jakeman Designer: Miles Hitchen Typeset by MPS Limited, Chennai, India

List of Contributors

Michael Abecassis Northwestern University Feinberg School of Medicine, Chicago, IL, United States; Oakland University William Beaumont School of Medicine, Royal Oak, MI, United States

Valentina Benedetti IRCCS—Istituto di Farmacologiche “Mario Negri,” Bergamo, Italy

Ahmed Abed Tenon Hospital, Paris, France; Sorbonne University, Paris, France

Timothy A. Bertram NC, United States

University of Pavia, Pavia, Italy

Massimo Abelli

Mario Alessiani University of Pavia Medical School, IRCCS San Matteo Hospital Foundation, Pavia, Italy Aynaa Alsharidi Canada

University Health Network, Toronto, ON,

Fiorella Altruda

University of Turin, Torino, Italy University of Pisa, Pisa, Italy

Tor Vergata University Hospital, Rome, Tor Vergata University Hospital, Rome,

Maria L. Angelotti University of Florence, Florence, Italy Robert J. Applegate Wake Forest School of Medicine, Winston-Salem, NC, United States Anthony Atala Wake Forest Institute for Regenerative Medicine, Winston-Salem, NC, United States Chiara Attanasio Napoli, Italy

IIT@CRIB Istituto Italiano di Tecnologia,

David Axelrod Lahey Hospital Burlington, MA, United States

and

Medical

Center,

Yanik Bababekov Massachusetts General Hospital, Boston, MA, United States Joydeep Basu States

University of Barcelona, Barcelona, Spain

Joshua Blake Division of Nephrology and Program in Developmental and Stem Cell Biology, The Hospital for Sick Children, and Departments of Paediatrics and Physiology, University of Toronto, Toronto, Ontario, Canada Lauren Brasile

Andrea Angeletti Recanati Miller Transplant Institute and Immunology Institute, Icahn School of Medicine at Mount Sinai, New York, NY, United States

Roberta Angelico Italy

RegenMedTX LLC, Winston-Salem,

Ugo Boggi University of Pisa, Pisa, Italy

Gabriella Amorese

Mario Angelico Italy

Ariela Benigni IRCCS—Istituto di Ricerche Farmacologiche “Mario Negri,” Bergamo, Italy

Oriol Bestard

Sarwat Ahmad University of Maryland School of Medicine, Baltimore, MD, United States

Ricerche

Tengion, Inc., Winston-Salem, NC, United

Francesca Becherucci Meyer Hospital, Florence, Italy

Children’s

University

P. Matthew Belford Wake Forest School of Medicine, Winston-Salem, NC, United States Enrico Benedetti University of Illinois Hospital & Health Sciences System, Chicago, IL, United States

BREONICS, Inc., Albany, NY, United States

Jonathan S. Bromberg University of Maryland School of Medicine, Baltimore, MD, United States Sophie Brouard Universite´ de Nantes, Nantes, France; Institut de Transplantation Urologie Ne´phrologie (ITUN), Nantes, France; CIC Biotherapy, Nantes, France Matthew Brovold Wake Forest Institute for Regenerative Medicine, Winston-Salem, NC, United States George W. Burke III University of Miami Miller School of Medicine, Department of Surgery, Miami Transplant Institute, Miami, FL, United States; University of Miami Miller School of Medicine, Diabetes Research Institute, Miami, FL, United States Mirela Busic

Ministry of Health, Zagreb, Croatia

Zeeshan Butt Northwestern University Feinberg School of Medicine, Chicago, IL, United States Silvia Caddeo Politecnico di Torino, Polytechnic University of Turin, Turin, Italy Stefano Calzone Politecnico di University of Turin, Turin, Italy Josep M. Campistol Barcelona, Spain

Hospital

Irene Carmagnola Politecnico di University of Turin, Turin, Italy Fiona Carty Ireland

Torino, Clinic

of

Torino,

Polytechnic Barcelona, Polytechnic

National University of Ireland, Maynooth,

Diego Castanares-Zapatero St Luc University Hospital, Universite´ Catholique de Louvain (UCL), Brussels, Belgium

xvii

xviii

LIST OF CONTRIBUTORS

Weill Cornell Medical College, New

Christos E. Chadjichristos Tenon Hospital, Paris, France; Sorbonne University, Paris, France

Letizia De Chiara York, USA

Brooke E. Chambers University of Notre Dame, Notre Dame, IN, United States

Johannes W. De Fijter Leiden University Medical Center, Leiden, The Netherlands

Sindhu Chandran University of California, San Francisco, CA, United States

Gloria de la Rosa Madrid, Spain

Christos Chatziantoniou Tenon Hospital, Paris, France; Sorbonne University, Paris, France

Francis L. Delmonico MA, United States

Ashton Chen Wake Forest Baptist Health, Winston-Salem, NC, United States

Junhong Deng Wake Forest Institute for Regenerative Medicine, Winston-Salem, NC, United States; Guangzhou University, Guangdong, China

Linda Chen University of Miami Miller School of Medicine, Department of Surgery, Miami Transplant Institute, Miami, FL, United States Xiwu Chen States

Vanderbilt University, Nashville, TN, United

Valeria Chiono Politecnico di University of Turin, Turin, Italy

Torino,

Polytechnic

Vanderbilt University, Nashville, TN,

Manuel Chiusa United States

Gaetano Ciancio University of Miami Miller School of Medicine, Department of Surgery, Miami Transplant Institute, Miami, FL, United States

Corinne Deurdulian Keck School of Medicine of USC, Los Angeles, CA, United States Abritee Dhal Wake Forest Institute for Regenerative Medicine, Winston-Salem, NC, United States Paolo Dionigi University of Pavia Medical School, IRCCS San Matteo Hospital Foundation, Pavia, Italy Beatriz Domı´nguez-Gil Madrid, Spain Marek Drozdzik Poland

National Transplant Organization,

Pomeranian Medical University, Szczecin,

Jean-Claude Dussaule Tenon Hospital, Paris, France; Sorbonne University, Paris, France; Saint-Antoine Hospital, Paris, France

San Salvatore Hospital of L’Aquila, L’Aquila,

Lauren Edgar Wake Forest School of Medicine, WinstonSalem, NC, United States

Elisabeth Coll Spain

Torino,

National Transplant Organization, Madrid,

Christine Collienne St Luc University Hospital, Universite´ Catholique de Louvain (UCL), Brussels, Belgium Robert B. Colvin Massachusetts General Hospital, Boston, MA, United States Monica Cortinovis IRCCS Mario Negri Pharmacological Research, Bergamo, Italy A.

Harvard Medical School, Boston,

Polytechnic

Gianluca Ciardelli Politecnico di University of Turin, Turin, Italy Gino Coletti Italy

National Transplant Organization,

Benedict Cosimi Massachusetts Boston, MA, United States

Institute

General

for

Hospital,

Paolo Cravedi Icahn School of Medicine at Mount Sinai, New York, NY, United States; Recanati Miller Transplant Institute and Immunology Institute, Icahn School of Medicine at Mount Sinai, New York, NY, United States Giuseppe D’Amico University of Illinois Hospital & Health Sciences System, Chicago, IL, United States Stefano Da Sacco GOFARR laboratory for Organ Regenerative Research and Cell Therapeutics, Children‘s Hospital Los Angeles, Los Angeles, CA, United States; University of Southern California, Los Angeles, CA, United States Richard Danger Universite´ de Nantes, Nantes, France; Institut de Transplantation Urologie Ne´phrologie (ITUN), Nantes, France Jacques Dantal

Nantes University Hospital, Nantes, France

Alan J. Davidson New Zealand

The University of Auckland, Auckland,

Maria Francesca Egidi

University of Pisa, Pisa, Italy

Hany El Hennawy Wake Forest Baptist Medical Center, Winston-Salem, NC, United States Karen English Ireland

National University of Ireland, Maynooth,

Matthew J. Everly Terasaki Angeles, CA, United States

Research

Institute,

Los

Sharmila Fagoonee University of Turin, Torino, Italy; Institute of Biostructure and Bioimaging (IBB)-CNR, Torino, Italy Elvira Smeralda Famulari

University of Turin, Torino, Italy

Alan C. Farney Wake Forest Baptist Health, WinstonSalem, NC, United States Marina Figliuzzi IRCCS—Istituto di Farmacologiche Mario Negri, Bergamo, Italy Marialuisa Framarino-dei-Malatesta of Rome, Rome, Italy

Ricerche

University Sapienza

David E. Fumo University of Toledo Medical Center, Toledo, OH, United States; Alliance for Paired Donation, Perrysburg, OH, United States Elena Gagliardini IRCCS—Istituto di Farmacologiche “Mario Negri,” Bergamo, Italy

Ricerche

Lorenzo Gallon Northwestern University, Chicago, IL, United States Sanjay K. Gandhi Wake Forest School of Medicine, Winston-Salem, NC, United States

xix

LIST OF CONTRIBUTORS

Michael D. Gautreaux Wake Forest School of Medicine, Winston-Salem, NC, United States Anna Geraedts Netherlands

Maastricht University, Maastricht, The

Domenico Giannese

Deepali Kumar Canada

University of Pisa, Pisa, Italy

Pier C. Giulianotti University of Illinois Hospital & Health Sciences System, Chicago, IL, United States Michael S. Goligorsky New Valhalla, NY, United States

York

Medical

Kengo Kidokoro University of Southern California, Los Angeles, CA, United States; Kawasaki Medical School, Kurashiki, Japan

College,

Adam Griesemar Columbia University College of Physicians and Surgeons, New York, NY, United States Josep M. Grinyo´ University of Barcelona, Barcelona, Spain Angelika C. Gruessner SUNY Upstate Medical University, Syracuse, NY, United States; University of Arizona, Tucson, AZ, United States Rainer W.G. Gruessner SUNY Upstate Medical University, Syracuse, NY, United States; University of Arizona, Tucson, AZ, United States Bulang He Sir Charles Gairdner Hospital, Perth, Western Australia; The University of Western Australia, Perth, Australia

University Health Network, Toronto, ON,

Michael A. Kutcher Wake Forest School of Medicine, Winston-Salem, NC, United States Quirino Lai

University of L’Aquila, L’Aquila, Italy

Pierre-Franc¸ois Laterre St Luc University Hospital, Universite´ Catholique de Louvain (UCL), Brussels, Belgium Ce´leste Lebbe´ Public Assistance - Paris Hospitals, Paris, France; Paris Diderot University, Paris, France; SaintLouis Hospital, Paris, France Christophe Legendre Public Assistance - Paris Hospitals, Paris, France; Paris Descartes University, Paris, France; Necker Hospital, Paris, France Rachel Lennon University of Manchester, Manchester, United Kingdom; Manchester Academic Health Science Centre (MAHSC), Manchester, United Kingdom

Eliot Heher Massachusetts General Hospital, Boston, MA, United States

Peng Li Wake Forest Institute for Regenerative Medicine, Winston-Salem, NC, United States; Nantong University, Nantong, China; The Neural Regeneration Co-innovation Center of Jiangsu Province, Nantong, China

Bing Ho Northwestern University Feinberg School of Medicine, Chicago, IL, United States

Jen-Jar Lin Wake Forest Baptist Health, Winston-Salem, NC, United States

Sarah A. Hosgood University of Cambridge, Cambridge, United Kingdom

Melissa H. Little Murdoch Children’s Research Institute, Melbourne, VIC, Australia; University of Melbourne, Melbourne, VIC, Australia

Kiyohiko Hotta Massachusetts General Hospital, Boston, MA, United States Atul Humar Canada

University Health Network, Toronto, ON,

H. David Humes Innovative BioTherapies, Inc, Ann Arbor, MI, United States; University of Michigan Medical School, Ann Arbor, MI, United States Giuseppe Iaria Italy

Tor Vergata University Hospital, Rome,

Barbara Imberti IRCCS—Istituto di Farmacologiche “Mario Negri,” Bergamo, Italy

Ricerche

Juan Carlos Izpisua Belmonte Salk Institute for Biological Studies, La Jolla, CA, United States Ina Jochmans Belgium

University

Hospitals

Leuven,

Leuven,

Ravi Katari Wake Forest School of Medicine, WinstonSalem, NC, United States Panagiotis Kavvadas Tenon Hospital, Paris, France Tatsuo Kawai Massachusetts General Hospital, Boston, MA, United States Carlos Kengla Wake Forest Baptist Winston-Salem, NC, United States

Medical

Center,

Xiongbing Lu Wake Forest Institute for Regenerative Medicine, Winston-Salem, NC, United States; Nanchang University, Nanchang, China John W. Ludlow Tengion, Inc., Winston-Salem, NC, United States Beatriz Mahı´llo Spain

National Transplant Organization, Madrid,

Rosalinde Masereeuw Utrecht University, Utrecht, The Netherlands Rafael Matesanz National Madrid, Spain Mirjana S. Matovinovic Zagreb, Croatia

Transplant

Merkur

Benedetta Mazzinghi Meyer Hospital, Florence, Italy

Organization,

University

Children’s

Hospital, University

Serge Cedrick Mbiandjeu Toya IRCCS Istituto di Ricerche Farmacologiche Mario Negri, Centro Anna Maria Astori, Science and Technology Park Kilometro Rosso, Bergamo, Italy Scott McEwen Case Western Reserve University School of Medicine, Cleveland, OH, United States Fabio Melandro Sapienza University of Rome, Rome, Italy

Tristan Keys Wake Forest University School of Medicine, Winston-Salem, NC, United States

Loredana Melchiorri San Salvatore Hospital of L’Aquila, L’Aquila, Italy

Amritha Kidiyoor Wake Forest Baptist Medical Center, Winston-Salem, NC, United States

Madhav C. Menon Icahn School of Medicine at Mount Sinai, New York, NY, United States

xx

LIST OF CONTRIBUTORS

Majid Mirzazadeh Wake Forest University School of Medicine, Winston-Salem, NC, United States Samantha Montag Oakland University William Beaumont School of Medicine, Royal Oak, MI, United States Robert A. Montgomery NYU Langone Transplant Institute, New York, NY, United States Virginie Montiel St Luc University Hospital, Universite´ Catholique de Louvain (UCL), Brussels, Belgium Nuria Montserrat Institute for Bioengineering of Catalonia (IBEC), Barcelona, Spain Marina Morigi IRCCS Istituto di Ricerche Farmacologiche Mario Negri, Centro Anna Maria Astori, Science and Technology Park Kilometro Rosso, Bergamo, Italy

Laura Perin GOFARR laboratory for Organ Regenerative Research and Cell Therapeutics, Children‘s Hospital Los Angeles, Los Angeles, CA, United States; University of Southern California, Los Angeles, CA, United States University of Pisa, Pisa, Italy

Vittorio Perrone

Paul Persad Wake Forest School of Medicine, WinstonSalem, NC, United States Ja´nos Peti-Peterdi University of Southern California, Los Angeles, CA, United States Astgik Petrosyan GOFARR laboratory for Organ Regenerative Research and Cell Therapeutics, Children‘s Hospital Los Angeles, Los Angeles, CA, United States; University of Southern California, Los Angeles, CA, United States Andrea Pietrabissa

University of Pavia, Pavia, Italy

Christian C. Morrill Utah State University, Logan, UT, United States; Wake Forest Baptist Health, WinstonSalem, NC, United States

Christopher J. Pino Innovative BioTherapies, Inc, Ann Arbor, MI, United States

Michel Mourad St Luc University Hospital, Universite´ Catholique de Louvain (UCL), Brussels, Belgium

Jacques Pirenne University Hospitals Leuven, Leuven, Belgium

Sean V. Murphy Wake Forest Baptist Medical Center, Winston-Salem, NC, United States

Francesco Pisani

Patricia Murray Kingdom

University of Liverpool, Liverpool, United

University of L’Aquila, L’Aquila, Italy

Rutger J. Ploeg University of Oxford, Oxford, United Kingdom University of La Laguna, Tenerife, Spain

Esteban Porrini

Tiziana Nardo Politecnico di University of Turin, Turin, Italy

Torino,

Polytechnic

Ambra Pozzi Vanderbilt University, Nashville, TN, United States

Paolo A. Netti IIT@CRIB Istituto Italiano di Tecnologia, Napoli, Italy; CRIB, Universita´ degli studi di Napoli Federico II, Napoli, Italy

Alberto Pugliese Diabetes Research Institute, Department of Medicine, Division of Diabetes, Endocrinology and Metabolism, Department of Microbiology and Immunology, Miami, FL, United States

Mark Nguyen University of California, San Francisco, CA, United States Michael L. Nicholson University Cambridge, United Kingdom John M. O’Callaghan Kingdom

of

Cambridge,

University of Oxford, Oxford, United

Linda Ohler George Washington University Transplant Institute, Washington, DC, United States Giuseppe Orlando Wake Forest University Health Sciences, Winston-Salem, NC, United States; Wake Forest Baptist Health, Winston-Salem, NC, United States; Wake Forest School of Medicine, Winston-Salem, NC, United States Kenji Osafune Center for iPS Application (CiRA), Kyoto, Japan

Cell

Research

and

Tetsu Oura Massachusetts General Hospital, Boston, MA, United States Anna Peired

University of Florence, Florence, Italy

Andrea Peloso Wake Forest Institute for Regenerative Medicine, Winston-Salem, NC, United States; University of Pavia, Pavia, Italy Zhenzhen Peng New Zealand

The University of Auckland, Auckland,

Norberto Perico IRCCS Mario Negri Pharmacological Research, Bergamo, Italy

Institute

for

Luigi Pugliese

University of Pavia, Pavia, Italy

Ton J. Rabelink Leiden University Medical Center, Leiden, The Netherlands Teresa Rampino

University of Pavia, Pavia, Italy

Michael A. Rees University of Toledo Medical Center, Toledo, OH, United States; Alliance for Paired Donation, Perrysburg, OH, United States Marlies E.J. Reinders Leiden University Medical Center, Leiden, The Netherlands Andrea Remuzzi IRCCS—Istituto di Ricerche Farmacologiche Mario Negri, Bergamo, Italy; University of Bergamo, Bergamo, Italy Giuseppe Remuzzi IRCCS Mario Negri Institute for Pharmacological Research, Bergamo, Italy; Azienda Socio Sanitaria Territoriale Papa Giovanni XXIII, Bergamo, Italy; University of Milan, Milan, Italy Anne Riquier-Brison University of Southern California, Los Angeles, CA, United States Raquel G. Roca University of Illinois Hospital & Health Sciences System, Chicago, IL, United States Jeffrey Rogers Wake Forest Baptist Winston-Salem, NC, United States Paola Romagnani

Medical

Center,

University of Florence, Florence, Italy

xxi

LIST OF CONTRIBUTORS

Ivy A. Rosales Massachusetts General Hospital, Boston, MA, United States

Bart Stubenitsky St. Antonius Ziekenhuis, Nieuwegein, The Netherlands

Norman D. Rosenblum Division of Nephrology and Program in Developmental and Stem Cell Biology, The Hospital for Sick Children, and Departments of Paediatrics and Physiology, University of Toronto, Toronto, Ontario, Canada

Riccardo Tamburrini Wake Forest School of Medicine, Winston-Salem, NC, United States

Cinzia Rota IRCCS Istituto di Ricerche Farmacologiche Mario Negri, Centro Anna Maria Astori, Science and Technology Park Kilometro Rosso, Bergamo, Italy

Qizhi Tang University of California, San Francisco, San Francisco, CA, United States Ekamol Tantissattamo Oakland University William Beaumont School of Medicine, Royal Oak, MI, United States

Piero Ruggenenti Mario Negri Institute for Pharmacological Research, Bergamo, Italy; Azienda Socio Sanitaria Territoriale Papa Giovanni XXIII, Bergamo, Italy

Masayuki Tasaki Massachusetts General Hospital, Boston, MA, United States

Francesca Ruini Politecnico di University of Turin, Turin, Italy

Elena Ticozzelli

Torino,

Polytechnic

Junichiro Sageshima University of California, Davis, Department of Surgery, Division of Transplantation, Sacramento, CA, United States; Wake Forest Baptist Health, Winston-Salem, NC, United States Fadi El Salem Icahn School of Medicine at Mount Sinai, New York, NY, United States Shaifali Sandal QC, Canada

McGill University Health Centre, Montreal,

Veronika Sander New Zealand

The University of Auckland, Auckland,

Ilaria Santeramo University United Kingdom

of

Liverpool,

Liverpool,

Renato M. Santos Wake Forest School of Medicine, Winston-Salem, NC, United States Minnie Sarwal University of California, San Francisco, CA, United States Jigesh Shah Massachusetts General Hospital, Boston, MA, United States Aneesha A. Shetty Northwestern University Feinberg School of Medicine, Chicago, IL, United States Lorenzo Silengo University of Turin, Torino, Italy Eric Siskind University of Maryland School of Medicine, Baltimore, MD, United States Anton Skaro Northwestern University Feinberg School of Medicine, Chicago, IL, United States; Oakland University William Beaumont School of Medicine, Royal Oak, MI, United States Renaud Snanoudj Paris, France

Public Assistance - Paris Hospitals,

Shay Soker Wake Forest Institute for Regenerative Medicine, Winston-Salem, NC, United States Andrew M. South Wake Forest Baptist Health, WinstonSalem, NC, United States Goce Spasovski University Ss Cyril and Methodius, Skopje, Macedonia Robert J. Stratta Wake Forest Baptist Medical Center, Winston-Salem, NC, United States Charles Strom Tufts University School of Medicine, Boston, MA, United States

Hisham Tchelepi Keck School of Medicine of USC, Los Angeles, CA, United States University of Pavia, Pavia, Italy

Opas Traitanon Northwestern University, Chicago, IL, United States; Thammasart University Hospital, Pathumthani, Thailand Matias Trillini IRCCS—Mario Negri Pharmacological Research, Bergamo, Italy

Institute

for

Ivo Tzvetanov University of Illinois Hospital & Health Sciences System, Chicago, IL, United States Marı´a O. Valentı´n National Transplant Organization, Madrid, Spain Flavio Vincenti University of California, San Francisco, CA, United States Fabio Vistoli University of Pisa, Pisa, Italy Jelle Vriend Radboud Institute for Molecular Life Sciences, Radboud University Medical Center, Nijmegen, The Netherlands Stephen J. Walker Wake Forest Institute for Regenerative Medicine, Winston-Salem, NC, United States; Wake Forest University Health Sciences, Winston-Salem, NC, United States Jason A. Wertheim Northwestern University Feinberg School of Medicine, Chicago, IL, United States; Jesse Brown VA Medical Center, Chicago, IL, United States David F. Williams Wake Forest Institute of Regenerative Medicine, Winston-Salem, NC, United States Bettina Wilm University of Liverpool, Liverpool, United Kingdom Martijn J. Wilmer Radboud Institute for Molecular Life Sciences, Radboud University Medical Center, Nijmegen, The Netherlands Rebecca A. Wingert University of Notre Dame, Notre Dame, IN, United States Xavier Wittebole St Luc University Hospital, Universite´ Catholique de Louvain (UCL), Brussels, Belgium Yun Xia Salk Institute for Biological Studies, La Jolla, CA, United States; Nanyang Technological University, Singapore Christodoulos Xinaris IRCCS—Istituto di Farmacologiche “Mario Negri,” Bergamo, Italy

Ricerche

xxii

LIST OF CONTRIBUTORS

Kazuhiko Yamada Columbia University College of Physicians and Surgeons, New York, NY, United States

Yuanyuan Zhang Wake Forest Institute for Regenerative Medicine, Winston-Salem, NC, United States

Takashi Yokoo The Jikei University School of Medicine, Tokyo, Japan

David X. Zhao Wake Forest School of Medicine, WinstonSalem, NC, United States

Shinya Yokote The Jikei University School of Medicine, Tokyo, Japan Maarten L. Zandvliet Leiden University Medical Center, Leiden, The Netherlands

Lihui Zhao Northwestern University Feinberg School of Medicine, Chicago, IL, United States; Oakland University William Beaumont School of Medicine, Royal Oak, MI, United States

Roy Zent Vanderbilt University, Nashville, TN, United States

Susan Y. Zhao Wake Forest Institute for Regenerative Medicine, Winston-Salem, NC, United States

Biographies

Giuseppe Orlando Dr. Orlando is an abdominal organ transplant surgeon scientist. His main achievements have been in the field of steroid-free immunosuppression, immunosuppression minimization, and clinical tolerance after liver transplantation. More recently, his research interest has switched towards the bioengineering, regeneration, and repair of transplantable organs. He has been successful in developing clinically relevant models for organ bioengineering consisting of scaffolds produced through the decellularization of porcine or human organs. He has been instrumental in popularizing the concept of recycling discarded human organs for tissue engineering purposes and is currently proposing the use of discarded human kidneys and pancreas as platforms for kidney and endocrine pancreas bioengineering and regeneration. The ultimate goal of these investigations is to provide “organson-demand” through the development of technologies that will eventually address the two most urgent needs in organ transplantation, namely, the identification of a new and potentially inexhaustible source of organs and immunosuppression-free transplantation. His literature output aims at merging the field of organ transplantation to regenerative medicine, and supports the idea that organ transplantation should transition towards a regenerative medicine-focused type of research with organ bioengineering and regeneration becoming the new Holy Grail for modern transplantation. This idea generates from the firm belief that no other field of health science has as keen an interest in investing in regenerative medicine as does organ transplantation; for our future, more than the future of any other field of health sciences, is being shaped by regenerative medicine. He has authored more than 200 research papers, review articles, book chapters, and books. He regularly serves as reviewer and board member or editor for numerous journals, as well as lecturer and moderator at national and international transplant and regenerative medicine conferences and symposia. This book follows his first book, Regenerative Medicine Technologies Applied to Organ Transplantation. He is the recipient of the 2017 Rising Star in Transplantation Award bestowed by the American Society of Transplant Surgeons. Giuseppe Remuzzi Professor of Nephrology, Director of the Department of Medicine of the Ospedali Riuniti di Bergamo (Papa Giovanni XXIII Hospital), Italy and Director of the Division of Nephrology and Dialysis of the same hospital. He also directs the Negri Bergamo Laboratories of the “Mario Negri” Institute for Pharmacological Research, a group of basic scientists and clinicians devoted to the study of human renal diseases and their corresponding animal models from the perspective of pathophysiology and therapeutic intervention. He touched major advances in many areas of nephrology. For example, his studies have led to new insights into many disorders, including the interactions between platelets and endothelium, pathophysiology of glomerular diseases, and the factors that influence the progressive loss of kidney function. Work focused on improving the outlook for patients with end stage renal disease. Giuseppe Remuzzi pays tribute to the work of pioneers such as Barry Brenner, who delved deep into the processes behind glomerular function and their possible reversibility. Early work on the use of angiotensin-converting enzyme inhibitors to slow the decline of glomerular filtration rates proved dialysis was avoidable, not inevitable. Studies on immunologic mechanisms that influence the survival of transplanted organs, understanding of immunologic tolerance in the disorders that are linked to autoimmunity, and finally, genetic diseases of the kidney have also been areas of investigation. Concerned by kidney donation shortages and deploring the current practice of discarding suboptimal donor kidneys, his team has shown that transplanting

xxiii

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BIOGRAPHIES

such kidneys in pairs is feasible and has set up an international effort to validate this approach. Giuseppe Remuzzi is investigating the kidney’s ability to regenerate itself. He has authored and coauthored more than 1201 scientific articles, reviews, and monographs and serves on editorial boards of numerous journals. He is member of the International Advisory Board of The Lancet and was an Editorial Board member of the New England Journal of Medicine from 1995 to 2013. During his professional career he received the International Society of Nephrology (ISN) Jean Hamburger Award (World Congress of Nephrology (WCN) 2005, Singapore), the John P. Peters Award (American Society of Nephrology (ASN) 2007, San Francisco), and the ISN AMGEN Award (WCN 2011, Vancouver). In November 2011, he won the Third Edition of the International Award “Luis Hernando” assigned by the In˜igo Alvarez de Toledo Renal Foundation (FRIAT) in Madrid, Spain. Since June 2013 he has been President of the ISN for the period 2013 15. David F. Williams Professor Williams has had 48 years experience in biomaterials, medical device and tissue engineering, working mostly at the University of Liverpool, United Kingdom, where he was ultimately Professor of Biomaterials and Biomedical Engineering, Director of the UK Centre for Tissue Engineering, and Senior Pro-Vice Chancellor of the University. During his career he has published over 30 books and 400 papers; his latest book, Essential Biomaterials Science, was published by Cambridge University Press in June 2014. He was Editor-in-Chief of Biomaterials, the world’s leading journal in this field between 2000 and 2014. He has received the major awards from the United States, European, and Indian societies of biomaterials including the Founders Award of the US Society for Biomaterials in 2007, and received the prestigious Acta Biomaterialia Gold Medal in 2012. In 1999 he was elected as a Fellow of the Royal Academy of Engineering and is a Foreign Fellow of the Indian National Academy of Engineering and a Fellow of the American Institute of Medical and Biological Engineering, all in recognition of his contributions to engineering in medicine. He was global President of the Tissue Engineering & Regenerative Medicine International Society from 2013 to 2015. He was a scientific advisor to several divisions of the European Commission during the 1990s and wrote several opinions on which European actions were taken. He has, over the last 20 years, given evidence in several major product liability and patent legal cases in the United States, Europe, and Australia. Professor Williams left the University of Liverpool in 2007. While retaining the title of Emeritus Professor at Liverpool, he is currently Professor and Director of International Affairs, Wake Forest Institute of Regenerative Medicine, North Carolina, USA. In addition, he is a Visiting Professor in the Christiaan Barnard Department of Cardiothoracic Surgery, Cape Town, South Africa; and a Guest Professor at Tsinghua University, Beijing, an Advisory Professor at Shanghai Jiao Tong University and Honorary Professor at Sichuan University, Chengdu, in China. He is Visiting Chair Professor of Biomedical Materials, Taipei Medical University, Taiwan. In Cape Town, along with Professor Peter Zilla, the current Christiaan Barnard Professor of Surgery, he has formed a company that will produce low-cost but high-technology medical devices that can be used with minimally invasive procedures to treat young adults in Subsaharan Africa who are suffering from rheumatic heart disease but currently have no therapies available to them.

Kidney Transplantation, Bioengineering, and Regeneration INTRODUCTION End stage organ disease has long provided significant challenges for the medical profession. Treatment options vary from organ to organ, largely depending on the principal functions of the organs themselves, and in many situations major developments and successes have been achieved over recent decades. Nevertheless, the challenges persist and we are far from a position in which we can be comfortable in our provision of clinically effective and socially acceptable procedures for the majority of patients presenting with these diseases. Irrespective of the organ, the potential treatment options include transplantation, mechanical or physical assist devices, and organ regeneration. Thus, with end stage heart failure, there is the technically very feasible heart transplantation; the expensive, demanding, but quite successful ventricular assist or total artificial heart; or the experimental stem cell therapy for myocardial regeneration. With end stage renal disease, there again is the transplantation option, with various forms of dialysis, the potential for bioartificial kidneys, and scaffold-based tissue engineering of the kidney. This book attempts to place these transplantation, bioengineering, and regenerative approaches for the kidney into perspective. The fundamental driving forces behind the search for improved options are the worldwide mismatch between the number of patients requiring a transplant and the number of donor kidneys available; the lack of improvement over several decades in the outcomes of dialysis therapies; and the increasing number of elderly patients with end stage kidney disease, and possibly comorbidities, who are less receptive to either conventional transplantation or dialysis. The treatment of kidney disease involves a great deal more than removal of waste products from blood by some filtration method, but even more than that, the paradigms of renal replacement therapies have to subsume a wide variety of economic, ethical, and societal issues as well as biological, physiological, pharmacological, clinical, and bioengineering challenges. We have attempted to bring all of these relevant aspects together in this one volume. As Editors, we first of all have to thank the many contributors who accepted our invitations eagerly and produced an excellent collection of authoritative chapters. We also express our appreciation to the staff of Elsevier, initially Jeff Rossetti and then Fenton Coulthurst for overseeing the extensive task of coordinating the publication of the book. Giuseppe Orlando, Giuseppe Remuzzi and David F. Williams

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C H A P T E R

1 Epidemiology of End-Stage Renal Failure: The Burden of Kidney Diseases to Global Health Matias Trillini1, Norberto Perico1 and Giuseppe Remuzzi1,2,3 1

IRCCS—Mario Negri Institute for Pharmacological Research, Bergamo, Italy 2Azienda Socio Sanitaria Territoriale Papa Giovanni XXIII, Bergamo, Italy 3University of Milan, Milan, Italy

1.1 THE GLOBAL BURDEN OF NONCOMMUNICABLE DISEASES Over the last century, communicable diseases have been the focus of global health strategies because they easily spread beyond national borders and have been extraordinarily unequal across the globe. These diseases threatened the lives of millions of people particularly in low- and middle-income countries with underdeveloped health care systems.1,2 Thus targeting these diseases was essential to resolve major global health issues. Lately, improvements in sanitation, prevention strategies, and medical science have helped to markedly reduce infectious disease burden and related causes of death.1 3 By 1990, noncommunicable diseases (NCDs) had already overtaken communicable diseases as the leading cause of mortality and disability worldwide.4 7 In 2008, NCDs accounted for nearly 36 million of the total global deaths (57 million).7,8 There is a consensus among public health professionals and policymakers that this epidemiological shift towards NCDs is globally established and it will continue to grow in the coming decades.1,4 12 Four main NCDs make the largest contribution to the current global burden of NCDs’ mortality.5,7 11 Major cardiovascular diseases take the largest proportion, with more than 17 million deaths/year worldwide, followed by cancers, respiratory diseases, and diabetes mellitus.5,8,10,13 The Global Burden of Disease study reported that in 2013 cardiovascular deaths accounted for one-third of total deaths globally, leading with ischemic heart disease, ischemic stroke, and hemorrhagic stroke.6 Even though declines in age-specific death rates from cardiovascular diseases represented the most common epidemiologic pattern worldwide, predictions for the future are based on aging and growth of populations that clearly outweigh improvements in treatment and prevention.14 Cancer is the second largest contributor to deaths worldwide with more than 8 million deaths in 2013.15 Morbidity and mortality due to cancer is predicted to increase in the next few decades due to the high prevalence of certain risk factors and because of improving survival of the elderly. Though some cancers have falling age-standardized trends, their decreasing rates do not significantly reduce the number of global absolute cases, which determine greater challenges in low resources settings, particularly in countries in transition.15,16 Chronic respiratory diseases affect hundreds of millions of people worldwide with major impact in morbidity and mortality. More than 4 million people die prematurely from chronic respiratory diseases each year.17 Asthma and chronic obstructive pulmonary disease (COPD) are associated with occupational exposures or air pollution and other conditions; however, cigarette active and passive smoking is by far the most important risk factor for COPD.18,19 Smoking impact has been generally decreasing and tobacco-attributable deaths

Kidney Transplantation, Bioengineering, and Regeneration. DOI: http://dx.doi.org/10.1016/B978-0-12-801734-0.00001-1

5

© 2017 Elsevier Inc. All rights reserved.

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1. THE BURDEN OF KIDNEY DISEASES AND END-STAGE RENAL FAILURE

are projected to decline by almost 10% between 2002 and 2030 in high-income countries, but to double to approximately 7 million in low- and middle-income countries, which have the highest incidence of smokers among young men.8,12 Diabetes mellitus represents a well-known established pandemic. Its prevalence has been increasing steadily in the last few decades both in developed and developing countries.20,21 From 1990 to 2013, worldwide mortality and years lived with disability due to diabetes significantly increased when both all ages and age-standardized rates were considered.6,22 Uniform epidemiological research has forecasted that diabetes mellitus prevalence is expected to at least double by the 2030s with a maximum increase in India that will reach almost 80 million diabetics followed by China and the United States.5,12,20,23,24 Overall, the rising trends in NCDs will have a significant economic impact with an outstanding projected burden that will surpass US$20 trillion by 2030.25 27 Therefore, NCDs are among the most severe threats to global economic development.5 Although recent attention has focused on the epidemiological weight of these aforementioned main NCDs, most of the global NCDs burden (55%) arises from other NCDs that have been largely neglected and, in many cases, cause more health burden through chronic disability rather than premature death. Among the most important are musculoskeletal disorders, depression, substance use disorders, liver cirrhosis, congenital diseases, hemoglobinopathies, road injuries, and chronic kidney disease (CKD).28 A large body of literature investigating CKD and particularly end-stage renal disease (ESRD) has been published in recent years. Although there is definitive evidence of the impact of kidney disease on the poor global health outcomes, it is still not recognized as a separate target.29 This chapter focuses on the epidemiology of CKD and ESRD, and their impact as key determinant contributors to the poor global health outcomes.

1.2 GLOBAL INCIDENCE AND PREVALENCE OF CHRONIC KIDNEY DISEASE AND END-STAGE RENAL DISEASE CKD describes a permanent state of progressive loss of kidney function and/or kidney damage that develops over a period of months to years. ESRD, the last stage of CKD, is the irreversible deterioration of renal function to an extent that is incompatible with life without renal replacement therapy (RRT)30 in the form of dialysis or transplantation.31 Because of several conditions determined by the status of the disease itself and characteristics of the population studied, there is uncertainty on how to measure CKD incidence and prevalence in the general population.32 34 Epidemiological studies on CKD have three major limitations: lack of agreement on formulas to estimate incidence and prevalence in the early stages of the disease, consideration of CKD as solely defined by estimated glomerular filtration rate (eGFR) not including albuminuria, and uneven and scarce data on advanced CKD stages because data on ESRD is retrieved from registries that only consider population on RRT.5,31,35,36 Worldwide applicability of screening approaches undertaken in developed countries is unclear.37 A recent systematic literature review of the methodology used in reporting CKD prevalence in Europe found considerable drawbacks in epidemiological studies. Among the most important limitations were significant diversity in population sample selection methods, lack of description of methods used to assess the condition, and heterogeneity on reporting results.38

1.2.1 Population Rate and Time Trends Not all CKD is represented by the ESRD population. Patients on RRT depict only the tip of the iceberg whereas the greater proportion of individuals in the CKD stages 1 4 account for approximately 90% of the CKD population. Thus, a proper assumption of the global burden of pre-RRT CKD disease is difficult to obtain and only rough estimates exist.5,31,37,39,40 In the last few decades, large efforts have been made to improve detection of early, advanced, and terminal renal disease worldwide.41 There is current consensus among epidemiologists and nephrologists that both nondialysis and dialysis-dependent CKD are relatively common in the general population.42,43 CKD incidence and prevalence are not equally distributed worldwide and a large gap still stands regarding epidemiology among the poor, minority populations, and developing countries, where projected trends have the fastest raises because

I. KIDNEY TRANSPLANTATION

1.2 GLOBAL INCIDENCE AND PREVALENCE OF CHRONIC KIDNEY DISEASE AND END-STAGE RENAL DISEASE

7

of the high population and/or rapid growth of the elderly.29,36,37,44 46 Prevalence seems to be increasing particularly in older individuals partly because of an increasing prevalence of diabetes and hypertension.32 On the other hand, unlike previous massive increasing trends in a frail population that were the standard for almost three decades, since 2012, a flattened pattern has been present on incidence rates of CKD in the United States and other developed countries.29,39 However, although incidence rates in the United States leveled off and showed a fewer total number of new incident cases for the years 2011 and 2012 when compared to 2010, the prevalence of ESRD still continues to rise.47 In 2012, in the United States, 402,514 ESRD patients were receiving hemodialysis therapy, 40,605 were being treated with peritoneal dialysis, and 175,978 had a functioning graft.47 CKD prevalence in the rest of the world has also achieved epidemic proportions, reaching 10% 13% of the populations in Norway, Taiwan, Iran, Japan, South Korea, China, Canada, and India.39,48 As for ESRD, worldwide prevalence ranges from 2447 cases pmp (patients per million population) in Taiwan to 10 cases pmp in Nigeria. In Latin America the ESRD prevalence ranges from 1019 pmp in Uruguay to 34 pmp in Honduras, a difference that may also reflect the relationship with the gross national product.5 Much less is known in Africa, with the highest prevalence of ESRD in Tunisia (713 pmp) and Egypt (669 pmp). A recent study showed that the prevalence and incidence of global maintenance dialysis due to ESRD have increased 1.7 and 2.1 times from 1990 to 2010, respectively.49 Thus, CKD population varies enormously around the globe and has been expanding at a rate of 7% per year with a current worldwide population of B2 million in ESRD who are alive only because they have access to one form or another of RRT.5,29,49 However, this population represents less than 10% of those who need it.

1.2.2 Age and Gender Declines in fertility, but mainly health system improvements, have increased average life expectancy and boosted the global phenomenon of population aging.50 Progressive nephrosclerosis due to the aging process might partially contribute to CKD development; however, the GFR decline associated with normal aging is not only explained by histological changes, so the manner in which age interacts with other risk factors for CKD has yet to be fully elucidated.51 54 Nevertheless, so far the prevalence of CKD has been considered strongly age-related, even within the same study population. A systematic review of studies performed in the United States, Europe, and Asia that considered middle- and old-aged subjects ($30 years old) showed that CKD prevalence could vary from 1.5% to 43.3%.42 CKD prevalence stages 1 4 assessed with eGFR and albuminuria in US population ranges from 6.44% among 20- to 39-year-old young adults to almost 50% in subjects aged 70 years and older.55 Other population-based studies and national cohort surveys performed in developed countries found that CKD prevalence invariably increased when the eldest subgroups were considered, even if the overall number could vary among them.56 59 Data concerning age-related CKD epidemiology in developing countries are scarce and narrowed by the scant health systems.60 A population-based study in Thailand found age-specific CKD prevalence near 50% for those aged 70 or older, results that are similar to industrialized countries and other Asian population-based studies.61 In sub-Saharan Africa, CKD is more prevalent among young adults.62 Results from a cross-sectional pilot study in Democratic Republic of Congo also showed higher CKD prevalence in young adults when compared with developed countries.63 Little is known about the clinical course and outcome of older individuals with CKD. Available data is inconclusive and current knowledge does not allow clinicians to reliably distinguish between those whose CKD will and will not progress.64 A prospective cohort study in the United States, which included more than 1000 adults aged 65 and older and with a median follow-up of more than 9 years, found that patients with CKD (GFR ,60 mL/minute/1.73 m2) were 13-fold and 6-fold more likely to die from any cause and from CV disease related causes than from ESRD, respectively.65 Since the competing risk for death at older ages is high, the majority of older adults with CKD will not develop ESRD. Some authors suggest that because of existing considerable heterogeneity in outcomes among patients of different ages and similar levels of eGFR, a uniform CKD stage-based approach promoted by the majority of practice guidelines could not be adequate.66,67 In contrast, other investigators found that, regardless of age, the presence of CKD increased mortality risk and consequently supported the use of current guidelines.68 Thus, further studies are needed in order to understand age-related changes in kidney function and clinical outcomes, which in turn will allow proper strategies to screen CKD and target therapies objectively. Although few specific studies analyzed the role of gender on CKD, differences between men and women have been published by several authors.69 71 In the United States, the National Health and Nutrition Evaluation

I. KIDNEY TRANSPLANTATION

8

1. THE BURDEN OF KIDNEY DISEASES AND END-STAGE RENAL FAILURE

Survey (NHANES, 1999 2012) has shown that overall age-adjusted CKD (stages 1 4) prevalence in adults changes from 14.8% to 13.5% and 15.9%, for males and females respectively, when gender was considered.55 A Norwegian 10-year population-based study found that CKD stage 3 was more prevalent in women than in men; still, male gender had a negative effect on both decline on GFR and hazard of renal failure.72 Two community-based studies, one in Taiwan in an area with a high prevalence of dialysis and the other in China including rural and urban populations, found that independent of age the prevalence of CKD was higher in women than in men.46,51 When ESRD is considered, Japanese men have higher dialysis incidence and prevalence than women and are also younger when started on RRT programs.69 Also in the United States, the adjusted incidence rate of ESRD is higher in men than in women (57.8% vs 42.2%, respectively).47 A metaanalysis of more than 10,000 patients with nondiabetic renal disease matched for blood and lipid levels showed that men had faster renal disease progression when compared to women.73 Although the “protective” effect favoring women has been a matter of debate, most of the evidence in animal and human studies confirmed the negative trend in renal disease progression in the male gender.72,74 Gender-based genetic variability has been linked to differences in blood pressure in both black and white individuals.75 Other authors suggested that as well as systolic blood pressure, hormonal status, lifestyle, and/or the prevalence of metabolic syndrome may also contribute to the gender difference in the age-related decline in GFR.70,74

1.2.3 Race/Ethnicity and Minorities Though they are distinct, race and ethnicity are rarely detached concepts in epidemiology and clinical practice. Because this overlap extends to medical literature, we consider race and ethnicity as a unique term for their assessment as risk factors that foster CKD progression and affect the burden of ESRD. Although ESRD incidence and prevalence have increasing trends in almost all races, they have the highest rates in racial/ethnic minorities in most epidemiological studies.76 Despite having similar rates for CKD in early stages, ESRD rates for racial/ethnic minorities are 1.5 4.0 times those of age-adjusted for whites.77 In the United States, African Americans have the highest incidence adjusted rate of ESRD, almost triple that of other races; among them, those living in the Southeast have a higher-than-average risk for developing ESRD. The higher prevalence of diabetes in African Americans may explain these differences. Hispanics, Asians, and Native Americans also have increased incidence rate trends of ESRD when compared to whites. Nevertheless, in recent years, incidence trends of ESRD across races in the United States have shown a reduction pattern or at least plateaued, except for Asian ethnicity, which has continued to increase.78,79 Other well-known minorities, such as Pacific Islanders, have a higher incidence of treated ESRD compared to Caucasians.80 A multiethnic population-based study in Singapore including three major Asian ethnic groups— Chinese, Malaysians, and Indians—found that the overall prevalence of CKD was higher in Malaysians compared to Indians and Chinese. Also, overall prevalence of CKD was higher than other Asians and Western populations but not for CKD stages 3 5.81 Less is known about ESRD epidemiology in diverse ethnicities across Africa and the Middle East because of paucity of studies, but differences could be regarded as a consequence of disparities in socioeconomic conditions more than race/ethnicity diversity. The factors underlying the epidemiological differences in CKD and ESRD in different races/ethnicities and minorities are not completely understood. Potential confounders could be the different prevalence of risk factors for CKD progression like hypertension or diabetes, other chronic or infectious diseases, genetic susceptibility, environmental factors, and inequalities in access to health resources, among others.76,77 Of note, among dialysis-treated ESRD patients, ethnic minorities are less likely to be listed for transplantation, wait longer for a transplant, and are less likely to receive allografts.76

1.2.4 Developing Countries and Socioeconomic Status The current literature has emphasized how disadvantaged populations and low socioeconomic status communities of developing countries suffer a disproportionate burden of nondialysis-dependent CKD and ESRD. Most of these differences are the consequence of direct stress factors associated with societal and health system level inequities, but also in part related to epigenetic alterations that through metabolic, inflammatory, and hormonal pathways can influence the expression of CKD and CKD risk factor genes and signaling.82 Complicated urinary tract infections, renal tuberculosis, parasitic diseases like schistosomiasis and leishmaniasis, HIV-associated nephropathy (HIVAN), hepatitis C, and other infectious diseases with glomerular involvement are

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REFERENCES

9

major epidemiological problems in developing countries and may contribute to the burden of chronic nephropathies. In these settings, also acute kidney injury often leads to ESRD due to the uncontrolled use of nonsteroidal antiinflammatory medications, as well as of traditional medicines in the primary-care environment.5,83,84 Diabetes prevalence is more strongly associated with poverty, and diabetes and diabetic nephropathy are increasing at alarming rates in developing countries.85 By 2030, more than 70% of patients with ESRD will live in low-income countries of Asia and Africa.86 Eight of the 10 largest countries in the world (China, India, Indonesia, Brazil, Pakistan, Bangladesh, Nigeria, and Russia) until recently have been classified as “developing countries.” Most of them have the highest population growth rates worldwide and a great part of their population living in extreme poverty. With important variations depending on the regions, China and India have high rates of annual ESRD prevalence growths, but only 10% 15% of all their ESRD patients are on maintenance RRT.36 However, epidemiological data on ESRD from developing countries are underrepresented, since renal registries only include patients on chronic RRT for whom maintenance dialysis or transplantation are available and/or affordable. Thus, ESRD incidence and prevalence in these settings may depend more on the level of wealth and health care system/conditions than true epidemiology variations.5 Not all CKD/ESRD epidemiology differences associated with socioeconomic status are confined by national boundaries. Two different studies in the United States showed that neighborhood and individual poverty are associated with increased risk of CKD and progression towards ESRD.87 Other studies in the United Kingdom, Germany, Sweden, Denmark, and Australia found association between economically disadvantaged populations and higher incidence of ESRD.88 Even in developing countries, low access to health care and scant resources increase the gap within their population and may create a bidirectional relationship between poverty and CKD/ESRD.86

CONCLUSIONS Evidence of the importance of CKD to public health and its contribution to the global burden of major NCDs has been accumulating, but there is little equity worldwide. A more concerted, strategic, and multisectorial approach, underpinned by solid research, is essential to help reverse the negative trend in the incidence of CKD and its risk factors, not just for a few beneficiaries but on a global health equity program. Thus, a pragmatic approach to reduce the global burden of renal diseases has to be adopted. For that, well-defined screenings of communities or high-risk populations followed by intervention programs have to be initiated, especially in lowand middle-income countries. Moreover, the International Society of Nephrology has urged WHO member states to recognize kidney diseases as a major NCD requiring the development of specific policies for effective awareness, early detection, and treatment, as part of the WHO action plan for the prevention and control of NCDs. There is an urgent need to establish surveillance networks for kidney disease in low- and middle-income countries, and incorporate simple, inexpensive strategies for preventing kidney disease and its complications in government health programs as part of their global strategy to improve public health. Some examples are the National Health Program in Uruguay, which has already incorporated CKD into its NCD prevention programs, and the Strategic Network of Health Services against CKD in Mexico. All these efforts will create major health gain, save lives, and minimize the present health inequity, which arises especially from the elevated costs of maintenance RRT if end-stage kidney disease is not prevented.

References Schlipko¨ter U, Flahault A. Communicable diseases: achievements and challenges for public health. Public Health Rev 2010;32:90 119. Cohen ML. Changing patterns of infectious disease. Nature 2000;406(6797):762 7. Murray CJL, Lopez AD. Measuring the global burden of disease. N Engl J Med 2013;369(5):448 57. Gwatkin DR, Guillot M, Heuveline P. The burden of disease among the global poor. Lancet 1999;354(9178):586 9. Perico N, Remuzzi G. Chronic kidney disease: a research and public health priority. Nephrol Dial Transplant 2012;27(Suppl. 3):iii19 26. Naghavi M, Wang H, Lozano R, et al. Global, regional, and national age sex specific all-cause and cause-specific mortality for 240 causes of death, 1990 2013: a systematic analysis for the Global Burden of Disease Study 2013. Lancet 2015;385(9963):117 71. 7. Beaglehole R, Bonita R, Alleyne G, et al. UN High-level meeting on non-communicable diseases: addressing four questions. Lancet 2011;378 (9789):449 55. 8. Alwan A. Global status report on noncommunicable diseases. World Health Organization; 2010. Available from: http://www.who.int/nmh/ publications/ncd_report_full_en.pdf. 1. 2. 3. 4. 5. 6.

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9. Global health and aging 2011. World Health Organization. Available from: http://www.who.int/ageing/publications/global_health/en/. 10. Roura LC, Arulkumaran SS. Facing the noncommunicable disease (NCD) global epidemic The battle of prevention starts in utero The FIGO challenge. Best Pract Res Clin Obstet Gynaecol 2015;29(1):5 14. 11. Global action plan for the prevention and control of noncommunicable diseases 2013 2020. World Health Organization. Available from: http://apps.who.int/iris/bitstream/10665/94384/1/9789241506236_eng.pdf. 12. Mathers CD, Loncar D. Projections of global mortality and burden of disease from 2002 to 2030. PLoS Med 2006;3(11):e442. 13. Smith SC, Collins A, Ferrari R, et al. Our time: a call to save preventable death from cardiovascular disease (heart disease and stroke). Glob Heart 2012;7:297 305. 14. Roth GA, Forouzanfar MH, Moran AE, et al. Demographic and epidemiologic drivers of global cardiovascular mortality. N Engl J Med 2015;372(14):1333 41. 15. Fitzmaurice C, Dicker D, Pain A, et al. The global burden of cancer 2013. JAMA Oncol 2015;1(4):505 27. 16. Popat K, McQueen K, Feeley TW. The global burden of cancer. Best Pract Res Clin Anaesthesiol 2013;27(4):399 408. 17. Ferkol T, Schraufnagel D. The global burden of respiratory disease. Ann Am Thorac Soc 2014;11(3):404 6. 18. Pauwels RA, Rabe KF. Burden and clinical features of chronic obstructive pulmonary disease (COPD). Lancet 2004;364(9434):613 20. 19. Burney P, Jarvis D, Perez-Padilla R. The global burden of chronic respiratory disease in adults. Int J Tuberc Lung Dis 2015;19(1):10 20. 20. Guariguata L, Whiting DR, Hambleton I, Beagley J, Linnenkamp U, Shaw JE. Global estimates of diabetes prevalence for 2013 and projections for 2035. Diabetes Res Clin Pract 2014;103(2):137 49. 21. Hu FB, Satija A, Manson JE. Curbing the diabetes pandemic: the need for global policy solutions. JAMA 2015;313(23):2319 20. 22. Vos T, Barber RM, Bell B, et al. Global, regional, and national incidence, prevalence, and years lived with disability for 301 acute and chronic diseases and injuries in 188 countries, 1990 2013: a systematic analysis for the Global Burden of Disease Study 2013. Lancet 2015;386(9995):743 800. 23. Kaveeshwar SA, Cornwall J. The current state of diabetes mellitus in India. Australas Med J 2014;7(1):45 8. 24. Wild S, Roglic G, Green A, Sicree R, King H. Global prevalence of diabetes estimates for the year 2000 and projections for 2030. Diab Care 2004;27(5):1047 53. 25. The growing danger of non-communicable diseases: acting now to reverse course. Conference Edition 2011. The World Bank. Available from: http://siteresources.worldbank.org/HEALTHNUTRITIONANDPOPULATION/Resources/Peer-Reviewed-Publications/ WBDeepeningCrisis.pdf. 26. Alleyne G, Binagwaho A, Haines A, et al. Embedding non-communicable diseases in the post-2015 development agenda. Lancet 2013;381 (9866):566 74. 27. Beaglehole R, Bonita R, Horton R, et al. Priority actions for the non-communicable disease crisis. Lancet 2011;377(9775):1438 47. 28. Lopez AD, Williams TN, Levin A, et al. Remembering the forgotten non-communicable diseases. BMC Med 2014;12(1):200. 29. Couser WG, Remuzzi G, Mendis S, Tonelli M. The contribution of chronic kidney disease to the global burden of major noncommunicable diseases. Kidney Int 2011;80(12):1258 70. 30. Roderick P. Epidemiology of end-stage renal disease. Clin Med 2002;2(3):200 4. 31. Anand S, Bitton A, Gaziano T. The gap between estimated incidence of end-stage renal disease and use of therapy. PloS One 2013;8(8): e72860. 32. Levey AS, Coresh J. Chronic kidney disease. Lancet 2012;379(9811):165 80. 33. Remuzzi G, Benigni A, Finkelstein FO, et al. Kidney failure: aims for the next 10 years and barriers to success. Lancet 2013;382 (9889):353 62. 34. Caskey FJ, Kramer A, Elliott RF, et al. Global variation in renal replacement therapy for end-stage renal disease. Nephrol Dial Transplant 2011;26(8):2604 10. 35. Levin A, Stevens PE. Early detection of CKD: the benefits, limitations and effects on prognosis. Nat Rev Nephrol 2011;7(8):446 57. 36. Jha V, Wang AY-M, Wang H. The impact of CKD identification in large countries: the burden of illness. Nephrol Dial Transplant 2012;27 (Suppl. 3):iii32 8. 37. Jha V, Garcia-Garcia G, Iseki K, et al. Chronic kidney disease: global dimension and perspectives. Lancet 2013;382(9888):260 72. 38. Bru¨ck K, Jager KJ, Dounousi E, et al. Methodology used in studies reporting chronic kidney disease prevalence: a systematic literature review. Nephrol Dial Transplant 2015;30(Suppl. 4):iv6 16. 39. Perico N, Remuzzi G. Need for chronic kidney disease prevention programs in disadvantaged populations. Clin Nephrology 2015;83(7 Suppl. 1):42 8. 40. El Nahas AM, Bello AK. Chronic kidney disease: the global challenge. Lancet 2005;365(9456):331 40. 41. Levey AS, Atkins R, Coresh J, et al. Chronic kidney disease as a global public health problem: approaches and initiatives - a position statement from Kidney Disease Improving Global Outcomes. Kidney Int 2007;72(3):247 59. 42. Zhang Q-L, Rothenbacher D. Prevalence of chronic kidney disease in population-based studies: systematic review. BMC Public Health 2008;8(1):117. Available from: http://dx.doi.org/10.1186/1471-2458-8-117. 43. Levey AS, Andreoli SP, DuBose T, Provenzano R, Collins AJ. Chronic kidney disease: common, harmful and treatable World Kidney Day 2007. Am J Nephrol 2007;27(1):108 12. 44. Cravedi P, Sharma SK, Bravo RF, et al. Preventing renal and cardiovascular risk by renal function assessment: insights from a cross-sectional study in low-income countries and the USA. BMJ Open 2012;2(5):e001357. 45. Rhee CM, Kovesdy CP. Spotlight on CKD deaths—increasing mortality worldwide. Nat Rev Nephrol 2015;11(4):199 200. 46. Zhang L, Wang F, Wang L, et al. Prevalence of chronic kidney disease in China: a cross-sectional survey. Lancet 2012;379(9818):815 22. 47. United States Renal Data System. USRDS annual data report: epidemiology of kidney disease in the United States. National Institutes of Health; 2015. Available from: www.usrds.org. 48. Stenvinkel P. Chronic kidney disease: a public health priority and harbinger of premature cardiovascular disease. J Intern Med 2010;268 (5):456 67. 49. Thomas B, Wulf S, Bikbov B, et al. Maintenance dialysis throughout the World in Years 1990 and 2010. J Am Soc Nephrol 2015;26:2621 33. 50. Beard JR, Officer A, de Carvalho IA, et al. The World report on ageing and health: a policy framework for healthy ageing. Lancet 2016;387:2145 54.

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51. Lin M-Y, Chiu Y-W, Lee C-H, et al. Factors associated with CKD in the elderly and nonelderly population. Clin J Am Soc Nephrol 2013;8:33 40. 52. Glassock RJ, Rule AD. The implications of anatomical and functional changes of the aging kidney: with an emphasis on the glomeruli. Kidney Int 2012;82(3):270 7. 53. Zu¨rbig P, Decramer S, Dakna M, et al. The human urinary proteome reveals high similarity between kidney aging and chronic kidney disease. Proteomics 2009;9(8):2108 17. 54. Glassock RJ, Oreopoulos DG. Aging and chronic kidney disease. Nephron Clin Pract 2011;119(Suppl. 1):c1. Available from: http://dx.doi. org/10.1159/000328007. 55. NHANES. Age-Adjusted Prevalence of CKD Stages 1 4 by Gender 1999 2012. Available from: http://www.cdc.gov/diabetes/programs/initiatives/kidney.html. 56. Gambaro G, Yabarek T, Graziani MS, et al. Prevalence of CKD in northeastern Italy: results of the INCIPE study and comparison with NHANES. Clin J Am Soc Nephrol 2010;5:1946 53. 57. van Blijderveen JC, Straus SM, Zietse R, Stricker BH, Sturkenboom MC, Verhamme KM. A population-based study on the prevalence and incidence of chronic kidney disease in the Netherlands. Int Urol Nephrol 2014;46(3):583 92. 58. Juutilainen A, Kastarinen H, Antikainen R, et al. Comparison of the MDRD Study and the CKD-EPI Study equations in evaluating trends of estimated kidney function at population level: findings from the National FINRISK Study. Nephrol Dial Transplant 2012;27(8):3210 17. 59. Arora P, Vasa P, Brenner D, et al. Prevalence estimates of chronic kidney disease in Canada: results of a nationally representative survey. CMAJ 2013;185:E417 23. 60. Perico N, Bravo RF, De Leon FR, Remuzzi G. Screening for chronic kidney disease in emerging countries: feasibility and hurdles. Nephrol Dial Transplant 2009;24(5):1355 8. 61. Ingsathit A, Thakkinstian A, Chaiprasert A, et al. Prevalence and risk factors of chronic kidney disease in the Thai adult population: Thai SEEK study. Nephrol Dial Transplant 2010;25(5):1567 75. 62. Arogundade FA, Barsoum RS. CKD prevention in Sub-Saharan Africa: a call for governmental, nongovernmental, and community support. Am J Kidney Dis 2008;51(3):515 23. 63. Sumaili EK, Krzesinski J-M, Zinga CV, et al. Prevalence of chronic kidney disease in Kinshasa: results of a pilot study from the Democratic Republic of Congo. Nephrol Dial Transplant 2009;24(1):117 22. 64. Tonelli M, Riella M. Chronic kidney disease and the aging population. Nephrol Dial Transplant 2014;29(2):221 4. 65. Dalrymple LS, Katz R, Kestenbaum B, et al. Chronic kidney disease and the risk of end-stage renal disease versus death. J Gen Intern Med 2011;26(4):379 85. 66. Moynihan R, Glassock R, Doust J. Chronic kidney disease controversy: how expanding definitions are unnecessarily labelling many people as diseased. BMJ 2013;347:f4298. 67. O’Hare AM, Choi AI, Bertenthal D, et al. Age affects outcomes in chronic kidney disease, J Am Soc Nephrol 2007;18(10):2758 65. 68. Hallan SI, Matsushita K, Sang Y, et al. Age and association of kidney measures with mortality and end-stage renal disease. JAMA 2012;308(22):2349 60. 69. Iseki K. Gender differences in chronic kidney disease. Kidney Int 2008;74(4):415 17. 70. Halbesma N, Brantsma AH, Bakker SJ, et al. Gender differences in predictors of the decline of renal function in the general population. Kidney Int 2008;74(4):505 12. 71. Carrero JJ. Gender differences in chronic kidney disease: underpinnings and therapeutic implications. Kidney Blood Press Res 2010;33 (5):383 92. 72. Eriksen BO, Tomtum J, Ingebretsen OC. Predictors of declining glomerular filtration rate in a population-based chronic kidney disease cohort. Nephron Clin Pract 2010;115(1):c41 50. 73. Neugarten J, Acharya A, Silbiger SR. Effect of gender on the progression of nondiabetic renal disease a meta-analysis. J Am Soc Nephrol 2000;11(2):319 29. 74. Silbiger S, Neugarten J. Gender and human chronic renal disease. Gend Med 2008;5:S3 10. 75. Norris K, Nissenson AR. Race, gender, and socioeconomic disparities in CKD in the United States. J Am Soc Nephrol 2008;19(7):1261 70. 76. Feehally J. Ethnicity and renal disease. Kidney Int 2005;68(1):414 24. 77. Harawa NT, Norris KC. The role of ethnic variation and CKD. Clin J Am Soc Nephrol 2015;10:1708 10. 78. Saran R, Li Y, Robinson B, et al. US Renal Data System 2015 Annual Data Report: epidemiology of kidney disease in the United States. Am J Kidney Dis 2016;67(3 Suppl. 1):S1 434. 79. Palmer AT, Lewis J. Racial differences in chronic kidney disease (CKD) and end-stage renal disease (ESRD) in the United States: a social and economic dilemma. Clin Nephrol 2010;74:S72 7. 80. Collins JF. Kidney disease in Maori and Pacific people in New Zealand. Clin Nephrol 2010;74(Suppl.1):s61 5. 81. Sabanayagam C, Lim SC, Wong TY, Lee J, Shankar A, Tai ES. Ethnic disparities in prevalence and impact of risk factors of chronic kidney disease. Nephrol Dial Transplant 2010;25(8):2564 70. 82. Nicholas SB, Kalantar-Zadeh K, Norris KC. Socioeconomic disparities in chronic kidney disease. Adv Chronic Kidney Dis 2015;22(1):6 15. 83. Swanepoel CR, Wearne N, Okpechi IG. Nephrology in Africa—not yet uhuru. Nat Rev Nephrol 2013;9(10):610 22. 84. Stanifer JW, Jing B, Tolan S, et al. The epidemiology of chronic kidney disease in sub-Saharan Africa: a systematic review and metaanalysis. Lancet Glob Health 2014;2(3):e174 81. 85. Hossain MP, Goyder EC, Rigby JE, El Nahas M. CKD and poverty: a growing global challenge. Am J Kidney Dis 2009;53(1):166 74. 86. Garcı´a-Garcı´a G, Jha V. World Kidney Day 2015: CKD in disadvantaged populations. Am J Kidney Dis 2015;65(3):349 53. 87. Young BA. The interaction of race, poverty, and CKD. Am J Kidney Dis 2010;55(6):977 80. 88. Patzer RE, McClellan WM. Influence of race, ethnicity and socioeconomic status on kidney disease. Nat Rev Nephrol 2012;8(9):533 41.

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C H A P T E R

2 Transplant Programs Worldwide and the Spanish Miracle Rafael Matesanz, Beatriz Domı´nguez-Gil, Elisabeth Coll, Beatriz Mahı´llo, Gloria de la Rosa and Marı´a O. Valentı´n National Transplant Organization, Madrid, Spain

2.1 INTRODUCTION In 2010, the World Health Assembly adopted Resolution WHA 63.22, urging Member States “to strengthen national and multinational authorities and/or capacities to provide oversight, organization and coordination of donation and transplantation activities, with special attention to maximizing donation from deceased donors and to protect the welfare of living donors with appropriate health-care services and long-term follow-up.”1 The Resolution was adopted 2 months after participants in the third World Health Organization (WHO) Global Consultation on Organ Donation and Transplantation (Madrid, Spain), called governments to take responsibility in the progress towards self-sufficiency in transplantation, and therefore in satisfying the transplantation needs of their patients, by using resources within their own patient population.2 Self-sufficiency should encompass strategies targeted both to decrease the burden of chronic diseases treatable with transplantation and to increase organ availability, with priority given to donation from the deceased. Initiatives for self-sufficiency should have a solid ethical basis respectful to the WHO Guiding Principles on Human Cell, Tissue and Organ Transplantation3 and the Declaration of Istanbul on Organ Trafficking and Transplant Tourism.4 These WHO straightforward messages were launched to address the universal shortage of organs; the more than 115,000 solid organ transplants performed globally every year barely cover 10% of the transplant needs and there are huge disparities in transplantation activities observed across countries.5 Differences in deceased donation greatly account for the extreme variability in access to transplantation therapies. Disparities in deceased donation are evident even when comparing countries with a high level of development, ranging between nonexistent to more than 30 deceased donors per million population (pmp) (Fig. 2.1).6 In this scenario, Spain occupies a worldwide privileged position, with the highest deceased donation rate ever recorded for a large country, and reaching a maximum 40 donors pmp in 2015. Transplantation activities in the country are well over 100 transplant procedures pmp, something hard to imagine for the majority of countries throughout the world. With around 14 donors pmp at the end of the eighties, the activity in Spain was at the mid-low position when compared to other European countries. The increase in deceased donation (Fig. 2.2), and consequently in the number of solid organ transplants (Fig. 2.3), resulted from the implementation of a set of measures, mainly of an organizational nature, altogether internationally named as the Spanish Model of Organ Donation and Transplantation.7,8 These measures were adopted after the Spanish National Transplant Organization (Organizacio´n Nacional de Trasplantes—ONT) was created in 1989. ONT was conceived as a technical agency embedded within the Ministry of Health, and in charge of overseeing and coordinating donation and transplantation activities in the country. The Spanish system is founded on two basic principles: a proper organization around the process of donation after death, along with innovation and continuous adaptation to the change.

Kidney Transplantation, Bioengineering, and Regeneration. DOI: http://dx.doi.org/10.1016/B978-0-12-801734-0.00002-3

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© 2017 Elsevier Inc. All rights reserved.

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2. ORGAN DONATION: THE SPANISH MIRACLE

FIGURE 2.1 Deceased donation activity (rates per million population) in the world. Donors after brain death and donors after circulatory death. The absolute number of deceased donors is shown in brackets. Year 2014. DBD, donation after brain death; DCD, donation after circulatory death; pmp, per million population. Source: WHO-ONT Global Observatory on Organ Donation and Transplantation. I. KIDNEY TRANSPLANTATION

2.2 THE SPANISH MODEL OF ORGAN DONATION AND TRANSPLANTATION

15

FIGURE 2.2 Deceased donation activity (absolute number and rate per million population) in Spain. Years 1989 2015. pmp, per million population. Source: Organizacio´n Nacional de Trasplantes.

FIGURE 2.3 Solid organ transplantation activity in Spain. Absolute numbers. Years 1989 2015. Source: Organizacio´n Nacional de Trasplantes.

2.2 THE SPANISH MODEL OF ORGAN DONATION AND TRANSPLANTATION Measures implemented in the country were developed on an appropriate healthcare, legal and technical background. The Spanish healthcare system is a public one with a universal coverage of the population, and universal access to transplantation. Technically, the country counts on extraordinarily prepared, enthusiastic, innovative, and motivated transplant teams. The Spanish Law on Transplantation was first enacted in 1979 and contained the

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2. ORGAN DONATION: THE SPANISH MIRACLE

TABLE 2.1 Main Elements of the Spanish Model of Organ Donation and Transplantation Donor coordination network at three levels: national, regional, hospital. Special profile of donor coordinators • • • •

Medical doctors, mainly critical care physicians, supported by nurses. Part-time dedication to the transplant coordination activities. Independence from the transplant teams. Appointed by and reporting to the hospital medical director. Main objective: deceased donation. Progressively more involved in: promotion, training and education, relation with the mass media, management of resources, research. Donor Coordinators inside the hospitals. Central Office (ONT) as a support agency. Quality Assurance Program in the deceased donation process: continuous clinical chart review of deaths at intensive care units of procurement hospitals. Two phases: internal and external audit. Great effort in medical training through different types of courses. Targets: donor coordinators, intensive care physicians and nurses, emergency and urgency physicians and nurses, other healthcare professionals. Close attention to the mass media with a special communication policy. Hospital reimbursement for donation and transplantation activities.

basic elements of any western transplant law.9 An opting-out system for consent to donation was devised and has been in place since then. However, the presumed consent policy is not strictly applied in practice; relatives are always approached and still have the final veto.10,11 Political competencies in the country are transferred to 17 Autonomous Regions, so any national initiative has to reach an interregional consensus, and this also applies to the field of donation and transplantation. This particular political situation has determined a mode of work in the field that aims at the cohesion of the system and the sharing of best practices. The core principle of the Spanish system is a very specific organizational approach to the process of donation after death, to ensure the systematic identification of opportunities for organ donation and their transition to actual organ donation. The main elements of the Spanish Model are summarized in Table 2.1 and described below.

2.3 DONOR COORDINATION NETWORK The coordination of donation activities has been structured at three different but related levels: national (ONT), regional (17 Regional Coordination offices), and hospital. The first two levels act as an interface between the technical and the political strata, and in support of the process of deceased donation. These two levels are appointed by and report to the national and regional health-care authorities, respectively. Any national decision on donation and transplantation activities is agreed upon by the National Transplant Committee of the Inter-territorial Health-Care Council, which comprises the ONT as chair and the 17 Regional Coordinators. The hospital level of coordination is composed of a network of hospitals specifically authorized for organ procurement activities by the relevant authorities, where the process of organ donation and procurement is developed. The network of donor hospitals grew from less than 20 in 1989 to 118 in 1992, a rapid evolution which reflects the significant efforts made by the system and the political support received in its initial years. The network has continued to increase, today comprising 186 hospitals throughout the country.

2.4 SPECIFIC PROFILE OF THE DONOR COORDINATOR As specified in the national legislation, each hospital authorized for organ procurement activities must count on a dedicated donor coordination team, variable in its composition, but always led by a figure with a very specific profile, a key element of the Spanish system.12 Donor coordinators are in-house professionals, members of staff at the procurement hospital concerned. They are nominated by and report to the medical direction of the hospital and the corresponding Regional Coordinator. Most of the donor coordinators are involved in donation activities on a part-time basis, which enables them to be appointed even at hospitals with a low deceased donor potential.

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2.6 QUALITY ASSURANCE PROGRAM IN DECEASED DONATION

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Most notably, the majority of donor coordinators are intensive care physicians who develop their daily work at those units where about 12% of deaths occur in persons with a clinical condition consistent with brain death.13 This feature has been the driving force for the Spanish intensive care community to consider organ donation as a routine component of end-of-life care. The following examples make evident the embracement of deceased donation by the intensive care community. The Spanish Society of Intensive, Critical Care Medicine and Coronary Units (Sociedad Espan˜ola de Medicina Intensiva, Crı´tica y Unidades Coronarias—SEMICYUC) specifically acknowledges organ donation as its responsibility at its Ethics Code of Practice.14 In consequence and in cooperation with ONT, this Society supports the continuous training of intensive care physicians in organ donation and procurement activities from the outset of their clinical practice. Moreover, SEMICYUC lists a set of indicators related to donor identification and conversion of potential into actual organ donors as key indicators of quality in intensive care.15

2.5 ONT AS A CENTRAL AGENCY IN SUPPORT OF THE SYSTEM Besides its key role as organ sharing office, ONT acts as an agency in support of the network of procurement hospitals and the successful development of the deceased donation process. In close cooperation with the Regional Coordination Offices, ONT issues the regulatory framework of practice, provides advice for the designation of appropriate professionals as in-hospital donor coordinators, ensures continuous professional training and public education, designs national protocols and programs to promote organ donation and improve practices, and monitors activities to identify opportunities for improvement. The support provided by ONT and some Regional Offices is particularly important for small hospitals, with more difficulties to develop the process of deceased donation on their own.

2.6 QUALITY ASSURANCE PROGRAM IN DECEASED DONATION The Quality Assurance Program in Deceased Donation has been in place since 1999, and has inspired national, regional, and local strategies for continuous improvements.13 So far focused on the process of donation after brain death (DBD), the program aims at monitoring the potential donor pool, evaluating performance in deceased donation and identifying areas where improvement is possible. The program is based on a continuous audit of clinical charts of patients dead in intensive care units (ICUs). It includes an internal audit performed by donor coordinators locally. Information compiled during the internal audit is reported to ONT, which provides national indicators of reference to the network (Table 2.2). With differences between hospitals with and without neurosurgical activities, as a mean 12% of patients dead in the Spanish ICUs die in conditions consistent with brain death. Main reasons why these potential DBD donors do not transition to actual DBD donors are medical unsuitability (25%) and consent declined to proceed with organ recovery (13%). Failure to identify and refer potential DBD donors barely accounts for 3% of donor losses. Hospitals that deviate from these national indicators are prompted to take measures for improvement. The program also includes external audits carried out following a specific methodology described in detail elsewhere.13 At a national level, this component of the program has offered undisputed evidence that improvement is possible. With 21,065 ICU deaths externally audited between 2001 and 2011, 2635 persons with a brain death condition were identified in the clinical chart review, of which 6% were never referred to the donor coordinator. Twenty-three percent of potential donors were considered medically unsuitable for donation, although in 11% of these cases, medical contraindications were deemed inappropriate by external observers. Hemodynamic instability leading to an early cardiac arrest and refusals to organ donation were the reasons behind 2% and 14% of losses, respectively, some considered avoidable. The program reveals that the number of actual donors in the country could be 21% higher if all potential donors were identified and preventable losses avoided.13 External audits also represent great opportunities for exchanging best practices and releasing recommendations for improvement to the donor coordination team and the hospital managers of the audited hospitals.

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2. ORGAN DONATION: THE SPANISH MIRACLE

TABLE 2.2 Data From the Spanish Quality Assurance Program in Deceased Donations. Years 1999 201316 ICU deaths audited

276,356

PD audited

33,310

Actual DBD donors

19,168

DBD POTENTIAL % PD/Hospital deaths

2.2

% PD/ICU deaths

12.3

LOSSES IN THE PROCESS % PD not referred to the donor coordination/PD

3.4

% Medical Contraindications/PD (including not referred cases)

24.8

% Maintenance problems/PD (including not referred cases)

2.9

% Family refusals/PD

12.9

% Judicial refusals/PD

0.3

% Brain death diagnosis not confirmed/PD

0.1

% Lack of suitable recipients/PD

0.6

% Logistic problems/PD

0.4

OVERALL PERFORMANCE % Actual DBD donors/PD

56.8

DBD, donation after brain death; ICU, intensive care unit; PD, potential DBD donor. Potential DBD donor, Person with a clinical condition consistent with brain death.

2.7 CONTINUOUS PROFESSIONAL TRAINING Training is an essential component of the Spanish system. The Spanish government and the regional authorities have promoted regular courses targeted to all professionals who directly or indirectly participate in the process of deceased donation—donor coordinators, intensive and emergency care professionals, neurologists, judges, coroners, and journalists. Since 1991, over 16,000 professionals have been trained through these courses in Spain.

2.8 CLOSE COOPERATION WITH THE MEDIA The objective of constructing a positive social climate towards donation and generating society’s trust in the system has not been achieved through any sort of promotional campaign, but through a close cooperation with the mass media.17,18 The communication policy of ONT and its network is based on four basic principles: (1) a 24-hour telephone for consultation; (2) easy and permanent access to the media; (3) connection with journalists built through dedicated meetings aimed at learning about mutual needs; (4) delivery of messages with no intermediaries. These measures have led the media to handle information about donation and transplantation appropriately, and allowed the system to efficiently neutralize any negative news.

2.9 REIMBURSEMENT OF DONATION AND PROCUREMENT ACTIVITIES Finally, as with any other medical activity performed within the public health-care system, hospitals are reimbursed for their procurement activities. The corresponding regional healthcare authorities allocate a specific budget to cover both the human and material resources needed for the effective development of these activities at every hospital.19

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2.10 CURRENT CHALLENGES IN ORGAN DONATION IN SPAIN

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2.10 CURRENT CHALLENGES IN ORGAN DONATION IN SPAIN Despite the important transplant activity performed annually in Spain (Fig. 2.3), the transplantation needs of the population are still far from being adequately met.20 The prevalence and incidence of end-stage renal disease in the country is of 500 and 150 cases pmp, respectively.21 With an estimated 20% of prevalent and 30% 40% of incident patients being candidates to kidney transplantation, the kidney transplant activity should be as high as 150 160 kidney transplants pmp to meet the demand, which greatly exceeds the 60 procedures pmp reached in the year 2015. Six to eight percent of patients waiting for nonkidney transplants die each year in the country.20 This is possibly an underestimation of the true mortality on the waiting list, since a similar percentage of patients are withdrawn from the list every year, in many instances due to a deterioration of their clinical condition. The chronic inability to adequately satisfy the transplant needs is challenged by substantial epidemiological changes in the country and by improvements in the care of critical patients in general terms and neurocritical patients in particular. Mortality related to cerebrovascular diseases and traffic accidents has fortunately decreased in Spain over the years, leading the country to hold one of the lowest mortalities relevant to organ donation in Europe.22 The decrease in the occurrence of events potentially leading to devastating brain injuries, as a result of transport and work safety measures, along with a best approach to the treatment of these events, explain such a decrease in mortality. The extended use of decompressive craniectomy and other neurosurgical interventions has also contributed to a progressive decline in the pool of potential DBD donors in the country. According to data from the Quality Assurance Program in Deceased Donation, the incidence of brain death has evolved from 65 cases pmp in 2001 to less than 50 pmp in the very last years (Fig. 2.4). Changes have also occurred in the type of care provided to critical patients at the end-of-life, in accordance with new guidance issued by the relevant professional societies.23 Far from what the Ethicus study described for Southern European countries at the beginning of the century, where deaths in the ICU following the withdrawal of life-sustaining therapy (WLST) were rare,24 at present more than 30% of deaths in the Spanish ICUs and close to 20% of deaths in patients with a devastating brain injury follow the decision to WLST because this is no longer in the best clinical interests of the patient.25,26 Until recently, controlled donation after circulatory death (cDCD) was not possible in the country, since the framework for its practice had not been developed yet and a moratorium existed since 1996 on this particular activity.27

FIGURE 2.4 Potential and actual donors after brain death per million population in Spain. Years 2001 13. Potential donor after brain death: Person with a clinical condition consistent with brain death (medical contraindications included). Source: Organizacio´n Nacional de Trasplantes.

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2.11 THE 40 DONORS PER MILLION POPULATION PLAN The situation described above prompted the system to consider new strategies to meet the demand of organs for transplantation and to ensure that the option of organ donation was posed in all circumstances of death. In 2008, ONT devised the 40 donors pmp Plan, establishing this objective in deceased organ donation to all Autonomous Regions in the country.28 The objective was not arbitrary. When comparing performance between regions and hospitals, profound differences could be observed, not easily explained by disparities in the pool of possible donors.29 These differences suggested that there was still large room for improvement within the Spanish system. Actions under development to yield a level of activity almost achieved for the country as a whole and already surpassed by several Spanish Regions (Fig. 2.5) are summarized below.

2.12 PROMOTING THE IDENTIFICATION OF POSSIBLE ORGAN DONORS OUTSIDE OF THE ICU AND CONSIDERING ELECTIVE NONTHERAPEUTIC INTENSIVE CARE Early identification and referral of persons with a devastating brain injury in whom further treatment has been deemed futile (possible donors) to consider elective nontherapeutic intensive care can substantially modify the pool of potential donors. In recent years, a group of hospitals had reached an outstanding performance of over 60 70 donors pmp, levels that challenged any previous assessment of the deceased donor potential in the country. However, former estimations of the DBD pool were focused exclusively on the ICU. Many factors determine the number of persons who finally die with a devastating brain lesion within an ICU and in conditions consistent with brain death.30 Some of these factors are not easily modifiable, such as the availability of ICU

FIGURE 2.5 Deceased donors per million population in Spanish regions. Year 2015. Source: Organizacio´n Nacional de Trasplantes.

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resources. However, admission criteria to the ICU and initiation/continuation of intensive care when this is deemed futile are acknowledged as variable but modifiable factors, very much dependent upon the level of professional knowledge and ownership of the deceased donation activity. In 2009, ONT promoted a benchmarking study targeted to identify best performing hospitals in the country in the different phases of the DBD process.31 The study identified 10 hospitals with outstanding results in the identification and referral of possible organ donors to the ICU to facilitate organ donation. Qualitative questionnaires pointed out critical factors for success at this stage—these hospitals had devised a pathway of care for the neurocritical patient inclusive of the consideration of organ donation when the responsible professional or team shifted from active treatment to palliative and end-of-life care. When prognosis was ominous and further treatment deemed futile by the responsible physician, organ donation was discussed with the relatives along with the need to initiate/continue intensive care with the aim of facilitating organ donation. The practice was a routine reflected at local protocols, well accepted and properly monitored. This finding was reflected in a Guide of Best Practices in Organ Donation released to the network with specific recommendations for the implementation of this principle in practice.32 Further work focused on the quantitative and qualitative description of possible organ donors who currently die outside of the ICU. The first national assessment was performed in the context of a study jointly undertaken by ONT and the Spanish Society of Emergency Care (Sociedad Espan˜ola de Medicina de Urgencias y Emergencias—SEMES). Possible organ donors accounted for 1 out of 2000 patients attended to at the emergency department of procurement hospitals—69% of these possible donors admitted to the hospital through the emergency department finally died outside of the ICU (in the emergency room itself or at the hospital ward) with no consideration of organ donation. More recently, the ACCORD-Spain study assessed end-of-life care practices relevant to organ donation at 68 hospitals in the country.26 Almost 40% of possible organ donors were never admitted to the ICU because further treatment was considered futile and because the option of organ donation had not been facilitated (Fig. 2.6). Notably, 43% of these patients were octogenarian and/or had significant comorbidities, and most were not ventilated at the moment when the decision was made not to continue intensive treatment. The study pointed out

FIGURE 2.6 Scenario that best describes the type or care provided at the end-of-life to patients dying as a result of a devastating brain injury in Spain. ICU, intensive care unit; WLST, withdrawal of life-sustaining therapy. Source: ACCORD Spain study. The study compiled information on 1970 possible donors aged 1 month-85 years identified in 68 of the 186 hospitals authorized for organ procurement activities in the country during the period 11/1/2014 4/30/2015.

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some of the many challenges of elective nontherapeutic intensive care—the frequent need of initiating elective ventilation, the uncertainties about contraindications to organ donation that might become evident once the decision has been made to continue intensive care, summed up to the difficulties of anticipating that brain death will occur within a reasonable period of time. The practice and the challenges of elective nontherapeutic intensive care have become components of the national curriculum for donor coordinators, intensive and emergency care professionals in the country.33,34 Jointly with SEMES, ONT has recently produced recommendations on the role of emergency care professionals in organ donation.35 A working group designated by ONT and SEMICYUC is now preparing guidelines for elective nontherapeutic intensive care targeted to harmonize and improve practices in this particular setting. Today the contribution of elective nontherapeutic intensive care to actual donation rates is evident. ACCORDSpain has revealed that 24% of organ donors in the country have been patients admitted to the ICU with the aim of incorporating organ donation into their end-of-life care pathway.26

2.13 FOSTERING THE USE OF ORGANS FROM EXPANDED CRITERIA AND NONSTANDARD RISK DONORS 2.13.1 Expanded Criteria Donors In the context of the fortunate progressive reduction in the number of deaths due to traffic accidents over the years, if criteria for organ donation had remained unaltered, the deceased donation activity in Spain would have dramatically decreased. However, the Spanish coordination and transplantation system has progressively adopted more flexible criteria for donor selection. As a result, the number of aged donors, mostly dead as a result of cerebrovascular accidents, has sustainably increased in Spain (Fig. 2.7). In 2015, more than 50% of deceased

FIGURE 2.7 Age group of deceased organ donors in Spain. Years 2000 15. Source: Organizacio´n Nacional de Trasplantes.

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organ donors were aged 60 years or older, falling under the age criterion that defines the expanded criteria donor kidney at the UNOS.36 Moreover, 30% of deceased donors were over 70 years, and 10% over 80 years. An “old for old” allocation strategy has been devised from the very beginning, whereby aged kidneys are preferentially allocated to aged recipients irrespective of HLA mismatch, as also performed in other European programs.37,38 More than 40% of the deceased kidney, 51% of the liver, 7% of the heart and 23% of the lung transplant procedures are performed with organs recovered from donors aged $ 60 years.20 Discard rates of organs recovered from expanded criteria donors, however, remain high. Further analysis and work should lead the system to conclude whether organ discard is based on objective factors determinant of posttransplant outcomes and to determine the role of, e.g., ex vivo perfusion techniques in better assessing organ viability.

2.13.2 Nonstandard Risk Donors The Quality Assurance Programme in Deceased Donation reveals that 25% of persons in a brain death condition are not considered medically suitable organ donors, though some of these medical contraindications are inappropriately established by the donor coordinator or the treating physician.13 While safety of the process is an unquestionable professional standard, a scientific analysis of the risks should deeply guide decisions about transplantation at a moment of shortage. The ageing of the donor population is also inherently linked to progressively more difficult assessments of donor suitability. ONT has a 24-hour medical team available for donor coordinators to ask for a second opinion regarding the evaluation of potential donors. National Consensus Documents on the evaluation of organ donors to prevent the transmission of neoplastic diseases and with regards to infections have also been important elements in meeting this need.39,40 These documents have been the bases for recommendations on donor suitability at the Council of Europe level.41 More recently, in order to clearly define safety limits in the use of organs for transplantation, a specific national registry on the follow-up of recipients transplanted from nonstandard risk donors (i.e., history of malignancy or active infection) has been developed by ONT. Information provided by this registry will indeed contribute to increasing the level of evidence to guide future risk-benefit assessments.

2.14 DEVELOPING THE FRAMEWORK FOR THE PRACTICE OF DONATION AFTER CIRCULATORY DEATH Contrary to what is described for other countries, donation after circulatory death (DCD) in Spain had been classically focused on what the First International Workshop on the so-called Non Heart Beating Donation, held in Maastricht in 1995, defined as category II or uncontrolled DCD (uDCD) donation from persons who have been declared dead following an unsuccessfully resuscitated cardiac arrest.42 This particular type of DCD implies a very important logistical effort, both inside and outside of the hospital, and a smooth cooperation between the different stakeholders involved.43 Legal, ethical, and the aforementioned difficulties have precluded many countries to embark on this type of donation despite efforts to emulate this program in other realities.44 Even within Spain, because of the degree of sophistication required, the activity was initially limited to three Spanish cities— A Corun˜a, Barcelona, and Madrid. Following national recommendations, and support by national and local authorities, other programs have been initiated,45 with a positive impact upon the number of uDCD donors, although the activity has stabilized thereafter (Fig. 2.8). At present, there are 11 uDCD programs in the country yielding an important number of kidneys with impressive results.46 48 Liver transplants from uDCD donors have been made possible with a normothermic regional perfusion approach which leads to reasonable results close to those obtained with livers from DBD donors.49,50 Results of lung transplantation from uDCD donors are comparable to those obtained with DBD donors.51,52 The contribution of ex vivo preservation strategies to the validation of these lungs is currently under study. cDCD had not been devised as an option in our country for many years. Until recently, a moratorium was in place on the use of organs from this type of donor and no regulatory framework had been established for its practice. The aforementioned changes to end-of-life care were the main reason to reconsider the position of the country towards cDCD. A National Consensus Conference on DCD set down the basis for this practice in Spain and was followed by the development of a new legislation that accommodated this possibility and established the minimum requisites for its development.12,53 Along with institutional support and efforts in professional training and public education the results have been impressive (Fig. 2.8). Today more than 50 hospitals in the

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FIGURE 2.8 Donation after circulatory death in Spain. Absolute numbers and rate per million population. Maastricht categories. Years 1995 2015. Source: Organizacio´n Nacional de Trasplantes.

country are embarked in cDCD which already contributes to 10% of the overall deceased donation rates and has had a substantial impact on the kidney, but also the liver and lung transplantation activity, with excellent posttransplant results.54,55

2.15 PROMOTING LIVING KIDNEY DONATION WHILE ENSURING COMPREHENSIVE DONOR PROTECTION AND CARE Living donation remained a rather anecdotal activity in Spain for many years, something to be understood in the global context of a country with an emerging and soon robust deceased donation program during the 1990s. Only a few highly experienced centers maintained a certain level of living kidney donation during those years. However, living kidney donation is now considered an essential element in dealing with the transplantation needs of patients. The evidence of excellent results of living kidney transplantation,56 the knowledge gained about the short-, mid-, and long-term safety of the donor as long as an appropriate framework of donor care is provided,57 60 and the incorporation of novel surgical approaches for donor nephrectomy have yielded a progressive change in the conception of living kidney donation in the country. This occurred alongside changes in international institutions such as the Council of Europe, which progressed from restrictive principles61 to the consideration of new strategies to increase the living donor pool, such as altruistic donation or kidney pair exchange.62,63 Variable experience in living transplantation between centers, living kidney donation not being offered as an alternative for ESRD patients, and ABO and HLA incompatibility were recognized obstacles in our system to living kidney donation.64,65 Training courses aimed at multidisciplinary teams of professionals (such as nephrologists, urologists, donor coordinators and nurses) from transplantation hospitals, dialysis centers and outpatient clinics have been developed over the last years. Donor coordinators are being incorporated as figures facilitating the process and covering the informative needs of patients and their relatives. The Spanish Society of Nephrology and ONT have issued comprehensive professional guidelines on living kidney donation and transplantation.66 A solid program of kidney pair exchange has been developed with more than 150 kidney transplant procedures performed since 2009.67 A program of altruistic donation is already in place, which, combined with the crossover

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FIGURE 2.9 Living kidney transplantation activity in Spain. Absolute number and rate per million population. Years 1991 2015. Source: Organizacio´n Nacional de Trasplantes.

donation program, has led to increased possibilities of living kidney transplantation.68 Living donation is finally developed under a framework of comprehensive donor protection and care. As a result of all these initiatives, living kidney transplantation has progressively increased, today representing 13% of all kidney transplant procedures performed in the country (Fig. 2.9).

2.16 FINAL REMARKS Organization around the process of deceased donation is the key to success for the Spanish system. This approach has been totally or partially replicated by other countries and regions resulting in a progression in the pursuit of self-sufficiency in transplantation through deceased organ donation. New challenges are being confronted effectively by the system: the transplantation needs of our population are expected to increase, while the potential of donation might continue decreasing in the upcoming years, particularly for DBD. Novel strategies to adapt to this changing scenario are being explored and successfully implemented in Spain. Finally, the pillars of the deceased and living donation program and the measures developed to increase organ availability are respectful with fundamental international ethical standards—which are solid elements of the Spanish Model per se.

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6. 2013 Report of the Global Observatory on Donation and Transplantation. Global Observatory on Donation and Transplantation website. ,https://view.publitas.com/ont/20151215_basic_slides_2013_con_datos_de_libya_de_2011/page/1. [accessed March 2016]. 7. Matesanz R, Domı´nguez-Gil B. Strategies to optimize deceased organ donation. Transplant Rev 2007;21:177 88. 8. Matesanz R, Domı´nguez-Gil B, Coll E, de la Rosa G, Marazuela R. Spanish experience as a leading country: what kind of measures were taken? Transpl Int 2011;24(4):333 43. 9. Ley 30/1979, de 27 de octubre, sobre extraccio´n y trasplante de o´rganos. ,https://www.boe.es/buscar/doc.php?id 5 BOE-A-197926445. [accessed March 2016] [in Spanish]. 10. Matesanz R, Domı´nguez-Gil B. Pros and cons of a regulated market in organs. Lancet 2009;374(9707):2049. 11. Fabre J, Murphy P, Matesanz R. Presumed consent: a distraction in the quest for increasing rates of organ donation. BMJ 2010;341:c4973. 12. Real Decreto 1723/2012, de 28 de diciembre, por el que se regulan las actividades de obtencio´n, utilizacio´n clı´nica y coordinacio´n territorial de los o´rganos humanos destinados al trasplante y se establecen requisitos de calidad y seguridad. ,https://www.boe.es/diario_boe/txt.php?id 5 BOE-A-2012-15715. [accessed March 2016] [in Spanish]. ´ lvarez J, Araiz J, et al. Continuously evaluating performance in deceased 13. de la Rosa G, Domı´nguez-Gil B, Matesanz R, Ramo´n S, Alonso-A donation: the Spanish quality assurance program. Am J Transplant 2012;12(9):2507 13. 14. Cabre´ Pericas LL, Abizanda Camposb R, Baigorri Gonza´lez F, Blanch Torra L, Campos Romero JM, Iribarren Diarasarri S, et al. Code of ethics of the Spanish Society of Intensive Care, Critical and Coronary Units (SEMICYUC)]. Med Intensiva 2006;30(2):68 73 [Article in Spanish]. 15. Quality Indicators in Critically Ill Patients. Update 2011. ,http://www.semicyuc.org/temas/calidad/indicadores-de-calidad. 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Health at a glance 2014. ,http://www.oecd-ilibrary.org/social-issues-migration-health/health-at-a-glance-2015_health_glance-2015-en. [accessed March 2016]. 23. Monzo´n Marı´n JL, Saralegui Reta I. Abizanda i Campos R, Cabre´ Pericas L, Iribarren Diarasarri S, et al. Treatment recommendations at the end of the life of the critical patient. Med Intensiva 2008;32(3):121 33 [Article in Spanish]. 24. Sprung CL, Cohen SL, Sjokvist P, Baras M, Bulow HH, Hovilehto S, et al. End-of-life practices in European intensive care units: the Ethicus Study. JAMA 2003;290(6):790 7. 25. Herna´ndez-Tejedor A, Martı´n-Delgado MC, Cabre´ L, Algora A, EPIPUSE study group. Limitation of life-sustaining treatment in patients with prolonged admission to the ICU. Current situation in Spain as seen from the EPIPUSE Study. Medicina Intensiva 2015;39(7):395 404 [Article in Spanish]. 26. Report on the ACCORD Spain Study. ,http://www.ont.es/infesp/Paginas/ProyectosenMarcha.aspx. [access March 2016] [in Spanish]. 27. Matesanz R. Documento de consenso espan˜ol sobre extraccio´n de o´rganos de donantes en asistolia. Nefrologı´a 1996;16(Suppl. 2):48 53 [Article in Spanish]. 28. Matesanz R, Marazuela R, Domı´nguez-Gil B, Coll E, Mahillo B, de la Rosa G. The 40 donors per million population plan: an action plan for improvement of organ donation and transplantation in Spain. Transplant Proc 2009;41(8):3453 6. 29. Coll E, Miranda B, Domı´nguez-Gil B, Martı´n E, Valentı´n M, Garrido G, et al. Organ donors in Spain: evolution of donation rates per regions and determinant factors. Med Clin (Barc) 2008;131(2):52 9 [Article in Spanish]. 30. Cuende N, Cuende JI, Fajardo J, Huet J, Alonso M. Effect population aging on the international organ donation rates and the effectiveness of the donation process. Am J Transplant 2007;7(6):1526 35. 31. Matesanz R, Coll E, Domı´nguez-Gil B, de la Rosa G, Marazuela R, Arra´ez V, et al. Benchmarking in the process of donation after brain death: a methodology to identify best performer hospitals. Am J Transplant 2012;12(9):2498 506. 32. Good practice guidelines in the process of organ donation. ,http://www.ont.es/publicaciones/Paginas/Publicaciones.aspx. [accessed March 2016]. 33. Matesanz R. Papel de los Servicios de Urgencias y Emergencias en la donacio´n de o´rganos. Emergencias 2010;22:68 71 [Article in Spanish]. 34. Escudero D, Otero J. Intensive care medicine and organ donation: exploring the last frontiers? Med Intensiva 2015;39(6):373 81. 35. El profesional de urgencias y el proceso de donacio´n. Recomendaciones ONT-SEMES. ,http://www.ont.es/infesp/Paginas/ DocumentosdeConsenso.aspx. [accessed March 2016] [in Spanish]. 36. Port FK, Bragg-Gresham JL, Metzger RA, Dykstra DM, Gillespie BW, Young EW, et al. Donor characteristics associated with reduced graft survival: an approach to expanding the pool of kidney donors. Transplantation 2002;74(9):1281 6. 37. Frei U, Noeldeke J, Machold-Fabrizii V, Arbogast H, Margreiter R, Fricke L, et al. Prospective age-matching in elderly kidney transplant recipients--a 5-year analysis of the Eurotransplant Senior Program. Am J Transplant 2008;8(1):50 7. 38. Arns W, Citterio F, Campistol JM. ‘Old-for-old’--new strategies for renal transplantation. Nephrol Dial Transplant 2007;22(2):336 41. 39. Criterios de seleccio´n del donante de o´rganos respecto a la Transmisio´n de infecciones. 2a edicio´n. 2004. ,http://www.ont.es/infesp/ % Paginas/DocumentosdeConsenso.aspx. [accessed March 2016] [in Spanish]. ´ 40. Criterios para prevenir la transmisio´n de enfermedades neopla´sicas en la donacio´n de Organos. ,http://www.ont.es/infesp/Paginas/ DocumentosdeConsenso.aspx.; 2006 [accessed March 2016] [in Spanish].

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41. Guide to the quality and safety of organs for transplantation. 5th Edition. ,https://www.edqm.eu/en/organ-tissues-cells-transplantation-guides-1607.html. [accessed March 2016]. 42. Kootstra G, Daemen JH, Oomen A. Categories of non-heart-beating donors. Transplant Proc 1995;27:2983 4. 43. Domı´nguez-Gil B, Duranteau J, Mateos A, Nu´n˜ez JR, Cheisson G, Corral E, et al. Uncontrolled Donation after Circulatory Death: European practices and recommendations for the development and optimization of an effective programme. Transplant Int 2015. Available from: http://dx.doi.org/10.1111/tri.12734. 44. Domı´nguez-Gil B, Haase-Kromwijk B, Van Leiden H, Neuberger J, Coene L, Morel P, et al. Current situation of donation after circulatory death in European countries. Transpl Int 2011;24(7):676 86. 45. Pe´rez-Villares JM, Lara-Rosales R, Pino-Sa´nchez F, Fuentes-Garcı´a P, Gil-Pin˜ero E, Osuna Ortega A, et al. Alpha code. The start of a new non-heart beating donor program. Med Intensiva 2013;37(4):224 31. 46. Sa´nchez-Fructuoso AI, Marques M, Prats D, Conesa J, Calvo N, Pe´rez-Contı´n MJ, et al. Victims of cardiac arrest occurring outside the hospital: a source of transplantable kidneys. Ann Intern Med 2006;145(3):157 64. 47. de Gracia MC, Osorio JM, Pe´rez-Villares JM, Galindo P, Ruiz MC, Pe´rez-Marfil A, et al. A new program of kidney transplantation from donors after cardiac death in Spain. Transplant Proc 2012;44(9):2518 20. 48. Miranda-Utrera N, Medina-Polo J, Pamplona M, de la Rosa F, Rodrı´guez A, Duarte JM, et al. Donation after cardiac death: results of the SUMMA 112 - Hospital 12 de Octubre Program. Clin Transplant 2013;27(2):283 8. 49. Fondevila C, Hessheimer AJ, Ruiz A, Calatayud D, Ferrer J, Charco R, et al. Liver transplant using donors after unexpected cardiac death: novel preservation protocol and acceptance criteria. Am J Transplant 2007;7(7):1849 55. 50. Jime´nez-Galanes S, Meneu-Diaz MJ, Elola-Olaso AM, Pe´rez-Saborido B, Yiliam FS, Calvo AG, et al. Liver transplantation using uncontrolled non-heart-beating donors under normothermic extracorporeal membrane oxygenation. Liver Transpl 2009;15(9):1110 18. 51. De Antonio DG, Marcos R, Laporta R, Mora G, Garcı´a-Gallo C, Ga´mez P, et al. Results of clinical lung transplant from uncontrolled nonheart-beating donors. J Heart Lung Transpl 2007;26(5):529 34. 52. Rodrı´guez A, del Rı´o F, Fuentes ME, Naranjo S, Moradiellos J, Go´mez D, et al. Lung transplantation with uncontrolled non-heart-beating donors; donor prognostic factor and immediate evolution post transplant. Arch Bronconeumol 2011;47(8):403 9. 53. Documento de Consenso Nacional sobre Donacio´n en Asistolia. ,http://www.ont.es/infesp/Paginas/DocumentosdeConsenso.aspx.; An˜o 2012 [accessed March 2016] [in Spanish]. 54. Hessheimer AJ, Domı´nguez-Gil B, Fondevila C, Matesanz R. Controlled donation after circulatory determination of death in Spain. Am J Transplant 2016. Available from: http://dx.doi.org/10.1111/ajt.13762. 55. Memoria de donacio´n en asistolia 2014. ,http://www.ont.es/infesp/Paginas/Memorias.aspx. [access March 2016] [in Spanish]. 56. Terasaki PI, Cecka JM, Gjertson DW. Takemoto S. High survival rates of kidney transplants from spousal and living unrelated donors. N Engl J Med 1995;333(6):333 6. 57. Delmonico FL. A report of the Amsterdam Forum on the Live Kidney Donor: data and medical guidelines. Transplantation 2005;79(Suppl. 6): S53 66. 58. Segev DL, Muzaale AD, Caffo BS, Mehta SH, Singer AL, Taranto SE, et al. Perioperative mortality and long-term survival following live kidney donation. JAMA 2010;303(10):959 66. 59. Ibrahim HN, Foley R, Tan L, Rogers T, Bailey RF, Guo H, et al. Long-term consequences of kidney donation. N Engl J Med 2009;360 (5):459 69. 60. Cozzi E, Biancone L, Lo´pez-Fraga M, Nanni-Costa A. Long-term outcome of living kidney donation: Position paper of the European Committee on Organ Transplantation, Council of Europe. Transplantation 2016;100(2):270 1. 61. Conclusions of the 3rd Conference of European Health Ministers (Paris, 16-17 November 1987). Council of Europe website. ,http:// www.coe.int. [access March 2016]. 62. Resolution CM/Res (2008) 6, on transplantation of kidneys from living donors who are not genetically related to the recipient. Council of Europe website. ,http://www.coe.int. [access March 2016]. 63. Resolution CM/Res(2013)56 on the development and optimisation of live kidney donation programmes and its Explanatory memorandum. ,https://www.edqm.eu/en/organ-transplantation-recommendations-resolutions-74.html#Council of Europe and CD P TO framework. [access March 2016]. 64. Domı´nguez-Gil B, Pascual J. Living donor renal transplantation in Spain: a great opportunity. Nefrologia 2008;28(2):143 7. 65. Valentı´n MO, Domı´nguez-Gil B, Martı´n Escobar E, Matesanz Acedos R. Not indicating live transplants is a poor practice. Nefrologia 2009;29(5):379 81. 66. Guı´a SEN-ONT sobre trasplante renal de donante vivo. ,http://www.ont.es/infesp/Paginas/DocumentosdeConsenso.aspx. [access March 2016]. 67. Progama de donacio´n renal cruzada. ,http://www.ont.es/infesp/Paginas/DocumentosdeConsenso.aspx. [access March 2016] [in Spanish]. 68. Protocolo de donacio´n renal altruista. ,http://www.ont.es/infesp/Paginas/DocumentosdeConsenso.aspx. [access March 2016] [in Spanish].

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C H A P T E R

3 The Deceased Kidney Donor Hany El Hennawy and Jeffrey Rogers Wake Forest Baptist Medical Center, Winston-Salem, NC, United States

3.1 BRAIN DEATH Death is an irreversible, biological event that consists of permanent cessation of the critical functions of the organism as a whole.1 Brain death is defined as the permanent absence of cerebral and brainstem functions. Trauma and subarachnoid hemorrhage are the most common events leading to brain death. Others causes include intracerebral hemorrhage, hypoxic ischemic encephalopathy, and ischemic stroke.2 4 The diagnosis of brain death is made by neurologic examination, provided that the underlying etiology is capable of causing neuronal death, and confounding processes such as drug intoxication, poisoning, metabolic derangements, and hypothermia have been excluded.5,6 The neurologic examination must demonstrate coma, no brain-generated response to external stimuli, and absent brainstem reflexes, including response to painful stimuli above the neck, or other brainoriginating movements, such as seizures, decerebrate or decorticate posturing. Other physical findings include absent pupillary light reflex, pupils that are midposition or dilated (4 9 mm), absent corneal reflexes, absent oculovestibular reflexes (caloric responses), absent jaw jerk, absent gag, sucking, or rooting reflexes, absent cough with tracheal suctioning, and apnea as demonstrated by an apnea test.5,7 The number and the expertise of the examiners required to make a brain death varies by state and country.5,6 The examiner making the diagnosis of brain death should be familiar with the clinical criteria and comfortable in performing all aspects of the examination.8 Moreover, the examiner should be someone other than the treating physician and someone other than the physician involved in the recovery of organs for transplantation.9 11 The duration of observation required to determine brain death varies extensively among countries.11 A follow-up evaluation after 24 hours was initially required for brain death diagnosis in the United States, but subsequent requirements were made age dependent. A 48-hour evaluation interval is required for infants aged 7 days to 2 months, 24 hours for those older than 2 months to 1 year, and 12 hours for those between 1 and 18 years.5 However, rates of organ donation have been noted to decrease with longer intervals between examinations.12 Ancillary tests are required to diagnose brain death when clinical criteria cannot be applied and to supplement the clinical examination in young children. Tests of brain blood flow, especially those of brain perfusion, are the most reliable “stand alone” laboratory examinations when a clinical diagnosis is not possible.13 For patients with primary supratentorial or hypoxic brain injury aged 2 years or more, repeat clinical examinations or one complete examination combined with an ancillary test are equally accurate. Ancillary tests are rarely used by physicians based at the treating hospital (31.1%) but are frequently utilized by external care providers (93.4%). The risk of death due to permanent cardiac arrest before completion of the brain death examination has been shown to increase approximately sevenfold when a neurological or neurosurgical consultation with ancillary studies was not performed.6,11,13 Cerebral blood flow tests include four vessel cerebral angiography, transcranial Doppler, magnetic resonance angiography, computed tomographic angiography, and nuclear medicine radionuclide scanning.14 Electrophysiologic tests used in the diagnosis of brain death include electroencephalogram (EEG) and evoked potentials. Electrocerebral inactivity may be confirmed when a 30-minute good quality EEG recording shows complete electrocerebral silence, defined as no cerebral activity greater than 2 uV, having first ruled out the possible influence of sedative drugs, metabolic disorders, or hypothermia.15 It is essential to support the family of a patient with a progressively worsening severe acute brain injury,

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and to do this with the utmost understanding of the patient’s ultimate hopelessness for meaningful recovery. Any conversation should start with a clarification of the catastrophic nature of the illness and with establishing the “point of no return.” When brainstem reflexes are lost and the patient has become apneic, family members should be appropriately informed. Communicating with family members also necessarily involves a discussion regarding the potential suitability for organ donation,16 although the highest success rates for obtaining consent for organ donation are achieved by trained requestors whom are not involved with the medical care of the potential donor.17

3.2 DECEASED DONATION In 2012, 17,305 kidney transplants were performed in the United States, 11,535 of which were from deceased donors. 340 fewer kidney transplants were performed in 2012 than in 2011. The number of kidney transplants has remained relatively stable since 2005, paralleling a leveling off of the end stage renal disease (ESRD) incidence rate. The waiting list continues to grow, with a seven percent increase from 2011 to 2012, reaching 81,981 patients at the end of 2012.18 Rates of deceased donation increased slightly in 2011, to 22.4 donors per million population. Deceased donor transplant rates remained steady in 2011, at 2.5 per 100 patient years on dialysis19 and at 5.65 per 1000 deaths.18 Since 2005, deceased donation rates by race have been highest among Blacks, reaching 27% in 2011, compared to 9.3% and 8.4% among Native Americans and Asians. Perhaps more concerning than relatively unvarying donation rates is that an increasing number of kidneys recovered for transplant are discarded. The discard rate has increased steadily from 12.7% in 2002 to 17.9% in 2011. The percentage of expanded criteria donor (ECD) and intended dual kidneys recovered but not transplanted in the United States remains significantly high (42% and 20%, respectively).20 This is particularly striking when compared to the 8% overall kidney discard rate reported by the Eurotransplant Consortium.21 The most common donor-specific reasons for kidney discard after procurement were biopsy findings (37.3%), poor organ function (9.2%), and anatomic abnormalities (7.1%).22 The odds of kidney discard are 12 times higher when biopsy shows .20% glomerulosclerosis (GS) than if GS is ,5%.23 In a death-censored adjusted Cox model, Tanriover et al. demonstrated that GS 15% 50% was not associated with higher risk of allograft failure compared to a reference group with GS , 15%. Overall, GS does not appear to be an accurate histologic indicator of subsequent renal function.20 In the future, more uniform and inclusive biopsy report system (vessels, glomeruli, tubules, and interstitium) and a better understanding of the significance of various histopathologic findings will be necessary to reduce discard rates and increase transplant rates of recovered organs.20 Furthermore, in 2011, kidneys were not recovered from 9% of donors in whom at least one other organ was recovered for transplant, primarily due to poor organ function and donor medical history. The increasing rates of kidney discard and nonrecovery undoubtedly reflect a deceased donor pool that is clearly aging and has more associated risks for graft failure. Improvements in organ donation, utilization, and allocation are needed to maximize the benefits of transplant from an aging and relatively static donor pool.19

3.3 EXPANDING THE DONOR POOL The ongoing organ shortage crisis challenges the transplant community to maximize and optimize the use of kidneys from all consented deceased donors. The fact that the waiting list continues to grow in the face of a relatively unchanged donor pool requires an ongoing reappraisal of the limits of acceptability when considering whether or not a recovered kidney can or should be transplanted. The annual mortality of a patient on the waiting list is approximately 6% and as high 10% in diabetic candidates. When the risk of waitlist mortality is weighed against the mortality related to a potential transplant recipient’s age and other comorbidities, it is clear that the amount of time different patients can tolerate waiting for a kidney transplant is highly variable. More aggressive utilization of “marginal” kidneys takes into account that, in appropriately selected recipients, more timely transplantation and improved patient survival in exchange for decreased long-term graft survival may represent a worthwhile trade-off. The next several sections focus on various strategies which are being utilized in ongoing efforts to maximize the rates of recovery and transplantation of deceased donor kidneys.

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3.3.1 Expanded Criteria Donors Over the past two decades, there has been a shift toward increasing numbers of older donors and older recipients in kidney transplantation, with cerebrovascular events now the leading cause of brain death in deceased donation.24 The aging recipient and donor population have inevitably resulted in increased transplantation of kidneys from older donors. The value of transplanting such kidneys was previously questioned because of concerns over decreased graft survival and poorer predicted outcomes.25 27 In October 2007, The United Network of Organ Sharing (UNOS) defined the term “expanded criteria donor” (ECD) as a donor age 60 years and older or 50 59 years old with at least two of the following criteria: history of hypertension, terminal serum creatinine $ 1.5 mg/ dL, and cerebrovascular cause of death. This definition was derived from a Scientific Registry of Transplant Recipients (SRTR) retrospective analysis of primary deceased donor adult kidney transplant recipients which demonstrated that ECD kidneys had a 70% higher risk of graft failure compared to a reference group of kidneys from donors aged 10 to 39, without hypertension, cerebrovascular cause of death, or terminal serum creatinine 1.5 mg/ dL.25 27 Optimal use of ECD kidneys continues to be debated, although there is general agreement that these kidneys should be used to improve patient access to transplantation when life expectancy on dialysis is shorter than the expected waiting time for a kidney, particularly in older patients and diabetics.28 Although there are reports of inferior graft survival and poorer intermediate-term results, Stratta et al. have contended that ECD kidneys are defined by suboptimal nephron mass and that appropriate donor and recipient selection may optimize outcomes with ECD kidney transplantation.24 Transplantation of ECD kidneys into selected “low risk” and “low functional need” recipients, specifically older patients with low body mass index (BMI), and low immunologic risk, has achieved acceptable intermediate-term outcomes, with graft survival comparable to a group of concurrently transplanted standard criteria donor (SCD) kidneys. ECD kidney graft survival has improved with era, possibly due to introduction of mycophenolate and subsequent decreases in calcineurin inhibitor doses and target levels. Unfortunately, kidney quality and graft survival is highly variable within the ECD category, with some ECD kidneys having better functional outcomes and superior graft survival compared to SCD kidneys. Consequently, a continuous kidney donor risk index (KDRI) was proposed in 2009, combining donor and transplant variables to quantify graft failure risk.29 There was a considerable overlap in KDRI distribution according to ECD and SCD classification. The graded impact of KDRI on graft outcome is thought to provide a more useful decision-making tool compared to simply categorizing donors as ECD or SCD. KDRI forms the basis of the kidney donor profile index (KDPI), which is utilized to stratify deceased donor kidneys according to expected graft survival in the newly implemented kidney allocation system in the United States, discussed in more detail later in this chapter. KDPI includes 10 donor factors (age, height, weight, ethnicity, history of hypertension and diabetes, cause of death, serum creatinine level, hepatitis C status, and DCD), with lower scores associated with increased graft longevity. It is anticipated that use of KDPI rather than ECD criteria will facilitate matching expected graft survival with recipient survival, thereby optimizing allocation of graft life years.

3.3.2 Donation After Circulatory Death The increasing use of kidneys from controlled donation after circulatory death (DCD) donors has the potential to positively impact organ recovery and transplant rates. The United Kingdom, the Netherlands, and Belgium now have very successful DCD donor programs with 7.0 9.5 DCD donors per million population in 2013. The United States, Australia, Spain, and Croatia also have well-developed DCD programs with 2.1 3.8 DCD donors per million population. However, many countries have no or limited experience with DCD donors and would benefit from this important source of deceased donor kidneys.30 Although overall donation rates have not changed significantly in recent years, the percentage of transplants performed from donation after cardiac death (DCD) donors in the United States has increased steadily from 1.4% in 1998 to 15.8% in 2011.22 Compared with kidneys recovered from donation after brain death donors (DBD), DCD kidneys have a higher incidence of delayed graft function (DGF) and are more vulnerable to the detrimental effect of prolonged preservation time.30 DCD kidneys are subjected to variable periods of warm ischemia after withdrawal of life support, followed by declaration of death by cardiocirculatory arrest with subsequent organ procurement.31 Warm ischemia is known to be associated with acute tubular necrosis, irreversible cell damage, and reduced graft survival after kidney transplantation. Despite the warm ischemia associated with DCD donation, numerous studies have demonstrated comparable short- and intermediate-term graft survival rates between brain dead non-ECD kidneys and DCD non-ECD kidneys. DGF is more common in kidneys from DCD donors, with an incidence of 25% 90%.32 Older donor age, and more specifically ECD status, appears to adversely affect kidney graft survival. In a study by Farney et al., DCD-ECD kidney graft survival at 3 years was 48%

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compared to 79% for non-ECD-DCD kidney.32 It has been suggested that utilizing DCD-ECD kidneys as dual kidney transplants could potentially improve outcomes. Interestingly, several studies have shown that DGF does not have the same detrimental effect on graft survival after transplantation of DCD kidneys as it does after transplantation of DBD kidneys.33 35 Two different studies of machine preservation reported opposite conclusions regarding the efficacy of machine preservation in reducing the incidence of DGF after DCD kidney transplantation, with an incidence of DGF 50% in both studies.36,37 Early experience with extracorporeal support to reduce ischemic injury and DGF is promising, although application of this technology is limited to date.

3.3.3 Kidneys From Donors With Acute Kidney Injury Until relatively recently, kidneys from donors with acute kidney injury (AKI) were almost universally declined for transplantation because outcomes were expected to be poor. A landmark series by Anil Kumar et al. in 2006 compared the outcome of 55 kidneys transplanted from donors with AKI with 55 concurrent and matched recipients of SCD kidneys and 55 concurrent and matched recipients of ECD kidneys.38 AKI kidneys were accepted for transplantation from donors aged ,50 years with no history of kidney disease and a pretransplant kidney biopsy without evidence of significant chronic changes. Three-year patient and graft survival was 90% and 90% in the AKI group, 100% and 89% in the SCD group, and 83% and 66% in the ECD group. Rates of biopsy proven acute rejection were comparable among groups; however, chronic allograft nephropathy was more prevalent in the ECD group. Mean serum creatinine levels were 1.9, 1.9, and 2.2 mg/dL in the AKI, SCD, and ECD groups, respectively (SCD and AKI vs ECD, P5.04). This study demonstrated that transplantation of kidneys from selected donors with AKI provides comparable graft survival and function to non-AKI donors, despite a higher DGF rate, and that transplantation of kidneys from appropriately selected AKI donors may help expand the donor pool. In 2009, Kayler et al. reviewed SRTR data from 1995 to 2007 to study the outcome and utilization of kidneys from deceased donors with AKI.39 The relative risk for graft failure in ECD recipients significantly increased with increasing serum creatinine. Among potential SCD donors, elevated serum creatinine was identified as a strong independent risk factor for kidney discard. However, when kidney transplantation was performed, elevated donor terminal creatinine was not a risk factor for graft failure. This study clearly underscored the fact that potentially transplantable kidneys are being discarded on the basis of elevated serum creatinine, and that a more aggressive approach to transplanting kidneys from donors with terminal AKI may increase the number of deceased donor kidneys available for transplantation. Others studies have similarly reported good results with transplantation of kidneys from donors with AKI, with long-term graft survival rates and functional outcomes comparable to non-AKI kidneys despite a significantly higher incidence of DGF with AKIs kidneys.40 42

3.3.4 Dual Kidney Transplants The use of kidneys “marginal” or ECD donors is an appealing strategy to expand the pool of organs available for transplantation. Concerns over the limited life span of “marginal” kidneys have led some to perform dual kidney transplantation (DKT) based on the concept that poor long-term outcomes associated with ECD kidneys may be the consequence of a discrepancy between the functional nephron mass provided by the graft and the metabolic requirements of the recipient.43,44 Simultaneous transplantation of two kidneys into one recipient, with appropriate donor and recipient selection, could increase the transplanted nephron mass and could improve long-term function and graft survival compared to single kidney transplantation (SKT) from a “marginal” donor. In some cases, DKT might even produce acceptable outcomes with donor kidneys thought to be unsuitable for SKT, which would otherwise be discarded. Selection of optimal candidates for DKT is important because it is a more extensive procedure and is typically performed in elderly recipients who tend to have less physiologic reserve than younger recipients. Appropriate recipient selection is critical to achieving good outcomes with DKT. While it is possible to perform DKT in elderly recipients, it is important to select patients with adequate cardiac reserve and without extensive iliac artery atherosclerosis. Selecting patients with BMI less than 30 kg/m2 may be preferable from a technical standpoint and may also help improve functional outcomes by maximizing nephron mass in recipients with lower muscle mass and lower metabolic demand.43 Remuzzi et al. used a scoring system to determine criteria for DKT versus SKT. The authors reported excellent short- and long-term results after allocation of kidneys from donors .60 years of age based on a preimplant biopsy 12-point histologic scoring system proposed by the Dual Kidney Transplant Group that assesses vessels, glomeruli, tubules and interstitium. Their results suggest that the histologic evaluation of donor kidneys serves to reduce variability in predicting outcomes.45 Unfortunately, the absence of UNOS data

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on features other than GS limits the ability to validate the usefulness of histopathology in predicting outcomes. Several reports have described improved graft function or survival with the use of DKT rather than SKT. Criteria for choosing to perform DKT have generally included older donor age and one or more other parameters, such as preimplant kidney biopsy, donor estimated creatinine clearance, kidney weight, machine perfusion characteristics and length of preservation time.44,46,47 Klair et al., used SRTR data of de novo kidney transplant recipients of adult deceased donor kidneys from 1995 to 2010 to examine outcomes of SKT and DKT stratified by KDRI group # 1.4 (n 5 49,294), 1.41 1.8 (n 5 15,674), 1.81 2.2 (n 5 6523) and .2.2 (n 5 2791). They noticed that DKT of kidneys with KDRI .2.2 was associated with significantly better overall graft survival compared to SKT with KDRI .2.2. Moreover, DKT was associated with significantly decreased odds of DGF (top 2 KDRI categories) and significantly decreased odds of 1-year serum creatinine level .2 mg/dL (top 3 KDRI categories). Furthermore, among SKT and DKT from KDRI .2.2 there were 16.1 and 13.9 graft losses per 100 patient follow-up years, respectively. Additionally, there were no differences in overall patient survival with the use of DKT with kidneys from any KDRI category. However, there was a trend toward a survival benefit with each increasing category of KDRI. They concluded that KDRI . 2.2 is a useful discriminatory cut-off for the determination of graft survival benefit with the use of DKT. It is noteworthy that the benefit of increased graft years was less than half that of SKT from donors in the same KDRI range.43 Over the past decade, the surgical technique DKT has evolved from a bilateral iliac fossa approach (either intraperitoneal or extraperitoneal) to an extraperitoneal unilateral approach. This shortens operating time by limiting iliac artery and vein dissection to one side and preserves the contralateral iliac vessels should retransplantation become necessary in the future. Despite the increased duration and technical complexity of DKT, surgical complication rates have been shown to be comparable to those of SKT.48

3.3.5 Kidneys From Small Pediatric Donors There has been controversy regarding the optimal use of kidneys from young pediatric donors ever since the first pediatric en bloc kidney transplant into an adult was performed in 1972.49 Concerns regarding long-term graft survival and function, particularly with kidneys from donors younger than 12 months of age, as well as a general reluctance to separate pediatric kidneys for transplantation into two recipients, have historically limited use of these kidneys. Reports of an increased incidence of vascular and urinary complications and higher rates of acute cellular rejection and DGF are often cited as the primary deterrents to transplantation of small, pediatric, deceased donor kidneys into adults.50 However, several studies have suggested that, when initial complications are avoided, pediatric kidneys may be considered excellent rather than marginal quality grafts for transplantation into adults.51 53 Pediatric en bloc kidneys have double the number of nephrons, and several studies have shown better long-term function than living donor kidney transplants, presumably due to the increased functional glomerular reserve these kidneys possess.54 Sharma et al. reported excellent long-term outcomes after pediatric en bloc kidney transplantation from donors weighing less than or equal to 15 kg, which were comparable to results following living donor kidney transplant.55 Separation of en bloc pairs has been suggested when the renal allograft measures greater than 6 cm in length and the donor weight is greater than 14 kg. Other series have shown that kidneys from donors 1 to 3 years old and/or weighing 9 15 kg can be successfully transplanted en bloc, and that those from donors more than 3 years old and/or weighing more than 15 kg were best transplanted as single grafts.56 A study by Bresnahan et al. showed better results using en bloc kidneys from donors younger than 5 years old compared with single grafts from donors weighing more than 15 kg. However, recipients of previous transplants, prolonged ischemia time, black recipients, and those with a BMI greater than 24 kg/m2 were considered risk factors for poor results.57 A recent analysis of UNOS data demonstrated that long-term outcomes of pediatric en bloc kidney transplants (donor younger than 5 years of age) were superior to matched recipients of solitary pediatric kidney transplants (donor younger than 5 years of age) and SCD adult kidney recipients.58 The ongoing disparity between static supply and increasing demand for organs has prompted some surgeons to explore the limits of acceptability of SKT from small pediatric donors into two recipients. Despite a higher incidence of posttransplantation vascular and urological complications, long-term graft survival after single KT (in weight-matched pediatric donors and selected adult recipients) was comparable with that after SCD kidney transplantation.59 Laurence et al. constructed a decision analysis model to predict outcome in life years for patients with ESRD on the waiting list, depending on whether they received en bloc or solitary pediatric transplants.60 At all recipient ages, the projected life years of both recipients of a solitary transplant exceeded the projected life years of an en bloc kidney transplant. Only recipients of solitary kidney transplants from donors weighing less than 10 kg had an estimated net loss of life years. In a review of OPTN data, Sureshkumar et al. also found that the graft failure risk of solitary pediatric kidneys was

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consistently lower when the donor weight exceeded 10 kg.61 Some studies have shown that kidneys from small donors may be at increased risk for late graft failure if they are transplanted into large recipients, and therefore, the relative size of the donor and recipient should be taken into account.62 In conclusion, these studies indicate that more liberal transplantation of kidneys from pediatric donors may expand the donor pool. Solitary rather than en bloc transplantation of kidneys from donors weighing more than 10 kg offers more cumulative graft years and maximizes organ utilization without compromising outcomes.

3.3.6 Centers for Disease Control and Prevention High-Risk Kidneys The use of kidneys from donors designated by the Centers for Disease Control and Prevention (CDC) as being at higher risk for transmission of viral infections is being increasingly recognized as another alternative for expanding the donor pool. The risk of transplanting organs from such donors is the possibility of transmitting potentially life threatening viral infections, such as human immunodeficiency virus, hepatitis C, and hepatitis B, rather than the potential for decreased graft survival associated with “marginal” donors. Unfortunately, transplantation of kidneys from CDC high-risk donors remains controversial because of limited data on risks and outcomes. The reported incidence of viral transmission with CDC high-risk donors is actually quite low63,64 and may even be less than the risk of viral infection associated with long-term hemodialysis.65 Despite these facts, CDC high-risk donors remain underutilized because of physician and patient perception that the risk of viral transmission is greater than the data would indicate. Introduction of nucleic acid testing has significantly narrowed the window between the time of infection and detection, and recent broader implementation of this testing should further minimize the risks of viral transmission with transplantation. A recent, single center, retrospective study of 50 recipients of kidneys from CDC high-risk donors revealed no instances of seroconversion with nearly 1 year of follow-up.66 Donors in this study were younger and less like to be ECD. Willingness to accept a kidney from a CDC high-risk donor was associated with significantly shorter time on the wait list prior to transplantation. Education of transplant physicians, surgeons, and prospective transplant recipients regarding the actual risks and benefits associated with using kidneys from CDC high-risk donors is necessary so that informed decisions can be made and so that these potentially lifesaving organs are appropriately utilized.

3.3.7 New Kidney Allocation System in the United States After more than a decade of ongoing development and revision via a consensus driven process, a new kidney allocation system was implemented in the United States in December 2014. The major areas of concern with the previous allocation system that are addressed in the new system are as follows: (1) Some potentially transplantable kidneys were being discarded because of how donors were classified and how organs were offered. Although an increase in donation rates was obviously desirable, it was felt that more transplants could still be performed by making better use of existing organs. (2) There was no maximum life year benefit in the previous system, in which there were often significant mismatches between the projected longevity of kidneys and the expected survival of recipients. As a result, some patients with long expected survival who received less than ideal kidneys required retransplantation, thereby lowering the chances for others to receive a first transplant. (3) Access to transplantation was found to be highly variable among candidates. In many cases, highly sensitized patients were waiting much longer than average for a transplant. Several new policy changes in the new allocation system have been implemented to address these concerns, with the intent of enhancing recipient survival, making better use of available kidneys, and increasing transplant opportunities for highly sensitized patients.67 69 The key components of the system are: (1) KDPI and EPTS: The Kidney Donor profile Index is a formula that has been established to estimate the expected graft survival of a donor kidney. Estimated Posttransplant Survival (EPTS) is a second formula which is used to estimate the likely benefit a specific patient would derive from transplantation. The system provides consideration for the 20% of kidneys with the best KDPI (longest estimated graft survival) to the 20% of candidates estimated by EPTS to have the longest time to benefit from a kidney transplant. For the remaining 80% of candidates, organ allocation remains similar to the previous system unless the patient falls into one of the “hard-to-match” categories. KDPI provides more detailed information than previous classifications of SCD and ECD, and it is believed that a graded rather than binary assessment of expected graft survival will improve kidney utilization and increase transplant rates. (2) Blood type subgroup matching: Blood type B is relatively uncommon and is present in only about 16% of kidney transplant candidates, many of whom are ethnic minorities. Although patients with blood type B could receive kidneys from blood type

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O donors, this practice would reduce access to deceased donor kidneys for blood type O recipients who can only receive blood type O kidneys. It has been shown that donors with the A2 subtype of blood type A are compatible with blood type B recipients and provide good outcomes after kidney transplantation. In the new system, kidneys from donors with blood type A2 can be offered to blood type B candidates on a national basis in order to improve access and reduce waiting time for blood type B candidates. (3) Immune sensitivity matching: For many years, the kidney allocation system has offered some additional priority in the form of extra allocation points for candidates with a panel-reactive antibodies (PRA) of 80% or higher, since these patients tend to wait longer for transplant because of increased difficulty obtaining an organ from an HLA compatible donor. However, in the previous system, no extra points were awarded as PRA increased, nor were points given to sensitized patients with PRA ,80%. Based on the results of a time-to-kidney offer analysis according to PRA, the new system gives priority points to sensitized patients using a sliding scale, starting at a PRA of 20%. The most highly sensitized patients (PRA 98%, 99%, and 100%) receive significantly more priority than they did previously. It is expected that this will provide more equitable access to transplant for a group of patients with the longest waiting times as a result of compatibility obstacles. (4) Dialysis waiting time: In the previous system, the duration of time on the waiting list was the predominant factor in prioritization of organ offers. Whereas some patients have regular access to medical care and are promptly placed on the waiting list when they begin dialysis or have a glomerular filtration rate of ,20 mL/minute, other patients reach ESRD and may be on dialysis for many years prior to being placed on the waiting list. Since waiting time is a priority factor, patients who are listed late for a transplant will likely be on dialysis longer than patients listed at an earlier stage of renal failure. Many patients who are not listed promptly have socioeconomic disadvantages. Consequently, in the new allocation system, waiting time points begin when a candidate is placed on the waiting list and has a glomerular filtration rate or creatinine clearance # 20 mL/minute or has started dialysis. For candidates listed after starting dialysis, waiting time is backdated to the date of dialysis initiation.

3.4 CONCLUSION Ongoing efforts continue to expand the deceased donor kidney pool by maximizing organ donation and utilization as well as improving organ allocation. Ideally, these strategies should increase transplant rates, optimize life years gained from transplantation, and minimize the need for retransplantation. Despite the progress that has been made, the demand for kidney transplantation rapidly continues to outpace organ availability. Ongoing advances in our understanding of immunologic and nonimmunologic mechanisms of allograft failure and less reliance on nephrotoxic immunosuppression regimens are paramount to optimizing long-term graft survival in deceased donor transplantation. Increasing the number of living donor transplants performed via desensitization protocols, ABO incompatible transplantation, kidney paired exchange (discussed elsewhere in this book), and other novel strategies, will be critical in order to make more deceased donor kidneys available for candidates who do not have the opportunity to receive a living donor kidney. Tolerance induction, cell transplantation, bioartificial organ technology, and other advances in regenerative medicine continue to represent the frontiers of transplantation science, and may ultimately be the best hope for clinical solutions that will one day mitigate the widening disparity between organ supply and demand.

References 1. Wijdicks EF. The diagnosis of brain death. N Engl J Med 2001;344:1215 21. 2. Goudreau JL, Wijdicks EF, Emery SF. Complications during apnea testing in the determination of brain death: predisposing factors. Neurology 2000;55:1045 8. 3. Saposnik G, Bueri JA, Maurino J, Saizar R, Garretto NS. Spontaneous and reflex movements in brain death. Neurology 2000;54:221 3. 4. Wijdicks EF, Rabinstein AA, Manno EM, Atkinson JD. Pronouncing brain death: contemporary practice and safety of the apnea test. Neurology 2008;71:1240 4. 5. Practice parameters for determining brain death in adults (summary statement). The Quality Standards Subcommittee of the American Academy of Neurology. Neurology 1995;45:1012 4. 6. Wijdicks EF, Varelas PN, Gronseth GS, Greer DM. Evidence-based guideline update: determining brain death in adults: report of the Quality Standards Subcommittee of the American Academy of Neurology. Neurology 2010;74:1911 18. 7. Citerio G, Murphy PG. Brain death: the European perspective. Semin Neurol 2015;35:139 44. 8. Booth CM, Boone RH, Tomlinson G, Detsky AS. Is this patient dead, vegetative, or severely neurologically impaired? Assessing outcome for comatose survivors of cardiac arrest. JAMA 2004;291:870 9. 9. Baumgartner H, Gerstenbrand F. Diagnosing brain death without a neurologist. BMJ 2002;324:1471 2.

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10. Shemie SD, Doig C, Dickens B, et al. Severe brain injury to neurological determination of death: Canadian forum recommendations. CMAJ 2006;174:S1 13. 11. Wijdicks EF. Brain death worldwide: accepted fact but no global consensus in diagnostic criteria. Neurology 2002;58:20 5. 12. Lustbader D, O’Hara D, Wijdicks EF, et al. Second brain death examination may negatively affect organ donation. Neurology 2011;76:119 24. 13. Hoffmann O, Masuhr F. Access to brain death diagnostics. Nervenarzt 2014;85:1573 81. 14. Ala TA, Kuhn MJ, Johnson AJ. A case meeting clinical brain death criteria with residual cerebral perfusion. Am J Neuroradiol 2006;27:1805 6. 15. Szurhaj W, Lamblin MD, Kaminska A, Sediri H. EEG guidelines in the diagnosis of brain death. Neurophysiol Clin 2015;45:97 104. 16. Kompanje EJ. Families and brain death. Semin Neurol 2015;35:169 73. 17. Rodrigue JR, Cornell DL, Howard RJ. Organ donation decision: comparison of donor and nondonor families. Am J Transplant 2006;6:190 8. 18. Saran R, Li Y, Robinson B, Ayanian J, et al. US Renal Data System 2014 Annual Data Report: Epidemiology of Kidney Disease in the United States. Am J Kidney Dis 2015;65:A7. 19. Collins AJ, Foley RN, Chavers B, et al. US Renal Data System 2013 Annual Data Report. Am J Kidney Dis 2014;63:A7. 20. Tanriover B, Mohan S, Cohen DJ, et al. Kidneys at higher risk of discard: expanding the role of dual kidney transplantation. Am J Transplant 2014;14:404 15. 21. Vinkers MT, Smits JM, Tieken IC, de Boer J, Ysebaert D, Rahmel AO. Kidney donation and transplantation in Eurotransplant 2006-2007: minimizing discard rates by using a rescue allocation policy. Prog Transplant 2009;19:365 70. 22. Matas AJ, Smith JM, Skeans MA, et al. OPTN/SRTR 2011 Annual Data Report: kidney. Am J Transplant 2013;13(Suppl. 1):11 46. 23. Sung RS, Christensen LL, Leichtman AB, et al. Determinants of discard of expanded criteria donor kidneys: impact of biopsy and machine perfusion. Am J Transplant 2008;8:783 92. 24. Stratta RJ, Rohr MS, Sundberg AK, et al. Intermediate-term outcomes with expanded criteria deceased donors in kidney transplantation: a spectrum or specter of quality? Ann Surg 2006;243:594 601. 25. Metzger RA, Delmonico FL, Feng S, Port FK, Wynn JJ, Merion RM. Expanded criteria donors for kidney transplantation. Am J Transplant 2003;3(Suppl. 4):114 25. 26. Ojo AO, Hanson JA, Meier-Kriesche H, et al. Survival in recipients of marginal cadaveric donor kidneys compared with other recipients and wait-listed transplant candidates. J Am Soc Nephrol 2001;12:589 97. 27. Port FK, Bragg-Gresham JL, Metzger RA, et al. Donor characteristics associated with reduced graft survival: an approach to expanding the pool of kidney donors. Transplantation 2002;74:1281 6. 28. Pascual J, Zamora J, Pirsch JD. A systematic review of kidney transplantation from expanded criteria donors. Am J Kidney Dis 2008;52:553 86. 29. Rao PS, Schaubel DE, Guidinger MK, et al. A comprehensive risk quantification score for deceased donor kidneys: the kidney donor risk index. Transplantation 2009;88:231 6. 30. Summers DM, Watson CJ, Pettigrew GJ, et al. Kidney donation after circulatory death (DCD): state of the art. Kidney Int 2015;88:241 9. 31. Abt PL, Fisher CA, Singhal AK. Donation after cardiac death in the US: history and use. J Am Coll Surg 2006;203:208 25. 32. Farney AC, Hines MH, al-Geizawi S, Rogers J, Stratta RJ. Lessons learned from a single center’s experience with 134 donation after cardiac death donor kidney transplants. J Am Coll Surg 2011;212:440 51. 33. Ojo AO, Wolfe RA, Held PJ, Port FK, Schmouder RL. Delayed graft function: risk factors and implications for renal allograft survival. Transplantation 1997;63:968 74. 34. Singh RP, Farney AC, Rogers J, et al. Kidney transplantation from donation after cardiac death donors: lack of impact of delayed graft function on post-transplant outcomes. Clin Transplant 2011;25:255 64. 35. Yarlagadda SG, Coca SG, Formica Jr. RN, Poggio ED, Parikh CR. Association between delayed graft function and allograft and patient survival: a systematic review and meta-analysis. Nephrol Dial Transplant 2009;24:1039 47. 36. Jochmans I, Moers C, Smits JM, et al. Machine perfusion versus cold storage for the preservation of kidneys donated after cardiac death: a multicenter, randomized, controlled trial. Ann Surg 2010;252:756 64. 37. Watson CJ, Wells AC, Roberts RJ, et al. Cold machine perfusion versus static cold storage of kidneys donated after cardiac death: a UK multicenter randomized controlled trial. Am J Transplant 2010;10:1991 9. 38. Anil Kumar MS, Khan SM, Jaglan S, et al. Successful transplantation of kidneys from deceased donors with acute renal failure: three-year results. Transplantation 2006;82:1640 5. 39. Kayler LK, Garzon P, Magliocca J, et al. Outcomes and utilization of kidneys from deceased donors with acute kidney injury. Am J Transplant 2009;9:367 73. 40. Greenstein SM, Moore N, McDonough P, Schechner R, Tellis V. Excellent outcome using “impaired” standard criteria donors with elevated serum creatinine. Clin Transplant 2008;22:630 3. 41. Lin NC, Yang AH, King KL, Wu TH, Yang WC, Loong CC. Results of kidney transplantation from high-terminal creatinine donors and the role of time-zero biopsy. Transplant Proc 2010;42:3382 6. 42. Molina M, Apaza J, Gonzalez Monte E, et al. Results of kidney transplantation from deceased donors with acute kidney injury. Transplant Proc 2015;47:42 4. 43. Klair T, Gregg A, Phair J, Kayler LK. Outcomes of adult dual kidney transplants by KDRI in the United States. Am J Transplant 2013;13:2433 40. 44. Nardo B, Bertelli R, Cavallari G, et al. Analysis of 80 dual-kidney transplantations: a multicenter experience. Transplant Proc 2011;43:1559 65. 45. Remuzzi G, Cravedi P, Perna A, et al. Long-term outcome of renal transplantation from older donors. N Engl J Med 2006;354:343 52. 46. Kayler LK, Mohanka R, Basu A, Shapiro R, Randhawa PS. Single versus dual renal transplantation from donors with significant arteriosclerosis on pre-implant biopsy. Clin Transplant 2009;23:525 31.

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47. Ruggenenti P, Perico N, Remuzzi G. Ways to boost kidney transplant viability: a real need for the best use of older donors. Am J Transplant 2006;6:2543 7. 48. Ekser B, Furian L, Broggiato A, et al. Technical aspects of unilateral dual kidney transplantation from expanded criteria donors: experience of 100 patients. Am J Transplant 2010;10:2000 7. 49. Meakins JL, Smith EJ, Alexander JW. En bloc transplantation of both kidneys from pediatric donors into adult patients. Surgery 1972;71:72 5. 50. Modlin C, Novick AC, Goormastic M, Hodge E, Mastrioanni B, Myles J. Long-term results with single pediatric donor kidney transplants in adult recipients. J Urol 1996;156:890 5. 51. Dharnidharka VR, Stevens G, Howard RJ. En-bloc kidney transplantation in the United states: an analysis of united network of organ sharing (UNOS) data from 1987 to 2003. Am J Transplant 2005;5:1513 17. 52. Merkel FK, Matalon TA, Brunner MC, et al. Is en bloc transplantation of small pediatric kidneys into adult recipients justified? Transplant Proc 1994;26:32 3. 53. Nghiem DD, Hsia S, Schlosser JD. Growth and function of en bloc infant kidney transplants: a preliminary study. J Urol 1995;153:326 9. 54. Sureshkumar KK, Reddy CS, Nghiem DD, Sandroni SE, Carpenter BJ. Superiority of pediatric en bloc renal allografts over living donor kidneys: a long-term functional study. Transplantation 2006;82:348 53. 55. Sharma A, Fisher RA, Cotterell AH, King AL, Maluf DG, Posner MP. En bloc kidney transplantation from pediatric donors: comparable outcomes with living donor kidney transplantation. Transplantation 2011;92:564 9. 56. Borboroglu PG, Foster 3rd CE, Philosophe B, et al. Solitary renal allografts from pediatric cadaver donors less than 2 years of age transplanted into adult recipients. Transplantation 2004;77:698 702. 57. Bresnahan BA, McBride MA, Cherikh WS, Hariharan S. Risk factors for renal allograft survival from pediatric cadaver donors: an analysis of united network for organ sharing data. Transplantation 2001;72:256 61. 58. Bhayana S, Kuo YF, Madan P, et al. Pediatric en bloc kidney transplantation to adult recipients: more than suboptimal? Transplantation 2010;90:248 54. 59. Sharma A, Ramanathan R, Behnke M, Fisher R, Posner M. Single pediatric kidney transplantation in adult recipients: comparable outcomes with standard-criteria deceased-donor kidney transplantation. Transplantation 2013;95:1354 9. 60. Laurence JM, Sandroussi C, Lam VW, Pleass HC, Eslick GD, Allen RD. Utilization of small pediatric donor kidneys: a decision analysis. Transplantation 2011;91:1110 13. 61. Sureshkumar KK, Patel AA, Arora S, Marcus RJ. When is it reasonable to split pediatric en bloc kidneys for transplantation into two adults? Transplant Proc 2010;42:3521 3. 62. Nakatani T, Uchida J, Yamazaki T, et al. Cadaveric renal transplantation from a non-heart-beating pediatric donor into adult recipients. Urol Int 2003;70:216 18. 63. Kucirka LM, Namuyinga R, Hanrahan C, Montgomery RA, Segev DL. Provider utilization of high-risk donor organs and nucleic acid testing: results of two national surveys. Am J Transplant 2009;9:1197 204. 64. Kucirka LM, Singer AL, Segev DL. High infectious risk donors: what are the risks and when are they too high? Curr Opin Organ Transplant 2011;16:256 61. 65. Freeman RB, Cohen JT. Transplantation risks and the real world: what does ‘high risk’ really mean? Am J Transplant 2009;9:23 30. 66. Lonze BE, Dagher NN, Liu M, et al. Outcomes of renal transplants from Centers for Disease Control and Prevention high-risk donors with prospective recipient viral testing: a single-center experience. Arch Surg 2011;146:1261 6. 67. Bray RA, Gebel HM. The new kidney allocation system (KAS) and the highly sensitized patient: expect the unexpected. Am J Transplant 2014;14:2917. 68. Friedewald JJ, Samana CJ, Kasiske BL, et al. The kidney allocation system. Surg Clin North Am 2013;93:1395 406. 69. Pondrom S. What you need to know about the new Kidney Allocation System. Am J Transplant 2014;14:1470.

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C H A P T E R

4 The Living Donor Andrea Pietrabissa, Luigi Pugliese, Massimo Abelli, Elena Ticozzelli and Teresa Rampino University of Pavia, Pavia, Italy

4.1 BACKGROUND The modern history of organ transplantation began on December 23, 1954, with the first identical-twin kidney transplantation. Previous attempts at transplanting a kidney in humans had invariably failed due to the immunological barrier, with little understanding of the rejection mechanism and no immunosuppressive drugs available. The medical and ethical issues behind the first live donation were raised and partially addressed by the pioneer kidney transplant team at Peter Bent Brigham Hospital in Boston, which comprised Drs. John P. Merrill (Head of Nephrology), J. Hartwell Harrison (Chief of Urology), Gustave Dammin (Pathologist-in-Chief), and Joseph E. Murray, the chief surgeon who was awarded the Nobel prize in 1990. In his autobiography, Dr. Murray recalls their awareness of being about to compromise the physician’s injunction to “do no harm,” and the difficulty of dealing with such an unprecedented situation, including the need to inform the patients and their family about the potential risks of an unknown procedure.1 At the same time, without the alternative of dialysis, not yet as effective as it is today, they were committed to attempting the transplant, as the patient was otherwise doomed to die. Apart from the commitment of the transplant team, it was also the determination of the donor twin to save his brother’s life that forced everybody towards the dawn of the new era. “On the eve of the operation,” Murray tells us, “the recipient twin wrote an urgent note to his brother: get out of here and go home.” The donor twin, in a return note, replied: “I am here and I am going to stay.” The success of the procedure demonstrated that organ transplantation could be lifesaving, despite the fact that at the time it was a solution only for those whose immune systems were identical (Fig. 4.1). Although many things have changed since 1954, and over one million patients have since been successfully transplanted, the unique circumstances of the live donor operation remains a challenge for the patients involved, their families, and transplant surgeons. Some of the original ethical issues of living donation are still under debate, while new ones have arisen along the way.2 The use of a kidney from a living donor represents the best possible option of treatment for most patients with end-stage chronic renal disease. Extensive evidence has now proven that live donor kidney transplantation is superior to dialysis and to deceased donor kidney transplantation, yielding longer graft and patient survival, better quality of life and higher cost-effectiveness.3,4 Relative to dialysis, the life expectancy of a transplanted patient, whether from a living or deceased donor, has been shown to be significantly longer, with the procedure also granting patients more control over their lifestyle. Apart from the freedom from the time spent on dialysis, a functioning transplant results in a better perception of health on the part of the patient and entails no dietary restrictions. With regard to transplants, the use of a live donor has many advantages over a deceased donor. The transplant procedure can be planned, which eliminates the need to be on a waiting list, and makes it possible to transplant patients before they start dialysis treatment—the so-called “pre-emptive transplantation.” Avoidance of dialysis is not only beneficial for the patient’s quality of life, but also prevents physical deterioration that can affect the results of a subsequent kidney transplant as well as the long-term survival of the graft. The possibility

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FIGURE 4.1 Dr. John P. Merrill (left) explains the workings of a then-new machine called an artificial kidney to the Herrick twins, after their successful kidney transplant. From http://www.kjonline.com/news/transplant-just-one-chapter-of-first-organ-donors-life_2011-01-01.html (Free use).

to schedule the transplant operations, both for the donor and the recipient, usually also means that the best surgical teams are employed, during daytime hours, thus minimizing the risk of technical failures due to lack of experience or fatigue. And in fact, the immediate results of a live donor kidney transplant are superior to that of a cadaveric graft. The delayed graft function, which occurs in 50% of cadaveric grafts, is seen in only 5% of live donor kidneys. The rejection rate in the immediate posttransplant period is also reduced after live donor transplantation. But what truly makes live donor kidney transplantation superior to transplantation from cadaveric kidneys is the resulting long-term graft survival. Ten years after the transplant, a cadaveric kidney has a 50% chance to still be functioning, whereas the same proportion of live donor kidneys will still be functioning 20 years after the transplant. And that number rises to 35 years in the case of an excellent genetic match between the donor and a sibling recipient. Obviously, these advantages have to be weighed against the risk run by the donor.5,6 Every surgery entails immediate risks, and the long-term risks related to the function of the remaining kidney, including the possibility of developing mono-kidney related diseases, must also be carefully considered.7 This applies even when selecting apparently healthy donor candidates. The recent appearance of altruistic donors,8 or Good Samaritan donors—complete strangers who donate an organ to an unknown recipient—poses new ethical questions as well on the practice of living organ donation. The rate of living kidney donation has increased worldwide since 1995, following the introduction of the laparoscopic technique by Ratner and Kavoussy.9 The new procedure, which replaced the previous lumbotomy incision, significantly reduced the surgical trauma to the donor, without compromising the quality of the graft. As such, it was effective in overcoming the medical barriers to living kidney donation, and elicited new interest on the part of surgeons and nephrologists. Several modifications of the original technique have subsequently been proposed,10,11 including the hand-assisted approach,12 the retroperitoneoscopic procedure,13 the roboticassisted technique,14 and the single-incision technique.15 In addition, in the attempt to further reduce the invasiveness of kidney procurement, a transvaginal delivery of the graft has been suggested in selected cases.16 Although in the last 5 years the figures have shown a decline in the rate of living kidney donation, for a number of reasons, more than one-third of kidney transplants performed in the United States still come from live donors. And new recommendations have recently been released to improve access to, and optimize the education and care of potential donors in order to push their numbers back up.17

4.2 DONOR SELECTION Nephrologists and transplant surgeons are challenged with the often-conflicting responsibility to preserve the health of individual donors while respecting their autonomy in deciding to donate. In April 2004, over 100 transplant surgeons and nephrologists met in Amsterdam to develop an international standard of care for the live donor.18 The Amsterdam Forum Guidelines state that prior to donation, the live kidney donor must receive a complete medical

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and psychosocial evaluation, and appropriate informed consent must be obtained. All donors must have standard tests performed to ensure that donating a kidney will not significantly affect their health, screening for the following conditions: hypertension, obesity, dyslipidemia, urine analysis for protein and blood, diabetes, stone disease malignancy, urinary tract infection, pulmonary and cardiovascular risk, alcohol and tobacco abuse. Living kidney donation guidelines have similarly been formulated by national transplantation associations worldwide, though significant variability is common with respect to donor age, body mass index (BMI), and hypertension.19 The overall donor outcome has proven to be excellent, leading to an extension of donor acceptance criteria in many transplant centers. In the United States, the vast majority of centers now accept donors with older age, vascular anomalies, or a BMI above 30 kg/m2 (in other words class I obesity). Donors with mild hypertension on a single-drug treatment, and women of childbearing age and an ASA of class 2 or higher are also increasingly being accepted as potential donors in Europe too.20 In 2014, the European Best Practice Guidelines group (EBPG) issued new guidelines on the evaluation and selection of kidney donors.21 Among the core recommendations, the group advised that the donor be evaluated by a physician who is not part of the transplant team and is not involved in the daily care of the recipient, as well as by a psychologist. It was also recommended that the simultaneous presence of more than one risk factor (hypertension, obesity, proteinuria, impaired glucose tolerance, hematuria) preclude donation. Under their guidelines, a BMI above 35 kg/m2 is considered a contraindication to donation, as is a diagnosis of diabetes mellitus. Old age in itself is not a contraindication to donating a kidney. As a consequence of such changes in the eligibility criteria for living kidney donation, it is possible that the long-term consequences of nephrectomy could pose new problems regarding donor safety. While it seems reasonable to accept in selected instances one of these extended criteria, a combination of two or more is likely to be high-risk for the donor and should be avoided. In general, any extended criteria donors should be referred to high-volume centers.22

4.3 SURGICAL TECHNIQUES Historically, donor nephrectomy began as an open technique, through a lumbotomy approach, defined as a 15 20 cm loin incision. Its associated prolonged convalescence and risk of long-term morbidity were likely disincentives to donate. In fact, several randomized trials have now shown there to be a shorter convalescence, less pain, and better quality of life after a laparoscopic donor nephrectomy when compared with the open technique.23 25 Nonetheless, a survey published in 2012, involving 12 European countries and 97 transplant centers, showed that 37 centers (39%) still use the open approach, with 31 employing the technique exclusively.20 Some of this conservatism may have to do with the newness of the laparoscopic approach; indeed, its adoption by transplant surgeons may initially lead to an increase in donor risk, potentially compromising the safety of an established procedure.7

4.3.1 Laparoscopic Technique The technique of the laparoscopic donor nephrectomy was first described in 1995 at the Johns Hopkins Medical Center.9 The basic steps have not changed since the original report, where the kidney was approached transperitoneally, and removed through a Pfannenstiel incision, thus providing a superior cosmetic result. The left kidney is usually preferred, in the absence of functional or anatomical barriers, due to the longer vein. It is recommended to over-hydrate the patient during the operation to overcome the depressive effect of CO2 pneumoperitoneum on renal perfusion. With the patient in the flank position and using 3 4 trocars, the left colon is reflected medially to expose the anterior aspect of the kidney. The hilum is dissected first, leaving the Gerota’s fascia and the retroperitoneal attachment intact to facilitate suspension of the kidney and ideal exposure of its vessels. The ureter is mobilized, paying attention to its delicate vascular supply, and is followed until it crosses the common iliac artery. Any “stripping” of the distal ureter should be avoided, as this will result in posttransplant urologic complications. Leaving the gonadal vein in situ does not seem to lead to increased ureteric complications, and can prevent postoperative testicular pain in the donor.26 The gonadal vein is followed to trace the left renal vein, which is then isolated from adventitial attachments. At that point, the left adrenal vein, the lumbar veins and the gonadal vein are divided. The use of clips to secure these vessels should be weighed carefully against the possibility that a clip on the renal vein might later interfere with the Endo GIA during the final steps of the nephrectomy. For this reason we prefer to ligate the venous collaterals on the renal vein side or use radio

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FIGURE 4.2 Operative setting of a live donor laparoscopic nephrectomy. Position of the three trocars and the Pfannenstiel incision.

frequency activated devices to control these relatively small vessels.27 The renal artery is exposed with a cautery hook dissection by dividing the lymphatic and sympathetic tissue that surrounds the vessel. Care must be taken to avoid injury to early branching of the artery, which is the most common cause of massive intraoperative bleeding and conversion.28 If the artery shows signs of vasospasm, topical papaverine will usually reverse it.29 Once the main renal vessels have been fully exposed, including polar arteries if present, the kidney is mobilized from its fatty attachments. The upper pole is the most demanding area to free, and one must be careful to preserve the left adrenal gland and not damage the renal artery, which can run very close to this structure. Visualization of the upper pole can be difficult, but this area can be reached with a dissecting instrument by directly pushing on the kidney capsule. Occasional superficial tears on the kidney might result from this maneuver. Once the full mobilization of the kidney is accomplished, a 7 10 cm Pfannenstiel incision is made for extraction, leaving the peritoneum intact (Fig. 4.2). A 15-cm specimen retrieval bag is inserted through a small opening in the peritoneum of the delivery incision and the kidney is preloaded, after division of the ureter and gonadal vessels, to improve the exposure of the renal vessels and reduce the warm ischemia time. Then an endovascular GIA is fired across the artery, followed by the vein. The use of a noncutting stapler (Endo-TA) can be considered in order to increase the length of the vessel, particularly that of the right renal vein. The bag is then closed and the peritoneum layer at the Pfannenstiel incision enlarged with finger traction to extract the kidney. The arterial and venous stumps are cut below the rows of staplers’ clips and the kidney is flushed on a back table with cold perfusion. Before division of the renal vessels, systemic heparin is generally administered, although its real value has recently come under question.30 The use of locking polymer clips for management of the renal hilum has been associated with major postoperative hemorrhage, which has occasionally resulted in donor death.31 33 The rationale for adopting clips to secure the main renal vessels lies in the desire to preserve as much artery length as possible, particularly when this vessel shows early branching; in such a case, indeed, the use of the endoGIA often results in two or more arteries being reimplanted, and is also more expensive.34 However, since 2005, there have been multiple reports of severe hemorrhage due to the slippage of polymer clips, to the point that a Class II recall was issued by the FDA in 2006 on the use of Hem-o-lok (Teleflex, Limerick, Pennsylvania, USA) polymer clips on the renal artery in laparoscopic donor nephrectomy. Nonetheless, clips continue to be used, posing an unjustified risk to the donor.35,36 Sufficient evidence has now been provided to support the exclusive use of transfixing methods to secure the renal artery during donor nephrectomy, through the use of either endovascular staplers or intracorporeal suturing. Stapler misfiring has also been reported, but this might partially be related to the use of metallic clips around the main renal vessels, a practice that should be avoided by using intracorporeal knot-tying, bipolar coagulation or radio frequency devices to secure the tributaries of the renal vein.

4.3.2 Hand-Assisted Technique Hand-assisted laparoscopic donor nephrectomy was introduced in 200137 to enhance the safety of the pure laparoscopic procedure. The hand-assisted technique (HALS) approach, in fact, has the theoretical advantage of

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offering direct finger control in case of intraoperative hemorrhage, helping to dissect the renal pedicle by providing optimal exposure and reducing the warm ischemia time, thanks to the quick extraction of the kidney, grasped by the intracorporeal hand. The hand-access device can be inserted through a Pfannenstiel incision. Some surgeons will consequently prefer to remove the right kidney, as the left (nondominant) hand can be ergonomically inserted through this incision. Alternatively, the assistant’s hand will be used when the left kidney is preferred. Other technical options entail the insertion of the hand-access device in the midline in the subxiphoid region or in the right subcostal area. These technical variations can result in poorer cosmetic outcomes. Comparisons of donor outcomes following HALS and pure laparoscopy have failed to show significant differences in intraoperative or early postoperative morbidity, leaving the choice between the two techniques to patient and surgeon preference. At this stage, approximately half of the transplant centers in the United States and two-thirds in Europe11 prefer HALS as opposed to pure laparoscopy. The use of HALS may indeed aid those unfamiliar with the advanced laparoscopic technique, providing a shorter learning curve.38

4.3.3 Retroperitoneoscopic Technique A posterior approach to the kidney via both pure laparoscopy and HALS has recently been reported.39,40 Avoiding entry into the peritoneal cavity can prevent potential postoperative complications, such as intestinal occlusion secondary to adhesions or internal hernia, and limits the occurrence of injury to the colon and spleen. However, the small working space reduces visibility, particularly when suction is used to clean the field of vision. In addition, extraction of the kidney is achieved through a high-flank incision, which reduces the cosmetic advantage of the laparoscopic approach. This technique is less commonly performed, also because transplant surgeons are more familiar with the transperitoneal approach to control the renal vascular pedicle. The retroperitoneoscopic technique proves particularly useful when the donor has previously undergone major abdominal surgery and is therefore likely to have multiple adhesions that might limit the exposure of the kidney.

4.3.4 Mini-Open Nephrectomy Some centers, fearing the potential morbidity of laparoscopy, have modified the open donor nephrectomy technique in the attempt to achieve some of the benefits associated with the minimally-invasive approach, while mitigating the risks.41 The mini-open nephrectomy is performed extraperitoneally through a 6 10 cm high-flank incision, external to the rectus muscle. Specialized sets of long, curved surgical instruments and retractors are occasionally used to facilitate dissection in the limited working space. The technical steps do not differ from those of a standard open nephrectomy.42 45 It has obvious limitations when performed in obese donors.

4.3.5 Single-Site Technique Recent technical advances have focused on further reducing the morbidity of laparoscopic surgery and improving cosmesis. Single-access surgery has been extensively adopted for cholecystectomy, but also for advanced procedures including colorectal surgery and solid organ removal. The “scarless” philosophy of this technical variation calls for performing surgery through a single opening, typically located at the umbilicus, where a natural scar is already present. A single-site donor nephrectomy was first reported by Gill in 2009.46 Procuring a viable renal allograft with current single-access technology poses a clear technical challenge, which limits its application to selected donors who meet specific anatomical criteria. Additionally, the potential benefit of a no-scar approach should carefully be weighed against the ensuing risk of incisional hernia. In other procedures, no advantage in postoperative pain could be seen when comparing this technique to full laparoscopy, and even the cosmetic advantage was questioned.47 New technical improvements will have to be developed, likely with the incorporation of robotics, before single-access or possibly transvaginal donor procurement may be considered as safe alternatives to standard laparoscopy (Fig. 4.3).

4.3.6 Transvaginal Extraction and NOTES Despite significant interest in NOTES (Natural Orifices Transluminal Endoscopic Surgery), its application to kidney donor procedures is currently limited to the extraction of the graft as the final step of a laparoscopic procedure (Hybrid NOTES).48 The technique was first reported by Montgomery,49 who proved its feasibility in a

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FIGURE 4.3 Operative set-up for single-access robotic surgery. Reproduced with permission from A. Pietrabissa.

hysterectomized donor. Later, the procedure was shown to be reproducible in the presence of a normal uterus.50 Alcaraz and his team from Barcelona have performed the largest series of transvaginal kidney extractions in living donors.16 When comparing the outcomes of their first 20 patients to what is expected with the conventional laparoscopic technique, no difference was noted in operative time, blood loss, or length of hospital stay, neither was any sexual dysfunction observed after donation. The presence of a distensible and elastic vagina is now a prerequisite to be eligible for the procedure, as well as the absence of varicosity of the uterine vessels, which was the cause of severe bleeding in one patient. A transvaginal trocar can be employed at the beginning of the operation to assist in retracting the colon and exposing the kidney; it should be placed in the midline to avoid damage to the uterine vessels, followed by digital tearing of the vaginal wall shortly after/just before graft extraction. The principal advantage of transvaginal extraction is the avoidance of the 5 10 cm Pfannenstiel incision to deliver the kidney. While the cosmetic benefit seems evident, a clear reduction in postoperative pain perception remains uncertain, since most donors require little or no analgesic even with the conventional laparoscopic approach. At this stage, perhaps it is an illusion to hope for further containment of postoperative pain as a consequence of technical refinement. The possibility of a true NOTES donor nephrectomy, performed completely through a transvaginal route, without the assistance of instruments inserted through abdominal trocars, is only theoretical. New technology has to be developed to overcome the challenge of controlling the instruments and optics that must travel in a parallel path from the deep pelvis to the upper pole of the kidney. Perhaps this will become possible only with the employment of flexible robotic systems, capable of separating the physical challenge from the resulting ergonomic problems. Early prototypes of robotic single-site platforms have proven effective for basic transumbilical procedures, such as cholecystectomy and simple hysterectomy; however, their use for NOTES operations has not yet been reported.

4.3.7 Use of Robotic Technology The robotic da Vinci Surgical System has been used in almost every area of abdominal surgery, including living kidney donation. The rationale of adding the cost of the robot to an already expensive procedure is to further minimize the associated risk for the donor. The possibility of more efficient suturing, the three-dimensional high-definition view, and the dexterity provided by wristed instruments with seven degrees of freedom51 can all potentially enhance the safety of the nephrectomy. In fact, the majority of conversions during laparoscopic live donor nephrectomies have been associated with intraoperative bleeding, resulting from arterial and venous tearing during dissection. With the endo-wristed robotic instruments, which allow for a more gentle approach to the renal vascular pedicle, there is the possibility to reach and dissect posterior branches of the main vessels with little trauma to the surrounding tissue. This may in turn reduce the risk of intraoperative bleeding, and should it occur, the easy placement of vascular sutures can limit its consequences. Suturing the tributaries of the renal vein with transfixion stitches, instead of clipping, prevents possible misfiring of the endoGIA during the final division of the renal vessels. Both a pure laparoscopic as well as a HALS technique have been proposed with the aid of robotic technology.52 During the final steps of the procedure, which entail endoGIA division of the renal pedicle and extraction

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of the kidney through the suprapubic incision, the robot is usually undocked and the procedure is resumed as in conventional laparoscopy. No evidence has been provided to date that the use of the da Vinci Surgical System is capable of reducing donor morbidity. A very large series of patients will have to be entered in a prospective study for that to be shown.

4.4 OUTCOME AND COMPLICATIONS OF THE SURGERY FOR THE DONOR There are good reasons for the donor surgeon to have a poor night’s sleep before a scheduled donor nephrectomy. The first live donor procurement was in fact nearly a misadventure. Retired transplant surgeon Nicholas L. Tilney, in his memoirs,53 recalls how the vascular clamp on the short arterial stump of Ronald Herrick—the first identical-twin donor—slipped off, and the surgeon, Dr. J.H. Harrison, managed to control the heavy bleeding. The patient’s recovery was uneventful, and the rest is history. Living donor nephrectomy raises the question of how to weigh the surgeon’s imperative to do no harm against patients’ individual choice to put themselves at risk in order to benefit others. Donor mortality can never be completely eliminated, and surgeons caring for donors must always do their best to mitigate this risk.54 In general, the 90-day risk of death following live donor nephrectomy is considered to be 3.1 per 10,000 donors (about one death every 3000 donors).55 This figure is more or less equivalent to the risk of dying in a car accident within any given 12-month period and, as such, seems to be acceptable for the public. Notably, at least eight donor deaths have occurred in the era of laparoscopic procurement, all related to the use of clips to secure the renal artery or vein.56 Most of these fatal hemorrhages occurred following closure of the surgical incisions, in the recovery room or in the surgical ward, meaning that the surgeon was apparently satisfied with the final hemostasis achieved in the operative field. The hemorrhage was therefore caused by a late slippage of the clips, minutes or hours after their placement.28 An additional unspecified number of serious, though nonfatal, hemorrhagic complications have also occurred following the use of nontransfixing methods to secure the renal vascular pedicle. The safest way to secure the renal artery and vein appears to be the use of staplers, either endoGIA or endoTA, should the surgeon need to preserve a longer segment of these vessels. This recommendation has been stressed by several journal articles, and was recently circulated by the American Society of Organ Transplantation to its members and US transplant centers.35 In general, major postoperative complications, i.e., Clavien grade 3 or higher, have been reported to occur in between 2.9%7 and 5.8%57 of donors. These complications include nonfatal hemorrhage, injury to the spleen or pancreas, perforation of the colon or small bowel, and postoperative intestinal occlusion. The most common complication of donor nephrectomy is infection of the extraction wound, which can lead to the late development of an incisional hernia. Although some physiologic changes inevitably occur in donors after nephrectomy, overall these do not result in significant long-term morbidity or a shorter life expectancy, when compared with nondonors. Gestational hypertension and preeclampsia are however more likely to be diagnosed in kidney donors than in matched nondonor controls.58 Information on this potential risk should be included in clinical practice guidelines, and donors of childbearing age should be informed accordingly. The most significant potential risk for donors may be the progressive deterioration of renal function. In the general non-Black population, the lifetime probability of end-stage renal disease (ESRD) is about 3%.59 The risk for a live donor to develop ESRD, and therefore require a kidney transplant, is surprisingly not any higher; however, following donation, ESRD tends to appear about 10 years earlier. To date, there have been about 325 living kidney donors who have been put on the US deceased donor kidney waiting list.60 Diabetes causes more postdonation ESRD than all other renal diseases combined. For the donor, maintaining normal body weight postdonation will therefore reduce this risk and help preserve renal function. Yearly checks of donor renal function, blood glucose levels and blood pressure are recommended to allow for early detection and possible early treatment of common conditions (such as hypertension or diabetes) capable of deteriorating the function of the residual kidney.

4.5 LIFE EXPECTANCY OF THE DONOR Two long-term studies analyzing outcomes of kidney donors were published between 2009 and today.6,61 One of them, following 80,000 live kidney donors dating back to 1994, found donor survival to be similar to that of

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the general control population, matched for age, sex, and race. However, it would be reasonable to anticipate some potential risk of reduced survival for the donor when more accurate prospective analyses are performed on the current donor population. Indeed, the typical living kidney donor of the 1990s was significantly different from the donors that are accepted today.62,63 In light of the increasing incidence of diabetes in the general population, it is possible that more donors will eventually develop progressive loss of renal function in the decades to come, even if the absolute increase in the 15-year incidence of ESRD from donation was recently reported to be below 0.5%.64 In addition, today’s donors are likely to be older, heavier, and have higher blood pressure than 20 years ago. Most donor programs around the world no longer have an upper age limit, and controlled hypertension and moderate obesity (BMI between 30 and 35) are not seen as absolute contraindications to donation. These factors will possibly have some bearing on the future life expectancy of current donors. Several studies have shown that donors are willing to accept higher risks for those close to them.2 Clearly, this raises the ethical issue of autonomy, and whether donors, once properly informed, should be allowed to accept added risk or not. The threshold for acceptable risk may also vary between transplant centers and between clinicians. In the end, donor consent and autonomy are necessary, but not sufficient on their own to proceed with kidney donation.18,65 Donor autonomy, in other words, cannot overrule medical judgment and decision-making. Accordingly, the British Transplantation Society guidelines2 state that when a fully informed donor wishes to proceed with a nephrectomy that involves risks of mortality or morbidity greater than what the transplant team finds acceptable, they are under no obligation to proceed. Referral for a second opinion would be appropriate in this case.

References 1. Murray JE. Surgery of the soul: reflections on a curious career. USA: Boston Medical Library. Science History Publications; 2012. 2. Maple NH, Hadjianastassiou V, Jones R, et al. Understanding risk in living donor nephrectomy. J Med Ethics 2010;36:142 7. 3. Wolfe RA, Ashby VB, Milford EL, et al. Comparison of mortality in all patients on dialysis, patients on dialysis awaiting transplantation, and recipients of a first cadaveric transplant. N Eng J Med 1999;341:1725 30. 4. Hariharan S, Johnson CP, Bresnahan BA, et al. Improved graft survival after renal transplantation in the United States, 1988 to 1996. N Eng J Med 2000;342:605 12. 5. Glannon W. Underestimating the risk in living kidney donation. J Med Ethics 2008;34:127 8. 6. Ibrahim HN, Foley R, Tan L, et al. Long-term consequences of kidney donation. N Eng J Med 2009;29:459 69. 7. Mjoen G, Oyen O, Holdaas H, et al. Morbidity and mortality in 1022 consecutive living donor nephrectomies: benefits of a living donor registry. Transplantation 2009;88:1273 9. 8. Ferrari P, Weimar W, Johnson RJ, et al. Kidney paired donation: principles, protocols and programs. Nephrol Dial Transplant 2014;7:309. 9. Ratner LE, Ciseck LJ, Moore RG, et al. Laparoscopic live donor nephrectomy. Transplantation 1995;15:1047 9. 10. Piros L, Langer RM. Laparoscopic donor nephrectomy technique. Curr Opin Organ Transplant 2012;17:401 5. 11. Banga N, Nicol D. Techniques in laparoscopic donor nephrectomy. BJUI 2012;110:1368 73. 12. Rajab A, Pelletier RP. The safety of hand-assisted laparoscopic living donor nephrectomy: the Ohio State University experience with 1500 cases. Clin Transplant 2015;29:204 10. 13. Tokodai K, Takayama T, Amada N, et al. Retroperitoneoscopic living donor nephrectomy: short learning curve and our original hybrid technique. Urology 2013;82:1054 8. 14. Tzvetanov I, Bejarano-Pineda L, Giulianotti PC, et al. State of the art of robotic surgery in organ transplantation. World J Surg 2013;37:2791 9. 15. Desai MM, Berger AK, Brandina R, et al. Laparoendoscopic single-site surgery: initial hundred patients. Urology 2009;74:805 12. 16. Alcaraz A, Musquera M, Peri L, et al. Feasibility of transvaginal natural orifice translumenal endoscopic surgery-assisted living donor nephrectomy: is kidney vaginal delivery the approach of the future?. Eur Urol 2011;59:1019 25. 17. La Pointe Rudow D, Hays R, Baliga P, et al. Consensus conference on best practices in live kidney donation: recommendations to optimize education, access, and care. Am J Transplant 2015;15:914 22. 18. Delmonico F. Council of the transplantation society. A report of the Amsterdam forum on the care of the live kidney donor: data and medical guidelines. Transplantation 2005;79:S53 66. 19. Arunachalam C, Garrues M, Biggins F, et al. Assessment of living kidney donors and adherence to national live donor guidelines in the UK. Nephrol Dial Transplant 2013;28:1952 60. 20. Klop WJK, Dols LFC, Kok NFM, et al. Attitudes among surgeons towards live-donor nephrectomy: a European update. Transplantation 2012;94:263 8. 21. Abramowicz D, Chocat P, Claas FHJ, et al. European renal best practice guideline on kidney donor and recipient evaluation and perioperative care. Nephrol Dial Transplant 2014;0:1 8. 22. Ahmadi AR, Lafranca JA, Claessens LA, et al. Shifting paradigms in eligibility criteria for live kidney donation: a systematic review. Kidney Int 2015;87:31 45. 23. Kok NF, Lind MY, Hansson BM, et al. Comparison of laparoscopic and mini incision open donor nephrectomy: single blind, randomised controlled clinical trial. MBJ 2006;333:221. 24. Andersen MH, Mathisen L, Oyen O, et al. Postoperative pain and convalescence in living kidney donors-laparoscopic versus open donor nephrectomy: a randomized study. Am J Transplant 2006;333:221.

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25. Oyen O, Andersen M, Mathisen L, et al. Laparoscopic versus open living-donor nephrectomy: experiences from a prospective, randomized, singe-center study focusing on donor safety. Transplantation 2005;79:1236. 26. Shirodkar SP, Gorin MA, Sageshima J, et al. Technical modification for laparoscopic donor nephrectomy to minimize testicular pain: a complication with significant morbidity. Am J Transplant 2011;11:1031 4. 27. Pietrabissa A, Boggi U, Moretto C, et al. Laparoscopic and hand-assisted laparoscopic live donor nephrectomy. Semin Laparosc Surg 2001;8:161 7. 28. Janki S, Verver D, Klop KWJ, et al. Vascular management during live donor nephrectomy: an online survey among transplant surgeons. Am J Transplant 2015;15(6):1701 7. 29. Zacherl J, Bock S, Feussner H, et al. Periarterial application of papaverine during laparoscopic donor nephrectomy improves early graft function after kidney transplantation in pigs. Surg Endosc 2004;18:417 20. 30. Friedersdorff F, Wolff I, Deger S, et al. No need for systemic heparinization during laparoscopic donor nephrectomy with short warm ischemia time. World J Urol 2011;29:561 6. 31. Friedman AL, Peters TG, Jones KW, et al. Fatal and nonfatal hemorrhagic complications of living kidney donation. Ann Surg 2006;243:126 30. 32. Baumert H, Ballaro A, Arroyo C, et al. The use of polymer (Hem-o-lok) clips for management of the renal hilum during laparoscopic nephrectomy. Eur Urol 2006;49:816 19. 33. Goh YS, Cheong PS, Lata R, et al. A necessary step toward kidney donor safety: the transition from locking polymer clips to transfixion techniques in laparoscopic donor nephrectomy. Transplant Proc 2014;46:310 13. 34. Jellison FC, Shah SK, Mashni Jr JW, et al. Vessel length following laparoscopic donor nephrectomy: impact of vascular ligation technique on allograft vessel length. J Endourol 2008;22:973 7. 35. Friedman AL, Peters TG, Ratner LE. Regulatory failure contributing to deaths of live kidney donors. Am J Transplant 2012;12:829 34. 36. Simforoosh N, Sarhangnejad R, Basiri A, et al. Vascular clips are safe and a great cost-effective technique for arterial and venous control in laparoscopic nephrectomy: single-center experience with 1834 laparoscopic nephrectomies. J Endourol 2012;26:1009 12. 37. Tokuda N, Nakamura M, Tanaka M, et al. Hand-assisted laparoscopic live donor nephrectomy using newly produced LAP DISC: initial three cases. J Endourol 2001;15:571 4. 38. Gaston KE, Moore DT, Pruthi RS. Hand-assisted laparoscopic nephrectomy: prospective evaluation of the learning curve. J Urol 2004;171:63 7. 39. Kohei N, Kazuya O, Hirai T, et al. Retroperitoneoscopic living donor nephrectomy: experience of 425 cases at a single center. J Endourol 2010;24:1783 7. 40. Gjertsen H, Sandberg AK, Wadstro¨m J, et al. Introduction of hand-assisted retroperitoneoscopic living donor nephrectomy at Karolinska University Hospital Huddinge. Transplant Proc 2006;38:2644 5. 41. Antcliffe D, Nanidis TG, Darzi AW, et al. A meta-analysis of mini-open versus standard open and laparoscopic living donor nephrectomy. Transplant Int 2009;22:463 74. 42. Kim SI, Rha KH, Lee JH, et al. Favorable outcomes among recipients of living-donor nephrectomy using video-assisted minilaparotomy. Transplantation 2004;77:1725 8. 43. Shenoy S, Lowell JA, Ramachandran V, et al. The ideal living donor nephrectomy “mini-nephrectomy” through a posterior transcostal approach. J Am Coll Surg 2002;194:240 6. 44. Mital D, Coogan CL, Jensik SC. Microinvasive donor nephrectomy. Transplant Proc 2003;35:835 7. 45. Redman JF. An anterior extraperitoneal incision for donor nephrectomy that spares the rectus abdominis muscle and anterior abdominal wall nerves. J Urol 2000;164:1898 900. 46. Desai MM, Berger AK, Brandina R, et al. Laparoendoscopic single-site surgery: initial hundred patients. Urology 2009;74:805 12. 47. Marks JM, Phillips MS, Tacchino R, et al. Single-incision laparoscopic cholecystectomy is associated with improved cosmesis scoring at the cost of significantly higher hernia rates: 1-year results of a prospective randomized, multicenter, single-blinded trial of traditional multiport laparoscopic cholecystectomy vs single-incision laparoscopic cholecystectomy. J Am Coll Surg 2013;216:1037 47. 48. Kaouk JH, Khalifeh A, Laydner H, et al. Transvaginal hybrid natural orifice transluminal surgery robotic donor nephrectomy: first clinical application. Urology 2012;80:1171 5. 49. Allaf ME, Singer A, Shen W, et al. Laparoscopic live donor nephrectomy with vaginal extraction: initial report. Am J Transplant 2010;10:1473 7. 50. Pietrabissa A, Abelli M, Spinillo A, et al. Robotic-assisted laparoscopic donor nephrectomy with transvaginal extraction of the kidney. Am J Transplant 2010;10:2708 11. 51. Petros FG, Angell JE, Abaza R. Outcomes of robotic nephrectomy including highest complexity cases: largest series to date and literature review. Urology 2015;86:1352 8. 52. Giacomoni A, Di Sandro S, Lauterio A, et al. Evolution of robotic nephrectomy for living donation: from hand-assisted to totally robotic technique. Int J Med Robot 2014;10:286 93. 53. Tilney NL. Transplant: from myth to reality. New Heaven: Yale University Press; 2003. 54. Morrissey PE, Monaco AP. Living kidney donation: evolution and technical aspects of donor nephrectomy. Surg Clin North Am 2006;86:1219 35. 55. Segev DL, Muzaale AD, Caffo BS, et al. Perioperative mortality and long-term survival following live kidney donation. JAMA 2010;10:959 66. 56. Dekel Y, Mor E. Hem-o-lok clip dislodgment causing death of the donor after laparoscopic living donor nephrectomy. Transplantation 2008;86:887. 57. Richstone L, Seideman C, Baldinger L, et al. Conversion during laparoscopic surgery: frequency, indications and risk factors. J Urol 2008;180:855 9. 58. Garg AX, McArthur E, Lentine KL. Gestational hypertension and preeclampsia in living kidney donors. Donor Nephrectomy Outcomes Research (DONOR) Network. N Engl J Med 2015;372:1469 70.

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59. Steiner RW, Ix JH, Rifkin DE, et al. Estimating risks of de novo kidney diseases after living kidney donation. Am J Transplant 2014;14:538 44. 60. Ross LF. Living kidney donors and ESRD. Am J Kidney Dis 2015;30:S0272-6386(15)00539-9. 61. Muzaale AD, Massie AB, Wang MC, et al. Risk of end-stage renal disease following live kidney donation. JAMA 2014;311:579 86. 62. Reese PP, Bloom RD, Feldman HI, et al. Mortality and cardiovascular disease among older live kidney donors. Am J Transplant 2014;14:1853 61. 63. Tong A, Chapman JR, Wong G, et al. Living kidney donor assessment: challenges, uncertainties and controversies among transplant nephrologists and surgeons. Am J Transplant 2013;13:2912 23. 64. Lam NN, Lentine KL, Garg AX. End-stage renal disease risk in live kidney donors: what have we learned from two recent studies? Curr Opin Nephrol Hypertens 2014;23:592 6. 65. Ethics Committee of the Transplantation Society. The consensus statement of the Amsterdam Forum on the Care of the Live Kidney Donor. Transplantation 2004;78:491 2.

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C H A P T E R

5 Criteria for Kidney Allocation in the United States Linda Ohler George Washington University Transplant Institute, Washington, DC, United States

5.1 INTRODUCTION: HISTORY OF KIDNEY ALLOCATION As solid organ transplantation developed in the United States, there initially was no oversight or organizational structure for identifying suitable donors for recipients with end organ diseases. Early in the 20th century there were reports of organs being transplanted from corpses and animals in Russia, France, and the United States, but without success beyond a few weeks.1 Between 1951 and 1953, Dr. David Hume and his colleagues reported transplantation of kidneys from deceased donors who had succumbed to cardiac death. There were no brain death laws at that time. Aside from cortisone, there were no immunosuppressive medications and the longterm outcomes of these kidney transplants were poor. Dr. Hume reported that, “ACTH and Cortisone did not appear to exert any pronounced beneficial effect on the survival of human renal transplants.”2 Dr. Joseph Murray performed the first successful kidney transplant in the United States in 1954. The transplant was performed between identical twins and immunosuppression was not an issue in this case. The recipient survived for 8 years and his twin brother survived 56 years.1 Based on the early reported outcomes of this case several other successful transplants occurred between identical twins. In 1962, the first successful deceased donor kidney transplant was also performed by Dr. Murray and his colleagues in Boston.3 Because there was no structured organ allocation system in the early days of transplantation, deceased donors were arranged by surgeons and nurses who would call hospitals and ask if there were any potential donors whose kidneys could be used for patients with end stage renal disease. In 1969, Drs. Hume and Amos founded the South Eastern Organ Procurement Foundation (SEOPF) in Richmond, Virginia, which was one of seven organ procurement organizations funded by the Federal Government.4 Originally known as the South Eastern Regional Organ Procurement Program (SEROPP), the name was changed in 1975 to SEOPF. The goal of this organization was to improve transplant outcomes through matching and sharing of kidneys for transplantation. In 1976 SEOPF developed a computer system they called the United Network for Organ Sharing. This new system for communication was made available for any transplant center in the United States who wished to register their patients and match them with potential kidney donors.4 In 1984 Congress passed the National Organ Transplant Act (NOTA). This new law (PL 98-507) prohibited the sale of organs, established grants for organ procurement organizations, and called for a national system of organ sharing.5 In preparation for passage of NOTA, a committee was formed between staff at SEOPF and members of the American Society of Transplant Surgeons (ASTS) to write the articles of incorporation of UNOS and establish it as a 501c3.4 This was in preparation for the newly formed 501c3 to apply for the contract with the United States Department of Health and Human Services (HHS) that would implement NOTA. HHS granted the contract to UNOS, which it has held for the past 30 years. With this contract the OPTN has provided the United States with policies for allocation of each organ transplanted, a system for organ placement, a process for data collection, and public and professional education regarding organ transplantation. The OPTN, under the direction and

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administration of UNOS, has coordinated close to 500,000 deceased donor transplants. Since 1986 UNOS has grown considerably from the original staff of five to a current staff of more than 300. In 1985, the information technology (IT) department for UNOS consisted of one person and a 32-bit microprocessor. By 2014 the IT department at UNOS had grown to more than 130 personnel.6 Currently UNOS has 22 Committees, which are composed of transplant professionals in the community. These committees develop policies to provide equitable organ allocation and to ensure patient safety.6 The UNOS Board of Directors is the governing body that oversees policy development. Each organ system has a committee such as the Kidney Committee, Thoracic Committee, Liver Committee, etc. Also part of NOTA, the OPTN required a process and program for data analysis which is performed by the Scientific Registry for Transplant Recipients (SRTR). This contract is currently held by the Minneapolis Medical Research Foundation in Minnesota. OPTN contracts are renewed every 3 years. NOTA was also charged with creating a multidisciplinary task force that would examine organ donation and transplantation in the United States. This resulted in the Uniform Anatomical Gift Act, which established organ and tissue donation as the right of any individual 18 years old and over.5

5.2 METHODS OF EARLY ORGAN SHARING The Omnibus Budget Reconciliation Act of 1986 required that all transplant centers and organ procurement organizations become part of the OPTN. This was the beginning of establishing a national system for organ allocation and sharing. Prior to implementation of the Omnibus Budget Reconciliation Act, the sharing of kidneys for transplantation was accomplished on a personal phone call basis, or with twins. Once liver and heart transplantation began demonstrating signs of success, the need for deceased organs became more prevalent and the necessity for a defined, protocol driven national system became even more evident. In the early 1970s procurement groups were beginning to form around transplant centers that were performing organ transplants such as kidney, liver, and heart. For example, there was SEOPF in Richmond, Virginia; the Regional Organ Procurement Agency (ROPA) in Los Angeles, California and in Pennsylvania there was the Delaware Valley Transplant Program in Philadelphia and the Pittsburgh Transplant Foundation.4,7,8 Public education was a mission of each of the procurement programs developing across the United States as the need for deceased organs increased. Public awareness and education became a critical part of increasing organ donation. In 1982, Don Denny of Pittsburgh collaborated with NATCO, the organization of transplant professionals, to help organize the allocation system through a phone communication system at 412-24-ALERT. This system provided the names and locations of patients in need of an organ transplant. The recording was updated daily. Other attempts were being made to systemize organ allocation. Howard Nathan and his colleagues in Philadelphia were developing a national system to rank the severity of illness of patients in need of a transplanted organ. The higher the number, the more critical the need was for a transplant. A rank of 4 was the highest and indicated the most urgent need. A status 3 patient was hospitalized but not in critical condition.7 Status 2 and 1 patients were not hospitalized. This was the first attempt at developing a status system for allocation of organs in the United States. As patient outcomes were improving for each organ transplanted, the demand for organs increased steadily. Patients began requesting transplantation as an option for their end organ disease processes. Successes were now being reported in lung transplantation and the need for a national system increased even more.9

5.3 EVOLUTION OF NATIONAL ORGAN ALLOCATION SYSTEMS With the transplant community call for increases in deceased organs for transplantation and for equitable distribution of the organs, UNOS, the OPTN contractor, set out to develop consensus-based policies through the committee structures within UNOS. Committees were focused not only on ways to increase organ donation but also to organize systems for ranking potential transplant recipients on the basis of critical need and outcomes that evaluated graft and patient survival. The initial Kidney Allocation System (KAS) was based on observations that long-term graft survival was best in a national sharing of 6 antigen matched or zero antigen mismatched kidneys.5 When this matching system did not result in a candidate for a deceased donor kidney, the kidney could then be allocated locally based on ABO compatibility and a point system that included length of waiting time on the UNOS list, presensitization, and quality of HLA matching.5 In this allocation scheme, children were given preference and received extra points based on their age at the time of UNOS listing.5

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A positive final crossmatch for any potential recipient would create the need for the organ to be allocated to the next potential candidate. Allocation was applied to transplant hospitals within OPO in which the donor was identified. If no potential recipients matched with the specific donor, then allocation was expanded to the region. With still no suitable candidates, the kidney was then offered nationally. While this system was originally planned to be a national allocation system for all deceased donor kidneys, local jurisdictions developed exceptions or variances that were also known as Alternative Local Units (ALUs) with special sharing agreements. These variances may have constituted subsections of an OPO, an entire state, or UNOS regions. They were considered alternative point systems within an OPO, state, or region and gave greater weight to factors within the allocation system such as time on the waiting list, presensitization, or any other strategy deemed important for that region or state. Some OPOs gave special consideration for diabetic candidates who required a kidney pancreas transplant, allowing a donor kidney to be allocated with a recovered pancreas from the same deceased donor. Any variance or exception to the allocation policy required ratification by the UNOS Board of Directors and required periodic review to ensure the variance continued to be patient driven rather than transplant center driven.5 During this time UNOS committees overseeing allocation of hearts, livers, and lungs began developing allocation systems prioritizing patients based on the medical condition of the candidate; however, no priority system was allocated to kidney candidates since all had access to dialysis.

5.3.1 Allocation Criteria: Concerns for Equitable Distribution In 1991, a report from the HHS Inspector General’s office described what many in the transplant community had expressed concerns about. This report quantified the issue with data stating that Blacks on the UNOS kidney list were waiting 13.9 months compared with 7.6 months for Caucasians.10 The report also noted that highly sensitized patients waited almost 4 times as long for a kidney transplant as other patients. In addition to these disparities, it was noted that geography also entered into the waiting times with some patients waiting over 18 months at centers while others waited less than 6 months.10 As we learned more about HLA tissue typing, trends began to demonstrate the disparity in using 6 antigen matching and zero antigen mismatching. It was reported that Blacks received only 22% of deceased donor kidneys in 1990 while 31% of those waiting for a suitable donor kidney were Black.10 Kasiske et al.11 noted the frequencies of ABO blood groups and HLA antigens are two factors that can impact on the rate of transplanting Blacks on the kidney transplant list. For instance, twice as many Blacks have ABO Blood type B as compared to Caucasians. Because ABO compatibility is a requirement for organ allocation, those with blood type B must wait for a kidney from a blood type B donor. Racial differences among HLA antigens also influence matching donors and potential recipients. The majority of 6 antigen HLA matching has been reported to be among those from within the same race. These facts have influenced the long waiting periods for Blacks on the transplant list. Gaston et al. advised in 1993 that, “if racial equity in renal transplantation was to be achieved, alternative allocation strategies must be formulated that address the interests of all potential recipients.”12 The point system had been added in an attempt to correct inequities for patients who were difficult to match due to race and high levels of preformed antibody. Starzl et al.13 had demonstrated the use of the point system in 1986 with a cohort of 270 deceased kidney donors at the University of Pittsburgh. A stratification system was set up in a computer using wait time, antigen matching, preformed antibodies, and medical urgency which was defined as exhausted access sites for dialysis patients. Logistic practicality was also included in the stratification system and was defined as the ease and rapidity with which a transplant could be performed. This factor considered ischemic time of the donor and availability of the candidate in terms of close proximity to the transplant center.12 Of the 253 kidney transplant recipients who met the study’s selection criteria, 247 kidneys were transplanted into recipients with the highest point score. The conclusion was that the use of a point system provided a more objective system for kidney allocation.12 In 1993 The California Transplant Donor Network (CTDN) received a variance that allowed local kidney allocation based solely on waiting time. There were no considerations for HLA typing during this 6-year study that included 1301 locally allocated kidneys. This cohort was compared to 37,858 concurrent kidney transplants performed nationally with allocation based on HLA matching. Interestingly there was no difference in the outcomes between the groups at 1, 3, 5, and 10 years posttransplant. The local kidney recipients within this OPO area had less HLA matching but shorter ischemic times.14 The authors concluded that a kidney allocation scheme that does not include points for HLA matching makes distribution more equitable among minorities.14

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Studies continued to demonstrate the pros and cons of current allocation systems and to call for more equitable distribution of kidneys. Issues of geography were often thought to be a result of the variances within OPOs and transplant centers. Many of the OPOs and transplant centers with variances studied those variances to determine if the variance impacted on overall outcomes that addressed factors such as race, ABO, HLA matching and highly sensitized patients.

5.3.2 Strategies to Increase Equity Over the past several years the OPTN/UNOS Kidney Transplantation Committee has worked diligently to develop what are thought to be more equitable changes to the KAS. Several strategies were considered in an attempt to provide eligible candidates with the most equitable distribution of kidneys for transplantation. Factors reviewed as part of this transition to a new system included geography, ABO, and degree of sensitization. Considerations for this new process for kidney allocation was being planned and restructured for 6 years before it was formalized and released to the transplant community for comment on February 16, 2011.15 The comment period was opened until April 1, 2011. During this period more than 260 comments were received including those from professional groups such as the National Kidney Foundation, American Society of Transplantation, the ASTS, and the American Society of Nephrology. Each group provided feedback and recommendations for additional changes. The major concerns at that time were about the potential for age discrimination and lack of changes to geographical boundaries for kidney distribution. Changes have been made in the kidney allocation policy over the years and include such changes summarized in Table 5.1. In 2003 the OPTN Board of Directors charged the Kidney Transplantation Committee with reviewing the current allocation system for limitations and developing improvements. In the process two public forums took place in Dallas, Texas (2007) and St. Louis (2009) whereby recommendations for improvement were received with some incorporated into the new KAS. A concept document was released for public comment in 2011 and in 2013 the OPTN Board of Directors approved the revised KAS. This new system went into effect on December 4, 2014. TABLE 5.1 Major Changes in Kidney Allocation Criteria 1989 2014 Year

Policy/Criteria Changes

1989

Points added for sensitized candidates

1995

Points added for waiting time

1996

Points assigned for living donors who develop end stage renal disease

1998

Points added for pediatric candidates

2003

HLA-B matching points eliminated Kidney candidates begin to accrue waiting time in inactive status

2004

Prioritized donors aged ,35 years to pediatric candidates after 0 mismatch, high CPRA, prior living donors and before paybacks

2009

Eliminated 0 mismatch for nonsensitized adults

2009

Change from PRA to Calculated PRA (CPRA)

2014

Use of Kidney Donor Profile Index (KDPI) to estimate deceased donor kidney’s expected survival (eliminates classifications of standard or expanded criteria donors)

2014

Accrual of waiting time for kidney transplantation determined by dialysis start date rather than date of listing for transplant or for those not on dialysis, accrual time begins on the date when GFR is documented to be less than or equal to 20 mL

2014

Eliminates payback system

2014

Prioritizes pediatric kidney transplant candidates to deceased donor kidneys with KDPI ,35%

2014

Eliminates regional variances in organ allocation

2014

Increases priority for sensitized patients

2014

Allows allocation of A2/A2B to B blood types to increase access to deceased donor kidneys

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The previous KAS was highly reliant on a candidate’s time on the waiting list. The new KAS addresses three major concerns about the current allocation system for kidneys: waste of usable kidneys, longevity matching, and inequities to access.15,16 Each of these concerns is discussed below. In developing a new allocation system, two ethical issues were considered: Equity and Utility. Utility is addressed in the new allocation system with the focus on improving outcomes. Equity is addressed with the focus on changes that improve access to kidney transplantation.17 The goals of the new allocation system have been planned to provide a more accurate estimate of graft and recipient outcomes by promoting posttransplant kidney function for kidney candidates with the longest potential survival. It is believed this improvement in matching will reduce differences based on racial/ethnic minority groups, pediatric candidates, and sensitized patients awaiting a suitable kidney donor.17

5.3.3 Waste or Discarding Usable Kidneys Waste or discarding usable kidneys has been a concern of the current allocation system because discard rates of kidneys have been higher than acceptable. Some discards have been related to high risk or extended criteria donors (ECD) but some standard criteria donor (SCD) kidneys have also been reported to have been discarded. In an attempt to decrease the discard of potentially usable kidneys, the new KAS offers kidneys with a Kidney Donor Profile Index (KDPI) of .85% to a wider geographic area. The use of KDPI in this situation has the potential to increase allocation of kidneys. The terms SCD, ECD, and Donation after Cardiac Death (DCD) will be replaced with the KDPI system that assesses donor risk index more accurately. In this new KAS program, kidneys at risk for discard could be offered as a Dual Kidney Transplant (DKT). Dual kidney transplantation allows the transplantation of both donor kidneys into a single recipient under certain circumstances such as older recipients. According to UNOS policy organ procurement organizations must allocate one kidney to one candidate unless the donor has at least two of the following criteria18: • • • • •

Donor age .60 Donor creatinine clearance ,65 mL/minute on admission Rising serum creatinine .2.5 mg/dL Donor comorbidities such as hypertension and diabetes Adverse renal histopathology

It has been reported that the use of two marginal or compromised kidneys result in more functioning nephrons as would be found in a single ideal kidney. Thus, use of the DKT may actually enhance transplant options and decrease the discard rate in the United States. Notably, the discard rate of organs in Europe was reported in one study as only 7.5% due to use of a rescue allocation policy that included more liberal donor criteria.19 In the United States it has been reported that over half of the expanded criteria donor kidneys have been discarded.20 The most recent data describes a 13% discard rate for organs recovered in the United States.21 With the new KAS use of the KDPI it is hoped this discard rate will decrease.

5.3.4 Inefficiencies in the Previous Allocation Systems Another concern that the new KAS policy addresses is the inefficiencies in the previous systems in terms of long-term patient and graft survival. Longevity matching can be achieved in the new KAS through the use of two scoring systems: The KDPI and the Estimated Posttransplant Survival (EPTS) Score. With the new KDPI, an estimate can be calculated for a deceased donor kidney’s quality and expected length of survival.18 Risk factors included in the calculations present clinicians with a Kidney Donor Risk Index (KDRI). Once the KDRI is calculated, the score is mapped from a relative risk scale to a cumulative percentage scale known as the KDPI.22 Factors for the calculation include the following clinical parameters and demographic considerations: • • • • • • •

Donor age Height and weight of the donor Ethnicity History of hypertension History of diabetes Cause of death Serum creatinine

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5. CRITERIA FOR KIDNEY ALLOCATION IN THE UNITED STATES

• Anti-HCV • Donor meets DCD criteria A higher KDPI percentage is associated with lower donor kidney quality whereas a lower KDPI percentage is associated with a high donor kidney quality.22 This scoring system helps to ensure the best quality donor kidneys are allocated to recipients who are likely to have a corresponding longevity rate. Longevity can be calculated for kidney transplant candidates using EPTS, which is a score developed to predict long-term outcomes in transplant recipients. The EPTS score is calculated for all adult candidates on the waiting list and can range from 0% to 100%. Calculations are based on 4 candidate factors23: • • • •

Time on dialysis Diabetes status Prior solid organ transplant Age of the candidate

A listed kidney transplant candidate with an EPTS score of 20% or less will be prioritized for kidneys from donors with KDPI scores of 20% or less before other candidates at local, regional, or national levels.23 An EPTS score of 20% estimates that a candidate is likely to survive longer than 80% of other recipients nationally. The EPTS score is not stagnant. It changes as the age of the candidate increases and the length of time on dialysis increases. Exceptions to the EPTS/KDPI system include candidates who are listed for multiorgan transplants, highly sensitized candidates and pediatric transplant candidates. Transplant programs must obtain informed consent from candidates who decided to accept donors with high KDPI scores of greater than 85%.

5.3.5 Overcoming Inequities of Access to Transplantation Much of the concern over allocation throughout the 50 plus years of kidney transplantation in the United States has surrounded the issue of access to transplantation. The new KAS system addresses this concern through the ABO system by allowing the A2/A2B donors to be allocated to blood type B candidates. Blood type B is most commonly seen in the Black race. In many parts of the United States candidates with blood group B wait longer than other ABO groups. Thus, finding a solution to this inequity required some investigation into the ABO subtypes. Because blood group A has many subtypes, it was determined that A1 and A2 can be less reactive than other subtypes, thus allowing non-A recipients to have successful kidney transplants from A2 donors. This strategy has been used successfully with both B and O recipients and has led to a reduction in waiting time for kidney patients.17,24,25 Candidates must be screened to determine that they have a low anti-A antibody titer that is acceptable for transplantation. Candidates must also sign a consent stating they have received education on the A2/A2B to B transplants and agree to participate in this process.

5.3.6 Accrual of Wait Time The new KAS allows candidates to accrue wait time based on the start date of dialysis.17,24 This requires source documentation of the start date with the use of form CMS 2728 to ensure accuracy of the dialysis start date entered into UNet. Thus, at the time a dialysis patient is registered on the UNOS list for transplantation, the candidate receives credit for time on dialysis. A patient who started dialysis 4 months prior to being listed will already have 120 days of accrued time at the time of UNOS registration. A patient who started dialysis 2 years prior to being registered with UNOS will receive approximately 730 days of wait time. Patients who are not on dialysis may be listed preemptively with the use of their documented glomerular filtration rate (GFR) of 20 mL or less. Any time a patient is listed for transplantation with UNOS, source documentation of the selection criteria used by the transplant team to list the candidate must be documented in the patient’s chart. In addition, source documentation must also be available demonstrating the start date of dialysis or the patient’s GFR at the time of listing. Preemptive listing for kidney transplantation is considered a best practice. Preemptively registered patients accrue time based on the date of listing in UNet. Accrual time for preemptive listing begins on the day of listing as opposed to the date of GFR testing. Pediatric candidates for kidney transplantation will also receive credit for their time on dialysis prior to being registered with UNOS. Thus the child’s dialysis start date is factored into accrual time on the wait list when registered for a kidney transplant with UNOS.26

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5.3.7 Sensitized Candidates The Calculated Panel of Reactive Antibodies or cPRA is being used in the new KAS to prioritize patients.26,27 When unacceptable antigens are identified by the HLA laboratory for a specific transplant candidate, the antigens are entered into the UNOS computer calculator. An established formula is used to calculate the cPRA using the unacceptable values that were entered. The cPRA is used to assess the number of incompatible donors for the candidate. If, at any time, additional unacceptable antigens are entered, the system automatically recalculates the cPRA value. Sensitized candidates with a cPRA value of 80% or higher receive points in the new KAS system.17 A cPRA score of 98% 100% must be reviewed and signed by the HLA laboratory director and the candidate’s transplant physician or surgeon to ensure the unacceptable antigens have been carefully assessed and documented in the patient’s medical record.26 The intent of this prioritization for sensitized patients in the new allocation system was to improve overall posttransplant survival and to improve access for highly sensitized patients. However, concerns have been expressed that this prioritization of highly sensitized patients does not decrease the risk for rejections and potential poor outcomes in this population.24,28 One problem identified is the fact that HLA DPB and HLA DQA antigens were not included in the simulation performed by the SRTR;28 Bray et al. found that the majority of candidates with cPRA .98% possessed antibodies against these two antigens. Prior to the new allocation system assigning priority to sensitized candidates, most individuals with cPRA .98% had prolonged wait times and a higher incidence of death while waiting for a suitable donor organ.29 With the new system these candidates have the highest priority for local, regional, and national sharing.26,28 Kucheryavaya et al. have reported that only 51% of HLA laboratories are reporting deceased donor DPB.30 It is unknown how many HLA laboratories are testing for DQA in deceased donors. With the majority of highly sensitized candidates exhibiting HLA, DPB, and DQA antibodies and more than half of deceased donors not being tested for these antigens, there is a high probability that there will be a high rate of positive crossmatches between these candidates and their respective donors.28 A recommendation is being made for mandatory testing of DPB and DQA for all deceased donors to help alleviate this identified impediment to equalizing the allocation of kidneys to highly sensitized transplant candidates.28

5.3.8 Additional Changes to the KAS System The payback system has been eliminated in the new KAS system. The payback system was implemented in the early 1990s with OPTN/UNOS Policy 3.5.5 for OPOs that receive a kidney from another OPO based on a zero antigen mismatch to payback a kidney of the same blood type to the national pool. It was found that payback kidneys had more cold ischemic times and a higher risk for delayed graft function. Therefore, the policy and practice have been eliminated.31 Variances have been eliminated as well. However, two variances were incorporated into the new KAS. The first variance is a committee-sponsored variance that allows for back-dating of dialysis time prior to listing patients for transplantation, and the other variance allows for A2 and A2B kidneys to be offered to patients with blood type B.32 Both variances were part of the plan to equalize distribution and allocation of kidneys.

5.4 CONCLUSIONS The new Kidney Allocation went into effect on December 4, 2014. Outcomes of the changes are being evaluated. The UNOS Kidney Allocation Committee spent considerable time and applied much thought and consideration to their plans to equalize the allocation of kidneys to disadvantaged candidates such as those with reported disparities in geography, race, and those who are highly sensitized from previous transplants, pregnancies, or blood transfusions. One unintended consequence has been identified within the highly sensitized population. Attempts are being made to address the problem by requiring HLA, DPB, and DQA on deceased donors. We will not know the full impact on the new system for a few years as data is collected in each of the areas where changes have been made. Initially, the system has certainly made significant strides in making changes to meet the needs of many candidates awaiting kidney transplantation.

References 1. Tilney Nicholas L. Transplant: from myth to reality. New Haven, CT: Yale University Press; 2003. 2. Hume DM, Merrill JP, Miller BF, Thorn G. Experiences with renal homotransplantations in the human: a report of 9 cases. J Clin Invest 1955;34(2):327 82.

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3. Merrill JP, Murray JE, Takacs FJ, Hager EB, Wilson RE, Dammin GJ. Successful transplantation of a kidney from a human cadaver. JAMA 1963;185:347 53. 4. Transplantation contributions: organ procurement and transplantation. ,http://johncmcdonald.org/transplantation-procurement.html. (accessed 24.05.15). 5. Neylan JF, Sayegh MH, Coffman TM, et al. The allocation of cadaver kidneys for transplantation in the United States: consensus and controversy. J Am Soc Nephrol 1999;10:2237 43. 6. UNOS Membership Network: committees. ,http://www.unos.org/donation/index.php?topic=network. (accessed 24.05.15). 7. Ohler L. Courage and character leaders and legends: an interview with Howard Nathan. Prog Transplant 2012;22(1):4 6. 8. Ohler L. Pacesetters and pathfinders: an interview with Barbara Schulman. Prog Transplant 2015;25(1):4 5. 9. Trulock EP, Egan TM, Kouchoukos NT, et al. Single lung transplantation for severe chronic obstructive pulmonary disease, Washington University Lung Transplant Group. Chest 1989;96(4):738 42. 10. Kusserow RP. The distribution of organs for transplantation: expectations and practices. ,https://oig.hhs.gov/oei/reports/oei-01-8900550.pdf. (accessed 30.05.15). 11. Kasiske BL, Neylan JF, Riggio RR, et al. The effect of race on access and outcome in transplantation. New Engl J Med 1991;324:302 7. 12. Gaston RS, Ayers I, Dooley LG, Diethelm AG. Racial equity in renal transplantation: the disparate impact of HLA based allocation. JAMA 1993;270(11):1352 6. 13. Starzl TE, Hakala TR, Tzakis A. A multifactorial system for equitable selection of cadaver kidney recipients. JAMA 1987;257:3073 5. 14. Hirose K, Cherikh WS, Tomlanovich S, et al. Allocating kidneys without points for HLA matching distributes organs more equitably without adversely affecting outcomes. Am J Transplant 2006;6(Suppl. s2):140 World Transplant Congress Abstract 221. 15. Ladin K, Hanto D. Rational rationing or discrimination: balancing equity and efficiency considerations in kidney allocation. Am J Transplant 2011;11:2317 21. 16. OPTN/UNOS Kidney Transplantation Committee Report to the Board of Directors. ,http://optn.transplant.hrsa.gov/converge/ CommitteeReports/board_main_KidneyTransplantationCommittee_7_1_2011_10_38.pdf. (accessed 31.05.15). 17. Friedewald JJ, Samana CJ, Kasiske BL, Israni AK, Stewart D, Cherikh W, et al. The kidney allocation system. Surg Clin North Am 2013;93:1395 406. 18. UNOS Policy 8.6 Double Kidney Allocation. ,http://optn.transplant.hrsa.gov/ContentDocuments/OPTN_Policies.pdf. (accessed 5.07.15). 19. Vinkers MT, Smits JM, Tieken IC, et al. Kidney donation and transplantation in Eurotransplant 2006-2007: minimizing discard rates by using a rescue allocation policy. Prog Transplant 2009;19(4):365 70. 20. Tanriover B, Mohan S, Cohen DJ, Radhakrishnan J, et al. Kidneys at higher risk for discard: expanding the role of dual kidney transplantation. Am J Transplant 2014;14:404 15. 21. Israni AK, Zaun DA, Rosendale JD, Snyder JJ, Kasiske BL. OPTN/SRTR 2013 annual data report: deceased organ donation. Am J Transplant 2015;15(Suppl. 2):1 13 ,http://dx.doi.org/10.1111ajt.13202. 22. A guide to calculating and interpreting Kidney Donor Profile Index. ,http://optn.transplant.hrsa.gov/ContentDocuments/ Guide_to_Calculating_Interpreting_KDPI.pdf. (accessed 31.05.15). 23. Organ Procurement and Transplantation Network. Estimated Post Transplant Survival Calculator (EPTS). ,http://optn.transplant.hrsa. gov/converge/resources/allocationcalculators.asp?index=82. (accessed 5.07.15). 24. Israni AK, Salkowski N, Gustafson S, Snyder JJ, Friedewald JJ, Formica RN, et al. New National allocation policy for deceased donor kidneys in the United States and possible effects on patient outcomes. J Am Soc Nephrol 2013;25:1842 8. 25. Hurst FP, Sajjad I, Elster EA, et al. Transplantation of A2 kidneys into B and O recipients leads to reduction in waiting time: USRDS experience. Transplantation 2010;89(110):1396 402. 26. UNOS Policy 8. Kidney Allocation. ,http://optn.transplant.hrsa.gov/ContentDocuments/OPTN_Policies.pdf. (accessed 5.07.15). 27. CPRA and Its Importance in organ Transplantation. ,https://www.unos.org/docs/CPRA_Patients.pdf. (accessed 31.05.15). 28. Bray RA, Brannon P, Breitenbach C, Bryan C, et al. The new OPTN kidney allocation policy: potential for inequitable access among highly sensitized patients. Am J Transplant 2015;15(1):284 5. 29. Bray R, Gebel HM. The new Kidney Allocation System (KAS) and the highly sensitized patient: expect the unexpected (Letter to the Editor). Am J Transplant 2014;14:2917. 30. Kucheryavaya A, Tyan D, Boyle G, Kiger D, et al. A substantial increase in reporting of HLA DPB typing of deceased donors in the U.S. Am J Transplant 2014;14:590. 31. American Society of Nephrology. Improvements to the kidney allocation system. What does it mean to eliminate the payback system? ,https://www.asn-online.org/policy/webdocs/deceased_donor.pdf. (accessed 5.05.15). 32. Organ Procurement and Transplantation Network. Information for OPOs preparing for new kidney allocation system. ,http://optn. transplant.hrsa.gov/news/information-for-opos-preparing-for-new-kidney-allocation-system/. (accessed 5.07.15).

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C H A P T E R

6 Strategies to Increase the Donor Pool Michael A. Rees1,2 and David E. Fumo1,2 1

University of Toledo Medical Center, Toledo, OH, United States 2Alliance for Paired Donation, Perrysburg, OH, United States

6.1 BACKGROUND For those with end-stage renal disease (ESRD), renal transplantation is not only more cost-effective, but it also provides an average of 10 extra years of life compared to dialysis.1 Unfortunately, the number of patients requiring a kidney transplant far exceeds the current supply (Fig. 6.1). The number of people on the deceased donor waitlist has increased by 82% over the last 10 years, and 7271 patients either died or became too sick to transplant while on the waiting list in 2012.2 Living donor kidneys have not only increased the supply, but have also been shown to have improved 5 and 10 year graft survival rates.2 However, as many as 35% of willing donor-recipient pairs will be ABO incompatible, and an additional 30% will be unable to donate for immunological reasons.3 Kidney paired donation (KPD) has overcome this immunological barrier by allowing recipients with willing but incompatible living donors to exchange kidneys with other incompatible pairs, such that each patient is able to receive a transplant from a

FIGURE 6.1 Number of people on the kidney transplant waitlist compared to the number of people transplanted.2

Kidney Transplantation, Bioengineering, and Regeneration. DOI: http://dx.doi.org/10.1016/B978-0-12-801734-0.00006-0

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6. STRATEGIES TO INCREASE THE DONOR POOL

compatible donor.4 Unfortunately, the number of living donor transplants has decreased from 6647 in 2004 to 5622 in 2012.2 Many attempts have been made to circumvent the organ shortage problem. The use of animal cells and organs has the longest history, with the first attempts to perform animal to human blood transfusions as early as the 17th century.5 Porcine Xenotransplantation currently holds the most promise, due to similarities in organ size, physiology, and the potential for genetic modification to correct molecular incompatibilities. Indeed, preclinical trials have shown that pig kidneys are capable of supporting life for weeks to months in primates.6 8 However, the inability to overcome strong humoral and cellular immune responses to porcine antigens, combined with concerns about the contraction of unknown infections in immunocompromised patients, places xenotransplantation several years into the future.9 Dialysis poses significant limitations for ESRD patients waiting or unqualified for transplantation. Hemodialysis is limited by noncontinuous treatment and dependence on dialysis centers, while peritoneal dialysis (PD) requires patients to be connected to a machine overnight. Advancements in technology have allowed the creation of wearable PD and HD devices, which are currently undergoing both preclinical and clinical trials.10 13 The ultimate advancement in dialysis technology would be an implantable artificial kidney, though issues of component miniaturization, blood flow and coagulation, waste elimination, and device lifespan have yet to be solved. Such a device may also prove to be cost-prohibitive.14 Dialysis is inherently limited in its capacity as a renal replacement, both due to the inadequacy of dialysis membranes and the inability to perform hemostatic, metabolic, and endocrine regulation. The development of bioartificial kidneys is a solution to this problem.15,16 Investigations range from constructing renal tubule assist devices using synthetic scaffolds and renal tubule epithelial cells, to recellularizing whole organ extracellular matrix scaffolds. Though promising, bioartificial renal replacement strategies come with their own complications, and their clinical use is years away. Unfortunately, not all attempts to overcome the kidney shortage problem have been positive. Transplant tourism, or the buying and selling of organs, has been a scourge on the field of organ transplantation. It has been estimated that as many as 10% of the 106,879 organs transplanted in 2010 were procured and transplanted through black markets, 68.5% of which were kidneys.17 Black markets thrive in developing countries such as the Philippines, China, India, and Pakistan, where wealthy patients are charged between $100,000 and $200,000 for a transplant. The majority of this money goes to the brokers, while donors are paid as little as $1000, exposed to dangerous conditions, and left with little to no follow-up care.18 Regulatory agencies such as the World Health Organization (WHO) developed policies to curtail organ trafficking.18 20 However, enforcement continues to be a problem, as the countries which transplant tourists originate from and travel to are largely responsible for policing organ trafficking.21 Thus, the development and enforcement of ethical and legal policies on transplantation remains a prominent concern in global health. The solution lies not just in the development and enforcement of policies, but in increasing the supply of deceased and living donor organs available.

6.2 CURRENT PRACTICES In order to discuss methods of increasing the supply of donor kidneys, it is necessary to begin with the development of legal and ethical standards that govern kidney transplantation. As early as the 1930s, deceased donor kidneys were being used to treat acute renal failure, but the lack of immunosuppression precluded graft survival. In 1954, the first successful living donor transplant was performed by Joseph Murray at Peter Bent Brigham Hospital on a pair of identical twins.22 The discovery and experimentation of 6-Mercaptopurine as an immunosuppressant began in the late 1950s, led by Sir Roy Calne (Fig. 6.2). In 1962 a deceased donor kidney graft survived for over a year, thus beginning the modern clinical era of kidney transplantation.23,24 In 1968, the Uniform Anatomical Gift act in the United States established legislation recognizing the power of individuals to make the decision while alive to donate their organs in the event of their untimely death. This act required donors to document the desire to donate one’s organs, or the ability of a power of attorney to make this decision in the event that no documentation exists. The ESRD Program of 1972 amended Medicare to include financial responsibility for dialysis and transplantation. In 1984, Congress passed the National Organ Transplant Act, which established regulations on organ procurement, mandated

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FIGURE 6.2 Sir Roy Calne with his dog, Loli, who was successfully immunosuppressed with 6-MP.

the development of a scientific registry to evaluate clinical outcomes, and prohibited the purchase of organs. Policies for the allocation of donor organs, discussed in depth in previous chapters, were established based on two guiding principles: Utility and Justice. To maximize these principles, organs would be allocated to patients with the best chance of benefiting, and those with the most urgent need or longest wait time. By 1990, yearly kidney transplant rates rose above 9000 in the United States, of which 2094 came from living donors.25 Despite advancements in immunosuppression, living kidney donation was still limited. Approximately onethird of willing donors are ABO incompatible with their intended recipients, while 30% of patients are highly sensitized and potentially HLA incompatible with their donors.26 28 In 1986 Felix Rapaport suggested that ABOand HLA-incompatible pairs could exchange kidneys in a practice known today as KPD4 (Fig. 6.3). The first KPD exchange was performed in South Korea in 1991, followed by an exchange in Switzerland in 1999.29,30 Though the first KPD exchange in the United States took place in 2000, it was not until the passage of the Charlie W. Norwood Act in 2007, which stated that paired donation is not considered valuable exchange, that KPD became legal. Today, paired donation accounts for approximately 10% of kidney donations in the United States.2 In reviewing the history of transplantation, the categories of donor kidneys have been defined. Deceased donors are currently the most numerous, accounting for approximately 7000 transplants annually. Living kidney donors account for approximately 5500 transplants per year, and come in several forms. Compatible donors are those that can give directly to their intended recipient. Incompatible donors cannot give to their intended recipient but, when entered into paired exchange programs, can swap kidneys with other incompatible pairs. The final source for kidneys is from nondirected donors (NDD), or people without an intended recipient who wish to give a kidney to anyone in need. In order to increase the pool of kidney donors, it will be critical to not only increase the number, but also the utility of all four sources of donor kidneys.

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FIGURE 6.3 Example of a two-way paired exchange.

6.3 DECEASED DONORS In 2001, a conference was held to discuss the growing organ shortage problem. At this time, it was noted that the deceased donor kidney discard rate was 15%, and as high as 50% for donors over the age of 60.31 The members of the kidney work group noticed that, while the size of the OPTN transplant waitlist in the United States had grown by 230% over the previous 10 years, the kidney waitlist in Spain had decreased by 28%.32 It was concluded that this effect was primarily due to the increased utilization of donors over the age of 60, and that adoption of this model in the United States could increase the number of donors by 38%.32,33 In collaboration with the OPTN, the final proposal developed by this conference defined a new class, “expanded criteria donors,” based on age $ 60 or age 50 59 with any two of the following conditions: Cerebrovascular accident as the cause of death, serum creatinine .1.5 mg/dL, or history of hypertension. These expanded criteria donors have a relative risk of graft failure of .1.7 compared with the reference group of “standard criteria donors (SCD),” who do not meet the criteria of ECDs.31 When this policy went into effect in late 2002, transplant candidates could choose whether or not they were willing to receive an ECD kidney if one were to become available. The effectiveness of this policy has been analyzed from several different perspectives. Because ECD kidneys have, by definition, a graft failure 1.7 times that of SCD kidneys, one would expect the graft and patient survival to be lower. Most studies found this to be the case, with ECD donor 1 and 5-year graft survival ranging from 81% to 83% and 48.6% to 64.1%, respectively, compared to 89.3% 91% and 65.2% 74% for non-ECD donors.33 37 However, and more importantly, recipients of ECD kidneys experienced a 5-year increase in survival compared to matched waitlist recipients who did not receive a kidney (Fig. 6.4).36 The obvious ethical question that arises is how to appropriately allocate these kidneys to both increase the donor pool and increase patient survival. In other words, which patients should be listed for ECD kidneys? In 2002 a work group entitled “Expanded Criteria Donor Kidneys: Who Should Get Them?” convened to answer this question. Utilizing data from the Scientific Registry of Transplant Recipients (SRTR), this group determined that ECD kidneys would provide the greatest benefit in patients older than 60 years old, older than 40 years old with diabetes mellitus, patients with failed vascular access, and patients whose expected wait time far exceeds their life expectancy.37 39 Sung et al. and Schold et al. both analyzed the effect of implementing these policies, and found that after 36 months there was a 16% increase in the number of ECD kidneys transplanted.37,40 Older recipients and patients with diabetes were significantly more likely to both be listed for and receive an ECD kidney after the policy took effect. Both groups found a minimal overall effect on wait times, but noted that wait

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FIGURE 6.4 Mortality risks in two groups of cadaveric renal transplant recipients relative to wait-listed dialysis patients.37

TABLE 6.1

Estimated Kidney Graft Survival Rates by Donor KDRI41 Estimated SINGLE kidney graft survival rates

KDPI (%)

KDPI

1 Year (%)

2 Years (%)

3 Years (%)

5 Years (%)

8 Years (%)

1

0.57

95.3

92.7

89.9

83.3

72.2

5

0.63

94.8

92.0

88.9

81.8

69.9

10

0.67

94.4

91.4

88.1

80.6

68.1

20

0.75

93.8

90.5

86.8

78.6

65.1

30

0.82

93.2

89.5

85.6

76.7

62.3

40

0.91

92.5

88.5

84.2

74.5

59.2

50

1.00

91.7

87.3

82.6

72.2

55.9

60

1.11

90.8

86.0

80.8

69.6

52.4

70

1.23

89.8

84.5

78.9

66.7

48.6

80

1.39

88.6

82.6

76.5

63.3

44.2

90

1.62

86.7

79.9

72.9

58.3

38.2

95

1.84

85.0

77.5

69.8

54.2

33.5

99

2.25

81.8

72.9

64.2

46.9

25.9

times were reduced for centers that showed discretion in the number of patients listed for ECD kidneys. They concluded that appropriate listing criteria will improve the effective utilization of these kidneys. In the most recent OPTN allocation policy the ECD criteria has been replaced with a Kidney Donor Profile Index (KDPI), which is a continuous metric based on 10 donor characteristics (Table 6.1).41 The KDPI ranges from 1% to 100%, such that a kidney with a KDPI of 1% would be predicted to have a graft survival better than 98% of all kidneys, while a kidney with a KDPI of 100% is among the bottom 1% of all kidneys in terms of graft survival.

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Survival distribution function

Survival distribution function

1.00

0.75

0.50

0.25

0.00

0.75

0.50

0.25

0.00 0

20

40

60

80

100

120

0

20

Time 1 STRATA:

40

60

80

100

120

Time 1 group-DCD

group-ECD

group-ECD/DCD

group-SCD

FIGURE 6.5 (Top) Graft survival by donor type, P 5 .0116. (Bottom) Patient survival by donor type, P 5 .2215.49

A second group of donors have also been utilized to increase the deceased donor pool. As opposed to donation after brain death (DBD), donation after cardiac death (DCD) refers to donors in whom cessation of cardiopulmonary function occurred before the organs were procured and do not meet the criteria for DBD. Controlled DCD donors are those whose hemodynamic stability and respiratory function were maintained until organ procurement, while uncontrolled DCD donors experienced cardiac arrest before consent for donation can be obtained. European countries have been using DCD kidneys since the early 1990s with excellent results.42 45 In 2005, a national conference on Donation after Cardiac Death was convened to determine DCD criteria, develop criteria to predict DCD candidacy, and create protocols to maximize the successful recovery and utilization of DCD organs in the United States. It was subsequently shown that, although DCD donor kidneys have significantly higher rates of delayed graft function (DGF), DCD recipients had no difference in graft survival, patient survival, or acute rejection episodes.46 50 However, and not unexpectedly, results for combined DCD-ECD kidneys were poor (Fig. 6.5).49 Though the use of DCD and high KDPI kidneys has increased from 17% to 30% since 1999, the total number of deceased donor kidneys has actually decreased.2 This shift away from SCD and DBD donors may be due to the increase in the number of donors with cerebrovascular disease compared to trauma donors, an improvement in life-saving interventions such as craniostomy, craniotomy, cooling, etc.51,52 It is possible that the benefits of current deceased donor practices have reached a peak, and explorations of additional measures are necessary to increase the donor pool. In the United States, one must choose to be an organ donor through an active consent process. If no choice is made, the status quo is that consent has not been given, and organs will not be removed unless a family member consents. It has been proposed that instead of the current “opt-in” policy, those that have not explicitly refused to be an organ donor would have given presumed consent. This “opt-out” policy has the potential to increase deceased donation rates. Abadie et al. compared opt-in and opt-out policies in 22 countries, and found that opt-out legislation increases the rate of donation, independent of confounding factors (Fig. 6.6).53 Their results suggest that presumed consent legislation could potentially increase the number of deceased donor kidneys by 25% 30%. Objections to opt-out legislation include concerns about violating liberty and autonomy, religious objections, and a fear of a negative public perception of the transplant community under such policies.54 56 It has also been argued that presumed consent may have no impact on donation, and could even decrease the number of living donors.57,58 Despite these arguments, many organs are lost in patients for whom no consent can be obtained, and it is reasonable to argue that at least some of the patients would have consented if given the opportunity. Presumed consent legislation has the potential to increase deceased donation, though it will necessitate a collaborative effort to inform the public, deter negative impressions, and minimize effects on living donation rates.54,59,60 Efforts have been made in recent years to increase the rates of deceased donor registrations. Studies have shown that some groups, in particular minorities, are less likely to donate.61 64 Programs run through the department of motor vehicles, universities, and workplaces have demonstrated success in targeting these groups, particularly if the interventions contained an interpersonal component.65,66 Indeed, the number of registered donors has increased from 60 million in 2006 to 124.6 million today.55,67 However, the same time period has not yielded

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6.3 DECEASED DONORS

Cadaveric donors per million population, 2002

40.00 35.00

Presumed consent countries Informed consent countries

30.00 25.00 20.00 15.00 10.00 6.00

Spain Austria Latvia Portugal Belgium USA Estonia Ireland France Italy Slovenia Finland Hungary Czech Republic Norway Denmark Canada UK Poland Netherlands Germany Sweden Australia Switzerland Israel New zealand Croatia Cyprus Lithuania Slovak republic Luxemburg Greece Turkey Bulgaria Romania Japan

0.00

Country

FIGURE 6.6 Cadaveric donation rates in 2002 by country.53

an equivalent increase in the number of deceased donors. The simple explanation for this is that not enough time has passed for the benefits of these efforts to come to fruition, and future years will see an increase in deceased donation. It is also possible that these new registrants, who would not previously have registered, are individuals whose family would likely have given consent anyway, such that a large group of hard to reach donors is still being missed.55 Future work should be directed toward evaluation of these campaigns, as well as efforts to target these hard to reach donors. Financial incentives for deceased donors have been suggested, but have not been attempted in the United States. However, serious ethical objections to such policies have been raised, including coercion, exploitation of the poor, violations to freedom and autonomy, and a concern about public opinion toward the transplant community.68 70 Possible mechanisms for donor reimbursements include tax incentives, healthcare premiums, and payments to family to defray funeral costs.71,72 Current discussion centers around what methods of compensation would be permissible within the current laws, as well as exploring new policies that would uphold ethical and legal standards.73 Center level factors may also have an effect on deceased donor utilization. Developing policies for financial support, quality assurance, improvement processes, and governing the use of donor organs can benefit transplant centers.74 Conversely, government oversight aimed at quality improvement metrics such as graft and patient survival are used to credential transplant centers, which may act as a disincentive to transplant highrisk donors.55,75 Recently, insurance companies have mandated specific periods of medical care coverage postoperatively, which can also deter transplant centers from utilizing high-risk donors. Because patients who receive a high-risk kidney still do better than patients on dialysis, it would likely be beneficial and costeffective to remove these disincentives.36,76 78 Creating a sliding scale of reimbursements and quality improvement/center certification metrics based on donor and recipient risk factors could increase the willingness of centers to utilize higher risk kidneys.77 The utilization of kidneys from patients with both documented infections and high infection risk is also an important topic. The ability to accurately test high-risk kidneys while balancing delays in transplanting these kidneys and unnecessary discards due to false-positive test results requires further investigation.55,79,80 Transplants from donors with known hepatitis C infection into serology-matched recipients has been shown to be both effective and safe.81,82 While positive long-term outcomes in HIV-positive transplant recipients have been demonstrated, these patients also drew from the pool of uninfected donors.83 85 Through an investigation of the National Inpatient Sample (NIS) and UNOS databases, Boyarasky et al. estimated that a potential pool of

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6. STRATEGIES TO INCREASE THE DONOR POOL

TABLE 6.2 Characteristics of Potential HIV Infected Organ Donors Identified in HIVRN 2000 0886* National estimate n 5 494 Range: 441 533 Mean age death (year), SD

48.3, 9.7

, 30 years old

2%

30 49 years old

56%

50 59 years old

30%

60 69 years old

12%

Gender Male

81%

Female

19%

Race/ethnicity Caucasian

42%

African American

37%

Hispanic

14%

Other/unknown

7%

HIV primary risk factor** MSM

42%

IDU

26%

MSI

5%

HET

21%

HEI

5%

Other/unknown

2%

Median CD4 (IQR)

396 (314 566)

Median VL (IQR)

50 (50 54)

Median creatinine (IQR)

1.0 (0.9 1.3)

HCV Positive

16%

Negative

28%

Unknown

56%

HBV Positive

18%

Negative

33%

Unknown

49%

*Clinical and HIV characteristics of potential HIVDD identified from HIVRN relevant to organ donation are presented. **MSM: men who have sex with men; IDU: injection drug users; MSI: MSM and IDU; HET: heterosexuals; HEI: HET and IDU.

500 600 HIV-infected donors per year could be created, from which HIV-positive patients could draw (Table 6.2).86 In 2013, the HIV Organ Policy Equity (HOPE) Act lifted the ban on the ability to transplant HIV infected organs into HIV-positive recipients. The benefits of this policy, as well as the need for further policies to increase the utilization of these kidneys, are areas of active research.87 89 I. KIDNEY TRANSPLANTATION

6.4 LIVING DONATION

67

6.4 LIVING DONATION According to the most recent SRTR annual report of 2012 data, the deceased donor waitlist increased from 88,910 to 92,885 between 2011 and 2012.2 Over 30,000 patients were added to the list, while only 16,000 patients were transplanted. Despite current and future efforts to increase deceased organ donation, deceased donation will inevitably hit a ceiling well short of the number needed to satisfy the waitlist. As this chapter is being written in 2015, 101,209 ESRD patients are waiting for a deceased donor’s kidney in the United States (Fig. 6.1).2 Thus, rather than relying on deceased donors to satisfy the growing need for suitable organs for transplantation, alternative solutions to the organ shortage are needed. In recent years, the largest growth area has been found in living donors, but this growth has been limited to nonbiologically-related kidney donors. Overall, living kidney donation peaked in 2004 at just under 7000, and has declined almost every year to a nadir of 5817 in 2014 (Fig. 6.7).25 In 2014 Testa et al. proposed a shift in the focus on deceased donor promotion from the government and transplant community to promote living donation as the preferred source of donor kidneys, based on three premises: (1) the growing gap between kidney supply and demand; (2) the vast potential of living donors compared to deceased donors; and (3) the superior outcomes for recipients of living donor kidneys.90 Objectors to such a policy express concern over the ethics of performing surgery on healthy individuals to benefit others, the ability of transplant professionals to properly present the risks of donation, and the role of government in promoting living donation.90 92 Thus, it is necessary to explore potential mechanisms to increase living donation and the ethical issues that arise.

FIGURE 6.7 Counts of Deceased Donor and Living Donor Transplants by Year, 1993 2012.25

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6. STRATEGIES TO INCREASE THE DONOR POOL

TABLE 6.3 Educational and Clinical Recommendations From the Consensus Conference on Best Practices in Live Kidney Donation99 Education Levels100 Highest priority • LDKT education of patients with advanced stages of CKD should occur repeatedly throughout disease progression and transplantation processes (e.g., at evaluation, waiting list, reevaluation) • Educate general nephrologists and primary care physicians about LDKT so patients have access to transplant education earlier in the disease process • Integrate essential components of LDKT content and processes across centers, to include comprehensive risk and benefit information about LKD, known fears or concerns about LKD, and opportunities for interaction between transplant candidates and LDKT recipients as well as with former living donors • Create a LKD Financial Toolkit, which includes a summary of LKD financial risks, estimation of costs, available financial resources for the donor, state tax laws pertaining to donation, and how the Medicare Cost Report can best be optimized by programs HIGH PRIORITY • Develop a philosophical approach that LDKT is the best option for most transplant candidates and reflect this philosophy in educational processes • Provide more culturally-tailored LDKT education to racial/ethnic minority patients, with historically lower LDKT rates, and their support systems • Provide patients and their caregivers with training about how to identify and approach potential living donors • Increase awareness of the National Living Donor Assistance Center among providers, patients, and potential living donors • Develop a process to ensure that transplant and dialysis team members attain competency in living donation risks, methods for communicating risks and benefits, and ways to provide guidance to transplant candidates on effective and ethical approaches to engaging potential donors • Improve and expand the use of technology to better educate patients • Implement an independent, national clearinghouse (e.g., website) for the general public and potential donors

6.5 DONOR AND RECIPIENT EDUCATION In order to improve living kidney donation rates, it is critical to increase the awareness and willingness of both donors and recipients to participate in living donor transplantation. Over the last 10 years, significant efforts have been directed toward identifying barriers experienced by both of these groups. Waterman et al. identified recipient barriers to living donor transplantation, including comfort with dialysis, lack of education about the benefits of living donation, concern for potential donors, and difficulty broaching the subject with potential donors.93 Recipients are particularly concerned with financial, emotional, and physical burdens that may be placed on donors, the prospect of future kidney disease in donors, and the potential of regret experienced by donors.93,94 Patients also expressed that asking someone to donate a kidney was difficult and overwhelming. Rodrigue et al. found that 56% of transplant candidates had a low willingness to ask for a kidney, which was particularly prevalent in nonwhite patients and patients with lower. However, the biggest worry for potential donors appears to be a concern for their recipient’s health, with anxiety about testing, surgery, pain, finances, recovery, and long-term health risks being secondary.95 Pradel et al. found that laparoscopic nephrectomy, a less invasive surgery with shorter recovery time and less postoperative pain, was more likely to influence recipients rather than donors regarding their willingness to participate in living kidney donation.96,97 Waterman et al. found that 23.1% of potential donors expressed a lack of understanding about testing and surgical procedures.95 They further showed that 79% of potential donors who utilized educational resources were “very comfortable” with donation, versus 58% who did not use educational resources (χ2 5 3.91, P 5 .05). In another study, Pradel et al. identified that satisfaction with information that dialysis patients received on living donation was strongly correlated with willingness to discuss living donor kidney transplantation (LDKT) with potential donors.98 In June 2014, a consensus conference was held to discuss best practices in live kidney donation.99 The work group on educational and clinical recommendations determined that patient education early and often in the disease process was of highest priority (Table 6.3).99 In a retrospective review of 304 transplant recipients, only 54% 66% of patients received transplant information from dialysis or primary care providers.101 Preemptive transplantation has numerous quality of life benefits, including decreased psychological stress, fewer dietary restrictions, and higher employment rates.102 104 Additionally, these patients may be more motivated to seek transplantation as an option to maintain their current quality of life. Therefore, it is critical

I. KIDNEY TRANSPLANTATION

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6.5 DONOR AND RECIPIENT EDUCATION

for chronic kidney disease (CKD) patients to be educated about transplant options before starting dialysis. Boulware et al. showed that implementation of an educational program, Talking about Live Kidney Donation, which included both educational materials and a social worker, increased the number of CKD patients who pursued transplantation conversations with family and physicians.105 Numerous educational interventions for ESRD patients, including nephrologist training, home-based education, and intensive educational programs that incorporate previous transplant recipients and specific living donor educators have demonstrated success in moving potential recipients through the transplant process.106 108 Transplant centers should implement such procedures, and employ living donor-specific coordinators to facilitate patient education.99 Improving donor education is equally important as increasing LDKT. A work group on potential living donor educational processes has made recommendations to transplant centers on how to ensure potential donors are fully informed of the process of LDKT, including implementing a living donor advocate, developing culturally competent home visits and web-based tools, and methods of discussing psychosocial and health risks of donation.99,109 112 Waterman et al showed that donors who contacted a transplant center for assessment had often resolved many of their concerns with donation, most commonly by speaking with family members, potential recipients, and though educational resources.95 Potential donors are increasingly utilizing internet resources for educational purposes. In a review of 86 websites, Moody et al. found that while the accuracy of presented information was high, 98% were written above the recommended reading level, an average of only 38% of recommended information was included (range 8% 76%), and information related to benefits and medical/psychological risks were consistently lacking (Table 6.4).113 The development of an independent, neutral, and standardized internet resource for donors and recipients should be created to strengthen the understanding and trust between the public and the transplant community.99 As discussed previously, transplant candidates have often found it difficult to ask potential donors for a kidney. While educated patients may have an easier time conversing with potential donors, Garonzik-Wang et al. have suggested that a “living donor champion” would feel more comfortable advocating on behalf of the patient.114 Fifteen transplant candidates were asked to identify a living donor champion, who was educated on the process of LDKT over a 6-month period that included five information sessions. As a result of these sessions, living donor champions demonstrated an increased comfort in approaching potential donors, and increased the number of donor inquiries, workups, and living donor transplants on behalf of their recipients compared to matched controls. Future studies should seek to validate the efficacy of living donor champions, and work to incorporate this procedure into the recipient education process.

TABLE 6.4

Website Coverage and Accuracy of Recommended Information on Living Donation Topics113 Of websites that covered the topic Amount of coverage (%)a

Accuracy (%)b

No. of sites which Topics in living kidney donation covered topic (n, (%))

Minimal (,20%)

Moderate (20% 50%)

Mostly Full .50% 50% 90% .90% (51% 80%) ( . 80%) incorrect correct correct

General information about kidney 86 (100%) failure and treatments

1

29

70

0

0

0

100

Short-term medical risks to donor 75 (87%)

16

39

36

9

0

2

98

Long-term medical risks to donor 41 (48%)

27

56

17

0

0

12

88

Psychological risks and long-term 39 (45%) course

41

43

8

8

0

2

98

Benefits to donor

35 (41%)

68

26

6

0

0

0

100

Risks and benefits to recipient

79 (92%)

16

60

24

0

0

0

100

Financial considerations

52 (60%)

33

40

23

4

0

0

100

Voluntarism

36 (42%)

39

36

19

6

0

0

100

Donor evaluation process

79 (92%)

10

56

33

1

0

0

100

a

The amount of coverage was defined by the mean percentage of checklist items covered by the website. Accuracy was defined by the mean percentage of items which were described correctly.

b

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6. STRATEGIES TO INCREASE THE DONOR POOL

6.6 UNRELATED DONORS AND SOLICITATION Positive results using unrelated kidney donors has obviated the restrictions on human leukocyte antigen matching and donor/recipient relationship.115,116 The number of unrelated donors increased from 27% of living donors in 2000 to 51% in 2012.2 While paired donation has accounted for a significant portion of this change, many donors are reaching out to social networks or making public solicitations for organs via social media, kidney matching websites, and public advertising. Likewise, altruistic NDD may make the decision to donate, and then seek out potential candidates using any of these sources. Outreach through religious communities is increasingly common. Renewal, a Brooklyn-based nonprofit group, facilitates as many as 30 kidney transplants a year by connecting recipients with potential donors within the Jewish community.117 Religious and spiritual motivations have prompted potential donors to direct kidneys to recipients within their community or found on the internet.118 120 Potential recipients using websites for organ solicitation in the form of recipient advertising venues, networks of potential donors, and social media pages have found success and comfort in this less intrusive method of asking for an organ.121,122 Matchingdonors.com, a registry that links potential donors with recipients, has facilitated numerous transplants, and utilizes an approach where NDD register their desire to donate for free and are provided with a list of recipient biographical summaries. These summaries are provided by potential recipients, who pay for the ability to post their summary for nondirected donor consideration. Some authors have raised ethical concerns about such an approach.123 Numerous other examples exist, and the use of community and public outreach has likely contributed to the increase in unrelated living donation rates. Ethical concerns have been raised regarding unrelated living donation. Unlike the donation of blood or bone marrow, kidney donation entails the potential for serious harm to the donor. While it is not difficult to fathom undertaking the risk of donation for a loved one, such an altruistic act for a stranger or mere acquaintance raises concerns among the transplant community. These arrangements may be influenced by coercion between employers and supervisors, emotional exploitation, the presence of donor psychosocial issues, and subverted payments for donation.124 127 Public solicitation poses risks to a fair allocation system, as organs would be distributed to those with the means and ability to present the most compelling story, not those in greatest need.127 This is particularly the case for deceased donor solicitation and directed deceased donation, which would allow candidates to receive a transplant ahead of those higher on the list. The OPTN has been tasked with developing strict policies regarding the allocation of deceased donor organs.20,115 Finally, the ability to direct donation allows potential donors to stipulate racial, religious, or ethnic requirements. Such discriminatory practices would be particularly difficult to prevent without prohibiting directed donation entirely.124,127 Contrary arguments appeal to donor autonomy to direct an organ to a recipient of choice, the ability of religious and community ties to be strong altruistic motivators, and the potential for solicitation to increase public awareness of the organ shortage problem.120,124,127,128 Transplant centers are increasingly willing to utilize unrelated donors in LDKT, though transplantation between strangers and of publicly solicited organs remains controversial (Table 6.5).126,129 With the current dearth of organs available for transplantation, the approach in the United States has been to avoid imposing carte blanche restrictions on donor/recipient relationships, as has occurred in other countries.130 In a 2006 policy, the OPTN stated that it “cannot regulate or restrict the ways relationships are developed in our society with respect to live organ donation, nor does it seek to do so. The obligation of the OPTN and its members is to uphold the provisions of the National Organ Transplantation Act (NOTA) that make it a felony ‘for any person to knowingly acquire, receive or otherwise transfer any human organ for valuable consideration for use in human transplantation.’”115 Rather, the OPTN has developed policies for the psychosocial evaluation of living donors to ensure that donation is undertaken voluntarily, without coercion, and that the donor is aware of the risks and benefits of their gift.131

6.7 DONOR INCENTIVES In Greek mythology, the Strait of Messina was flanked by two hazards: Scylla, a rock shoal, and Charybdis, a whirlpool (Fig. 6.8). One could not successfully navigate a safe distance from one without passing too close to the other. This choice between two evils is reminiscent of the conundrum of removing disincentives for living kidney donors. In attempting to provide reasonable compensation to increase the rates of living donation, thereby

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6.7 DONOR INCENTIVES

TABLE 6.5

Psychosocial Issues and Whether They Are Considered a Contraindication (Absolute, Relative) to Living Donation or Not126 N

Absolute

Relative

None

Knowledge of financial gain or reward

131

118 (89)

10 (8)

3 (2)

Active substance abuse/dependence

125

108 (86)

15 (12)

2 (2)

Active mental health problems or instability

127

96 (76)

31 (24)

0 (0)

Desire for secondary gain

126

49 (39)

66 (52)

11 (9)

Lack of disclosure to spouse/partner, or next-of-kin who may be affected most by donation

128

45 (35)

75 (59)

8 (6)

Motivated primarily by desire for medical care

125

38 (30)

66 (53)

21 (17)

Unrealistic expectations about donor experience

129

38 (29)

85 (66)

6 (5)

History of poor adherence to healthcare recommendations

127

28 (22)

91 (72)

8 (6)

Strained donor-recipient relationship

126

28 (22)

87 (69)

11 (9)

Lack of family or social support

129

26 (20)

90 (70)

13 (10)

Lack of healthcare coverage

125

19 (15)

53 (42)

53 (42)

Donation strongly advised by faith community leaders

127

17 (13)

69 (54)

41 (32)

Financial instability

131

7 (5)

94 (72)

30 (23)

No history of past altruistic behaviors

125

0 (0)

21 (17)

104 (83)

FIGURE 6.8 James Gillray, Britannia between Scylla and Charybdis (1793).

decreasing the burden of ESRD, the transplant community runs the risk of commodifying the human body. Discussions aimed at navigating these waters must include ethical, legal, and financial consideration, and the transplant community stands divided on many of these issues. Policies are currently in place in the United States to remove disincentives for living kidney donors, which include donor reimbursement for travel, housing, and lost wages.73,99 However, the line between and legality of financial incentives versus the removal of disincentives for living kidney donors is less clear. Proponents of incentivizing donors believe that such a system would increase donation, increase the pool of living donors, decrease the number of deaths due to renal failure, and cripple the economic support for the black market and

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6. STRATEGIES TO INCREASE THE DONOR POOL

FIGURE 6.9 Break-even point to society if all vendors and current donors were paid. Cost-effective point includes impact of gain in Quality of Life Years.133

transplant tourism.132 Furthermore, due to the difference in cost of transplantation compared to dialysis, increasing the pool of living donors would work to relieve the financial burden of ESRD. A financial analysis by Matas et al. revealed that transplantation has the potential to save between $90,000 and $269,000 per ESRD patient (Fig. 6.9).133 The authors posited that a kidney vendor program could break even while providing up to $90,000 to living kidney donors. Financial programs for kidney donors are currently being used abroad. In 2008 Israel adopted the Organ Transplantation Law, which includes specific clauses to remove disincentives by providing donor compensation for lost wages, transportation costs, medical and life insurance, and psychological consultations.134 Iran has adopted a program through which donors are both financially compensated and provided with healthcare.135 The introduction of these programs has resulted in an increase in kidney donation and a reduction in the transplant waitlist. Opponents of financial compensation believe that such a system would not only fail to increase the supply of available organs, but would also cause harm to donors and result in an immoral commodification of the human body.136 Those who adhere to this view feel that paying potential donors would exploit vulnerable populations (e.g., those with low socioeconomic status) and create income-based disparities in access to transplantation.68,137 Furthermore, donor incentives would put recipients at risk because organ vendors would have significant motivation not to disclose information that may put them at risk of being disqualified as a donor.136 The Iranian model demonstrates that paid donors were in poorer health and had lower social functioning scores than unpaid donors.135 Finally, there is concern that payments for donors would be viewed negatively by the public as commodification of the human body, which would lead to mistrust of the transplant community and decreased donation rates.68,137,138 In June 2014, the ASTS and AST held a workshop on increasing organ donation in the United States.73 It was agreed that the continued removal of donor disincentives should be the first priority. Members of this incentive work group went on to explore the possibility of providing incentives for living donors and suggest that a pilot project would work to test assumptions about the impact of incentives on living donation. Fisher et al. provide ethical justification of such a project, and challenge arguments claiming that incentives would exploit vulnerable populations, cause undue influence for donors, damage the doctor patient relationship, and negatively impact public opinion.138 They also discuss the need to strike a balance between providing adequate compensation and avoiding “toxic” incentive amounts that would risk causing undue influence. Opponents argue that pilot programs would reshape the attitudes of the population regarding their expectations of financial rewards, which would be solidified even if the project were abandoned.136 Furthermore, if the project failed to increase donation rates, proponents would be unlikely to abandon the attempt, but rather increase compensation rates without regard for the detrimental effects on donor autonomy. Finally, there is concern that financial programs in the United States would foster a return to unregulated organ markets abroad. In navigating through these challenges, the transplant community must pass between two perils. Doing so will require the conjoined effort of members of the transplant community, public policy experts, and members of congress, and must do so with the support of the general public.73

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6.8 IMMUNOLOGICAL BARRIERS

73

6.8 IMMUNOLOGICAL BARRIERS Despite the growing number of HLA sensitized candidates, recent national data shows that as few as 4.9% of living donor transplants have been performed on highly sensitized candidates (cPRA .80%) annually.2 Additionally, ABO incompatibility is present in 35% of potential donors.3 Desensitization has allowed for transplants to be performed across both blood group and HLA incompatibilities. Transplant centers utilize varying regimens of plasmapheresis and intravenous immunoglobin for antibody removal, and antibody synthesis blocking agents for antibody attenuation.139 141 Long-term follow up data demonstrates that ABOincompatible graft and patient survival is similar to compatible living donor outcomes, with the potential caveat of an increased risk of graft loss in the first 14 postoperative days.142 146 Patients who underwent desensitization across HLA-incompatibilities have had higher rejection rates, as well as graft and patient survival rates below the national average.147,148 However, Montgomery et al. demonstrated that HLA desensitization provides improved long-term patient survival compared to remaining on dialysis or waiting for a compatible kidney, providing compelling evidence that desensitization may be the best option for highly sensitized patients (Fig. 6.10).149 Several barriers to desensitization still need to be resolved. Morbidity and mortality rates, most prominently cardiac events and infections, are higher among recipients who undergo desensitization.147,149 151 Variations in HLA laboratory procedures, desensitization protocols, and center-specific assessment of the need for desensitization based on antibody strength have made comparing outcome data difficult.139,152 In a survey of 125 transplant centers representing 84% of the living kidney donor volume, 74% reported using desensitization (Table 6.6).152 However, only 24% of these centers performed ABO-incompatible transplants, and the number of incompatible transplants performed at each center was not quantified. Because desensitization carries the risk of reduced patient and graft survival, regulatory pressures may discourage centers from performing desensitization, particularly at smaller centers.152 Statistical derivation of expected outcomes must allow for incorporation of the reduced patient and graft survival inherent in desensitization in order for this approach to reach its full potential. Future work should be directed toward homogenizing laboratory and desensitization protocols, developing new pharmaceutical agents, and creating policies to increase utilization using survival on dialysis as a comparator rather than compatible transplantation, yet striving to maintain positive outcomes overall.

FIGURE 6.10

Survival benefit of desensitization in HLA-incompatible kidney recipients.149

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74 TABLE 6.6

6. STRATEGIES TO INCREASE THE DONOR POOL

Results of desensitization

Type

Performed?a

Desensitize?b

PP/IVIgc

IVIgc

Otherc

CD20d

PLNF

86/123

50/86

37/50

23/50

14/50

27/49

70%

58%

74%

46%

28%

55%

63/123

48/63

34/48

21/48

14/48

25/47

51%

76%

71%

44%

29%

53%

22/123

22/22

18/22

8/22

5/22

13/22

18%

100%

82%

36%

23%

59%

30/124

24/30

20/24

7/24

7/24

16/26

83%

29%

29%

62%

PFNC

PCC

ABOi

24%

e

80%

a

Numbers represent the percent on the basis of those from the overall cohort who answered the given question. For example, there were 86 centers that performed PLNF transplants, representing 70% of the 123 centers that responded to the set of questions about PLNF transplants. b Numbers represent the percent on the basis of those who reported performing the given modality. For example, of those 86 centers that performed PLNF transplants, 50 of 86 (58%) used preoperative desensitization in that situation. c Numbers represent the percent on the basis of those who reported using desensitization. For example, of those 50 centers that desensitized PLNF recipients, 37 (74%) used PP/ IVIg. d Numbers represent the percent on the basis of those who answered the question about anti-CD20 antibody. For example, of 49 centers reporting use of anti-CD20, 27 stated that they never used it in PLNF situations. e For ABOi transplants, the number represents those who precondition with A2, B, or A1 donors. Of 30 centers that performed ABOi transplants, 24 (80%) precondition one of those blood type donors and 15 (50%, not shown in table) reported always preconditioning for ABOi transplants.152 PP/IVIg, plasmapheresis and low-dose intravenous Ig; IVIg, high-dose intravenous Ig; PLNF, positive Luminex, negative flow crossmatch; PFNC, positive flow, negative cytotoxic crossmatch; PCC, positive cytotoxic crossmatch; ABOi, ABO incompatible.

6.9 KIDNEY PAIRED DONATION KPD represents the fastest growing source of transplantable kidneys by overcoming the immunological barriers that render approximately 1/3 of potential kidney donors incompatible with their intended recipient.3,153 159 First suggested by Felix Rappaport in 1986, KPD was first reduced to practice in South Korea in 1991.29,160 It wasn’t until nearly a decade later that the first kidney exchange was performed in the United States in 2000.161 For a variety of reasons, including concerns about the NOTA prohibition against donating an organ for valuable consideration, it was not until the passage of the Charlie Norwood Amendment of NOTA in 2007 that KPD began to gain traction in the United States.153,162 164 Of the more than 3500 KPD transplants performed to date, 70% have been performed in the last 5 years.165 In its simplest form, KPD involves 2 incompatible pairs exchanging kidneys, such that each patient receives a transplant from a compatible living donor (Fig. 6.11A).3,26,153,166 In 2001, UNOS region 1 began performing list exchange, in which an incompatible donor donated a living donor kidney to a recipient on the deceased donor kidney transplant waiting list, and in exchange, their intended incompatible recipient was given priority for a future deceased donor kidney (Fig. 6.11C).153,167 Concerns about disadvantaging blood type O candidates on the deceased donor waiting list limited the application of list exchange so that early kidney exchanges in the United States were primarily limited to simple two-way exchanges between pairs with willing but incompatible living donors. With the advent of computer-aided matching algorithms, it became apparent that exchanges between three or more willing but incompatible pairs would increase the number transplants possible.167,168 The incorporation of NDDs into KPD allowed for the creation of a transplant chain in which each donor pays their kidney forward to the next incompatible pair until no further matches exist in the current pool. In domino paired donation (DPD), this leftover donor is directed to the deceased donor waitlist (Fig. 6.11D),153 while nonsimultaneous extended altruistic donor (NEAD) chains allow for the leftover donor to become a “bridge donor” and be reentered into the pool to continue the chain at a later date (Fig. 6.11E).153,167,169,170 As with list exchange, initial concerns were raised that using NDDs in KPD would disadvantage blood type O waitlist candidates. However, the ability of all types of chains to provide additional transplant opportunities for patients with willing incompatible donors, previously relegated to the waitlist, has generally been accepted as beneficial.171 177 The United States has at least seven single and multicenter KPD registries that utilize matching algorithms to create potential kidney exchanges. Algorithms in general operate using optimization or rule-based hierarchical systems, each with distinct advantages and disadvantages, and there is no consensus on the “best” matching strategy.178

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6.9 KIDNEY PAIRED DONATION

75 FIGURE 6.11 A history of KPD from its original proposal by Felix Rapaport to the present day. (A) Two incompatible pairs swap kidneys in a two-way exchange. (B) A three-way exchange with no reciprocal exchanges so hard-to-match pairs are better matched. (C) In a list exchange, an incompatible donor donates to a wait-list candidate in exchange for wait-list priority for the recipient. (D) DPD pairs an altruistic donor with an incompatible recipient. The donor then continues the chain or donates to a wait-list candidate. (E) A compatible pair can join an exchange helping hard-to-match pairs. (F) NEAD chains are like domino chains except the last donor becomes a bridge donor and awaits more pairs to perpetuate the chain at a later time.153

Multiregional registries require complex levels of coordination between transplant centers, and various strategies have been adopted.179 181 The ability to overcome geographic barriers has been greatly aided by the ability to safely and efficiently ship kidneys nationally.182 185 Despite these advancements, KPD appears to have hit a plateau, and 2014 saw the first decrease in the number of KPD transplants performed compared to the previous year. Overcoming the multitude of challenges currently inhibiting KPD stands to have the biggest impact on both the quality of life and healthcare burden in patients with ESRD. The rapid dissemination of KPD among transplant centers that took place between 2006 and 2008 has slowed since that time.186 While the top 10% of KPD centers perform an average of 5.52 KPD transplants per 100 LDKTeligible patients, most centers perform less than one.186 In a survey of transplant center attitudes toward KPD, Clark et al. found that the most prominent barriers to KPD were (1) unequal quality of kidneys, (2) logistics of donor travel, (3) logistics of working with other centers and KPD registries, and (4) the dedication, training, and payment of staff to handle KPD programs.185 They further noted that the biggest barriers to implementing KPD were not the most frequently cited concerns (Fig. 6.12). Utilizing the knowledge obtained from high-performing KPD centers can help overcome many of the cited barriers of donor travel, dedicating and training KPD staff, and working with other transplant centers. Furthermore, KPD actually has the potential to increase the quality of kidneys by matching donor/recipient ages and decreasing HLA mismatches.186 If all transplant centers utilized KPD at the level of the highest performing KPD centers, an additional 1099 2000 transplants could be performed per year.186,187 Public support of KPD is generally high, with 85% of respondents to a national survey extremely or very willing to participate188. KPD pools have a distinctly lower proportion of blood type O donors and type AB recipients, since these two groups are always ABO compatible. It has been estimated that the incorporation of compatible pairs in KPD could double the proportion of matched pairs.166,189,190 This practice has been pejoratively called altruistically unbalanced paired kidney exchange (AUPKE), as compatible donors are asked to be doubly altruistic by not only

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FIGURE 6.12 Most frequently cited major and minor barriers to implementing kidney paired donation.185

donating a kidney, but to also forego donating to their compatible recipient for the benefit of an incompatible pair.174 Ross et al. further suggested that asking compatible pairs to participate in KPD represents a conflict of interest for the physician, who has a moral obligation to act in the best interests of the patient. Nonetheless, some of these same authors have subsequently conceded that there is equipoise regarding the incorporation of compatible pairs in kidney exchange.191 Modeling and execution of such exchanges have shown that compatible pairs have the potential to receive a higher quality kidney (younger or fewer HLA mismatches), and surveys suggest that approximately one-third of compatible pairs would be willing to participate.187,189,192 195 In 2006 Gentry et al. produced a consensus KPD matching statement, which includes participation of compatible pairs as a parameter to be considered in designing a national KPD matching program196,197. In 2012, a consensus conference was held to discuss the dynamic challenges inhibiting optimal adoption of KPD.178 Though competition among the KPD registries has inspired innovation, it has inadvertently led to fragmented implementation methods throughout the country. The consensus conference called for standardization and collaboration in implementing and executing KPD in the United States. One observation was the pattern in which transplant centers enroll incompatible pairs. Centers may withhold easy to match pairs, preferring to perform internal exchanges. While this may be logistically and financially beneficial for a particular transplant center, it is detrimental to the pool overall.154 Secondly, pairs can be enrolled in multiple registries to increase the probability of finding a match. While this is of benefit to the individual patient, it has the potential to create waste when a registry attempts to create an exchange using a pair that has already been matched or transplanted elsewhere.178,179,198 Perhaps one of the biggest challenges in KPD is the complexity of paying for donor evaluations, histocompatibility testing, administrative costs, nephrectomy and complication costs, and transportation costs.180,199 The Alliance for Paired Donation has received AHRQ grant funding to develop a standard acquisition charge model to alleviate these challenges.179 Many of the barriers to maximizing KPD could be overcome by the creation of a national registry. Several countries have implemented national KPD registries, including the Netherlands, South Korea, the United Kingdom, Canada, and Australia, with great success.156,159,200 202 The United States presents a unique challenge based on the shear number and geographic distribution of transplant centers and payers. Simulations by Segev et al. have suggested that a national optimized KPD program could result in more transplants, better HLA concordance, increased matching of highly sensitized patients, and shorter travel distances for donors.3 HLA standardization could be achieved by a single national tissue-typing laboratory. National KPD programs in Australia and the Netherlands have successfully employed a single-laboratory approach.158,203 However, these programs differ significantly from the United States in the number of centers, geographic area, presence of a single payer and the number of potential incompatible pairs that would need to be crossmatched. The national KPD programs in Canada and the United Kingdom have developed standardized crossmatch criteria, which may be a reasonable alternative for combating the overwhelming volume a single laboratory would have to deal with in the United States.159,200 Variations in HLA testing and reporting, unacceptable antigen levels, and crossmatching were discussed at the national consensus conference, and recommendations were made to streamline this process

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TABLE 6.7

77

Recommended Guidelines for KPD Histocompatibility Testing178

• HLA typing: Should be DNA based for HLA-A, -B, -C, -DRB1, -DRB3 5, -DQA1, -DQB1, -DPB1 loci and inclusive of certain specific alleles and common null alleles, as needed. Extended donor typing may be required depending upon antibody specificities • Antibody testing: Two methods should be used, at least one being a solid phase immunoassay. Antibody specificity should be confirmed by a single antigen assay. Assay limitations should be recognized and considered in interpretation. Antibody testing should be performed at least quarterly and after any proinflammatory/sensitizing event • Unacceptable antigens: Unacceptable antigens should be assigned based on the transplant center’s crossmatch acceptance criteria and should be updated whenever antibody tests indicate a change. There should be two levels of unacceptable antigens, high and low risk; possible listing of antigen combinations to address multiple, weak antibodies. Definition of sensitization should be based on the calculated panel reactive antibody • Virtual crossmatching: Correlation of antibody assays with transplant center risk criteria is essential. Labs should achieve 95% accuracy in crossmatch prediction. Labs should try to identify combinations of multiple, weak antibodies that could yield a positive crossmatch when a donor has all of the corresponding antigens • Crossmatching: Flow cytometric crossmatches are recommended for sensitized patients. Unexpected positive results should be resolved, and unacceptable antigens updated. Patients should be inactive until reasons for failed crossmatches have been resolved and unacceptable antigens are updated. Cryopreserved donor cells should be available for preaccepted, “exploratory” crossmatches • Exchange of specimens and data: There should be standardized practices for test requisitions, labeling, and shipment of shared samples. Data entry should be verified by two person audit, at least one of whom should have histocompatibility expertise • Communication: Histocompatibility Laboratory Directors should participate in the evaluation of proposed paired donation matches and be available to provide consultation. KPD programs should have a Histocompatibility Advisory Committee comprised of physicians, surgeons, coordinators, and histocompatibility experts to provide quality assurance review and facilitate logistical planning for testing

(Table 6.7).178 Finally, a national KPD program would require an analysis of the myriad of strategies employed by current KPD programs to generate potential exchanges to develop a single algorithm with an allocation rubric that maximizes both the quality and quantity of exchanges.166,175,190,195,196,204 207

6.10 SUMMARY Over the last 75 years, advancements in the field of renal replacement therapy have reversed the death sentence that was previously placed on ESRD patients. The question is no longer how do we treat this disease, but which modality should we utilize? Immunosuppressive therapies have broken down the immunological barriers to renal transplantation, while dialysis has provided a bridge for those waiting for a transplant. Changes in healthcare policy, public opinion, and allocation protocols have increased the number of both living and deceased kidney donors. Innovative thinking has led to the development and utilization of KPD and desensitization to further overcome donor-recipient incompatibility. More recently, the idea of incentivizing both living donors and deceased donor’s families has become an intriguing matter of discussion. However, despite these efforts, thousands die every year while waiting for a kidney. While bioengineering and genetic manipulation to allow for xenotransplantation and stem cell therapy has promise for the future, the immediate solution is to increase the supply of kidney donors. Doing so will require the collaborative effort of the transplant community, government, and policy makers, and the support of the general public. By continuing to explore new strategies to increase both living and deceased organ donation, observing what has proved beneficial abroad, and remaining vigilant over the ethical responsibilities to both donors and recipients, we can continue to work toward the goal of giving the gift of life to every person in need of a kidney.

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81. Veroux M, Corona D, Sinagra N, Giaquinta A, Zerbo D, Ekser B, et al. Kidney transplantation from donors with hepatitis C infection. World J Gastroenterol 2014;20(11):2801 9. 82. Abbott KC, Lentine KL, Bucci JR, Agodoa LY, Peters TG, Schnitzler MA. The impact of transplantation with deceased donor hepatitis cpositive kidneys on survival in wait-listed long-term dialysis patients. Am J Transplant 2004;4(12):2032 7. 83. Locke JE, Montgomery RA, Warren DS, Subramanian A, Segev DL. Renal transplant in HIV-positive patients: long-term outcomes and risk factors for graft loss. Arch Surg 2009;144(1):83 6. 84. Locke JE, Segev DL. Renal transplantation in HIV-positive recipients. Curr Infect Dis Rep 2010;12(1):71 5. 85. Stock PG, Roland ME. Evolving clinical strategies for transplantation in the HIV-positive recipient. Transplantation 2007;84(5):563 71. 86. Boyarsky BJ, Hall EC, Singer AL, Montgomery RA, Gebo KA, Segev DL. Estimating the potential pool of HIV-infected deceased organ donors in the United States. Am J Transplant 2011;11(6):1209 17. 87. Health R, Services Administration DoH, Human S. Organ procurement and transplantation: implementation of the HIV Organ Policy Equity Act. Final rule. Fed Regist 2015;80(89):26464 7. 88. Richterman A, Blumberg E. The challenges and promise of HIV-infected donors for solid organ transplantation. Curr Infect Dis Rep 2015;17(4):471. 89. Richterman A, Sawinski D, Reese PP, Lee DH, Clauss H, Hasz RD, et al. An assessment of HIV-infected patients dying in care for deceased organ donation in a United States urban center. Am J Transplant 2015;15(8):2105 16. 90. Testa G, Siegler M. Increasing the supply of kidneys for transplantation by making living donors the preferred source of donor kidneys. Medicine (Baltimore) 2014;93(29):e318. 91. Mandelbrot DA, Pavlakis M, Danovitch GM, Johnson SR, Karp SJ, Khwaja K, et al. The medical evaluation of living kidney donors: a survey of US transplant centers. Am J Transplant 2007;7(10):2333 43. 92. Starzl TE. Will live organ donations no longer be justified? Hastings Cent Rep 1985;15(2):5. 93. Waterman AD, Stanley SL, Covelli T, Hazel E, Hong BA, Brennan DC. Living donation decision making: recipients’ concerns and educational needs. Prog Transplant 2006;16(1):17 23. 94. Pradel FG, Mullins CD, Bartlett ST. Exploring donors’ and recipients’ attitudes about living donor kidney transplantation. Prog Transplant 2003;13(3):203 10. 95. Waterman AD, Covelli T, Caisley L, Zerega W, Schnitzler M, Adams D, et al. Potential living kidney donors’ health education use and comfort with donation. Prog Transplant 2004;14(3):233 40. 96. Flowers JL, Jacobs S, Cho E, Morton A, Rosenberger WF, Evans D, et al. Comparison of open and laparoscopic live donor nephrectomy. Ann Surg 1997;226(4):483 9. discussion 9 90. 97. Pradel FG, Limcangco MR, Mullins CD, Bartlett ST. Patients’ attitudes about living donor transplantation and living donor nephrectomy. Am J Kidney Dis 2003;41(4):849 58. 98. Pradel FG, Suwannaprom P, Mullins CD, Sadler J, Bartlett ST. Haemodialysis patients’ readiness to pursue live donor kidney transplantation. Nephrol Dial Transplant 2009;24(4):1298 305. 99. LaPointe Rudow D, Hays R, Baliga P, Cohen DJ, Cooper M, Danovitch GM, et al. Consensus conference on best practices in live kidney donation: recommendations to optimize education, access, and care. Am J Transplant 2015;15(4):914 22. 100. Rodrigue JR, Cornell DL, Kaplan B, Howard RJ. Patients’ willingness to talk to others about living kidney donation. Prog Transplant 2008;18(1):25 31. 101. Waterman AD, Barrett AC, Stanley SL. Optimal transplant education for recipients to increase pursuit of living donation. Prog Transplant 2008;18(1):55 62. 102. Dahabreh Z, Dimitriou R, Giannoudis PV. Health economics: a cost analysis of treatment of persistent fracture non-unions using bone morphogenetic protein-7. Injury 2007;38(3):371 7. 103. Hays R, Waterman AD. Improving preemptive transplant education to increase living donation rates: reaching patients earlier in their disease adjustment process. Prog Transplant 2008;18(4):251 6. 104. Tomasz W, Piotr S. A trial of objective comparison of quality of life between chronic renal failure patients treated with hemodialysis and renal transplantation. Ann Transplant 2003;8(2):47 53. 105. Boulware LE, Hill-Briggs F, Kraus ES, Melancon JK, Falcone B, Ephraim PL, et al. Effectiveness of educational and social worker interventions to activate patients’ discussion and pursuit of preemptive living donor kidney transplantation: a randomized controlled trial. Am J Kidney Dis 2013;61(3):476 86. 106. Rodrigue JR, Cornell DL, Lin JK, Kaplan B, Howard RJ. Increasing live donor kidney transplantation: a randomized controlled trial of a home-based educational intervention. Am J Transplant 2007;7(2):394 401. 107. Sullivan C, Leon JB, Sayre SS, Marbury M, Ivers M, Pencak JA, et al. Impact of navigators on completion of steps in the kidney transplant process: a randomized, controlled trial. Clin J Am Soc Nephrol 2012;7(10):1639 45. 108. Weng FL, Brown DR, Peipert JD, Holland B, Waterman AD. Protocol of a cluster randomized trial of an educational intervention to increase knowledge of living donor kidney transplant among potential transplant candidates. BMC Nephrol 2013;14:256. 109. Gill JS, Tonelli M. Understanding rare adverse outcomes following living kidney donation. JAMA 2014;311(6):577 9. 110. Hays RE, LaPointe Rudow D, Dew MA, Taler SJ, Spicer H, Mandelbrot DA. The independent living donor advocate: a guidance document from the American Society of Transplantation’s Living Donor Community of Practice (AST LDCOP). Am J Transplant 2015;15 (2):518 25. 111. Kaplan B, Ilahe A. Quantifying risk of kidney donation: the truth is not out there (yet). Am J Transplant 2014;14(8):1715 16. 112. Thiessen C, Kim YA, Formica R, Bia M, Kulkarni S. Written informed consent for living kidney donors: practices and compliance with CMS and OPTN requirements. Am J Transplant 2013;13(10):2713 21. 113. Moody EM, Clemens KK, Storsley L, Waterman A, Parikh CR, Garg AX, et al. Improving on-line information for potential living kidney donors. Kidney Int 2007;71(10):1062 70. 114. Garonzik-Wang JM, Berger JC, Ros RL, Kucirka LM, Deshpande NA, Boyarsky BJ, et al. Live donor champion: finding live kidney donors by separating the advocate from the patient. Transplantation 2012;93(11):1147 50.

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115. Delmonico FL. Commentary: the WHO resolution on human organ and tissue transplantation. Transplantation 2005;79(6):639 40. 116. Terasaki PI, Cecka JM, Gjertson DW, Takemoto S. High survival rates of kidney transplants from spousal and living unrelated donors. N Engl J Med 1995;333(6):333 6. 117. Wolf B. Do Chareidim Contribute Their ‘Fair Share’ of Organs. The Algemeiner. 2013 7/31/2013;Sect. Spirituality/Tradition. 118. Dixon DJ, Abbey SE. Religious altruism and the living organ donor. Prog Transplant 2003;13(3):169 75. 119. Henderson AJ, Landolt MA, McDonald MF, Barrable WM, Soos JG, Gourlay W, et al. The living anonymous kidney donor: lunatic or saint? Am J Transplant 2003;3(2):203 13. 120. Mueller PS, Case EJ, Hook CC. Responding to offers of altruistic living unrelated kidney donation by group associations: an ethical analysis. Transplant Rev (Orlando) 2008;22(3):200 5. 121. Costello KL, Murillo AP. “I want your kidney!” Information seeking, sharing, and disclosure when soliciting a kidney donor online. Patient Educ Couns 2014;94(3):423 6. 122. Williams ME. Internet organ solicitation, explained. Adv Chronic Kidney Dis 2006;13(1):70 5. 123. Neidich EM, Neidich AB, Cooper JT, Bramstedt KA. The ethical complexities of online organ solicitation via donor-patient websites: avoiding the “beauty contest”. Am J Transplant 2012;12(1):43 7. 124. Hanto DW. Ethical challenges posed by the solicitation of deceased and living organ donors. N Engl J Med 2007;356(10):1062 6. 125. Jowsey SG, Schneekloth TD. Psychosocial factors in living organ donation: clinical and ethical challenges. Transplant Rev (Orlando) 2008;22(3):192 5. 126. Rodrigue JR, Pavlakis M, Danovitch GM, Johnson SR, Karp SJ, Khwaja K, et al. Evaluating living kidney donors: relationship types, psychosocial criteria, and consent processes at US transplant programs. Am J Transplant 2007;7(10):2326 32. 127. Truog RD. The ethics of organ donation by living donors. N Engl J Med 2005;353(5):444 6. 128. Glazier AK, Sasjack S. Should it be illicit to solicit? A legal analysis of policy options to regulate solicitation of organs for transplant. Health Matrix Clevel 2007;17(1):63 99. 129. Spital A. Evolution of attitudes at U.S. transplant centers toward kidney donation by friends and altruistic strangers. Transplantation 2000;69(8):1728 31. 130. Biller-Andorno N, Agich GJ, Doepkens K, Schauenburg H. Who shall be allowed to give? Living organ donors and the concept of autonomy. Theor Med Bioeth 2001;22(4):351 68. 131. Dew MA, Jacobs CL, Jowsey SG, Hanto R, Miller C, Delmonico FL, et al. Guidelines for the psychosocial evaluation of living unrelated kidney donors in the United States. Am J Transplant 2007;7(5):1047 54. 132. Matas AJ, Hippen B, Satel S. In defense of a regulated system of compensation for living donation. Curr Opin Organ Transplant 2008;13 (4):379 85. 133. Matas AJ, Schnitzler M. Payment for living donor (vendor) kidneys: a cost-effectiveness analysis. Am J Transplant 2004;4(2):216 21. 134. Lavee J, Ashkenazi T, Stoler A, Cohen J, Beyar R. Preliminary marked increase in the national organ donation rate in Israel following implementation of a new organ transplantation law. Am J Transplant 2013;13(3):780 5. 135. Fallahzadeh MK, Jafari L, Roozbeh J, Singh N, Shokouh-Amiri H, Behzadi S, et al. Comparison of health status and quality of life of related versus paid unrelated living kidney donors. Am J Transplant 2013;13(12):3210 14. 136. Delmonico FL, Martin D, Dominguez-Gil B, Muller E, Jha V, Levin A, et al. Living and deceased organ donation should be financially neutral acts. Am J Transplant 2015;15(5):1187 91. 137. Jha V, Chugh KS. The case against a regulated system of living kidney sales. Nat Clin Pract Nephrol 2006;2(9):466 7. 138. Fisher JS, Butt Z, Friedewald J, Fry-Revere S, Hanneman J, Henderson ML, et al. Between Scylla and Charybdis: charting an ethical course for research into financial incentives for living kidney donation. Am J Transplant 2015;15(5):1180 6. 139. Sharif A, Alachkar N, Kraus E. Incompatible kidney transplantation: a brief overview of the past, present and future. QJM 2012;105(12):1141 50. 140. Gloor JM, Winters JL, Cornell LD, Fix LA, DeGoey SR, Knauer RM, et al. Baseline donor-specific antibody levels and outcomes in positive crossmatch kidney transplantation. Am J Transplant 2010;10(3):582 9. 141. Montgomery RA. Renal transplantation across HLA and ABO antibody barriers: integrating paired donation into desensitization protocols. Am J Transplant 2010;10(3):449 57. 142. Flint SM, Walker RG, Hogan C, Haeusler MN, Robertson A, Francis DM, et al. Successful ABO-incompatible kidney transplantation with antibody removal and standard immunosuppression. Am J Transplant 2011;11(5):1016 24. 143. Fuchinoue S, Ishii Y, Sawada T, Murakami T, Iwadoh K, Sannomiya A, et al. The 5-year outcome of ABO-incompatible kidney transplantation with rituximab induction. Transplantation 2011;91(8):853 7. 144. Genberg H, Kumlien G, Wennberg L, Berg U, Tyden G. ABO-incompatible kidney transplantation using antigen-specific immunoadsorption and rituximab: a 3-year follow-up. Transplantation 2008;85(12):1745 54. 145. Montgomery JR, Berger JC, Warren DS, James NT, Montgomery RA, Segev DL. Outcomes of ABO-incompatible kidney transplantation in the United States. Transplantation 2012;93(6):603 9. 146. Okumi M, Toki D, Nozaki T, Shimizu T, Shirakawa H, Omoto K, et al. ABO-incompatible living kidney transplants: evolution of outcomes and immunosuppressive management. Am J Transplant 2015;9(3):567 77. 147. Haririan A, Nogueira J, Kukuruga D, Schweitzer E, Hess J, Gurk-Turner C, et al. Positive cross-match living donor kidney transplantation: longer-term outcomes. Am J Transplant 2009;9(3):536 42. 148. Vo AA, Peng A, Toyoda M, Kahwaji J, Cao K, Lai CH, et al. Use of intravenous immune globulin and rituximab for desensitization of highly HLA-sensitized patients awaiting kidney transplantation. Transplantation 2010;89(9):1095 102. 149. Montgomery RA, Lonze BE, King KE, Kraus ES, Kucirka LM, Locke JE, et al. Desensitization in HLA-incompatible kidney recipients and survival. N Engl J Med 2011;365(4):318 26. 150. Baek CH, Yang WS, Park KS, Han DJ, Park JB, Park SK. Infectious risks and optimal strength of maintenance immunosuppressants in rituximab-treated kidney transplantation. Nephron Extra 2012;2(1):66 75. 151. Kahwaji J, Sinha A, Toyoda M, Ge S, Reinsmoen N, Cao K, et al. Infectious complications in kidney-transplant recipients desensitized with rituximab and intravenous immunoglobulin. Clin J Am Soc Nephrol 2011;6(12):2894 900.

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152. Garonzik Wang JM, Montgomery RA, Kucirka LM, Berger JC, Warren DS, Segev DL. Incompatible live-donor kidney transplantation in the United States: results of a national survey. Clin J Am Soc Nephrol 2011;6(8):2041 6. 153. Wallis CB, Samy KP, Roth AE, Rees MA. Kidney paired donation. Nephrol Dial Transplant 2011;26(7):2091 9. 154. Ashlagi I, Roth AE. Free riding and participation in large scale, multi-hospital kidney exchange. Theoretical Economics 2014;9(3):7. 155. Cole E, Malik S. Foundations and principles of the Canadian living donor paired exchange program. Can J Kidney Health Disease 2014;1(6):7. 156. de Klerk M, Keizer KM, Claas FH, Witvliet M, Haase-Kromwijk BJ, Weimar W. The Dutch national living donor kidney exchange program. Am J Transplant 2005;5(9):2302 5. 157. Delmonico FL, Morrissey PE, Lipkowitz GS, Stoff JS, Himmelfarb J, Harmon W, et al. Donor kidney exchanges. Am J Transplant 2004;4 (10):1628 34. 158. Ferrari P, Fidler S, Wright J, Woodroffe C, Slater P, Van Althuis-Jones A, et al. Virtual crossmatch approach to maximize matching in paired kidney donation. Am J Transplant 2011;11(2):272 8. 159. Johnson RJ, Allen JE, Fuggle SV, Bradley JA, Rudge C, Kidney Advisory Group UKTN. Early experience of paired living kidney donation in the United kingdom. Transplantation 2008;86(12):1672 7. 160. Park K, Lee JH, Huh KH, Kim SI, Kim YS. Exchange living-donor kidney transplantation: diminution of donor organ shortage. Transplant Proc 2004;36(10):2949 51. 161. Zarsadias P, Monaco A, Morrissey P. A pioneering paired kidney exchange. Student BMJ 2010;18:c1562. 162. An Act to amend the National Organ Transplant Act, S. 301, The Senate of the United States, 110th Congress, 1st Session Sess (2007). 163. Rees MA, Bargnesi D, Samy K, Reece L. Altruistic donation through the Alliance for Paired Donation. Clin Transpl 2009:235 46. 164. United S. National Organ Transplant Act: Public Law 98-507. US Statut Large 1984;98:2339 48. 165. Transplant: Transplant Year by Donor Relation (1988 2015) [Internet]. U.S. Department of Health and Human Resources. 2015 [cited 11/12/2015]. Available from: http://optn.transplant.hrsa.gov/converge/latestData/rptData.asp. 166. Roth AE, Sonmez T, Unver MU. Kidney Exchange. Quart J Econ 2004;119(2):32. 167. Roth AE, Sonmez T, Unver MU, Delmonico FL, Saidman SL. Utilizing list exchange and nondirected donation through ‘chain’ paired kidney donations. Am J Transplant 2006;6(11):2694 705. 168. Saidman SL, Roth AE, Sonmez T, Unver MU, Delmonico FL. Increasing the opportunity of live kidney donation by matching for twoand three-way exchanges. Transplantation 2006;81(5):773 82. 169. Rees MA, Kopke JE, Pelletier RP, Segev DL, Rutter ME, Fabrega AJ, et al. A nonsimultaneous, extended, altruistic-donor chain. N Engl J Med 2009;360(11):1096 101. 170. Montgomery RA, Gentry SE, Marks WH, Warren DS, Hiller J, Houp J, et al. Domino paired kidney donation: a strategy to make best use of live non-directed donation. Lancet 2006;368(9533):419 21. 171. Ashlagi I, Gilchrist DS, Roth AE, Rees M. NEAD Chains in Transplantation. Am J Transpl 2011;11(12):2. 172. Gentry SE, Segev DL, Montgomery RA. A comparison of populations served by kidney paired donation and list paired donation. Am J Transplant 2005;5(8):1914 21. 173. Melcher ML, Veale JL, Javaid B, Leeser DB, Davis CL, Hil G, et al. Kidney transplant chains amplify benefit of nondirected donors. JAMA Surg 2013;148(2):165 9. 174. Ross LF. The ethical limits in expanding living donor transplantation. Kennedy Inst Ethics J 2006;16(2):151 72. 175. Woodle ES, Daller JA, Aeder M, Shapiro R, Sandholm T, Casingal V, et al. Ethical considerations for participation of nondirected living donors in kidney exchange programs. Am J Transplant 2010;10(6):1460 7. 176. Ross LF, Rubin DT, Siegler M, Josephson MA, Thistlethwaite Jr. JR, Woodle ES. Ethics of a paired-kidney-exchange program. N Engl J Med 1997;336(24):1752 5. 177. Ross LF, Zenios S. Practical and ethical challenges to paired exchange programs. Am J Transplant 2004;4(10):1553 4. 178. Melcher ML, Blosser CD, Baxter-Lowe LA, Delmonico FL, Gentry SE, Leishman R, et al. Dynamic challenges inhibiting optimal adoption of kidney paired donation: findings of a consensus conference. Am J Transplant 2013;13(4):851 60. 179. Fumo DE, Kapoor V, Reece LJ, Stepkowski SM, Kopke JE, Rees SE, et al. Historical matching strategies in kidney paired donation: the 7year evolution of a web-based virtual matching system. Am J Transplant 2015;15(10):2533 786. 180. Mast DA, Vaughan W, Busque S, Veale JL, Roberts JP, Straube BM, et al. Managing finances of shipping living donor kidneys for donor exchanges. Am J Transplant 2011;11(9):1810 14. 181. Veale J, Hil G. National Kidney Registry: 213 transplants in three years. Clin Transpl 2010;333 44. 182. Montgomery RA, Katznelson S, Bry WI, Zachary AA, Houp J, Hiller JM, et al. Successful three-way kidney paired donation with crosscountry live donor allograft transport. Am J Transplant 2008;8(10):2163 8. 183. Segev DL, Veale JL, Berger JC, Hiller JM, Hanto RL, Leeser DB, et al. Transporting live donor kidneys for kidney paired donation: initial national results. Am J Transplant 2011;11(2):356 60. 184. Simpkins CE, Montgomery RA, Hawxby AM, Locke JE, Gentry SE, Warren DS, et al. Cold ischemia time and allograft outcomes in live donor renal transplantation: is live donor organ transport feasible? Am J Transplant 2007;7(1):99 107. 185. Clark E, Hanto R, Rodrigue JR. Barriers to implementing protocols for kidney paired donation and desensitization: survey of U.S. transplant programs. Prog Transplant 2010;20(4):357 65. 186. Massie AB, Gentry SE, Montgomery RA, Bingaman AA, Segev DL. Center-level utilization of kidney paired donation. Am J Transplant 2013;13(5):1317 22. 187. Bingaman AW, Wright FH, Murphey CL. Kidney paired donation in live-donor kidney transplantation. N Engl J Med 2010;363 (11):1091 2. 188. Segev DL, Powe NR, Troll MU, Wang NY, Montgomery RA, Boulware LE. Willingness of the United States general public to participate in kidney paired donation. Clin Transplant 2012;26(5):714 21. 189. Gentry SE, Segev DL, Simmerling M, Montgomery RA. Expanding kidney paired donation through participation by compatible pairs. Am J Transplant 2007;7(10):2361 70. 190. Roth AE, Unver MU, Sonmez T. A Kidney Exchange Clearinghouse in New England. Am Econ Rev Papers Prec 2005;95(2):5.

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191. Cuffy MC, Ratner LE, Siegler M, Woodle ES. Equipoise: ethical, scientific, and clinical trial design considerations for compatible pair participation in kidney exchange programs. Am J Transplant 2015;15(6):1484 9. 192. Bingaman AW, Wright Jr. FH, Kapturczak M, Shen L, Vick S, Murphey CL. Single-center kidney paired donation: the Methodist San Antonio experience. Am J Transplant 2012;12(8):2125 32. 193. Kranenburg LW, Zuidema W, Weimar W, Passchier J, Hilhorst M, de Klerk M, et al. One donor, two transplants: willingness to participate in altruistically unbalanced exchange donation. Transpl Int 2006;19(12):995 9. 194. Ratner LE, Rana A, Ratner ER, Ernst V, Kelly J, Kornfeld D, et al. The altruistic unbalanced paired kidney exchange: proof of concept and survey of potential donor and recipient attitudes. Transplantation 2010;89(1):15 22. 195. Hanto RL, Reitsma W, Delmonico FL. The development of a successful multiregional kidney paired donation program. Transplantation 2008;86(12):1744 8. 196. Roth AE, Sonmez T, Unver MU. Kidney paired donation with compatible pairs. Am J Transplant 2008;8(2):463. 197. gentry SE, Rees MA, Roth AE. Consensus kidney paired donation matching statement. Proposal for a National Kidney Paired Donation Program. Kidney Transplantation Committee, 2006 12/18/2006. Report No. 198. Liu W, Treat E, Veale JL, Milner J, Melcher ML. Identifying opportunities to increase the throughput of kidney paired donation. Transplantation 2015;99(7):1410 15. 199. Rees MA, Schnitzler MA, Zavala EY, Cutler JA, Roth AE, Irwin FD, et al. Call to develop a standard acquisition charge model for kidney paired donation. Am J Transplant 2012;12(6):1392 7. 200. Cole EH, Nickerson P, Campbell P, Yetzer K, Lahaie N, Zaltzman J, et al. The Canadian Kidney Paired Donation Program: a national program to increase living donor transplantation. Transplantation 2014;22(1):45 6. 201. Kute VB, Vanikar AV, Shah PR, Gumber MR, Patel HV, Modi PR, et al. Facilitators to national kidney paired donation program. Transpl Int 2013;26(5):e38 9. 202. Ferrari P, de Klerk M. Paired kidney donations to expand the living donor pool. J Nephrol 2009;22(6):699 707. 203. de Klerk M, Witvliet MD, Haase-Kromwijk BJ, Claas FH, Weimar W. A highly efficient living donor kidney exchange program for both blood type and crossmatch incompatible donor-recipient combinations. Transplantation 2006;82(12):1616 20. 204. Ashlagi I, Gilchrist DS, Roth AE, Rees MA. Nonsimultaneous chains and dominos in kidney- paired donation-revisited. Am J Transplant 2011;11(5):984 94. 205. Gentry SE, Montgomery RA, Swihart BJ, Segev DL. The roles of dominos and nonsimultaneous chains in kidney paired donation. Am J Transplant 2009;9(6):1330 6. 206. Montgomery RA. Living donor exchange programs: theory and practice. Br Med Bull 2011;98:21 30. 207. Veale J, Hil G. The National Kidney Registry: 175 transplants in one year. Clin Transpl 2011;255 78.

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C H A P T E R

7 Kidney Preservation Ina Jochmans1, John M. O’Callaghan2, Rutger J. Ploeg2 and Jacques Pirenne1 1

University Hospitals Leuven, Leuven, Belgium 2University of Oxford, Oxford, United Kingdom

7.1 PRINCIPLES OF KIDNEY PRESERVATION 7.1.1 Oxygen Deprivation As soon as circulation to the donor kidney diminishes and stops, cells become oxygen and nutrient deprived. A detrimental cascade of processes is started that ends in cellular injury and death. 1. Anaerobic metabolism is activated to maintain adenosine triphosphate (ATP) dependent membrane pumps that ensure intracellular ion composition (mainly of Na1, K1 and Ca21). ATP stores rapidly deplete since substrates for ATP production are not being delivered. 2. As membrane pumps fail, Na1 is no longer excreted and the intracellular environment becomes hyperosmolar. H2O enters the cell and causes cell swelling. 3. ATP-dependent Ca21 transport stops and Ca21 accumulates, both in the cytoplasm and the mitochondria. This cytosolic calcium will activate a large number of enzymes such as phospholipases, proteases, ATPases and endonucleases, leading to destruction of cellular integrity. The calcium overload also plays a role in the opening of the mitochondrial permeability transition pore after circulation is restored, which is a critical event in the progression of cell death.1,2 4. Lactate produced during anaerobic metabolism leads to an acidotic intracellular environment. Severe acidosis activates phospholipases and proteases, causing lysosomal damage and cell death.3 5. Reactive oxygen species (ROS) are formed when cells are deprived of oxygen and the stage is set for increased oxidative damage at the time or reperfusion.4,5 Reoxygenation will increase ROS production and the ROScoping mechanisms of the cell are flooded. ROS damage all components of the cell, including proteins, lipids, and DNA, leading to cell injury and death. From this, it is evident that kidneys need to be protected during the prolonged ischemic phase between retrieval and transplantation. One method of kidney preservation would be to sustain oxygen and nutrient supply in a near-physiological environment. In 1813, Julien-Jean-Cesar Le Gallois said, “if one could replace for the heart some kind of injection of artificial blood, either natural or artificially made . . . one could succeed easily in maintaining alive indefinitely any part of the body.”6 Although normothermic organ preservation is no longer science fiction (see Chapter 8: Ex-vivo Normothermic Perfusion in Renal Transplantation), intentional cooling of the kidney to 0 4 C is still the key factor for successful preservation.

7.1.2 Hypothermia Cooling the kidney by 10 C halves the metabolic rate, and cooling to 4 C reduces metabolism to about 5% 8% of the rate at normothermic temperatures. Cooling effectively increases organ viability. This is often referred to as the “temperature effect.” Simple cooling of dog kidneys with ice water sustained kidney viability and allowed successful transplantation after 12 hours.7 However, there are deleterious effects of rapid cooling itself Kidney Transplantation, Bioengineering, and Regeneration. DOI: http://dx.doi.org/10.1016/B978-0-12-801734-0.00007-2

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TABLE 7.1 Composition of the Most Commonly Used Preservation Solutions for Static Cold Storage SCS solutions EC UW

HOC

HTK

Celsior

IGL-1

HMP solution Belzer MPS

Electrolytes (mM) Na

10

30

84

15

100

120

100

K

115

120

84

9

15

25

25

Lactobionate Raffinose Hydroxyethyl starch

Citrate Mannitol Mannitol

Lactobionate Mannitol

Lactobionate Raffinose Polyethylene glycol

Glucose Gluconate Mannitol Ribose Hydroxyethyl starch

Citrate

Histidine

Histidine

Phosphate

Phosphate

Glutathione Adenosine

Adenine

Impermeant Glucose

Buffer

Phosphate Phosphate

ROS scavenger

Glutathione Allopurinol

Tryptophan

Nutrients

Adenosine

Ketoglutarate Glutamate

Belzer MPS, Belzer machine perfusion solution; EC, Euro-Collins; HMP, hypothermic machine perfusion; HOC, hyperosmolar citrate (also called Marshall’s solution); HTK, histidine-tryptophan-ketoglutarate; IGL-1, Institut Georges Lopez-1; SCS, static cold storage; UW, University of Wisconsin solution.

that superimpose on those of ischemia. Membranes lose their selective permeability as the phospholipid layer undergoes changes.8 Furthermore, membrane pumps are also inhibited by the cold, resulting in increased Na1 and Ca21 concentrations and several detrimental enzymes are released into the cytoplasm, causing cell injury and death.8

7.1.3 Preservation Solution As cooling can only slow down the detrimental effects of oxygen deprivation, and indeed some are even enhanced by the cold state, additional protection is needed during preservation. Preservation solutions were designed for this purpose. To effectively preserve, a cold preservation solution needs to contain: 1. A balanced electrolyte composition (Na1 and K1). There are in general two types of solutions, those with an intracellular electrolyte composition (i.e., high K1, low Na1) and those with an extracellular composition (i.e., low K1, high Na1). K1-rich solutions may result in a K1-overload upon organ reperfusion in the recipient. Furthermore, K1 can cause vasoconstriction making K1-rich solutions less desirable for use with machine perfusion preservation. 2. Impermeants to counteract cell swelling. These are saccharides (glucose, mannitol, sucrose, raffinose), anions (citrate, gluconate, lactobionate), and colloids (hydroxyethyl starch, polyethylene glycol). These large molecules remain in the interstitial space after equilibration with the perfusion solution, increasing osmotic pressure and as such counteracting cell swelling by diminishing the number of water molecules that enter the cell. 3. An adequate pH-buffer to prevent acidosis. Anaerobic metabolism continues in the cold and will result in acidosis. Mild acidosis (pH 6.9 7.0) inhibits lactate formation and as such offers a little cellular protection. But profound acidosis destabilizes the lysosomes causing cell death.3 The most commonly used buffers in preservation solutions are phosphate and histidine. 4. Antioxidants These may neutralize ROS such as allopurinol, glutathione, tryptophan. Table 7.1 summarizes the composition of the most commonly used preservation solutions.

7.2 STATIC COLD STORAGE Static cold storage (SCS) consists of a washout of the kidney with a cold preservation solution, first in situ and then a second rinse ex situ, after which the kidney is submerged in the solution and packed on ice. SCS is a simple, affordable preservation technique that allows unsupervised shipment of kidneys by any form of transport. It is the most prevalent preservation method in use today. Successful SCS hinges on the preservation solution used. Collins’ solution, introduced in 1969, was the first to be used.9 It was Euro-Collins’ (EC)—a modified Collins’ solution with omission of magnesium—that was implemented by the international organ sharing organization Eurotransplant in I. KIDNEY TRANSPLANTATION

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the 1970s.10 Shortly thereafter, hyperosmolar citrate solution (HOC), currently mainly used in Australia and the United Kingdom, was introduced by Ross and Marshall.11 In the late 1980s, Belzer and Southard developed the University of Wisconsin (UW) solution, containing hydroxyethyl starch and antioxidants.12 In Germany, Bretschneider presented histidine-tryptophan-ketoglutarate (HTK) for cardioplegia during cardiac surgery.13 Experimental work showed that HTK, with its powerful buffering capacity, could also be used for organ preservation.14 Institut George Lopez-1 (IGL-1) solution is a recent preservation solution that has built on UW, the major difference being an extracellular electrolyte composition and the use of polyethylene glycol as impermeant.15,16

7.2.1 Are All Static Cold Storage Preservation Solutions Equal? A number of randomized controlled trials (RCTs) have been performed over the years comparing the different SCS solutions for kidney preservation and these have been discussed in a recent systematic review.17 The use of both UW and HTK results in significantly lower delayed graft function (DGF) rates when compared to EC.18 There does not seem to be a difference in the incidence of DGF between the use of UW and HTK, as long as large volumes (10 15 L) of HTK are used and cold ischemic times are not prolonged.19 21 There are limited data with respect to graft survival and acute rejection rates, however both are likely influenced by the incidence of DGF.22 HOC has not been compared with either UW or HTK in a randomized controlled way. When choosing a SCS solution it is important to keep in mind that most of the time other (abdominal) organs will be retrieved from the same donor. In this light, analyses performed by the European Liver Transplant Registry show that preservation of the liver with HTK is an independent factor of graft loss.23

7.2.2 Is Static Cold Storage Too Simple? SCS has been the most prevalent form of kidney preservation ever since SCS preservation solutions became available. Around that time a turning point in the donor profile was also reached. The concept of brain death was penetrating the transplantation community and radically changed the process of organ donation. Donor death was no longer diagnosed on circulatory criteria (donation after circulatory death: DCD) but on neurological criteria (donation after brain death: DBD).24,25 The typical donor was young and suffered major trauma that led to brain death. The pristine kidneys from these donors could tolerate the detrimental effects of brain death and the following cold ischemic time in SCS quite well. However, since then our deceased donor profile has significantly changed again and continues to change. Median donor age has increased considerably. In the Eurotransplant region median donor age for kidneys has increased from 36 years in 1990 to 53 years in 2013 (Fig. 7.1).26 As increasing age correlates with comorbidities such as arterial hypertension, cardiovascular accidents, etc. we see that expanded criteria donor (ECD) kidneys are increasingly used.27 Furthermore, DCDs— both controlled and uncontrolled DCDs—have returned to clinical practice and are now an important contributor to the deceased donor pool in a number of countries, such as the United States, United Kingdom, the Netherlands, Belgium, France, and Spain.28 32 Not only has the use of DCDs increased exponentially, these donors are also becoming older, combining risk factors for decreased graft function. In Belgium, the Netherlands, and the United Kingdom, 60% of DCD donors are now older than 50 years, and 40% are older than 60 years.26,33 These higher-risk kidneys—ECD and DCD—are particularly susceptible to preservation-induced injury, DGF, and primary nonfunction (PNF) and can experience reduced long-term graft survival. SCS of higher-risk kidneys seems to have reached its limits and improved preservation is needed.

7.3 MACHINE PERFUSION PRESERVATION Hypothermic machine perfusion (HMP) was the first kidney preservation technique to be used clinically, before the availability of cold storage solutions. The pioneering work of Belzer in the 1960s, showing that cryoprecipitated plasma could be used to perfuse canine kidneys, led to the first successful human HMP-kidney transplant in 1968.34 37 HMP was commonly used, predominantly in the United States during the 1970s, but was quickly replaced by SCS after preservation solutions were shown to adequately preserve kidney grafts. It appeared that the use of large and nontransportable HMP units was no longer necessary. Nevertheless, Belzer continued his work on HMP and developed a synthetic HMP solution replacing his previous human albumin-containing solution. Belzer MPS could preserve canine kidneys for up to 5 days and is still the standard HMP solution today (Table 7.1).12,38 I. KIDNEY TRANSPLANTATION

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FIGURE 7.1 Median donor age of deceased donor kidneys in Eurotransplant between 1995 and 2013. Graph constructed using Eurotransplant data.26

FIGURE 7.2 Currently available hypothermic machine perfusion devices for clinical kidney preservation. Panel A: RM3 (Waters Medical systems, Birmingham, Alabama, USA), www.wtrs.com; panel B: LifePort Kidney Transporter (Organ Recovery Systems, Itasca, Illinois, USA), www.organ-recovery.com; panel C: Kidney Assist (Organ Assist, Groningen, the Netherlands); www.organ-assist.nl.

Thanks to technological advances, user-friendly, portable HMP devices are now commercially available (Fig. 7.2). In parallel with the changing donor profile, the use of HMP is increasing (Fig. 7.3).

7.3.1 Does Hypothermic Machine Perfusion Provide Better Preservation Than Static Cold Storage? Relatively few well-conducted RCTs have compared HMP to SCS. A meta-analysis of all 16 studies39 57 prospectively comparing HMP with SCS between 1971 and 2001 showed that HMP is associated with a relative risk of DGF of 0.80 (0.67 0.96) compared to SCS.58,59 No effect of HMP on 1-year graft survival was detected, however all studies were severely underpowered with respect to the likely impact on graft survival.58 Furthermore,

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FIGURE 7.3 Percentage of deceased donor kidneys preserved by hypothermic machine perfusion in the United States of America each year. Graph constructed using OPTN data.145

the evidence spreads out over decades during which SCS solutions, donor profile, outcome measures etc. have changed considerably. 7.3.1.1 Hypothermic Machine Perfusion in Deceased Donor Kidneys In 2009, the report of an international RCT in Eurotransplant comparing HMP to SCS of kidneys from all deceased donor types (standard criteria donors (SCD), ECD, controlled DCD) was published.60 DGF occurred in 21% of HMP-kidneys versus 27% in SCS-kidneys. Logistic regression showed that HMP reduced the risk of DGF (adjusted odds ratio (AOR) 0.57 (0.36 0.88)). If DGF developed, its duration was 3 days shorter after HMP (10 days vs 13 days, P 5 .04). PNF occurred in 2% of HMP-kidneys and in 5% of SCS-kidneys (P 5 .08). Graft survival was also improved by HMP (94% vs 90%, P 5 .04). Cox regression analysis showed that HMP reduced the risk of graft failure in the first year after transplantation (adjusted hazard ratio (AHR) 0.52 (0.29 0.93)). The 3-year follow-up data of this trial confirmed the improved graft survival of HMP-kidneys (91% vs 87%; AHR 0.60 (0.37 0.97)) (Fig. 7.4).61 7.3.1.2 Hypothermic Machine Perfusion of Expanded Criteria Donor Kidneys The beneficial effect of HMP might be more pronounced in ECD kidneys.27,62 Multivariate regression of the 91 randomised ECD kidney pairs in the Eurotransplant HMP trial showed that HMP reduced the risk of DGF compared with SCS (AOR 0.46; (0.21 0.99)). PNF was also lower in HMP compared to SCS-kidneys (3% vs 12%, P 5 .04).63 One-year graft survival was higher after HMP (92% vs 80%, P 5 .02) with an AHR of 0.35 (0.15 0.86). The 3-year graft survival advantage after HMP was maintained for ECD kidneys (86% vs 76%, AHR 0.38; (0.18 0.80) (Fig. 7.4).61 The presence or absence of DGF seems to have an impressive effect on graft survival, especially in SCS-ECD-kidneys. If DGF occurred in ECD kidneys that had undergone HMP, the 1-year graft survival was 10% lower than HMP-ECD-kidneys with immediate function, although this difference was not significant (94% vs 85%, P 5 .16). However, in SCS-kidneys that developed DGF, graft survival was significantly worse compared to when the graft functioned immediately (41% vs 97%, P , .0001). If only recipients of grafts that developed DGF were analyzed, there was a significant difference in 1-year graft survival between HMP and SCSkidneys (85% vs 41%, P 5 .003) (Fig. 7.4). 7.3.1.3 Hypothermic Machine Perfusion of Kidneys Donated After Circulatory Death Previous studies have suggested that HMP of DCD kidneys results in better early graft function and improved graft survival compared to SCS. However, other studies do not support this conclusion.39,64 67 A comprehensive meta-analysis failed to show a significant risk reduction of DGF in HMP-DCD-kidneys.58,59 Although the Eurotransplant HMP trial included DCD donors, 88% of inclusions were DBD kidneys. In a separate randomised extension of the Eurotransplant HMP trial, data from 82 Maastricht III DCD kidney pairs were analyzed.68 HMP

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FIGURE 7.4 Three-year graft survival of deceased donor kidneys included in the Eurotransplant HMP trial. Panel A shows graft survival in 672 kidney recipients, with a hazard ratio for graft failure in the machine-perfusion group of 0.60 (95% confidence interval, 0.37 0.97; P 5 .04). Panel B shows the post hoc analysis of a subgroup of 588 recipients of kidneys donated after brain death, with data split according to whether delayed graft function developed in the recipient. Delayed graft function was defined as the need for dialysis in the first week after transplantation. From Moers, C., Pirenne, J., Paul, A., & Ploeg, R.J. (2012). Machine perfusion or cold storage in deceased-donor kidney transplantation. New England Journal of Medicine, 366(8), 770 771 Copyright r (2012) Massachusetts Medical Society. Reprinted with permission.

reduced the risk of DGF (AOR 0.43 (0.20 0.89)). Of HMP-DCD-kidneys, 54% developed DGF compared to 70% of SCS-DCD-kidneys (P 5 .007). PNF occurred in two cases in each study arm. HMP did not result in an increased 1-year or 3-year graft survival (94% for HMP-kidneys vs 95% for SCS-kidneys). Although equal outcome might seem contradictory, this finding is in line with an increasing number of reports showing similar medium-term graft survival for DCD and DBD kidneys despite higher rates of DGF in DCD kidneys.31,69 72 An RCT analyzing outcome of 45 Maastricht III DCD kidney pairs in the UK showed no difference in DGF between HMP and SCS (58% vs 56%; P 5 .99; AOR 1.14 (0.38 3.49)).73 One kidney in the HMP group suffered from PNF. One-year graft survival was similar in both groups (93% in the HMP group and 98% in the SCS group, P 5 .3). The contradicting results between the two trials, currently the largest RCTs performed and the best available evidence, might be related to the setting in which HMP is used. In the Eurotransplant HMP trial, kidneys were placed on the HMP device at the donor center, immediately after retrieval, whereas in the UK HMP trial, those kidneys removed away from the transplant center were cold stored during transfer, after which HMP was started. As such, it could be that HMP needs to be used in a continuous setting to achieve a benefit. An ongoing RCT in the United Kingdom is duplicating the set-up of the Eurotransplant HMP trial with continuous HMP being compared to SCS (ISRCTN 50082383). 7.3.1.4 Meta-Analyses A number of meta-analyses have recently been published reviewing the evidence of HMP and SCS in kidney transplantation. Two studies analyzed data from all donor types and reported a decreased risk for DGF with HMP (AORs of 0.83 (0.72 0.96)74 and 0.81 (0.71 0.98)18). Two meta-analyses analyzed data from DCD kidneys and found a protective effect of HMP on DGF (AORs of 0.56 (0.36 0.86)75 and 0.64 (0.43 0.95)76). There is one meta-analysis that combined the data of HMP in ECD kidneys which also showed a protective effect of HMP on DGF (AOR 0.59 (0.54 0.66)77). Overall, even with the meta-analysis of prospective data, numbers have not been large enough to identify a benefit of HMP on outcomes that are relatively rare, such as PNF and 1-year graft loss. In studies that have individually identified a difference, this has largely been in ECDs.

7.3.2 Is Machine Perfusion Cost-Effective? Meta-analyses assessing the cost-effectiveness of HMP have not drawn any conclusions because of a lack of properly powered studies. Published economic evidence at the time was of poor quality and not based on randomized studies.58,78 An economic evaluation of the Eurotransplant HMP trial data combined the 1-year results based on the empirical data from the study with a Markov model with a 10-year time horizon.79 Short-term

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evaluation showed that HMP results in lower average costs in the first year posttransplant when compared to SCS and across all deceased donor types but mostly apparent in the ECD group, related to the increased 1-year graft survival of pumped ECD kidneys. The costs of dialysis and readmission were mainly responsible. The longterm analyses showed a similar benefit; the Markov model revealed cost savings of $86,750 per life-year gained in favor of HMP. The corresponding incremental cost-utility ratio was minus $496,223 per quality-adjusted lifeyear gained.

7.3.3 Does Timing or Duration of Hypothermic Machine Perfusion Matter? It is well known that renal vascular resistance (RR) falls during HMP, whilst perfusate flow increases.80,81 This has prompted the question of whether or not there is a required minimum duration of perfusion for any potential benefit of HMP to be evident. Recently published experimental studies have found that between 1 and 4 hours of HMP before reperfusion with blood could reduce RR and improve creatinine clearance compared to kidneys preserved by SCS alone.82,83 Then again, other studies have shown that despite reaching mean perfusate flows after 2 hours of perfusion, RR values continue to improve at 6 hours.80 As it is frequently necessary to transport organs many miles between donor and recipient, a beneficial effect of a short period of end-ischemic HMP sounds attractive and avoids the logistics of transporting machines. Nevertheless, there is some suggestion that when following long periods of SCS, relatively short periods of HMP (less than 4 hours) have either reduced or no benefit compared to continuous HMP throughout.84 Clinical trials that have shown significant benefits with HMP have used continuous HMP with the kidney being pumped for many hours, on average the majority of the total cold ischemic time (median 11 15 hours).60,63,68,85 Recent large registry analyses (over 90,000 kidneys) have shown that in the case of SCD kidneys, HMP reduces the risk of DGF compared to SCS regardless of a very short or very long cold ischemic time.86 In the same study, the risk of DGF was reduced for ECD kidneys only if the total cold ischemic time was greater than 6 hours, and in the case of DCD only if 6 24 hours.86 These discrepancies may be a function of the numbers available for power but if not, have important consequences. Furthermore, there is currently no good evidence to suggest that HMP allows for lengthening of cold ischemic times. As total cold ischemic time is a well-established predictor of DGF, a balance between minimizing cold ischemic time and any potential benefit of HMP is required.87 It seems that just a few hours of HMP, following SCS, can have a positive impact on early graft function compared to SCS alone as long as the total cold ischemic time is not extended. Currently an RCT comparing end-ischemic HMP with SCS in DBD kidneys is ongoing (ISRCTN 35082773). However, to the best of our knowledge, there are no clinical trials comparing continuous with end-ischemic HMP.

7.3.4 Does Machine Perfusion Predict Graft Viability and Quality? HMP allows the study of perfusion characteristics such as RR and perfusate biomarkers that might provide an assessment of kidney graft viability and quality before transplantation. 7.3.4.1 The Value of Renal Vascular Resistance Retrospective evidence suggests that RR and flow rate during HMP correlate with kidney graft function.58 However, in most of these studies, a selection bias was introduced because kidneys were systematically discarded based on arbitrarily defined parameter thresholds. Today, “poor” perfusion dynamics are still frequently used to discard kidneys even though their true prognostic value on graft outcome had never been studied until recently. Indeed, more than 15% of HMP-kidneys are discarded annually in the United States, partly based on elevated RR.88 In the Eurotransplant HMP trial, the preservation method was not revealed at the time of organ offer and HMP clinicians had no knowledge of the RR value. The decision to accept a given kidney was based solely on traditional donor data, making it possible to elucidate the true prognostic value of RR on PNF, DGF, and 1-year graft survival.89 Analysis showed that RR at the end of HMP was an independent risk factor for the development of DGF, independent of donor type (AOR 38.1 (1.56 934)). However, the predictive power of RR was low (AUC of the ROC curve 0.58). This means that, despite the association of RR and DGF, RR has a limited value in the prediction of DGF for a specific donor-recipient pair. RR was also a risk factor for 1-year graft failure but again with low predictive power (AHR 12.3 (1.11 136.9)). A retrospective study on RR in DCD kidneys showed that RR at the start of HMP is a risk factor for both DGF (AOR 2.34 (1.11 4.96)) and PNF (AOR 2.04 (1.36 3.06)) but again the predictive power was only moderate at best (0.61).90 Available evidence indicates that kidneys should not be discarded based on RR criteria alone.

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7.3.4.2 The Value of Perfusate Injury Biomarkers Retrospective data, systematically reviewed recently,91 also suggest that biomarker concentrations in the perfusate of HMP-kidneys correlate with graft outcome. The groups of Newcastle and Maastricht have previously determined the perfusate concentration of total glutathione-S-transferase (GST), heart-type fatty acid binding protein (H-FABP), and alanine-aminopeptidase and found higher concentrations in perfusate of discarded kidneys.92 94 Furthermore, lactate dehydrogenase (LDH) and GST concentrations correlated with warm ischemia time, and GST and H-FABP were higher in Maastricht II compared to Maastricht III DCD kidneys. However, these data are confronted with the same methodological design and selection bias as the RR data, and none of the previous studies investigated whether perfusate biomarkers were independently associated with graft outcome. In prospectively collected perfusate of the Eurotransplant HMP trial, GST, N-acetyl-β-D-glucosaminidase, and H-FABP were found to be independent predictors of DGF, but not of PNF or graft survival.95 The predictive power of these three biomarkers was also moderate at best (0.67, 0.64, and 0.64, respectively). A retrospective study in DCD kidneys showed that LDH and IL-18 perfusate concentrations were associated with PNF (AOR 1.001 (1.000 1.002) for both markers) but that the predictive power was poor (0.62 and 0.59).96 Thus, similar to RR, perfusate biomarkers alone should not lead to kidney discard. As there are multiple donor, preservation, and recipient factors influencing graft outcome, it is not surprising that RR and individual perfusate biomarkers are not sufficient on their own to be used as sole predictors of outcome. However, given the fact that they do independently correlate with DGF (RR, biomarkers) and with graft survival (RR), they are valuable in assisting clinicians in decision-making. Perhaps that implementation of these markers in existing quality scores for DGF and graft survival will increase their predictive power.97 99 Emerging technologies such as proteomics and metabolomics open new doors to investigating viability and quality assessment during HMP.

7.3.5 Can Hypothermic Machine Perfusion Be Used for Active Repair of the Injured Kidney? The capabilities of HMP to repair damaged organs and its use as a method to deliver a variety of therapies have been tested in several ways. The particular strength of this method is the ability to provide local effects without systemic exposure or uptake of drugs, cells, etc. 7.3.5.1 Reconditioning One potential use of HMP is in the reconditioning of organs prior to transplantation. Any mechanistic effect that HMP has on the kidney graft is not well understood, other than the drop in RR that is clearly evident.81 As a downstream effect there is some evidence from experimental kidney transplant studies that biomarkers of injury and of ischemia reperfusion injury are reduced by HMP.100 102 Kidneys preserved by HMP in these studies had improved tubular and renal cell function, and less protein excretion after reperfusion.100 There is some evidence that HMP improves ATP recovery, reduces perforin expression and proapoptotic signals.103 Potentially the upregulation of HIF-1α and altered expression of caspase proteins may play a protective role.104 The preserved expression of flow dependent genes might also be important.105 7.3.5.2 An Improved Preservation Solution Currently, Belzer’s HMP solution is used to perfuse kidneys. It is a gluconate-based perfusate that contains hydroxyethyl starch and, contrary to the SCS preservation solution, has a low K1 concentration to avoid vasoconstriction (Table 7.1). The constitution of the perfusion solution is likely to play an important role in the outcome of the graft after transplantation. Keeping in mind the mechanisms of ischemia reperfusion injury, it is possible that improved solutions or the addition of specific reagents targeting these mechanisms to the preservation solution could be developed. New solutions that resemble cell culture media (AQIX RS-I, Lifor) show potential.106,107 They contain amino acids, metabolic substrates, vitamins, salts, and organic buffers that make them ideal potential new solutions for HMP. 7.3.5.3 Drug Delivery HMP has proven to be a useful way to deliver potentially protective therapies to the kidney during preservation without systemic administration. Administration in this way permits adequate penetration of vascular compartments without altering perfusion dynamics and allows assessment of the degree of uptake during perfusion.108 So far experience with this method of delivery has included heparin conjugates that adhere to

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vascular endothelium.108 A water-soluble preparation of the drug Propofol has also been added to HMP with improved early graft function.109 These studies demonstrate the potential to administer therapies in this way; however, clinical results are yet to be reported. An ongoing clinical study aims to administer the inhibitor of complement, Mirococept, during HMP, which would act locally and not affect systemic complement activation (ISRCTN49958194). There are several chemical compounds including hydrogen sulfide, carbon monoxide, and nitrous oxide, which have antiapoptotic and vasodilatory effects when applied in experimental conditions.110 These could be applied during HMP. In experimental models of ischemia reperfusion injury, hydrogen sulfide administration during ischemia results in reduced TNF-alpha and IL-2 levels and neutrophil invasion with improved microvascular circulation and subsequently improved renal function.111 113 7.3.5.4 Gene-Therapy The transfection of genes targeted at inhibiting harmful pathways or stimulating protective ones is also a potential adjunct to HMP. Delivering gene-therapy during perfusion would mean that the vector (usually a virus) does not have to be administered systemically to the patient. Several gene-therapy targets have been identified which may improve ischemia reperfusion injury in transplanted kidneys.114 Potential targets to reduce acute rejection include genes that up-regulate IL-4, IL-10, IL-13, Fas-ligand or blockades of costimulatory molecules.114 Tolerogenic strategies and stimulation of regeneration are also potential avenues for gene-therapy that are now being explored.114 This technique is in the very early stages of investigation and so far there is limited evidence that renal cells can be transfected in the cold and during normothermic perfusion.115,116 7.3.5.5 Stem Cells HMP may allow the direct administration of mesenchymal stem cells (MSC) to the kidney during preservation.117 In a murine model of acute kidney failure the administration of MSC improved recovery from ischemia reperfusion injury and subsequent early kidney function.118 In a ground-breaking clinical study of living-related kidney transplants, the administration of MSC, at reperfusion and 2 weeks later, resulted in lower rates of acute rejection, less infection, and improved graft function at 1 year compared to IL-2 receptor antagonists.119 Subsequent studies have assessed the potential role of donor-derived MSC in a regimen to reduce immune suppression, administered to the recipient.120 Pretransplant administration of autologous MSC has also been tested in very small series, with some advantages over posttransplant administration.121 Further research is required to assess HMP as a potential method to deliver MSC directly to the organ, bypassing the difficulty of trafficking cells to the desired site. So far these studies have administered MSC under normothermic conditions only.

7.3.6 How Can Hypothermic Machine Perfusion Be Improved? 7.3.6.1 Effect of Temperature Traditionally both static storage and machine perfusion of kidney grafts has been done at ice-cold temperatures in order to slow down metabolism as much as possible. Machine preservation at temperatures below normothermia, but above ice-cold temperatures, has been investigated (subnormothermia) but the mechanism of action is not fully understood and the results so far in kidney transplantation are experimental only. A recent animal study in a DCD model found that kidneys stored by machine perfusion at 20 C had improved creatinine clearance compared to both SCS and oxygenated HMP.122 Given the higher metabolic demands at higher temperatures, it seems that provision for oxygenation and higher perfusate flow need to be made.122 Another experimental model of kidney preservation used “room temperature” perfusion with Lifor as perfusion solution. This type of preservation was associated with improved perfusate flow and reduced renal resistance during perfusion.123 Preservation at temperatures just below normal (30 C) with AQIX RS-I has been tested for short periods (2 hours).124 The same fluid was also tested as a potential subnormothermic flush before SCS in experimental studies.125 More work has been done with liver models of subnormothermic preservation at temperatures as diverse as 7 21 C, however the information available is still limited in its scope and applicability to clinical practice.126 Maintaining a kidney graft at temperatures closer to normothermia comes with ongoing metabolism at a faster pace that must be supported with oxygen, nutrients, and clearance of metabolites, making it a much more complex technique compared to HMP.127 The use of normothermic machine perfusion has also been extensively investigated over the past decade and has recently been introduced in the clinics.127 133 An in-depth review on normothermic machine perfusion of kidneys is given in Chapter 8, Ex-vivo Normothermic Perfusion in Renal Transplantation.

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7.3.6.2 The Addition of Oxygen Although the rational of using oxygen to sustain metabolic cell processes makes perfect sense, it is quite paradoxical that the majority of clinically applied preservation methods nowadays do not use oxygen. However, with the availability of high-tech perfusion platforms, the deteriorating quality of donor kidneys and the need to “repair” these higher-risk kidneys prior to transplantation, oxygenation is finding its way into transplant research. Increasing evidence suggests that kidneys preserved by HMP will consume oxygen, and that the level of oxygen consumption is correlated with postpreservation glomerular filtration rate.134 In that sense, adding oxygen to the preservation solution would be beneficial, especially when it comes to restoring cellular levels of ATP after the kidney has been exposed to ischemia.135 On the other hand, the presence of oxygen could potentially increase the production of radical oxygen species, thereby causing increased injury.136 The delicate balance between active oxygenation and antioxidant properties of the preservation solution will no doubt need to be taken into account. Oxygen can be delivered in a number of ways: by simple retrograde persufflation of oxygen directly through the renal vein (without necessarily using machine perfusion); during machine perfusion where oxygen can be dissolved into the perfusate; or by adding artificial oxygen carriers (e.g., acellular solution based of perfluorocarbons by Brasile et al.137 139, Hemo2Life140). The most recent studies have used technology in which a membrane oxygenator complements a more typical HMP device. There are animal auto-transplant studies published which have found indications for some benefit of oxygen provision, particularly if following a prolonged warm ischemic time.141 The major benefit seen so far is in the early kidney graft function; the study was unfortunately too small to comment on graft survival. A similar study however did not find such a stark difference in renal function when following a DBD model, with no first warm ischemic time.142 Other recent studies have compared oxygenated HMP against SCS, rather than HMP, again showing some improvement in early graft function.143,144 The use of oxygen during kidney preservation has recently been reviewed by Hosgood et al.135 As there is no clear clinical evidence to support widespread use of HMP with supplemental oxygenation, clinical trials with new technology are required. Ongoing trials of oxygenated HMP are investigating the benefit of end-ischemic oxygenated HMP versus SCS in ECD kidneys (ISRCTN63852508) and continuous oxygenated HMP versus HMP in DCD kidneys (ISRCTN32967929).

7.4 CONCLUSION SCS of the kidney in appropriate cold storage preservation solutions is still the most prevalent form of kidney preservation today. SCS is easy and affordable and can be used worldwide with acceptable results. However, with the increasing use of higher-risk kidneys we are pushing the boundaries of SCS. Technological advances, increased insight into ischemia reperfusion injury, the need to evaluate kidney quality and viability before transplantation, and the wish to repair injured organs has opened the field of machine perfusion preservation once more. For now, machine perfusion is mostly used in its hypothermic form. There is good evidence showing a reduction in DGF and survival benefit in ECD when kidneys are preserved by HMP compared to SCS. Numbers have not been large enough to identify a clear benefit of HMP on outcomes that are relatively rare, such as PNF and 1-year graft loss, despite meta-analysis of available data. Furthermore, more consistent good data are needed that focus on the benefit of HMP on graft function, including 6 and 12 months glomerular filtration rate. It appears that there is a minimum time the kidney needs to be pumped to benefit from HMP but we do not know the exact number of hours required. Other remaining questions are whether pumping should be continuous or whether a short period of (oxygenated) HMP before transplantation (end-ischemic HMP) is enough, and evidence has to be gained that HMP can be used to prolong cold ischemia. Increasing evidence suggests a benefit of oxygenated HMP, however this remains to be confirmed in clinical trials. It is also still unclear whether (sub)normothermic temperatures could improve outcomes. HMP provides additional insight on viability and quality of the kidney, however none of these data should be used as stand-alone tools to decide whether to accept or discard kidneys. Contrary to SCS, HMP offers the opportunity to repair the kidney by reconditioning, adding several drugs acting against ischemia reperfusion injury, or stem cells and this is an important challenge to be explored in preclinical and clinical trials. To date, we see a number of emerging techniques allowing in vivo and ex-vivo normothermic perfusion of organs showing good initial outcome after transplantation. In the coming years we will have to unravel which type of donor kidney will benefit most from what kind of combination of SCS, hypothermic and/or normothermic reperfusion technique—with or without active repair—to result in both optimal kidney function and longer graft survival.

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Watson PF, Morris GJ. Cold shock injury in animal cells. Symp Soc Exp Biol 1987;41:311 40. 9. Collins GM, Bravo-Shugarman M, Terasaki PI. Kidney preservation for transportation. Initial perfusion and 30 hours’ ice storage. Lancet 1969;2(7632):1219 22. 10. Eurotransplant International Foundation. Annual Report 1976. Leiden: Eurotransplant; 1976. 11. Ross H, Marshall VC, Escott ML. 72-hr canine kidney preservation without continuous perfusion. Transplantation 1976;21(6):498 501. 12. Hoffmann RM, Southard JH, Lutz M, Mackety A, Belzer FO. Synthetic perfusate for kidney preservation. Its use in 72-hour preservation of dog kidneys. Arch Surg 1983;118(8):919 21. 13. Bretschneider HJ. Myocardial protection. Thorac Cardiovasc Surg 1980;28(5):295 302. 14. Holscher M, Groenewoud AF. Current status of the HTK solution of Bretschneider in organ preservation. Transplant Proc 1991;23 (5):2334 7. 15. Codas R, Petruzzo P, Morelon E, et al. IGL-1 solution in kidney transplantation: first multi-center study. Clin Transplant 2009;23 (3):337 42. 16. Dondero F, Paugam-Burtz C, Danjou F, Stocco J, Durand F, Belghiti J. A randomized study comparing IGL-1 to the University of Wisconsin preservation solution in liver transplantation. Ann Transplant 2010;15(4):7 14. 17. O’Callaghan JM, Knight SR, Morgan RD, Morris PJ. Preservation solutions for static cold storage of kidney allografts: a systematic review and meta-analysis. Am J Transplant 2012;12(4):896 906. 18. O’Callaghan JM, Morgan RD, Knight SR, Morris PJ. Systematic review and meta-analysis of hypothermic machine perfusion versus static cold storage of kidney allografts on transplant outcomes. Br J Surg 2013;100(8):991 1001. 19. de Boer J, De Meester J, Smits JM, et al. Eurotransplant randomized multicenter kidney graft preservation study comparing HTK with UW and Euro-Collins. Transplant Int 1999;12(6):447 53. 20. Roels L, Coosemans W, Donck J, et al. Inferior outcome of cadaveric kidneys preserved for more than 24 hr in histidine-tryptophanketoglutarate solution. Leuven Collaborative Group for Transplantation. Transplantation 1998;66(12):1660 4. 21. Klaus F, Castro DB, Bittar CM, et al. Kidney transplantation with Belzer or Custodiol solution: a randomized prospective study. Transplant Proc 2007;39(2):353 4. 22. Pascual M, Theruvath T, Kawai T, Tolkoff-Rubin N, Cosimi AB. Strategies to improve long-term outcomes after renal transplantation. N Engl J Med 2002;346(8):580 90. 23. Adam R, Delvart V, Karam V, et al. Compared efficacy of preservation solutions in liver transplantation: a long-term graft outcome study from the European Liver Transplant Registry. Am J Transplant 2015;15(2):395 406. 24. Beecher H. A definition of irreversible coma. Report of the Ad Hoc Committee of the Harvard Medical School to Examine the Definition of Brain Death. JAMA 1968;205(6):337 40. 25. Selby R, Selby MT. Status of the legal definition of death. Neurosurgery 1979;5(4):535 40. 26. Eurotransplant. www.eurotransplant.org. 2015. 27. Metzger RA, Delmonico FL, Feng S, Port FK, Wynn JJ, Merion RM. Expanded criteria donors for kidney transplantation. Am J Transplant 2003;3(Suppl. 4):114 25. 28. Kootstra G, Daemen JH, Oomen AP. Categories of non-heart-beating donors. Transplant Proc 1995;27(5):2893 4. 29. Morrissey PE, Monaco AP. Donation after circulatory death: current practices, ongoing challenges, and potential improvements. Transplantation 2014;97(3):258 64. 30. Jochmans I, Darius T, Kuypers DR, et al. Kidney donation after circulatory death in a country with a high number of brain dead donors: 10-year experience in Belgium. Transplant Int 2012;25(8):857 66. 31. Summers DM, Watson CJ, Pettigrew GJ, et al. Kidney donation after circulatory death (DCD): state of the art. Kidney Int 2015;2:241 9. 32. Dominguez-Gil B, Haase-Kromwijk B, Van Leiden H, et al. Current situation of donation after circulatory death in European countries. Transplant Int 2011;24(7):676 86. 33. NHS Blood and Transplant: Transplant activity in the UK. 2013. http://www.uktransplant.org.uk/ukt/statistics 34. Belzer FO, Park HY, Vetto RM. Factors influencing renal blood flow during isolated perfusion. Surg Forum 1964;15:222 4. 35. Belzer FO, Ashby BS, Dunphy JE. 24-hour and 72-hour preservation of canine kidneys. Lancet 1967;2(7515):536 8. 36. Belzer FO, Ashby BS, Huang JS, Dunphy JE. Etiology of rising perfusion pressure in isolated organ perfusion. Ann Surg 1968;168(3):382 91. 37. Belzer FO, Ashby BS, Gulyassy PF, Powell M. Successful seventeen-hour preservation and transplantation of human-cadaver kidney. N Engl J Med 1968;278(11):608 10. 38. McAnulty JF, Ploeg RJ, Southard JH, Belzer FO. Successful five-day perfusion preservation of the canine kidney. Transplantation 1989; 47(1):37 41. 39. van der Vliet JA, Kievit JK, Hene RJ, Hilbrands LB, Kootstra G. Preservation of non-heart-beating donor kidneys: a clinical prospective randomised case-control study of machine perfusion versus cold storage. Transplant Proc 2001;33(1 2):847. 40. Kosieradzki M, Danielewicz R, Kwiatkowski A, et al. Rejection rate and incidence of acute tubular necrosis after pulsatile perfusion preservation. Transplant Proc 1999;31(1 2):278 9.

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41. Gage F, Ali M, Alijani MR, et al. Comparison of static versus pulsatile preservation of matched-paired kidneys. Transplant Proc 1997;29(8): 3644 5. 42. Veller MG, Botha JR, Britz RS, et al. Renal allograft preservation: a comparison of University of Wisconsin solution and of hypothermic continuous pulsatile perfusion. Clin Transplant 1994;8(2 Pt 1):97 100. 43. Matsuno N, Sakurai E, Tamaki I, Uchiyama M, Kozaki K, Kozaki M. The effect of machine perfusion preservation versus cold storage on the function of kidneys from non-heart-beating donors. Transplantation 1994;57(2):293 4. 44. Matsuno N, Kozaki M, Sakurai E, et al. Effect of combination in situ cooling and machine perfusion preservation on non-heart-beating donor kidney procurement. Transplant Proc 1993;25(1 Pt 2):1516 17. 45. Matsuno N, Sakurai E, Uchiyama M, Kozaki K, Tamaki I, Kozaki M. Use of in situ cooling and machine perfusion preservation for nonheart-beating donors. Transplant Proc 1993;25(6):3095 6. 46. Merion RM, Oh HK, Port FK, Toledo-Pereyra LH, Turcotte JG. A prospective controlled trial of cold-storage versus machine-perfusion preservation in cadaveric renal transplantation. Transplantation 1990;50(2):230 3. 47. Jaffers GJ, Banowsky LH. The absence of a deleterious effect of mechanical kidney preservation in the era of cyclosporine. Transplantation 1989;47(4):734 6. 48. Mendez R, Mendez RG, Koussa N, Cats S, Bogaard TP, Khetan U. Preservation effect on oligo-anuria in the cyclosporine era: a prospective trial with 26 paired cadaveric renal allografts. Transplant Proc 1987;19(1 Pt 3):2047 50. 49. Halloran P, Aprile M. A randomized prospective trial of cold storage versus pulsatile perfusion for cadaver kidney preservation. Transplantation 1987;43(6):827 32. 50. Heil JE, Canafax DM, Sutherland DE, Simmons RL, Dunning M, Najarian JS. A controlled comparison of kidney preservation by two methods: machine perfusion and cold storage. Transplant Proc 1987;19(1 Pt 3):2046. 51. Alijani MR, Cutler JA, DelValle CJ, et al. Single-donor cold storage versus machine perfusion in cadaver kidney preservation. Transplantation 1985;40(6):659 61. 52. Marshall VC. Renal preservation prior to transplantation. Transplantation 1980;30(3):165 6. 53. Toledo-Pereyra LH. Renal hypothermic storage with a new hyperosmolar colloid solution. Boletin de la Asociacion Medica de Puerto Rico 1983;75(8):347 50. 54. Beck TA. Machine versus cold storage preservation and TAN versus the energy charge as a predictor of graft function posttransplantation. Transplant Proc 1979;11(1):459 64. 55. Marshall VC, Ross H, Scott DF, McInnes S, Thomson N, Atkins RC. Preservation of cadaveric renal allografts-comparison of flushing and pumping techniques. Proc Eur Dial Transplant Assoc 1977;14:302 9. 56. Marshall VC. Problems in clinical organ preservation. Med J Aust 1977;2(11):361 4. 57. Sterling WA, Pierce JC, Hutcher NE, Lee HM, Hume DM. A comparison of hypothermic preservation with hypothermic pulsatile perfusion in paired human kidneys. Surg Forum 1971;22:229 30. 58. Wight J, Chilcott J, Holmes M, Brewer N. The clinical and cost-effectiveness of pulsatile machine perfusion versus cold storage of kidneys for transplantation retrieved from heart-beating and non-heart-beating donors. Health Technol Assess 2003;7(25):1 94. 59. Wight JP, Chilcott JB, Holmes MW, Brewer N. Pulsatile machine perfusion vs. cold storage of kidneys for transplantation: a rapid and systematic review. Clin Transplant 2003;17(4):293 307. 60. Moers C, Smits JM, Maathuis MH, et al. Machine perfusion or cold storage in deceased-donor kidney transplantation. N Engl J Med 2009;360(1):7 19. 61. Moers C, Pirenne J, Paul A, Ploeg RJ. Machine perfusion or cold storage in deceased-donor kidney transplantation. N Engl J Med 2012; 366(8):770 1. 62. Polyak MM, Arrington BO, Stubenbord WT, et al. The influence of pulsatile preservation on renal transplantation in the 1990s. Transplantation 2000;69(2):249 58. 63. Treckmann J, Moers C, Smits JM, et al. Machine perfusion versus cold storage for preservation of kidneys from expanded criteria donors after brain death. Transplant Int 2011;24(6):548 54. 64. Plata-Munoz JJ, Muthusamy A, Quiroga I, et al. Impact of pulsatile perfusion on postoperative outcome of kidneys from controlled donors after cardiac death. Transplant Int 2008;21(9):899 907. 65. Moustafellos P, Hadjianastassiou V, Roy D, et al. The influence of pulsatile preservation in kidney transplantation from non-heart-beating donors. Transplant Proc 2007;39(5):1323 5. 66. St Peter SD, Imber CJ, Friend PJ. Liver and kidney preservation by perfusion. Lancet 2002;359(9306):604 13. 67. Opelz G, Terasaki PI. Advantage of cold storage over machine perfusion for preservation of cadaver kidneys. Transplantation 1982;33(1):64 8. 68. Jochmans I, Moers C, Smits JM, et al. Machine perfusion versus cold storage for the preservation of kidneys donated after cardiac death: a multicenter randomized controlled trial. Ann Surg 2010;252(5):756 64. 69. Kokkinos C, Antcliffe D, Nanidis T, Darzi AW, Tekkis P, Papalois V. Outcome of kidney transplantation from nonheart-beating versus heart-beating cadaveric donors. Transplantation 2007;83(9):1193 9. 70. Brook NR, Waller JR, Nicholson ML. Nonheart-beating kidney donation: current practice and future developments. Kidney Int 2003;63(4): 1516 29. 71. Kootstra G, van Heurn E. Non-heartbeating donation of kidneys for transplantation. Nat Clin Pract Nephrol 2007;3(3):154 63. 72. Summers DM, Johnson RJ, Hudson A, Collett D, Watson CJ, Bradley JA. Effect of donor age and cold storage time on outcome in recipients of kidneys donated after circulatory death in the UK: a cohort study. Lancet 2012. 73. Watson CJE, Wells AC, Roberts RJ, et al. Cold machine perfusion versus static cold storage of kidneys donated after cardiac death: a UK multicenter randomized controlled trial. Am J Transplant 2010;10:1991 9. 74. Lam VW, Laurence JM, Richardson AJ, Pleass HC, Allen RD. Hypothermic machine perfusion in deceased donor kidney transplantation: a systematic review. J Surg Res 2013;180(1):176 82. 75. Deng R, Gu G, Wang D, et al. Machine perfusion versus cold storage of kidneys derived from donation after cardiac death: a metaanalysis. PLoS One 2013;8(3):e56368. 76. Bathini V, McGregor T, McAlister VC, Luke PP, Sener A. Renal perfusion pump vs cold storage for donation after cardiac death kidneys: a systematic review. J Urol 2013;189(6):2214 20.

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77. Jiao B, Liu S, Liu H, Cheng D, Cheng Y, Liu Y. Hypothermic machine perfusion reduces delayed graft function and improves one-year graft survival of kidneys from expanded criteria donors: a meta-analysis. PLoS One 2013;8(12):e81826. 78. Bond M, Pitt M, Akoh J, Moxham T, Hoyle M, Anderson R. The effectiveness and cost-effectiveness of methods of storing donated kidneys from deceased donors: a systematic review and economic model. Health Technol Assess 2009;13(38):1 156 iii iv, xi xiv 79. Groen H, Moers C, Smits JM, et al. Cost-effectiveness of hypothermic machine preservation versus static cold storage in renal transplantation. Am J Transplant 2012;12(7):1824 30. 80. Patel SK, Pankewycz OG, Nader ND, Zachariah M, Kohli R, Laftavi MR. Prognostic utility of hypothermic machine perfusion in deceased donor renal transplantation. Transplant Proc 2012;44(7):2207 12. 81. Jochmans I, Moers C, Smits JM, et al. The prognostic value of renal resistance during hypothermic machine perfusion of deceased donor kidneys. Am J Transplant 2011;11(10):2214 20. 82. Gallinat A, Paul A, Efferz P, et al. Hypothermic reconditioning of porcine kidney grafts by short-term preimplantation machine perfusion. Transplantation 2012;93(8):787 93. 83. Gallinat A, Efferz P, Paul A, Minor T. One or 4 h of “in-house” reconditioning by machine perfusion after cold storage improve reperfusion parameters in porcine kidneys. Transplant Int 2014;27(11):1214 19. 84. Hosgood SA, Mohamed IH, Bagul A, Nicholson ML. Hypothermic machine perfusion after static cold storage does not improve the preservation condition in an experimental porcine kidney model. Br J Surg 2011;98(7):943 50. 85. Gallinat A, Moers C, Treckmann J, et al. Machine perfusion versus cold storage for the preservation of kidneys from donors ./ 5 65 years allocated in the Eurotransplant Senior Programme. Nephrol Dial Transplant 2012;27(12):4458 63. 86. Gill J, Dong J, Eng M, Landsberg D, Gill JS. Pulsatile perfusion reduces the risk of delayed graft function in deceased donor kidney transplants, irrespective of donor type and cold ischemic time. Transplantation 2014;97(6):668 74. 87. Opelz G, Dohler B. Multicenter analysis of kidney preservation. Transplantation 2007;83(3):247 53. 88. Sung RS, Christensen LL, Leichtman AB, et al. Determinants of discard of expanded criteria donor kidneys: impact of biopsy and machine perfusion. Am J Transplant 2008;8(4):783 92. 89. Jochmans I, Moers C, Smits JM, et al. The prognostic value of renal resistance during hypothermic machine perfusion of deceased donor kidneys. Am J Transplant 2011;11:2214 20. 90. de Vries EE, Hoogland ER, Winkens B, Snoeijs MG, van Heurn LW. Renovascular resistance of machine-perfused DCD kidneys is associated with primary nonfunction. Am J Transplant 2011;11(12):2685 91. 91. Bhangoo RS, Hall IE, Reese PP, Parikh CR. Deceased-donor kidney perfusate and urine biomarkers for kidney allograft outcomes: a systematic review. Nephrol Dial Transplant 2012;27(8):3305 14. 92. Gok MA, Pelsers M, Glatz JF, et al. Use of two biomarkers of renal ischemia to assess machine-perfused non-heart-beating donor kidneys. Clin Chem 2003;49(1):172 5. 93. Gok MA, Pelzers M, Glatz JF, et al. Do tissue damage biomarkers used to assess machine-perfused NHBD kidneys predict long-term renal function post-transplant?. Clin Chim Acta 2003;338(1 2):33 43. 94. Daemen JW, Oomen AP, Janssen MA, et al. Glutathione-S-transferase as predictor of functional outcome in transplantation of machinepreserved non-heart-beating donor kidneys. Transplantation 1997;63(1):89 93. 95. Moers C, Varnav OC, van Heurn E, et al. The value of machine perfusion perfusate biomarkers for predicting kidney transplant outcome. Transplantation 2010;90(9):966 73. 96. Hoogland ER, de Vries EE, Christiaans MH, Winkens B, Snoeijs MG, van Heurn LW. The value of machine perfusion biomarker concentration in DCD kidney transplantations. Transplantation 2013;95(4):603 10. 97. Irish WD, Ilsley JN, Schnitzler MA, Feng S, Brennan DC. A risk prediction model for delayed graft function in the current era of deceased donor renal transplantation. Am J Transplant 2010;10:2279 86. 98. Irish WD, McCollum DA, Tesi RJ, et al. Nomogram for predicting the likelihood of delayed graft function in adult cadaveric renal transplant recipients. J Am Soc Nephrol 2003;14(11):2967 74. 99. Rao PS, Schaubel DE, Guidinger MK, et al. A comprehensive risk quantification score for deceased donor kidneys: the kidney donor risk index. Transplantation 2009;88(2):231 6. 100. Hosgood SA, Yang B, Bagul A, Mohamed IH, Nicholson ML. A comparison of hypothermic machine perfusion versus static cold storage in an experimental model of renal ischemia reperfusion injury. Transplantation 2010;89:830 7. 101. Vaziri N, Thuillier R, Favreau FD, et al. Analysis of machine perfusion benefits in kidney grafts: a preclinical study. J Transl Med 2011;9:15. 102. Codas R, Thuillier R, Hauet T, Badet L. Renoprotective effect of pulsatile perfusion machine RM3: pathophysiological and kidney injury biomarker characterization in a preclinical model of autotransplanted pig. BJU Int 2012;109(1):141 7. 103. La Manna G, Conte D, Cappuccilli ML, et al. An in vivo autotransplant model of renal preservation: cold storage versus machine perfusion in the prevention of ischemia/reperfusion injury. Artif Organs 2009;33(7):565 70. 104. Jani A, Zimmerman M, Martin J, et al. Perfusion storage reduces apoptosis in a porcine kidney model of donation after cardiac death. Transplantation 2011;91(2):169 75. 105. Gracia-Sancho J, Villarreal Jr. G, Zhang Y, et al. Flow cessation triggers endothelial dysfunction during organ cold storage conditions: strategies for pharmacologic intervention. Transplantation 2010;90(2):142 9. 106. Kay MD, Hosgood SA, Harper SJ, Bagul A, Waller HL, Nicholson ML. Normothermic versus hypothermic ex vivo flush using a novel phosphate-free preservation solution (AQIX) in porcine kidneys. J Surg Res 2011;171:275 82. 107. Gage F, Leeser DB, Porterfield NK, et al. Room temperature pulsatile perfusion of renal allografts with Lifor compared with hypothermic machine pump solution. Transplant Proc 2009;41(9):3571 4. 108. Sedigh A, Larsson R, Brannstrom J, et al. Modifying the vessel walls in porcine kidneys during machine perfusion. J Surg Res 2014; 191(2):455 62. 109. Snoeijs MG, Vaahtera L, de Vries EE, et al. Addition of a water-soluble propofol formulation to preservation solution in experimental kidney transplantation. Transplantation 2011;92(3):296 302. 110. Snijder PM, de Boer RA, Bos EM, et al. Gaseous hydrogen sulfide protects against myocardial ischemia-reperfusion injury in mice partially independent from hypometabolism. PLoS One 2013;8(5):e63291.

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111. Hunter JP, Hosgood SA, Patel M, Rose R, Read K, Nicholson ML. Effects of hydrogen sulphide in an experimental model of renal ischaemia-reperfusion injury. Br J Surg 2012;99(12):1665 71. 112. Zhu JX, Kalbfleisch M, Yang YX, et al. Detrimental effects of prolonged warm renal ischaemia-reperfusion injury are abrogated by supplemental hydrogen sulphide: an analysis using real-time intravital microscopy and polymerase chain reaction. BJU Int 2012;110 (11 Pt C):E1218 27. 113. Hosgood SA, Nicholson ML. Hydrogen sulphide ameliorates ischaemia-reperfusion injury in an experimental model of non-heartbeating donor kidney transplantation. Br J Surg 2010;97(2):202 9. 114. Sandovici M, Deelman LE, de Zeeuw D, van Goor H, Henning RH. Immune modulation and graft protection by gene therapy in kidney transplantation. Eur J Pharmacol 2008;585(2-3):261 9. 115. Yang B, Elias JE, Bloxham M, Nicholson ML. Synthetic small interfering RNA down-regulates caspase-3 and affects apoptosis, IL-1 beta, and viability of porcine proximal tubular cells. J Cell Biochem 2011;112(5):1337 47. 116. Brasile L, Stubenitsky BM, Booster MH, Arenada D, Haisch C, Kootstra G. Transfection and transgene expression in a human kidney during ex vivo warm perfusion. Transplant Proc 2002;34(7):2624. 117. Van Raemdonck D, Neyrinck A, Rega F, Devos T, Pirenne J. Machine perfusion in organ transplantation: a tool for ex-vivo graft conditioning with mesenchymal stem cells?. Curr Opin Organ Transplant 2013;18(1):24 33. 118. Lange C, Togel F, Ittrich H, et al. Administered mesenchymal stem cells enhance recovery from ischemia/reperfusion-induced acute renal failure in rats. Kidney Int 2005;68(4):1613 17. 119. Tan J, Wu W, Xu X, et al. Induction therapy with autologous mesenchymal stem cells in living-related kidney transplants: a randomized controlled trial. JAMA 2012;307(11):1169 77. 120. Peng Y, Ke M, Xu L, et al. Donor-derived mesenchymal stem cells combined with low-dose tacrolimus prevent acute rejection after renal transplantation: a clinical pilot study. Transplantation 2013;95(1):161 8. Available from: http://dx.doi.org/10.1097/TP.0b013e3182754c53 121. Perico N, Casiraghi F, Gotti E, et al. Mesenchymal stromal cells and kidney transplantation: pretransplant infusion protects from graft dysfunction while fostering immunoregulation. Transplant Int 2013;26(9):867 78. 122. Hoyer DP, Gallinat A, Swoboda S, et al. Subnormothermic machine perfusion for preservation of porcine kidneys in a donation after circulatory death model. Transplant Int 2014;27(10):1097 106. 123. Gage F, Leeser DB, Porterfield NK, et al. Room temperature pulsatile perfusion of renal allografts with Lifor compared with hypothermic machine pump solution. Transplant Proc 2009;41(9):3571 4. 124. Kay MD, Hosgood SA, Harper SJF, et al. Static normothermic preservation of renal allografts using a novel nonphosphate buffered preservation solution. Transplant Int 2007;20(1):88 92. 125. Kay MD, Hosgood SA, Harper SJ, Bagul A, Waller HL, Nicholson ML. Normothermic versus hypothermic ex vivo flush using a novel phosphate-free preservation solution (AQIX) in porcine kidneys. J Surg Res 2011;171(1):275 82. 126. Graham JA, Guarrera JV. “Resuscitation” of marginal liver allografts for transplantation with machine perfusion technology. J Hepatol 2014;61(2):418 31. 127. Hosgood SA, van Heurn E, Nicholson ML. Normothermic machine perfusion of the kidney: better conditioning and repair? Transplant Int 2014. 128. Patel M, Hosgood S, Nicholson ML. The effects of arterial pressure during normothermic kidney perfusion. J Surg Res 2014;191(2):463 8. 129. Hosgood SA, Nicholson ML. Ex vivo normothermic perfusion of declined human kidneys after inadequate in situ perfusion. Am J Transplant 2014;14(2):490 1. 130. Nicholson ML, Hosgood SA. Renal transplantation after ex vivo normothermic perfusion: the first clinical study. Am J Transplant 2013; 13(5):1246 52. 131. Hosgood SA, Nicholson ML. Normothermic kidney preservation. Curr Opin Organ Transplant 2011;16(2):169 73. 132. Hosgood SA, Nicholson ML. First in man renal transplantation after ex vivo normothermic perfusion. Transplantation 2011;92(7):735 8. 133. Hosgood SA, Barlow AD, Yates PJ, Snoeijs MG, van Heurn EL, Nicholson ML. A pilot study assessing the feasibility of a short period of normothermic preservation in an experimental model of non-heart beating donor kidneys. J Surg Res 2011;171(1):283 90. 134. Bunegin L, Tolstykh GP, Gelineau JF, Cosimi AB, Anderson LM. Oxygen consumption during oxygenated hypothermic perfusion as a measure of donor organ viability. ASAIO J 2013;59(4):427 32. 135. Hosgood SA, Nicholson HF, Nicholson ML. Oxygenated kidney preservation techniques. Transplantation 2012;93(5):455 9. 136. Fuller BJ, Lee CY. Hypothermic perfusion preservation: the future of organ preservation revisited? Cryobiology 2007;54(2):129 45. 137. Brasile L, Stubenitsky BM, Booster MH, Arenada D, Haisch C, Kootstra G. Hypothermia--a limiting factor in using warm ischemically damaged kidneys. Am J Transplant 2001;1(4):316 20. 138. Brasile L, Stubenitsky BM, Booster MH, et al. Overcoming severe renal ischemia: the role of ex vivo warm perfusion. Transplantation 2002;73(6):897 901. 139. Brasile L, Stubenitsky BM, Haisch CE, Kon M, Kootstra G. Repair of damaged organs in vitro. Am J Transplant 2005;5(2):300 6. 140. Thuillier R, Dutheil D, Trieu MT, et al. Supplementation with a new therapeutic oxygen carrier reduces chronic fibrosis and organ dysfunction in kidney static preservation. Am J Transplant 2011;11(9):1845 60. 141. Thuillier R, Allain G, Celhay O, et al. Benefits of active oxygenation during hypothermic machine perfusion of kidneys in a preclinical model of deceased after cardiac death donors. J Surg Res 2013;184(2):1174 81. 142. Gallinat A, Paul A, Efferz P, et al. Role of oxygenation in hypothermic machine perfusion of kidneys from heart beating donors. Transplantation 2012;94(8):809 13. 143. Maathuis M-HJ, Manekeller S, van der Plaats A, et al. Improved kidney graft function after preservation using a novel hypothermic machine perfusion device. Ann Surgery 2007;246(6):982 8. 144. Doorschodt BM, Schreinemachers MC, Florquin S, et al. Evaluation of a novel system for hypothermic oxygenated pulsatile perfusion preservation. Int J Artif Organs 2009;32(10):728 38. 145. OPTN. 2009 Annual Report of the U.S. Organ Procurement and Transplantation Network and the Scientific Registry of Transplant Recipients: Transplant Data 1999 2008. U.S. Department of Health and Human Services, Health Resources and Services Administration, Healthcare Systems Bureau, Division of Transplantation, Rockville, MD. 2009. http://optn.transplant.hrsa.gov

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C H A P T E R

8 Ex-vivo Normothermic Perfusion in Renal Transplantation Sarah A. Hosgood and Michael L. Nicholson University of Cambridge, Cambridge, United Kingdom

8.1 INTRODUCTION Since the 1970s, organ preservation has relied on the principle of cooling to reduce cellular metabolism and slow the injury processes.1 This allows the organ to be allocated nationally to the most suitable and best immunologically matched recipient. However, the boundaries of kidney transplantation are increasingly being extended and there is more reliance on the use of so-called marginal donors to accommodate the increasing demand.2,3 These kidneys present with higher incidences of early graft dysfunction4 and reduced survival compared to the traditional donor sources.5 The inferior outcome of these kidneys is directly influenced by the hypothermic temperatures during preservation.2,6 The widespread adoption of hypothermic preservation in organ transplantation was based on the early experimental studies by Lapchinsky in the Soviet Union in the 1950s,7 and the work of Carrel and Lindbergh in the 1930s.8
12 They showed that ischemic injury could be minimized by reducing the temperature. In 1963, Calne et al. used the concept of hypothermic temperatures to extend the preservation time and successfully transplanted canine kidneys after 12 hours of storage.13 This led to the universal application of hypothermic preservation in clinical organ transplantation. Hypothermic temperatures act to inhibit the enzymatic processes and prevent injury. There is a two- to threefold decrease in metabolism for every 10 C reduction in temperature.14,15 This slows the degrading processes, but at the same time the anaerobic environment leads to progressive damage. The intracellular pH is lowered, which causes lysosomal instability. The depletion of adenosine triphosphate (ATP) allows the build-up toxic metabolites, adenosine, inosine, and hypoxanthine. Inactivation of the NA1/K1 ATPase pumps allows the accumulation of calcium, sodium, and water within the cell. The damage caused by hypothermic conditions is evident upon reperfusion when normal circulation is restored to the organ.16,17 The downstream effects of ischemia reperfusion (I/R) injury results in the impairment of blood flow to the kidney and reduced urine output after transplantation.18 I/R injury is a major determinant of acute kidney injury (AKI) and delayed graft function (DGF).19,20 This also has important consequences on long-term graft survival. Large data series have shown that the cold ischemic time is an independent risk factor for graft failure.2,21 An alternative technique of preservation is to use normothermic temperatures, to support circulation and metabolism and prevent cellular deterioration.

8.2 THE ORIGIN OF NORMOTHERMIC PERFUSION The concept of organ preservation was first proposed by the French physician, scientist, and philosopher, Julien Jean Cesar le Gallois, in 1813.22 He stated that, “If one could replace for the heart some kind of injection of Kidney Transplantation, Bioengineering, and Regeneration. DOI: http://dx.doi.org/10.1016/B978-0-12-801734-0.00008-4

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artificial blood, either natural or artificially made . . . one could succeed easily in maintaining alive indefinitely any part of the body.” Alexis Carrel, a French surgeon and scientist in the early 1900s, was one of the first to study the effects of tissue preservation at both hypothermic and normothermic temperatures.8
12 In 1912, he won the Nobel Prize for his work on vascular sutures and the experimental transplantation of blood vessels and organs. He discovered that cooling allowed tissues to retain viability for longer periods compared to normal temperatures, and demonstrated the ability to transplant blood vessels after several days when stored in a specially designed solution (Locke’s solution). Nonetheless, he pursued the idea of keeping organs alive and functioning at warmer temperatures. With the help of Charles Lindbergh, an engineer and pilot, he developed the first organ perfusion device, in 1935. The system was capable of maintaining a sterile, pulsating circulation of fluid through a living organ. It was made up of three glass chambers, an organ chamber at the top, a pressure equalizing chamber in the middle, and fluid chamber on the bottom. A mixture of oxygen, carbon dioxide, and nitrogen was pulsated by compressed air into the perfusate through the equalizing chamber and into the organ. They perfused a variety of organs such as the thyroid, suprarenal gland, ovary, spleen, heart, and kidney from adult fowls and cats. The organs were perfused with a nutrient fluid designed to culture the organs which often contained blood, serum, or solutions containing proteinsplit products such as hemin, cysteine, insulin, thyroxine, glutathione, vitamin A, and ascorbic acid. This perfusion system allowed the organs to be studied in an ex vivo environment to acquire knowledge on the metabolic processes and nutrient requirements of individual organs.8
12 Towards the end of his career, his political and radical views were questioned, and his research at this time was disregarded by many. Nonetheless, his research pioneered the development of the extracorporeal perfusion systems that are widely used today in cardiac surgery and indeed for the last decade, to perfuse isolated organs at normothermic temperatures ex vivo.

8.3 TECHNOLOGY In the 1990s, driven by the shortage of donors and increasing use of what was then termed nonheart beating donor kidneys, an interest in normothermic perfusion techniques was renewed. Several groups identified a need for alternative techniques of preservation to allow more marginal organs to be used and to improve their outcome. Brasile et al. published a series of manuscripts detailing an acellular normothermic perfusion system.23
26 The exsanguinous metabolic support (EMS) system was a pressure-controlled device which included an oxygenator and pulsatile pump with controllers to maintain PaO2, PaCO2, pH, and temperature. Kidneys were perfused at a subnormal temperature of 32 C and mean arterial pressure of approximately 35 mmHg with an acellular culture-like medium solution. In 2006, our group (Hosgood & Nicholson) reported the use of an adapted pediatric cardiopulmonary bypass system incorporating a centrifugal pump, a membrane oxygenator, venous reservoir, and heat exchanger. Kidneys were perfused at a near physiological pressure (75 mmHg) and temperature (36 C) with a packed red cell based solution.27
29 A priming solution containing a crystalloid solution with mannitol, an infusion of a vasodilator (prostacyclin), and nutrient solution, provided a stable environment for the kidney. There are other isolated reports of systems that have adapted or modified hypothermic perfusion technology,30 or used custom-made devices with roller pumps and dialysis circuits31,32 to obtain optimal renal function. There is one commercially available system; “The Kidney Assist” made by a Dutch company “Organ Assist.” It is a transportable, oxygenated kidney perfusion system that can be used at hypothermic or normothermic temperatures. It is a pressure-controlled device which includes a rotary pump to deliver a pulsatile flow. A hollow fiber oxygenator is also included in the system to oxygenate the blood based solution. Despite the availability of this system there are no current reports of its application in kidney transplantation.

8.4 PERFUSION CONDITIONS 8.4.1 Artificial Solutions One of the crucial requirements of a normothermic perfusion medium is the inclusion of an oxygen carrier to ensure adequate aerobic metabolism. The first reports by Brasile et al. describing the use of an acellular normothermic solution included a perflourocarbon (PFC) emulsion (Perflubron) as the oxygen carrier.23 It was mixed with an enriched tissue culture-like medium containing essential and nonessential amino acids, lipids, and

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carbohydrates to provide nutritional support. PFCs are inert solutions that have a high capacity for dissolving oxygen.33 However, the first generation PFCs used at this time were relatively unstable solutions with variable half-lives and an inability to be sterilized. When administered intravenously they caused side effects such as anaphylaxis, hypotension, reduced platelet count, and complement activation.33 As a result they were not applied clinically. Historically, other artificial hemoglobin based oxygen carriers have also been used as preservation mediums.34 A stroma-free hemoglobin based solution was an effective oxygen carrier however it was found to cause toxic effects on the kidney. A more stable pyridoxalated hemoglobin-polyoxyethylene solution has proved to be more successful and Brasile et al. went on to replace the PFC in their perfusion medium with this solution.35 New more stable 2nd and 3rd generation PFCs are being developed, mainly as blood substitutes, and there is interest in their use as a kidney preservation medium. In a unique study, Humphreys et al. used a commercially made PFC (Oxygent) to provide oxygenation and reduce ischemic injury.36 Rabbit kidneys were perfused using a retrograde infusion through the urinary collecting system. These new generation PFCs could potentially be developed as normothermic preservation solutions. However, due to the complexity of manufacturing, the cost is considerable, which may limit their adaptation. Other novel solutions such as Lifor, an artificial preservation medium containing a nonprotein oxygen carrier, nutrients, and a growth factor have also been used for preservation at more normothermic temperatures.37 Gage et al. showed that porcine kidneys perfused with Lifor at room temperature had higher flow rates and lower levels of resistance compared to kidneys perfused with UW at room temperature or at 4 C.37 There was no information on the outcome of the kidney, however the higher perfusion flows suggested better preservation of the cellular structure. AQIX-RS-I is a nonphosphate buffered solution designed to support cellular activity at a normothermic temperature.38 The composition of AQIX-RS-1 reflects physiological ionic concentrations, osmolarity, and ion conductivity, to maintain the cell membrane and enzymatic processes. AQIX-RS-1 is capable of carrying oxygen in solution or when mixed with red blood cells. Studies in the heart have shown an advantage compared to a standard cardioplegia solution.39 In the kidney, our group showed that kidneys could be flushed and statically stored for 2 hours at 32 C using AQIX-RS-1 solution and therefore it has potential as a normothermic perfusion medium.38 Hemarina-M101 is a new extracellular hemoglobin derived from a marine invertebrate.40 It has been formulated into an oxygen carrier called Hemoxycarrier. It has a high affinity for oxygen accompanied with an antioxidant activity and has been added to cold storage (CS) solutions with favorable results. It functions over a wide range of temperatures (4
37 C), which makes it attractive as a future normothermic perfusion solution.

8.4.2 Blood Based Solutions Blood based solutions were previously considered to have their limitations with prolonged periods of perfusion causing gross hemolysis, platelet activation, and deterioration in the oxygen carrying capacity of the red blood cells.41 High intrarenal resistance and tissue edema was a significant problem. However, with the development of improved atraumatic centrifugal pumps and equipment used in cardiac bypass surgery, hemolysis is less of a problem. Modern pumps reduce the risk of stress and hemolysis and the membrane oxygenators enable filtration and highly efficient oxygenation.42 They provide a more natural environment and at present are more cost effective as a perfusate compared to the artificial solutions. The use of banked packed red cells has the added advantage of the absence of leukocytes, platelets, and complement, limiting neutrophil infiltration and the inflammatory response in the kidney during perfusion.8

8.4.3 Temperature A variety of temperatures have been used in kidney normothermic systems, ranging from 32 C to normal body temperature. They all appear to have successful outcomes but there have been no direct comparisons between the conditions. Schopp et al. proposed that the abrupt change in temperature from CS to reperfusion could lead to mitochondrial dysfunction and proapoptotic signal transduction.43 In a series of experiments with porcine kidneys they showed that a gradual controlled rewarming preserved renal function and reduced

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postischaemic injury to the mitochondria. This is a technique that could be simply applied during ex vivo normothermic perfusion (EVNP) and warrants further investigation.

8.5 END EX-VIVO NORMOTHERMIC PERFUSION Normothermic perfusion can be carried out for prolonged periods to avoid hypothermic injury. However, logistically this is difficult when kidneys are transported from donor to recipient centers. At present there is little evidence for the necessity to normothermically perfuse kidneys for a prolonged period. Much of the recent work has focused on using normothermic perfusion in conjunction with hypothermic techniques. This simplifies the logistical problems and also appears to be beneficial. A brief period of normothermic perfusion can upregulate protective mechanisms such as heat shock protein 70.44 This aids the repair process and primes the kidney in preparation for reperfusion. A central component of protection is the replenishment of ATP, which prevents the further breakdown of metabolites and restores normal cellular function. Brasile et al. found that although kidneys could be maintained for a significant period of time, up to 48 hours,45 a short period of ex vivo warm perfusion at the end of the preservation period can adequately resuscitate the kidney.46 Our group adopted this principle and demonstrated in porcine kidneys that 2 hours of end EVNP with leukocyte depleted blood could enhance the renal blood flow during reperfusion and reverse some of the detrimental effects of warm and cold ischemic injury.29 The technique was assessed in a porcine autotransplantation model and we found that animals that received a kidney that had been resuscitated using EVNP recovered function sooner than those undergoing hypothermic machine perfusion alone.47 The technique was concluded to be a safe and feasible method of kidney preservation and provided the evidence needed to translate the technique into clinical practice.

8.5.1 Clinical end Ex-Vivo Normothermic Perfusion Brasile et al. was the first to report the perfusion of human kidneys using the EMS system, however these were only used in an experimental setting and not transplanted.48 Based on the evidence from our experimental work, in 2011 our group reported the first case of EVNP followed by the transplantation of an extended criteria donor (ECD) kidney.49 The technology was identical to our experimental model except that a unit of oxygenated compatible packed red cells from the blood bank was used instead of leukocyte depleted autologous blood. The donor had an out-of-hospital cardiac arrest (1 hour) and based on the history, the kidney was declined by six transplant centers in the United Kingdom. On arrival at the recipient center the kidney had a short period of EVNP (35 minutes) after nearly 11 hours of cold ischemia. The recipient, a 55-year-old dialysis dependent lady, had slow graft function after the transplant but remained dialysis free. She had good graft function at 3 months posttransplant and at 4 years posttransplant her kidney function remains excellent. In the second report we demonstrated the feasibility and safety of the technique in a series of ECD kidneys.50 Using the same technique and technology, 17 kidneys underwent EVNP for just over 60 minutes prior to transplantation. The outcome was compared to a matched control group of 47 ECD kidneys which had CS alone. Only 1/17 (5.6%) patients in the EVNP group had DGF compared to 17/47 (36.2%) in the control group. A randomized controlled trial is planned to assess EVNP and will recruit 400 patients undergoing a DCD kidney transplantation (Maastricht Categories III & IV). Recruits will be allocated at random in a 1:1 ratio to either static CS plus 1 hour of EVNP or CS only. The primary outcome measure will be DGF defined as the need for dialysis in the first 7 days posttransplantation. Secondary outcome measures will include measures of early and longer-term graft function (12 months), length of hospital stay, graft and patient survival, and episodes of rejection.

8.6 INTERMEDIATE EX VIVO NORMOTHERMIC PERFUSION Reestablishing cellular function using EVNP can also be carried out at an earlier time point, e.g., in between periods of hypothermic preservation. Clinically this would be practical when access to theater may be delayed. In the 1980s, van Der Wijk et al. used this technique of intermediate normothermic perfusion to extend the preservation period.51 Canine kidneys were preserved for 144 hours using a combination of hypothermic and normothermic preservation techniques. Halfway through the preservation period kidneys were perfused with blood

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before returning to hypothermic preservation. Three and four hours of perfusion were deemed necessary to reverse the ischemic damage. In another report Rijkmans et al. was able to extend the preservation period to 6 days with a 3-hour period of normothermic perfusion in the middle.52 Although rather extreme durations of preservation, these studies support the use of normothermic perfusion in this way.

8.6.1 Clinical Intermediate Ex Vivo Normothermic Perfusion We reported a single case of intermediate EVNP in clinical kidney transplantation.53 The ECD kidney had 10 hours 30 minutes of cold ischemia before undergoing 60 minutes of EVNP using the same cardiopulmonary bypass technology. The kidney demonstrated a good level of renal function during perfusion. After EVNP, the kidney was flushed with cold preservation solution and repacked in ice. Due to ill health of the first intended recipient the kidney had a second period of cold ischemia (5 hours and 21 minutes). The kidney was transplanted without any complications and the recipient had immediate graft function. There were no untoward effects of the additional cold ischemic insult after EVNP, implying that this technique was feasible and safe. Although further evidence is needed, EVNP applied in this way may be an effective way to reduce the effects of cold ischemic injury or perhaps extend the length of preservation as shown in the early experimental models.

8.7 KIDNEY MANIPULATION/CONDITIONING Normothermic preservation provides a unique opportunity to manipulate or treat the kidney prior to transplantation. This ensures the direct administration of a therapy to the targeted organ and avoids unwanted side effects in the recipients that are associated with many therapies. Brasile et al. found that the protective gene hemeoxygenase-1 (HO-1) could be up-regulated within the kidney with the administration of cobalt protoporphyrin (CoPP) during normothermic perfusion even after significant periods of warm ischemia.54 In contrast, CoPP administered during hypothermic machine perfusion had no effect on the expression of HO-1. HO-1 is one of the three isoforms that catalyze the conversion of heme to biliverdin, free iron and carbon monoxide. These compounds reduce free radical injury and have antiinflammatory actions. Brasile et al. also demonstrated that kidneys could be repaired after 2 hours of warm ischemia with the addition of fibroblast growth factors during normothermic perfusion.55 They showed that cellular processes should be supported, and protein synthesis was evident during 24 hours of ex vivo perfusion of nontransplanted human kidneys. More recently they have explored the application a of nanobarrier membrane (NB-LVF4) during 3 hours of normothermic perfusion to delay the onset of rejection in canine kidneys.56 Delivering genes to block or stimulate protective pathways targeting acute and chronic rejection or ischemia reperfusion injury is an attractive therapy in transplantation.57 Brasile et al. demonstrated that the kidney could be effectively transfected during 24 hours of EVNP.45 They administered the recombinant adenovirus, Ad5, CMV5 GFP encoded with green fluorescence protein and found that effective transfection and synthesis occurred. The reporter gene GFP localized in the intima of the blood vessels, demonstrating the ability to deliver and target the vascular endothelium. RNA interference using a 21-nucleotide small interfering RNA (siRNA) is another approach to modulating and protecting the kidney.58,59 The introduction of a double-stranded RNA elicits the selective degradation of homologous mRNA transcripts. siRNA are capable of blocking gene expression in mammalian cells and have proved to be a potent and specific method of gene silencing.58,59 We have demonstrated siRNA’s ability to reduce caspase 3 expression and reduce some of the effects of reperfusion injury in the isolated kidney when added to the cold preservation solution.60 The effect maybe greater when administered under normothermic conditions, but it is likely that prolonged periods of EVNP are needed to ensure that the kidney is modified before reperfusion. Nonetheless, shorter periods of perfusion may be used to deliver the genes and target the later events in the reperfusion and immune cascade. Experimentally, we have also explored the use of gaseous molecules administered during EVNP to further ameliorate the effects of ischemic injury.61 Gaseous molecules such as nitric oxide donors or carbon monoxide in the form of soluble carbon monoxide-releasing molecules (CORMs) can enhance renal blood flow via the activation of soluble hem-containing guanylate cyclase to produce guanosine 30 , 50 -cyclic monophosphate (cGMP) in vascular smooth muscle, causing relaxation. In an ex vivo model, kidneys were treated with a carbon monoxidereleasing molecule (CORM-3). These kidneys had improved renal and tubular cell function and lower intra renal resistance after reperfusion compared to kidneys treated with a nitric oxide donor.61

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In the clinical setting there has been no reported use of the treatment of kidneys during normothermic perfusion. Nonetheless, with further development of the technology and more prolonged perfusion times, normothermic perfusion could be used to manipulate the kidney, targeting specific genes associated with ischemia reperfusion injury, rejection, or fibrosis.

8.8 RESUSCITATION EVNP may also be used to recover kidneys that have been declined for transplantation. Inadequate in situ perfusion at the time of retrieval is particularly common in kidneys from DCD donors. Inadequately perfused kidneys are likely to have additional warm ischemic injury due to incomplete cooling during retrieval.62,63 The microcirculation is also likely to be compromised and the cumulative damage caused by a period of hypothermic preservation can result in irreversible injury. Therefore, many of these kidneys are regarded as unsuitable for transplantation. We reported a single case of a pair of human kidneys declined for transplantation due to inadequate in situ perfusion.64 Both kidneys were retrieved from a 42-year-old DCD donor. The donor had no previous medical history and the kidneys were retrieved using a standard technique of in situ cooling via an aortic cannula and flush-out with cold University of Wisconsin (UW) solution. After the in situ flush both kidneys appeared inadequately perfused. An attempt was made to flush the kidneys on the back table but both failed to flush properly and they were deemed unsuitable for transplantation. The kidneys were offered for research and after approximately 9 hours, an attempt was made to flush the kidneys again. Some blood was cleared from each kidney but they remained patchy in appearance (Fig. 8.1A). The kidneys were then perfused using EVNP for 60 minutes. Both kidneys appeared pink and evenly perfused and the blood flow improved throughout perfusion (Fig. 8.1B). Each kidney also produced a significant amount of urine. A biopsy was taken after EVNP which showed significant donor-related changes and some evidence of acute tubular injury, but the nuclei appeared normal and there was no evidence of cortical necrosis in either kidney. Although the kidneys were not transplanted, this case shows the ability of EVNP to recover organs and assess the functional capacity of a kidney prior to transplantation.

8.9 VIABILITY/QUALITY ASSESSMENT Reestablishing renal function ex vivo is likely to be an informative measure of organ quality and acceptability for transplantation. Brasile et al. were able to measure renal function and metabolism during perfusion to assess the level of damage.65 They used a viability score based on the sum of three indices. Oxidative index: changes in oxygen consumption, Perfusion index: thresholds of pressure and flow, and Vascular index: changes in the platelet count during perfusion. Thresholds of oxygen consumption, perfusion pressure and flow, and levels of platelets measured in the perfusate were defined. A higher score indicated less damage. This score correlated with the degree of acute tubular necrosis (ATN) in canine kidneys after transplantation. Dogs with the least severe ATN had the highest viability score. Our group proposed an assessment of quality by measuring the level of function and macroscopic appearance.66 The assessment was made using a series of 74 human kidneys that were declined for transplantation. Each kidney underwent EVNP for 60 minutes as previously described. The red cell-based solution used to perfuse the kidneys allows any abnormalities or areas of inadequate perfusion to be visualized easily. We formulated a scoring system to assess the quality of the kidney based on a combination of the macroscopic appearance FIGURE 8.1 (A) A picture of a human kidney declined for transplantation due to inadequate in situ perfusion. (B) A picture of the same kidney after 60 min of ex vivo normothermic perfusion.

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TABLE 8.1

Potential Parameters to Assess Viability During Ex Vivo Normothermic Kidney Perfusion

Viability assessment Perfusion parameters

Renal function

Tubular function

Renal blood flow

Urine output

Filtration fraction

Intracellular enzymes/molecular markers AST, GGT, GST, LDH 1

Intrarenal resistance

Creatinine levels

Fractional excretion Na

Free iron

Oxygen consumption

Creatinine clearance

Total protein

NGAL

Acid base balance

Oxidative damage

AST, Aspartate aminotransferase; GGT, gamma-glutamyltransferase; GST, glutathione S-transferase; LDH, lactate dehydrogenase; NGAL, neutrophil gelatinaseassociated lipocalin.

and thresholds of renal blood flow and urine output. Renal blood flow was used as an index of vascular integrity and urine production as a simple index of glomerular filtration and tubular function. The combination of the perfusion parameters renal blood flow, urine output, and macroscopic assessment provided an overall measure of kidney quality. The scores ranged from 1, indicating the least injured and therefore the highest quality, to 5, the most injured and lowest quality kidneys. This scoring system was applied to a series of 36 marginal kidneys that underwent EVNP followed by transplantation at the same center. Kidneys with an EVNP assessment score of 3 had a significantly higher incidence of DGF (3 of 8) than those with a score of 1 (1 of 17) or 2 (0 of 11). Posttransplant renal function was also significantly worse in these kidneys, with higher serum creatinine levels and a lower eGFR at 12 months. This study suggests that EVNP could be a useful aid in the pretransplant assessment of marginal kidneys. There is also scope for EVNP to provide a more detailed assessment. The addition of other measures of renal and tubular function, histological evaluation or the use of biomarkers to determine the severity of renal injury may be advantageous in kidneys with higher EVNP assessment scores to aid the decision process67
70 (Table 8.1). Approximately 12% of kidneys in the United Kingdom and up to 18% of kidneys in the United States are donated and retrieved for transplantation but are subsequently declined due to concerns about their quality.71
73 This technology could be used to increase the number of kidney transplants and provide the transplanting surgeon with a more detailed appraisal of the kidney.

8.10 SUMMARY & CONCLUSION During the last decade the transplant community has endorsed alternatives to hypothermic techniques for organ preservation. Although the clinical use of normothermic perfusion in kidney transplantation pales in comparison to the expansion of heart, lung, and liver clinical programs, the results are extremely encouraging. The technique has generated a great deal of interest in the kidney transplant community and it is likely that other centers will adopt this technology in the near future. EVNP has many advantages over hypothermic techniques. Early indications suggest that a short resuscitation period can improve early kidney graft function. In the future it could be used to repair damaged organs by administering therapeutic agents, altering gene expression, and adding growth factors or stem cells directly to the kidney. The assessment of quality is also an important factor with many kidneys being retrieved but subsequently deemed unsuitable for transplantation. Ex vivo normthermic perfusion provides a comprehensive assessment of function and perfusion that can aid the surgeon in the decision process. This technology may help to increase the transplant numbers and prevent the unnecessary discard of organs. The advantages of EVNP techniques in kidney transplantation have been explored by a small number of surgeons and researchers. Without doubt this technology will advance over the next decade and have a wider impact in clinical kidney transplantation.

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4. 53. Hosgood SA, Nicholson ML. The first clinical case of intermediate ex vivo normothermic perfusion in renal transplantation. Am J Transplant 2014;14(7):1690
2. 54. Brasile L, Buelow R, Stubenitsky BM, Kootstra G. Induction of heme oxygenase-1 in kidneys during ex vivo warm perfusion. Transplantation 2003;76(8):1145
9. 55. Brasile L, Stubenitsky B, Haisch CE, Kon M, Kootstra G. Potential of repairing ischemically damaged kidneys ex vivo. Transplant Proc 2005;37(1):375
6. 56. Brasile L, Glowacki P, Castracane J, Stubenitsky BM. Pretransplant kidney-specific treatment to eliminate the need for systemic immunosuppression. Transplantation 2010;90(12):1294
8. 57. Sandovici M, Deelman LE, de Zeeuw D, van Goor H, Henning RH. Immune modulation and graft protection by gene therapy in kidney transplantation. Eur J Pharmacol 2008;585(2-3):261
9. 58. Zheng X, Zhang X, Feng B, et al. Gene silencing of complement C5a receptor using siRNA for preventing ischemia/reperfusion injury. Am J Pathol 2008;173(4):973
80. 59. Yang B, Elias JE, Bloxham M, Nicholson ML. Synthetic small interfering RNA down-regulates caspase-3 and affects apoptosis, IL-1 beta, and viability of porcine proximal tubular cells. J Cell Biochem 2011;112(5):1337
47. 60. Yang B, Hosgood SA, Nicholson ML. Naked small interfering RNA of caspase-3 in preservation solution and autologous blood perfusate protects isolated ischemic porcine kidneys. Transplantation 2011;91(5):501
7. 61. Hosgood SA, Bagul A, Kaushik M, Rimoldi J, Gadepalli RS, Nicholson ML. Application of nitric oxide and carbon monoxide in a model of renal preservation. Br J Surg 2008;95(8):1060
7. 62. Gok MA, Bhatti AA, Asher J, et al. The effect of inadequate in situ perfusion in the non heart-beating donor. Transpl Int 2005;18 (10):1142
6. 63. Snoeijs MG, Dekkers AJ, Buurman WA, et al. In situ preservation of kidneys from donors after cardiac death: results and complications. Ann Surg 2007;246(5):844
52. 64. Hosgood SA, Nicholson ML. Ex vivo normothermic perfusion of declined human kidneys after inadequate in situ perfusion. Am J Transplant 2014;14(2):490
1. 65. Stubenitsky BM, Booster MH, Brasile L, Araneda D, Haisch CE, Kootstra G. Pretransplantation prognostic testing on damaged kidneys during ex vivo warm perfusion. Transplantation 2001;71(6):716
20. 66. Hosgood SA, Barlow AD, Hunter JP, Nicholson ML. Ex-vivo normothermic perfusion - An innovative technology for quality assessment of marginal donor kidney transplants. Br J Surg 2015;102(11):1433. 67. Waller HL, Harper SJ, Hosgood SA, et al. Biomarkers of oxidative damage to predict ischaemia-reperfusion injury in an isolated organ perfusion model of the transplanted kidney. Free Radic Res 2006;40(11):1218
25. 68. Ruggenenti P, Perico N, Remuzzi G. Ways to boost kidney transplant viability: a real need for the best use of older donors. Am J Transplant 2006;6(11):2543
7. 69. Malyszko J, Lukaszyk E, Glowinska I, Durlik M. Biomarkers of delayed graft function as a form of acute kidney injury in kidney transplantation. Sci Rep 2015;5:11684. 70. Bhangoo RS, Hall IE, Reese PP, Parikh CR. Deceased-donor kidney perfusate and urine biomarkers for kidney allograft outcomes: a systematic review. Nephrol Dial Transplant 2012;27(8):3305
14. 71. Callaghan CJ, Harper SJ, Saeb-Parsy K, Hudson A, Gibbs P, Watson CJ, et al. The discard of deceased donor kidneys in the UK. Clin Transplant 2014;28(3):345
53. 72. Dare AJ, Pettigrew GJ, Saeb-Parsy K. Preoperative assessment of the deceased-donor kidney: from macroscopic appearance to molecular biomarkers. Transplantation 2014;97(8):797
807. 73. Matas AJ, Smith JM, Skeans MA, et al. OPTN/SRTR 2012 Annual Data Report: kidney. Am J Transplant 2014;14(Suppl. 1):11
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9 Kidney Transplant Recipient Surgery Fabio Vistoli1, Vittorio Perrone1, Gabriella Amorese1, Ugo Boggi1 and Giuseppe Orlando2 1

University of Pisa, Pisa, Italy 2Wake Forest University Health Sciences, Winston-Salem, NC, United States

9.1 INTRODUCTION In the past 30 years, the results of kidney transplantation have improved, especially because of prevention and treatment of acute rejection episodes, management of posttransplant complications, and a better knowledge of immunosuppressive medications. As a result, transplantation has become the mainstay and preferred treatment for patients of all ages with end-stage renal disease (ESRD). In the first part of the 20th century, the work of Alexis Carrel produced a tremendous progression in vascular surgical techniques and generated great interest in the possibility to replace diseased organs with new ones from a different individual. However, it was only in the second part of the same century that the era of human organ transplantation could begin, following progress in human and transplant immunology. The first successful transplant of a kidney between identical twin brothers was performed on December 23, 1954 in Boston.1 Today, kidney transplantation has become a common procedure performed all over the world. The basic principles of kidney transplant recipient surgery have remained fairly constant during the last 60 years, and the wide variety in individual specialist practices address the many different ways in which a solid technical result can be obtained. Also, the surgical procedure for renal transplantation has changed little from the original pelvic operation portrayed in 1951 by Kuss et al.2 It appears that the operation has become a standard and there is little requirement for further talks over the theme. Actually, the argumentation on this surgical technique has never stopped since its introduction, and in order to improve the surgical results, numerous parts of operative techniques have been changed and novel strategies have been designed. Additionally, in the contemporary immunosuppressive era, the number of surgical complications has increased and has presented some new elements. The indication for renal transplantation is irreversible renal failure that requires, or will soon require, dialysis, in any modality it is performed. Generally accepted contraindications are noncompliance, active malignancy, active infection, high probability of operative mortality, and unsuitable anatomy for technical success. The surgical act is always the key to obtaining a successful transplantation, and surgical strategies are continually measured with the morbidity and mortality of the patients. Today, kidney transplantation is the treatment of choice for patients with ESRD, with a low incidence of technical complications. Compared with dialysis, it is associated with increased patient survival and better quality of life, and it is cost effective.3 Herein we describe the standard operations performed today and present redesigned surgical approaches, together with the major surgical complications and the development of their detection tools and treatment strategies.

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9.2 PREPARATION OF THE RENAL GRAFT (SYNONYMOUS TO BENCH PROCEDURE OR BENCHING) The renal graft is prepared (5benched) before implantation at the back table (5bench). The kidney is placed in a plastic bag containing the preservation solution of choice, which sits in a bowl containing clean saline and ice (Fig. 9.1). During benching, the temperature of the fluid should range between 1 C and 4 C. Kidneys from living donors require short and little back-table preparation because most of the dissection has already been done during the nephrectomy. On the other hand, in case of en bloc kidney procurement, the degree of graft preparation could be longer and significant. We start the preparation by putting a mosquito clamp on the end of the ureter, maintaining it off to the side to prevent incidental damages. The renal vein(s) and the artery(ies) are then cleaned, and any side branches that do not enter the kidney are ligated. The last part of the preparation is dedicated to accurate lymphostasis of the fatty tissue around the renal hilum that is accurately ligated to reduce the risk of lymphorrea after graft reperfusion.

9.2.1 Preparation of the Renal Vein The length of the left renal vein is usually sufficient once the adrenal, lumbar, and gonadal branches are ligated (Fig. 9.1). It is not mandatory to dissect away the entire gonadal vein: Despite the presence of small tributary branches from the kidney and the ureter that could bleed, the simple ligation of both ends is sufficient to obtain a good hemostasis after reperfusion. When multiple renal veins are of similar size, they should be preserved, either by conjoining or by using a jump graft. In contrast, because the venous drainage communicates within the kidney, small accessory renal veins may be tied off. The right renal vein is shorter than the left renal vein and is often thinner, especially in its posterior aspect. In a living donor, a small cuff of donor cava can give thicker tissue and wider lumen to anastomose. In a cadaveric donor, when the kidneys are split, the entire remaining cava should be sent with the right kidney. This allows the use of cava to extend the right renal vein, often to a length equal to or exceeding the right renal artery. When inspecting the donor cava, first ensure that the suprarenal portion is intact, as it may be damaged during removal of the liver.

FIGURE 9.1 The upper left panel shows a right kidney sitting in the basin containing preservation solution. The upper right panel shows a living donor graft right after procurement. The lower panels show a right deceased donor graft before and after benching; the vena cava conduit has been reconstructed and will serve as new renal vein.

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The most common reconstruction technique is to cut the cava in line with the right renal vein and to then sew the superior and inferior parts of the cava with 5 0 or 6 0 Prolene suture. The left renal vein orifice is often used for anastomosis to the recipient. If the length is not sufficient, then the vena cava can be rotated in line with the right renal vein and only the superior opening sewn with a running suture. The lower opening of the vena cava can be anastomosed end to side directly to the recipient. Use of the full stump of the vena cava requires careful ligation of all lumbar venous branches coming from the cava and may result in a very wide anastomosis. After any reconstruction, testing of the vessels by irrigating with heparinized perfusion or saline solution should show any missed branches or large holes in the vessel, sutures or ligations that have to be sewn.

9.2.2 Preparation of the Renal Artery The renal artery orifice should be examined for injury: Especially flaps of intimal plaque or aneurysms, which may not be visible when the artery is not completely distended. The artery should be cleaned and followed up to the hilum, preserving any renal branches. In fact, all renal arteries are end arteries, so any branches that are ligated will result in a region of nonperfused renal parenchyma. This is important, in particular, for inferior pole branches, which often provide the only blood supply for the donor ureter. On the other hand, a superior pole branch may appear to be just an adrenal artery, yet in tracing its path could be possible to find out that it takes a turn into the renal parenchyma. When the renal artery does not have donor aorta attached (living donor or diseased cadaveric aorta) we prefer to put a stitch on the superior aspect of the vessel for orientation. When donor aorta is attached and healthy, a small rim of aorta can be preserved (the so called “Carrel patch”), which reduces trauma to the renal artery intima during anastomosis suture. Multiple renal arteries can be managed by a variety of techniques, according to number, size, relative length, presence and health of donor aorta, and distance along the aortic patch.4,5 In a cadaveric donor, the recommended approach is to use a patch of donor aorta that includes all the renal artery orifices. This needs a longer recipient arteriotomy, however not longer than 3 4 cm, alternatively, it can be reconstructed on the back table to still allow a shorter single recipient anastomosis. In the case the donor aorta is not available or is too diseased, arteries can be anastomosed individually, also the smaller the size, the more difficult the anastomosis. If two arteries are of similar length and size, they can be sewn together to form one single orifice. Each artery is spatulated facing each other and then the apices stitched together with 6 0 or 7 0 Prolene. The suture line can then be run up each side of the arteries to form a unique larger orifice. In the case of two renal arteries in which one is smaller and/or shorter than the other, an end-toside anastomosis of the smaller to the larger can be done with running or interrupted suture. A small, plastic cannula placed in the larger artery, faced in the side hole, can help to prevent suturing of the posterior wall.

9.2.3 Preparation of the Ureter The entire vascularization of the donor ureter comes from the donor renal arteries. For this reason, preservation of the ureteral blood supply is essential (Fig. 9.2). The best way to maintain donor ureter arterial supply is by accurately preserving the fat and adventitial tissue found in the triangle formed by the ureter, lower renal graft pole, and renal artery (or arteries). In fact in this space there are the small branches (coming from the renal artery) responsible for the arterial supply to the ureter. In the case of two ureters, they can be anastomosed individually on the vescica or conjoined together on the back table spatulating the end of both of them, suturing together at the apex, and then sewing the two different sides of the ureteral edge.

9.2.4 Completion of the Bench Preparation Once the vessels and ureter are prepared, the perirenal fat is removed and care is taken not to tear the renal capsule. Then, lymphostasis of the hilum is achieved with multiple silk ties, aimed at reducing the risk of lymphorrea after reperfusion. Importantly, all solid lesions that are accidentally identified while benching should be biopsied and sent for frozen section; as well, any large cyst should be deroofed (Fig. 9.3) and its margin secured with 4/0 PDS running suture, to ensure that no internal solid component is present, taking care of possible communication with the excretory renal system. All vessels should be flushed manually with cold heparinized perfusion solution to ensure that there are no leaks requiring ligation or suture.

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FIGURE 9.2 The upper left panel shows a normal ureter in a renal graft from a living donor. The periureteral tissue is adequate and well viable. The other panels show the case of a stripped ureter which was completely lacking any periureteral tissue. The organ was eventually discarded.

FIGURE 9.3 Post reperfusion appearance of a renal graft which had been refused by several centers due to the presence of a very large cyst in the lower pole. However, as the donor was otherwise excellent and the overall renal mass satisfactory, we accepted the kidney which was implanted after deroofing and placement of a 4/0 PDS running suture on the edge of the cyst in order to prevent bleeding. The graft reperfused extremely well and the recipient experienced initial graft function.

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9.3 STANDARD SURGICAL TECHNIQUE OF KIDNEY TRANSPLANTATION The standard kidney transplant procedure is the pelvic operation originally used by French surgeons Ku¨ss, Dubost, and Servelle and their associates in 1951 and refined subsequently by Murray and Harrison for the first successful kidney transplantation in 1954. The heterotopic pelvic approach has been accepted worldwide for its multiple advantages and is considered, up to today, the standard access.

9.3.1 Site The most common site for placing a standard kidney transplant is in the retroperitoneal iliac or pelvic fossa; either side can be used for either kidney but there is no universal agreement on the criteria for selecting the appropriate one to place the transplant. It has been proposed placing the donor kidney in the recipient’s contralateral side (e.g., left kidney to right side; right kidney to left side) so that the renal pelvis and ureter are anterior in case of future surgeries for ureteral reconstruction in the face of donor ureteral necrosis or stenosis are required. Others stated that the more important issue is to stay away from sites of previous transplants, other operations, or peritoneal dialysis catheters, although dissection on the right side is a little simpler. When both sides are equally available, most surgeons favor the right side because the right external iliac vessels are longer and more horizontal and the vein is usually more superficial compared to the left side, which facilitates the vascular anastomoses. Moreover, on the right side, in case the iliac vessels prove unsuitable, it is easier to move up to the aorta and inferior vena cava, while still remaining retroperitoneal. Rarely, if the pelvis is not feasible, an orthotopic transplant can be done by removing the left native kidney and anastomosing the donor vein to the recipient renal vein, the donor artery to the renal or splenic artery, and the donor ureter to the recipient ureter. With advances of surgical strategy and increase of clinical experience the idea of selecting the right iliac fossa as the favored site for the first transplantation has been widely acknowledged. Despite this, the ipsilateral serious atherosclerotic vascular disease or venous disorders (e.g., previous deep venous thromboses; femoral dialysis catheters) should be routinely excluded. The left iliac fossa is the preferred side when a simultaneous pancreas and kidney transplant is being performed or when the patient is a candidate for a future pancreas after kidney transplant. Vascular anastomoses sites are performed on the external or internal iliac artery and on the external iliac vein and ureteral anastomosis directly on the bladder. There are several practical advantages for these heterotopic choices. Staying out of the peritoneal cavity allows more rapid return of bowel function and any hemorrhage or urine leak is confined to a smaller nonabsorptive space, making diagnosis easier and more rapid. The kidney lies just under the skin without any interposed bowel, which simplifies ultrasound monitoring and percutaneous biopsy. Finally, the distance to the bladder is short, allowing for use of the better vascularized proximal part of the donor ureter for implantation. The peritoneal dialysis catheters and previous minor abdominal operation such as appendectomy, conventional herniorrhaphy are not absolute contraindications. It additionally evokes one issue for nephrologists in that the initial peritoneal dialysis catheter is on the left side for the potential renal recipients. The same argument is valid for femoral dialysis catheter (better on the left than on the right side), also if the best choice is to put the catheter on the jugular or axillary vein, to preserve both inguinal sides for transplantation. The standard Gibson or the “hockey stick” incision can stay away from most stoma of peritoneal dialysis catheters. Then again, the minor transperitoneal surgeries or little operations on abdominal wall for the most part yield limited adhesion at the place to perform the transplantation. On the other hand, the transplantation is not suggested at the side where there is a background of herniorrhaphy with propylene mesh or ipsilateral open operation of ureter and bladder. The propylene mesh results in an inflammatory reaction and connective tissue proliferation leading to fibrosis formation and a thick scar plate on the internal surface of lower abdominal wall, which could make the dissection of bladder difficult. Past ipsilateral pelvic surgeries for the most part preclude transplantation on that side because of remodeled anatomical characteristics and serious perivesical tissue proliferation. Great polycystic kidneys are difficult for surgeons, so it is better choose the side of the smaller kidney. However, bilateral greatly enlarged polycystic kidneys would make the transplant surgery extremely troublesome or impossible. In that event, right or bilateral nephrectomy may be considered. For the second transplantation recipients the kidney is implanted on the contralateral side, usually left side. The patient is positioned supine. In our practice we prefer the patient have both arms out, for easy access to anesthesia if additional venous or arterial access is required. We place a Foley catheter with a Y-shaped connector attached to a urinary bag and to a 1 L bag of saline containing iodine solution. A small volume of fluid is used to irrigate the bladder, especially if the patient is anuric. This clears debris of the bladder, which will later be opened into the retroperitoneum.

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9.3.2 Incision and Exposure The kidney transplant can be performed by means of a wide range of different routes, however two important issues must be considered when choosing the incision for a renal transplant: A good access to the iliac fossa and bladder and a low rate of wound related morbidity. Traditionally, three classic incisions have been suggested for kidney transplant surgery: pelvic Gibson incision, the hockey stick incision, and oblique incision. The pelvic Gibson incision is a curvilinear incision made in lower quadrant of the abdomen which provides atraumatic access to the iliac fossa and bladder for renal transplantation. Oblique incision and inverted J-shaped incision (hockey stick incision) are the other two more frequently used incisions. In terms of incidence of long-term complications, the oblique surgical incision is better than the hockey stick incision for lower incidence of hernia and abdominal wall relaxation and more favorable cosmetic results.6 Paramedian, midline incision, and even transverse incision were introduced in living kidney transplantation surgery for better cosmesis, although these cuts present the drawback of difficult exposure of the operative field and reserved for selected candidates only. When a Gibson incision is made, incision extends from the midline one finger’s breadth above the symphysis pubis to a point two finger’s breadth medial to the anterior superior iliac spine through skin and muscle layers. The external oblique muscle and fascia are divided in the line of the incision and split to the side of the wound. The internal oblique and transverse muscles are opened with the cautery in the line of the cut, or in a more helpful manner to separate the two layers of muscles on the intersection of the oblique muscles and the rectus sheath, which avoids the section of the internal oblique and transversus muscles. The latter method has two major advantages: First, it reduces the blood loss resulting from muscle wound surface during the transplantation; second, the muscle fibers could disrupt during closure because of high tension of the wound covering the graft. The pararectus division of muscles and aponeurosis facilitates wound closure and reduces the incidence of wound complications. The inferior epigastric vessels can be preserved or ligated and divided, but if there are multiple renal arteries, the inferior epigastric artery can be used for anastomosis to a lower polar renal artery. In women, the round ligament is usually transected, but in men an effort is made to preserve the cord. The spermatic cord has freed laterally and retracted medially. The cord may be transected to aid in exposure, but testicular atrophy and hydrocele may be more likely in that case. The exposure of iliac vessels is an easy process, however extension and stretching of the extraperitoneal space may tear off the peritoneum and eventually cause enterocele, an uncommon complication called “renal paratransplant hernia.” If a peritoneal defect is produced, it should therefore be closed immediately to prevent this complication. A self-retaining retractor is inserted to obtain optimal exposure, which allows the assistant to free both hands to assist the anastomoses. However, the position of the retractor should be checked carefully before fixing it because the inadvertent retractor injury is responsible of femoral nerve injury causing reversible muscle weakness or paralysis of hip flexion. The lymphatic structures along and over the vessels must be ligated with a nonabsorbable suture and divided, rather than cauterized, to prevent the later occurrence of lymphorrea causing lymphocele. Attention should be paid to identify the genitofemoral nerve often mistaken for a lymph vessel. The nerve lies on the medial edge of the psoas muscle, and a branch may cross the distal external iliac artery.

9.3.3 Vascular Reconstruction In general, the end-to-side venous anastomosis is performed first, and then the end-to-side arterial one (Fig. 9.4). Some may prefer to perform the arterial anastomosis first if the renal artery is anastomosed to the internal iliac artery. The internal iliac artery is not the preferred site for the arterial anastomosis compared with external iliac artery today, although it was once the classical pattern, especially in living kidney transplantation. Dissection of the internal iliac artery is not as simple as that of the external iliac artery. To obtain a good control of the internal iliac artery, a mobilization of a tract of the external and common iliac arteries is also needed, with the increase of the operative time and risk of surgical complications. The risk of end-to-end anastomosis site stenosis and erectile dysfunction is higher than that of end-to-side external iliac artery following the transplantation. Lastly, the short internal iliac artery and variation are common. In summary, today, end-to-end anastomosis to the internal iliac artery is not recommended. Transplant surgeons have developed different techniques for arterial and venous anastomoses since the first 3-point technique for an end-to-end allograft arterial anastomosis described in 1902 by Carrel. Most efforts have been made to reduce ischemic time and increase the quality of anastomosis. The traditional and universally used technique is the 2-point anastomosis, with initial sutures placed at either end of the venotomy or arteriotomy. Some place an anchor suture at the midpoint of the lateral wall to avoid posterior or anterior wall being caught up in the suture line. Another running anastomoses

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FIGURE 9.4 The upper left panel shows a benched left living donor kidney graft ready for implantation. The upper right panel shows the external iliac vessels after thorough dissection and preparation; notably, the vessels are mounted on two different vessel loops. The lower left panel shows the renal graft wrapped in a stockinette containing slush, that allows gentle handling and cooling of the kidney during the anastomosis. The right lower panel shows the renal vein clamped with a double curve vascular clamp.

technique (“1-suture, 1-knot technique”), which does not need to turn the kidney medial and lateral, has shown some advantages especially in obese patients and recipients with deep iliac fossa. Semisutureless anastomosis was performed both on artery and vein, using 4-stay sutures and several vascular clips for each anastomosis, without a continuous vascular suture.7 Other sutureless vascular anastomosis techniques using vascular clips or titanium ring pin staplers have been described and suggested as safe and time-saving in small series,8,9 but not one of them has become popular. Vascular occlusion during the anastomosis can be achieved with side-biting clamps (e.g., Satinsky, C-Clamp), bulldog clamps, Rommel tourniquets, or angled padded clamps (e.g., Fogarty). At the start of the vascular anastomosis it is worth pausing to ensure with the nurses that all suture material is ready and with anesthesia that preoperative medications (e.g., antibiotics, steroids, immunosuppressive induction drugs) have been given. We usually will give a “kidney cocktail” containing albumin, mannitol, and furosemide that runs in during the anastomosis and ask for a target mean arterial pressure of about 80 100 mmHg. 9.3.3.1 Venous Anastomosis As the vein is usually deeper, we prefer beginning with the venous anastomosis (Fig. 9.5). The renal graft vein is anastomosed end-to-side, usually to the external iliac vein using a continuous 6 0 or 5 0 monofilament nonadsorbable vascular suture following an appropriate external iliac venotomy. If the iliac vein is very deep, ligation and transection of the internal iliac vein can improve mobilization. We place a Satinsky side-biting vascular clamp and create a venotomy with a #11 scalpel blade. The interior of the vessel is flushed with heparinized saline and the opening cut to exact size with angled scissors. We routinely use 6 0 Prolene suture for the end to side anastomosis between renal vein and iliac vein, sewing and tying a single double-armed suture at the cranial end of the anastomosis and then sewing towards the caudal apex (Fig. 9.6). At this point, we routinely place a bulldog clamp across the renal vein and remove the iliac vein clamp. This allows early detection of venous anastomotic leakage and reduces the amount of hardware in what is sometimes a tight space. In rare conditions (e.g., thrombosis, hypoplasia of both iliac veins), the renal graft vein has to be anastomosed to other site. The inferior vena cava (infrarenal or infrahepatic) is the most common alternative, associated or not with a native nephrectomy. Portal venous drainage system, inferior mesenteric vein, superior mesenteric vein, even venous collaterals with large caliber due to thrombosis of the inferior vena cava and iliac veins (e.g., presacral collateral vein, left ovarian vein) have been used for renal graft vein drainage with satisfactory results.10 Short right renal vein, particularly from living donors, represent a technical challenge to the transplant surgeon. A satisfactory anastomosis can be achieved by complete mobilization of the recipient external iliac vein. Right renal vein extension using the inferior vena cava is an excellent option to elongate donor vein, when required, especially in obese recipients. These techniques are used in deceased kidney transplantation, but are not suitable for living donors. Extensive

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FIGURE 9.5 The upper and lower left panels show the venotomy before and after placement of the 5/0 prolene sutures that will be used to perform the anastomosis, as well as of the 6/0 prolene stay stitches that are placed at 6 and 12 o’clock in order to maintain the lumen open during the anastomosis. The upper right panel shows the placement of the sutures in the renal vein, while the lower right panel illustrates the suture while it is ran on the back wall of the veins.

FIGURE 9.6 The upper left panel shows completion of the posterior wall of the venous anastomosis. The upper right and lower left panels show an almost complete and complete anastomosis, respectively. Once the anastomosis is complete, it is tested by opening the vascular clamp after placement of a plastic bulldog clamp.

elongation of renal vein should be avoided either in live or deceased transplantation for prophylaxis of occurrence of renal vein thrombosis. However a number of techniques have been developed to elongate the short live donor vein: extension techniques using saphenous, gonadal vein, or superficial femoral vein grafts or a polytetrafluoroethylene graft have demonstrated good results. 9.3.3.2 Arterial Anastomosis The end-to-side arterial anastomosis is generally placed more proximally than the vein, usually performed using an appropriately trimmed cuff of aorta attached to the renal artery with a continuous 5 0 or 6 0

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FIGURE 9.7 The yellow arrow shows the renal artery. After completion of the venous anastomosis, a vascular clamp is placed on the external iliac artery (white arrow). In the upper left panel, the surgeon is about to do an arteriotomy. Afterward, an arterial punch (upper right panel) is used to create an adequately large breach (lower left panel) in the wall of the external iliac artery. At this point, the anastomosis is started by running the 6/0 prolene on the posterior wall.

monofilament vascular suture after an external iliac arteriotomy (Fig. 9.7). Great care should be taken in application of the arterial clamps to avoid detachment of vascular plaques. Endarterectomy is rarely needed. Some prefer to produce an opening with artery puncher in order to facilitate the anastomosis of renal arteries from living kidney donors or in cadaveric ones in absence of “Carrel patch.” To obtain a normal blood flow to the renal graft a careful suture performance is important: full-thickness suture of arterial wall in each stitch, in particular in arteriosclerotic recipients, plays a crucial role. The arterial clamp is placed on the external iliac artery, usually superior to the venous anastomosis. After incising the anterior surface of the artery with a #11 scalpel blade, we prefer to complete the opening with the same #11 scalpel or with scissors which cut all vessel wall layers equally, even if there is plaque. For the end-to-side anastomosis, we prefer 7 0 Prolene. If a Carrel patch is available, we will place 5 0 Prolene suture at cranial end of the anastomosis and sew along each side to the caudal apex. If it is a small artery with no patch, we usually prefer to sew a single suture circumferentially. In that case, care should be taken to not cinch the suture too tightly when tying the knot (“growth factor”) so that as the arteries fill with blood the anastomosis is not narrowed. There are various anastomosis patterns for kidneys with multiple arteries: Anastomosis of two arteries close together on an aortic patch of a left-sided deceased donor kidney is the simpler situation. If arteries are more than 2 cm apart, two separate anastomoses are recommended in order to minimize excessive stretching of the vessels and ultimately the risk for kinking and thrombosis. Double arteries to a right-sided kidney frequently make situating of the kidney troublesome without kinking one or the other artery, so the branches have to be shortened and two separate anastomoses performed. The most complicated cases are encountered in the living donor setting where the aortic cuff is absent, and multiple, short arteries are common. For double arteries, two separate parallel anastomoses to the external iliac artery are required. Sometimes the lower hilar artery or lower polar artery can be anastomosed to internal iliac artery or inferior epigastric artery in an end-to-end fashion. Very small accessory renal arteries at the upper pole can be ligated without problems. Arteries reconstruction during the back-table preparation before revascularization is an effective method. The advantages of this approach are that they preserve the small accessory renal arteries by an end-to-side or conjoined anastomosis to renal artery and reduce the operative time by simplifying the anastomosis on the recipient. 9.3.3.3 Reperfusion of the Kidney Graft Upon completion of the anastomosis, the vascular clamps are removed and the kidney reperfuses and pinks up almost immediately. It is vital for the patient to be adequately perfused and not have a low blood pressure (Fig. 9.8). The venous clamp is removed first followed by the arterial. If bulldogs were used on the artery, the distal clamp is removed first to test the anastomosis. The kidney is warmed with saline poured on the surface.

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FIGURE 9.8 After completion of the arterial anastomosis (upper left panel), the stockinette is cut off (upper right panel) and the bulldog first, and the vascular clamp thereafter, are removed.

Anastomotic bleeding can be carefully sutured if a large gap is present, but bleeding from needle holes will typically stop with time and mild pressure. Bleeding on the renal surface can be controlled with the electrocautery or the Argon beam coagulator. Attention should be paid to bleeding from small hilar arterial vessels which might be in spasm when the kidney is cold but will start bleeding as the kidney warms and perfuses. The kidney may pink up slowly in the face of ischemia-reperfusion injury, but if there is an anatomic line of demarcation between perfused and nonperfused kidney, immediately search for a possibly missed or transected arterial branch. To improve renal perfusion, especially in kidneys from cadaveric donors, somebody injects 5 mg of verapamil into the renal artery unless the patient is heavily beta blocked, in which case the combination of a calcium channel blocker may lead to heart block.

9.3.4 Urinary Tract Reconstruction Reconstruction of the urinary tract starts after a successful graft revascularization. There is a range of urinary tract reconstruction techniques. The standard procedure is the ureteroneocystostomy (Fig. 9.9). Pyeloureterostomy and ureteroureterostomy ordinarily are considered rescue techniques when the transplant ureter’s blood supply is not sufficient or the urinary bladder is hard to distinguish. No difference was found comparing the overall incidence of urological complications between ureteroureterostomy and ureteroneocystostomy in kidney transplantation. Ureteroureterostomy decreases the incidence of urine leakage and is considered a good first option for urinary tract reconstruction with a greater possibility of resolving a ureteral stenosis with endourology and no risk of reflux.11 In other comparison series the outcomes from routine pyeloureterostomy group were even better and also had the similar advantages as the ureteroureterostomy group.12 In any case, ureteroneocystostomy is still the favored choice of urinary tract remaking for most specialists in view of its advantages: it is a common method applied in general urological surgeries; profound dissection of native ureter and native nephrectomy are superfluous; it can be performed irrespective of the quality or presence of the native ureter, and it holds the possibility of change to a ureteroureterostomy or pyeloureterostomy if the implant fails. The location of ureteroneocystostomy is typically a few centimeters away from the vascular anastomoses, which facilitates the examination and remedy of a urinary complication during the reinterventions. 9.3.4.1 Ureteroneocystostomy There is a range of methods for ureteroneocystostomy, categorizable into transvesical or extravesical and antireflux or nonantireflux. The Leadbetter-Politano (LP) technique is the transvesical ureteroneocystostomy depicted

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FIGURE 9.9 The cystoureteral anastomosis is performed using two 5/0 PDS running sutures, on a protective 6 French double J stent. At the end, an antireflux valve is created using 3/0 PDS (in the form of either a running suture or multiple stitches).

by Murray et al. in 1954 for the first successful renal transplant. This strategy uses one cystostomy to get to the inside of the bladder and another cystostomy to reproduce another ureteric hole in a normal anatomic position. The ureter is tunneled in the submucosal space to avoid reflux. The extravesical ureteroneocystostomy was initially described by Witzel in 1896, then again by Gregoir in April 1961, and soon after by Lich et al., who published the method in November 1961.13 The Lich-Gregoir (LG) technique was intended to avoid a second cystostomy, yet hold an antireflux component. It makes a 2 3 cm submucosal tunnel with muscle backing of the ureter to give a valve effect. Other advantages were less bladder dissection, a shorter ureteral length, and no interference with native ureteral function. The LG technique was noted to be fast and actually less demanding to perform than the LP strategy. A few varieties of the LG implantation have been described, e.g., the utilization of running rather than interrupted sutures to make the ureteral-mucosal anastomosis; execution of a tunnel by submucosal blunt dissection rather than muscular imbrications; placement of a single horizontal Halsted stitch at the proximal apex of the bladder incision to the ureter to avoid tension at the acute angle of the anastomosis; position of an anchor stitch on the distal ureteral tip to the full thickness of the bladder, collapsing back the tip of the ureter to make a terminal cuff; incorporation of the muscular layer with the mucosal layer of the bladder in the anastomosis and the parallel-incision method with a submucosal tunnel made between the two parallel cuts in the lateral bladder. All of these “modified” Lich ureteroneocystostomies include extravesicular access, the formation of an antireflux tunnel, and an urothelial anastomosis. Another extravesical way to deal with ureteroneocystostomy that also incorporates an antireflux tunnel but does not have an urothelial anastomosis, is known as the U-stitch technique. This technique was demonstrated to abbreviate operative times even more than the LG method thanks to the elimination of the urothelial anastomosis. But it is associated with an augmented risk of urinary complications and abandoned in numerous institutions worldwide. The procedures without an antireflux mechanism are rarely described. Early comparisons have failed to demonstrate significant differences amongst reflux and antireflux methods, but most nonantireflux systems have been abandoned. The management of double ureters is similar to that of dual arteries. If there is a common part, a simpler ureteroneocystostomy can be performed as a single ureter. Two separate ureteroneocystostomy using LG technique is preferred when two ureters are not in one common sheath. The dual ureters can also be reconstructed on back table conjoining them into one common conduit to anastomose with bladder or native ureter. The bladder dissection is facilitated by filling the bladder via the Foley catheter and clamping the outflow. Minimal length of donor ureter that allows a tension free anastomosis should be used to minimize distal ischemia. We prefer the Lich-Gregoir or extravesical technique. The donor ureter is anastomosed to the recipient bladder mucosa using 6 0 PDS running anastomosis suture (ureteroneocystostomy) and to prevent reflux, the seromuscular layers is closed over it with interrupted 3 0 Vicryl sutures.

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9.3.4.2 Other Alternatives There are some uncommon types of urinary tract reconstruction strategy utilizing as a part of particular conditions (e.g., pyelopyelostomy in orthotopic renal transplantation; ureteroenterostomy into an intestinal conduit or an intestinal pouch; pyelovesicostomy). Regardless of what technique is utilized, a tension free and watertight anastomosis is essential.

9.3.5 Conclusion of the Surgery Hemostasis, reexamination and placement of drains are the steps taken before closing the wound. Accurate hemostasis is essential for each surgery particularly in a uremic patient. Attention should be paid to the vascular anastomosis site and renal pedicle region to search active bleeding from an unrecognized leak or an unligated vessel, which could be a reason for reoperation during the early postoperative period. The role of reexamination is to discover if there are some grave technical errors and correct them before the closure. Vessels have to be accurately checked: The quality of renal artery pulsation and the vascular tension of renal vein should be explored gently using fingers, modifying the position of the renal graft if there is a kinking or compression of long vessels. The ureter should be tension and burden free from structures after the graft is appropriately put. No urine leakage is allowed. Drain placement is another very important step before closure. The value of a baseline biopsy obtained before closure remains controversial, but it is, without any doubt, a risk factor for vascular complications. A capsulotomy of the transplanted kidney before closure has been abandoned in adult transplantation because it is of no use on the whole. Closure is less easy than incision making (Fig. 9.10). Muscular tension often is higher either from the additional renal graft or descending effect of muscle relaxant. Some centers routinely close muscles and aponeurosis with single-layer #1 polydioxanone (PDS) running suture, which reduces the closing time but is also a risk factor of wound complications. Our experience is to close the wound with twolayer #2 polypropylene nonabsorbable suture: interrupted stitches in muscle layer and interrupted stitches in the external oblique muscle fascia layer, which has been proved an appropriate method. A subcutaneous absorbable suture layer completes the wound closure. Attention should also be paid to avoid injuring the peritoneum or the transplanted ureter when closing the incision. Furthermore, excessive tension on the suture may lead to compression of the kidney or lead to defects resulting in wound complications.

FIGURE 9.10

Appearance of the surgical site at the end of the procedure.

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9.3.6 Stent, Catheter and Drains Double J stent capacity in renal transplants to significantly diminish ureteric complications is broadly accepted. One metaanalysis has shown that the prophylactic routine stenting in renal transplants is cost effective.14 Increased risk of urinary tract infection, additional cystoscopy and patient discomfort from bladder spasm is relatively unimportant and controllable and cannot balance the reduced incidence of ureteric complications, which can be a cause of graft loss. The optimal duration of prophylactic stenting has not been determined. Based on center preference, it is usually 2 6 weeks. In our practice we also use a Bracci catheter to selectively drain the urine produced by the grafted kidney. The catheter is placed during transplantation percutaneously and transvescically to stent the graft ureter anastomosis. In this way the urinary output from the grafted kidney can be selectively collected, measured, and analyzed during the posttransplant period. The catheter is pulled back and removed after 12 days. Acute vascular rejection usually occurs 1 2 weeks after transplantation, a selective graft catheterization is helpful for the patients to detect the early sharp reduction of graft urine output, a usual signal of acute rejection. A dwelling catheter is necessary for every kidney transplantation patient. It is important to maintain the catheter in an unobstructed condition during the early postoperative period. The reported duration time usually is 5 7 days; our practice is 5 days in case of double J ureter stenting and 14 days in case of Bracci catheter ureter stenting. There is controversy over the need and term of perigraft drains. Some authors recommended nondrains closure if hemostasis is satisfactory because drain tubes increase the infection risk in immunosuppressive patients. But most others support placing a closed suction retroperitoneal drain at the time of transplant and a considerable majority of them suggest removal of drains in 48 hours in case of infection. Based on our practice, we suggest a “three-drain policy” routinely for every transplant patient. The incidence of postoperative hematomas and lymphoceles in renal transplantation is higher than general surgeries’ for multiple reasons. In the early posttransplant period, bleeding is from the operative field. Even a week later, the spontaneous bleeding of graft can also develop a problematic hematoma. Moreover, most lymphoceles formations and urine leaks occur approximately 1 week after transplantation: Too early removal of drains increases the risk. The reason for three drains is based on the fact that there are basically three isolated dead spaces created by the allograft: over the upper pole and under the lower pole of the transplant kidney, and in the perivesical space. One lower drain often cannot drain the bleeding from the upper pole. We place one drain onto the upper pole of graft, another down the lower pole near to the vessels and a third one in the prevesical space, centimeters away from the ureter. The upper and lower poles drains usually are removed 4 5 days posttransplant or until drainage is less than 20 mL daily, the perivesical drain is routinely removed 1 day after the catheter removal if there is no evidence of urine leak or lymphorrhea.

9.4 SURGICAL CONSIDERATIONS IN PEDIATRIC RECIPIENTS AND DONORS 9.4.1 Pediatric Recipients In pediatric recipients, the standard surgical technique applied in adults conveys two primary disadvantages. In the first place, there is a size mismatch between the usable extraperitoneal space and adult sized grafted kidney. Second, the recipient artery might be small compared to the artery of the graft. This can make the vascular anastomosis more troublesome and may put at risk the blood pressure and flow required for the donor kidney to survive; the classic approach is to consider the same transplant technique as for adults if the child body weight is more than 20 kg. If weight is less than 20 kg, the right Gibson incision can be carried up to the costal margin to increase exposure of the right extraperitoneal space or can be used a transperitoneal incision. Some centers usually perform transperitoneal kidney transplantation in pediatric recipients under 5 years of age. Others centers perform extraperitoneal renal transplantation in children under 15 kg of body weight, which limits potential gastrointestinal complications and allows the confinement of potential surgical complications, such as bleeding and urinary leakage. When a transperitoneal approach is used, a midline incision from the xiphoid to the pubis is done and the posterior peritoneum is incised lateral to the ascending colon. Ligating and dividing two to three lumbar veins posteriorly is often necessary to facilitate the application of vascular occluding clamp. The terminal aorta is dissected free at its junction with the right or left common iliac artery. The donor artery is either anastomosed to the distal aorta to obtain the best arterial inflow, or with one of the common iliac arteries in an end-toside fashion using 5 0 or 6 0 monofilament vascular suture. The use of common iliac artery prevents a complete occlusion of the aorta, which is associated with temporary acidosis of both lower extremities. The donor vessels

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are often without the aortic cuff and may be spatulated to ensure a wider anastomosis and to avoid kinking. The ureter of an adult size kidney is usually long and wide enough to obtain a tension-free ureteroneocystostomy, however if it is too long it may kink or twist easily, and may even cause internal hernia; therefore, sometimes the long ureter should be shortened to obtain best results. Ureteral stenting is beneficial in avoiding urological complications, but special attention should be paid to the removing strategy because standard cystoscopy in adults is not suitable for younger children. It is a smart approach to attach the stent with the indwelling bladder or reservoir catheter and removing it as the catheter is pulled back. An antireflux strategy is needed for pediatric patients. Because a large number of recipients are a result of obstructive urological diseases due to outflow obstruction, small capacity or poor function of the bladder, which all predisposes to vesicoureteral reflux of the transplanted kidney.

9.4.2 Pediatric En-Bloc Transplant In small pediatric donors (kidney smaller than 8 cm), better results are obtained by transplanting both kidneys together, still attached to the aorta and inferior vena cava. During the preparation of the kidneys on the backtable, all lumber branches along the aorta and inferior vena cava are carefully ligated. The suprarenal segments of aorta and inferior vena cava are oversewn with Prolene. These kidneys often have very little hilar fat, and great care has to be taken to preserve the blood supply to both ureters.

9.5 ORTHOTOPIC KIDNEY TRANSPLANTATION Orthotopic kidney transplantation is only occasionally performed because of its difficult procedure with highly related morbidity. However, an expanding rate of patients with ESRD at present are not candidates for a heterotopic kidney transplant for reasons of associated severe vascular pathology, obesity, or retained iliac fossae from a previous kidney graft. The surgical technique comprises a retroperitoneal approach to the splenic hilum with a lumbotomy. To protect its whole length, the vein is ligated near the renal parenchyma including its bifurcation. The native renal artery is often restricted and cannot be utilized. The recipient’s urinary tract is dissected and protected. In most of the reported cases, renal graft revascularization was performed utilizing the recipient’s splenic artery and left renal vein. Artery revascularization is obtained with end-to-end anastomoses between graft renal artery and native splenic artery, renal artery or inferior mesenteric artery, or end-to-side anastomoses between graft renal artery and aorta. Vein revascularization is obtained with end-to-end anastomoses between graft renal vein and native renal vein or splenic vein or end-to-side anastomoses between graft renal vein and inferior vena cava. The excretory system is reconstructed using pyelo-pyelic anastomoses in most cases, and uretero-ureteral anastomoses, uretero-pyelic anastomoses, ureterocalicostomy in the others. Overall vascular complication rate is about 5.4%, and total urological complication rate is about 8.1%. No significant differences are observed between orthotopic and heterotopic transplant series when comparing overall patients and graft survival.15 Chapter 11, Orthotopic Kidney Transplantation, will discuss orthotopic kidney transplantation more in depth.

9.6 CONCLUSIONS Renal transplantation is the preferred therapy for patients with ESRD. Patient and graft survival rates are excellent but long-term outcomes have not shown much change. Surgical intervention is the first, critical step of a successful kidney transplant. Few grafts are lost due to severe surgical complications, which are frequently associated with technique errors. Meticulous surgical technique during transplantation may help to avoid the majority of preventable surgical complications and related morbidity and mortality. The long-term renal graft loss rate remains a concern as it has not improved much in the past three decades.16 Shortage of organs remains the major challenge, with about 100,000 patients waiting for a kidney transplant (https://www.unos.org/data/transplanttrends/#waitlists_by_organ, accessed on June 18, 2016). Continued efforts to improve surgical transplantation technique and above all to increase the donor pool are needed to alleviate the donor shortage.

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References 1. Guild WR, Harrison JH, Merrill JP, Murray J. Successful homotransplantation of the kidney in an identical twin. Trans Am Clin Climatol Assoc 1955;67:167 73. 2. Kuss R, Teinturier J, Milliez P. Some attempts at kidney transplantation in man. Mem Acad Chir (Paris) 1951;77(22 24):755 64. 3. Winkelmayer WC, Weinstein MC, Mittleman MA, Glynn RJ, Pliskin JS. Health economic evaluations: the special case of end-stage renal disease treatment. Med Decis Making 2002;22(5):417 30. 4. Orlando G, Gravante G, D’Angelo M, et al. A renal graft with six arteries and double pelvis. Transpl Int 2008;21(6):609 11. 5. Benedetti E, Troppmann C, Gillingham K, et al. Short- and long-term outcomes of kidney transplants with multiple renal arteries. Annals Surg 1995;221(4):406 14. 6. Nanni G, Tondolo V, Citterio F, et al. Comparison of oblique versus hockey-stick surgical incision for kidney transplantation. Transplant Proc 2005;37(6):2479 81. 7. Mital D, Foster PF, Jensik SC, et al. Renal transplantation without sutures using the vascular clipping system for renal artery and vein anastomosis--a new technique. Transplantation 1996;62(8):1171 3. 8. Jones JW. A new anastomotic technique in renal transplants reduces warm ischemia time. Clin Transplant 1998;12(1):70 2. 9. Ye G, Mo HG, Wang ZH, Yi SH, Wang XW, Zhang YF. Arterial anastomosis without sutures using ring pin stapler for clinical renal transplantation: comparison with suture anastomosis. J Urol 2006;175(2):636 40 discussion 40. 10. Wong VK, Baker R, Patel J, Menon K, Ahmad N. Renal transplantation to the ovarian vein: a case report. Am J Transplant 2008;8(5): 1064 6. 11. Nie Z, Zhang K, Huo W, Li Q, Zhu F, Jin F. Comparison of urological complications with primary ureteroureterostomy versus conventional ureteroneocystostomy. Clin Transplant 2010;24(5):615 19. 12. Timsit MO, Lalloue F, Bayramov A, et al. Should routine pyeloureterostomy be advocated in adult kidney transplantation? A prospective study of 283 recipients. J Urol 2010;184(5):2043 8. 13. Linn R, Ginesin Y, Bolkier M, Levin DR. Lich-Gregoir anti-reflux operation: a surgical experience and 5-20 years of follow-up in 149 ureters. Eur Urol 1989;16(3):200 3. 14. Mangus RS, Haag BW. Stented versus nonstented extravesical ureteroneocystostomy in renal transplantation: a metaanalysis. Am J Transplant 2004;4(11):1889 96. 15. Musquera M, Peri LL, Alvarez-Vijande R, Oppenheimer F, Gil-Vernet JM, Alcaraz A. Orthotopic kidney transplantation: an alternative surgical technique in selected patients. Eur Urol 2010;58(6):927 33. 16. Orlando G, Soker S, Stratta RJ. Organ bioengineering and regeneration as the new Holy Grail for organ transplantation. Ann Surg 2013;258(2):221 32.

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C H A P T E R

10 Robotic-Assisted Kidney Transplantation Ivo Tzvetanov, Pier C. Giulianotti, Giuseppe D’Amico, Raquel G. Roca and Enrico Benedetti University of Illinois Hospital & Health Sciences System, Chicago, IL, United States

10.1 INTRODUCTION Contemporary kidney transplantation is a process where the ultimate goal is to provide excellent graft function with no or minimal complications. The surgical procedure is a crucial part of this process. The traditional open approach was developed in the 1950s and has been performed with minimal changes for more than 60 years. The significant changes in the morbidity of the general population, and especially the epidemic prevalence of obesity for the last two decades, have posed new dilemmas for surgeons to resolve. At the same time, constantly increasing expectations in society regarding the healthcare outcomes have led to the development of quality improvement projects, which has made a high incidence of surgical complications unacceptable. The applications of minimally invasive approaches in many surgical specialties, by minimizing surgical trauma, have led to improved outcomes and higher patient satisfaction.1,2 Because of its higher complexity and necessary precision, kidney transplantation was not considered feasible with the conventional laparoscopic techniques. After the introduction of more advanced robotic technology, with its three-dimensional, higher resolution visual system and wrist-like, multidimensional instrument motions, the idea of minimally invasive kidney transplantation became real.3 After the first reports of the late 2000s,4 robotic-assisted kidney transplantation (RAKT) has slowly but steadily gained popularity throughout the world. Within the past 5 years this has become obvious in the increasing number of publications from the US, Europe, India, and Taiwan. The newest surgical manuals constantly include chapters on robotics in transplantation and the largest transplant forums have specialized sessions on the topic. Epidemiological data indicate that 20% 50% of patients on dialysis are obese. Obese patients have longer wait times until kidney transplantation (KT) and inferior patient outcome, to a great extent due to much higher incidence of wound complications and infections.5 8 If these complications could be avoided, obese patients could have the same outcomes as nonobese recipients. Our group at the University of Illinois in Chicago recognized this significant disparity and found the solution to some of the main problems posed by the obese recipients in the application of the robotic minimally invasive technique for KT. By minimizing the size of the incision and changing its anatomical location on the abdomen we were able to minimize wound complications and almost completely avoid wound infections. Other centers see advantages for nonobese patients also, including reduced level of pain and better cosmetics.

10.2 UNIVERSITY OF ILLINOIS’ EXPERIENCE The initiation of the robotic kidney transplant program at the University of Illinois in Chicago occurred in 2009, when we performed our first robotic-assisted transabdominal renal transplant (RAKT) from a diseased donor.9 After this first successful case we performed a series of RAKTs from living donors. Our only selection criterion was obesity (BMI . 30). Once the recipient has been accepted for transplantation if a living donor (LD) is Kidney Transplantation, Bioengineering, and Regeneration. DOI: http://dx.doi.org/10.1016/B978-0-12-801734-0.00010-2

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available, the surgical approach depends on the patient’s BMI. If the patient has a BMI , 30 kg/m2, an open surgical approach is chosen. Otherwise, if the patient is obese, with a BMI $ 30 kg/m2, the patient is approached by a minimally invasive robotic-assisted technique. Patients without LD are placed on the waiting list and if BMI $ 30 kg/m2, a weight-loss program is started. Once a deceased donor becomes available, obese patients undergo robotic-assisted surgery and nonobese patients undergo open surgery. Previous surgeries are not considered a contraindication to performing a roboticassisted procedure. The only exclusion criteria are severe atherosclerosis of the iliac vessels of the recipient or the graft (for a deceased donor). We do not consider age or immunologic risk status as a contraindication for RAKT.

10.2.1 Surgical Technique 10.2.1.1 Patient Positioning and Port Placement After induction of general anesthesia, a three-way Foley catheter is placed. This allows filling of the bladder with 100 150 mL of diluted Methylene blue solution after completion of the vascular anastomosis, which facilitates identification of the bladder and prevents spatial interference during the vascular suturing. The patient is positioned supine, with parted and flexed legs; shoulder block and tape are used to avoid patient sliding during the operation. After the patient is prepared in the sterile fashion, a 7 cm mid-line incision approximately 5 cm below the xiphoid process is made, and a hand access device is placed. Depending on the body habitus of the recipient, the location of this mid-line incision could be closer to the umbilicus in order to allow easier access to the surgical field for the bedside hand-assisting surgeon. After pneumoperitoneum is obtained at 15 mmHg, the laparoscopic ports are positioned in the following manner for right sided implantation: (1) 12 mm port for the 30 degrees robotic scope on the right side of the umbilicus; (2) 8 mm robotic port high in the right flank; (3) 8 mm robotic port in the left lower quadrant; (4) 12 mm port is then placed for assistance on the left side of the umbilicus between the camera and the left lower quadrant robotic port. Once the ports are placed, the patient is placed in a 30 degrees Trendelenburg position, with the right side elevated (for implantation to the right external iliac vessels). The robotic tower is docked into position from the patient’s right leg site, parallel and slightly diagonal to the body. 10.2.1.2 Graft Implantation and Reperfusion The right colon is mobilized, and right external iliac artery and vein exposed. The iliac vessels are dissected free, using a bipolar forceps and a hook electrocautery. In order to facilitate the exposure and the dissection around the external iliac vein, a vessel loop is used to retract the artery upwards. Another vessel loop is placed around the iliac vein to allow dissection of the posterior surface of the vein. If small venous branches are identified, they are suture ligated. Once the external iliac vessels are completely dissected free, two robotic bulldog clamps are used to occlude the external iliac vein proximal and distal. Robotic Potts scissors are used to create a venotomy to about 15 mm. A 12 cm, double-needle, 5 0 Gore-Tex suture with a knot in the middle is placed at the corner of the venotomy. Kidney graft is inserted in the abdominal cavity by the assisting surgeon and positioned parallel to the dissected iliac vessels. Previous marking of the renal vessels of the graft facilitates the extremely important step of initial orientation. Veno-venous anastomosis is completed in an end-to-side fashion with running suture (Fig. 10.1).

FIGURE 10.1 Venous anastomosis.

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If needed, interrupted stitches of 5 0 Prolene are used to reinforce the corners of the anastomosis. Subsequently, the external iliac artery is clamped between robotic bulldogs, and an oval window (proportional to the size of the renal artery of the graft) is made in the anterior wall of the artery with robotic scissors. To facilitate this precise step, a 5 0 Prolene stitch is placed trough the anterior wall of the external iliac artery and gentle pulling is applied. The arterial anastomosis is completed in an end-to-side fashion with a 12 cm double-needle 6 0 GoreTex suture with a knot in the middle (Fig. 10.2). Once vascular suturing is completed, venous clamps are removed first, followed by immediate removal of the arterial clamps. The reperfusion of the organ and hemostasis are verified and bleeding points secured with 6 0 Prolene suture. With meticulous back bench preparation of the graft, bleeding after reperfusion may be completely avoided. We routinely use a robotic fluorescence camera and intravenous injection of 3 mL Indocyanine green (ICG). This confirms the complete and homogeneous reperfusion of the graft. At this point, the pressure of the pneumoperitoneum is decreased to 10 mmHg to minimize the possible negative effect of high intraabdominal pressure on the graft perfusion. 10.2.1.3 Ureterocystoneostomy The urinary bladder is filled with diluted methylene blue solution in order to facilitate its identification. Distending the bladder at the beginning of the operation could cause spatial interference, especially in patients with preserved diuresis from the native kidneys. Once the dome of the bladder is localized, the muscular layers are incised and the bladder mucosa is prepared. The ureter is anastomosed to the bladder with 5-0 Monocryl running suture using typical antireflux technique, suturing full thickness of the ureteral wall with the mucosal layer of the bladder (Fig. 10.3). Utilization of a ureteral stent is optional. Upon completion of the anastomosis, the seromuscular layer is closed over the ureterocystostomy with 3-0 Vicryl suture to create an antireflux mechanism. At the end of the procedure, the minilaparotomy is closed with running 0 Polydioxanone suture. The 12 mm port sites are closed with an endosuture needle and 0 Vicryl. Skin incisions are closed cosmetically. Placement of drains is not necessary.

10.2.2 Kidney Biopsy When needed, we prefer to perform kidney biopsies under laparoscopic guidance, considering the intraperitoneal location of the graft. Ultrasound guided biopsy potentially can cause significant bleeding. The procedure is completed under general anesthesia and antibiotic prophylaxis is routinely given. Our preference for port positioning is one infra umbilical and one in the upper quadrant on the site of the graft. A Tru-cut biopsy needle, usually 18 G, is introduced through the abdominal wall and directed towards the upper pole of the graft. Bleeding from biopsy sites is controlled effectively with cauterization. The patient is observed for 6 hours and discharged home unless immediate initiation of treatment is needed. Using this technique we did not observed any complications.

FIGURE 10.3 FIGURE 10.2

Arterial anastomosis.

Uretero-vesical anastomosis with the utilization of a ureteral

stent.

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10.2.3 Our Data In 2009, our group reported the first fully RAKT.9 The recipient was a 29-year-old woman with a body mass index (BMI) of 41 kg/m2 who had been on hemodialysis for 5 years. The operative time was 223 minutes, and the blood loss was less than 50 cc. The kidney had immediate graft function. No perioperative complications were observed, and the patient was discharged on postoperative day 5 with normal kidney function. The RAKT was performed with the technique described above. The indication for a robotic approach was morbid obesity since higher body mass index (BMI) in kidney transplant recipients is associated with increased risk of surgical site infections which negatively impact graft survival.6 In 2013 we conducted a case-control study.10 From June 2009 to December 2011, a prospective cohort of 39 obese patients with ESRD underwent RAKT at University of Illinois Hospital & Health Sciences System. 28 of these patients completed a follow-up period of at least 6 months after transplant. We compared the early posttransplant outcome from these 28 patients with those of a retrospective cohort control group of 28 obese patients, who underwent standard open kidney transplantation prior to June 2009 at our institution. The mean BMI of the robotic group was 42.6 6 7.8 kg/m2 compared to 38.1 6 5.4 kg/m2 in the control group (P 5 .02). The mean cold ischemia time was 2.8 6 3.6 hours in the robotic group and 2 6 4.5 in the control group (P 5 .48), and the mean warm ischemia time was 47.7 6 7.8 minutes in the robotic group and 49.2 6 25.2 minutes in the control group. There were no surgical site infections (SSI) in the robotic group, while 28.6% (8/28) in the control group developed SSI (P 5 .004). This initial experience showed that RAKT may enable obese patients with ESRD to access KT and may thereby reduce health disparities in groups with a high prevalence of obesity and ESRD. Within the last 6 years we have applied this standardized technique for over 150 robotic-assisted kidney transplants in obese recipients. We were able to almost completely avoid wound infections, which reach more than 30% in open surgery for this patient population. The 1- and 3-year graft survival were 95% and 89%, respectively. The 1- and 3-year patient survival were 97% and 96%, respectively. Forty-one patients were at high immunological risk (data not published). Moreover, in 2015 we reported for the first time a combined robotic sleeve gastrectomy and RAKT.11 The patient was a 35-year-old woman with a BMI of 42 kg/m2 (96.8 kg) and ESRD. After 24 months follow-up the patient was well, with good kidney function and almost complete loss of the excess body weight.

10.2.4 Training and Learning Curve Robotic kidney transplantation is a highly complex operation. A surgeon planning to perform this procedure needs to have extensive experience with the robotic system and at the same time to be proficient in open transplantation. Frequent performances of more routine procedures, such as robotic cholecystectomy, hernia repair, bowel resections, etc. is very important in order to develop necessary ease with the surgical robot. In our institution we routinely perform robotic donor nephrectomies. Regular practice of vascular suturing in a “dry” robotic lab followed by animal models transplantation would be the optimal step. Multiple assistances and close observations cannot be overemphasized. The performance of the actual transplant has to start stepwise under the guidance of an experienced surgeon. The stages of the operation which are not time sensitive, such as vascular dissection and ureterocystoneostomy, have to be mastered first. The vascular implantation on well selected cases has to be done as the last step of the training. We consider that proficiency will be achieved after at least 20 fully completed cases.

10.3 RAKT IN THE WORLD The first partial RAKT was performed in France and reported in 2001.4 The recipient was undergoing a second transplant using a kidney procured from a deceased donor. The first graft had been transplanted through an open surgery. For RAKT in the left iliac fossa, the patient was placed in supine position with legs spread and flexed to allow rolling in the surgical cart. The assistant standing on the left side of the patient made an incision in the left lower quadrant and placed the self-retaining retractor after retraction of peritoneum. During the remaining part of the procedure the assistant surgeon’s role was to perform hemostasis, placing the vascular clamps and maintaining traction on the running sutures placed by the robot trough the incision. Besides a camera arm, two other instrument arms were used for arteriotomy, venotomy, vascular anastomosis, and ureteroneocystostomy. Cold ischemia time was 26 hours and 45 minutes. Operative time was 178 minutes. Vascular anastomosis was performed in 57 minutes and immediate graft function was achieved.

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The first transabdominal RAKT in Europe was performed by Boggi et al.12 in 2011. The recipient was a 37year-old woman with lupus nephritis, on dialysis for 32 months. She weighted 59 kg. The patient was positioned supine, with the right flank slightly elevated. The table was then tilted 25 degrees to the left, further elevating the right flank, and 15 degrees in Trendelenburg’s position. A 7 cm suprapubic incision was made along the previous Pfannenstiel incision and hand access device was inserted. The robot was placed on the patient’s right side, and a 0 degree telescope was used. The renal vein was anastomosed end-to-side to the common iliac vein using two half running sutures of 6-0 expanded Polytetrafluoroethylene. The same technique was used to create an end-toside arterial anastomosis between the renal artery and the common iliac artery. The uretero-vesical anastomosis was fashioned through the suprapubic incision using open surgical technique (Gregoir-Lich extravesical anastomosis). Before closure of the Pfannenstiel incision, the graft was covered by cecum and pelvic peritoneum with an attempt to keep it in the retroperitoneal location. The warm ischemia time was 51 minutes and immediate graft function was noted on release of vascular clamps. One day after transplant, the patient was ambulating and started oral intake. Pain was minimal, and no analgesia was required after 48 hours. Later, Menon et al.13 15 from Detroit collaborated with Ahlawat in Gurgaon, and Modi in Ahmedabad in establishing two kidney transplant programs in India. In 2014, they published a prospective study of 50 consecutive patients who underwent RAKT. The mean BMI was 24.1 kg/m2. The robot was docked between the splitlegs of the patient in a lithotomy position. A Gel-point port was used to seal the mid-line incision in the periumbilical region. The pelvic bed was cooled to 18 20 C with the introduction of 180 240 mL ice slush via modified Toomey syringes. A temperature probe was used to continuously monitor renal cooling. The graft was inserted into the abdomen through the Gel-point port incision. The vascular anastomosis and the ureteroneocystostomy were performed robotically. The vascular anastomosis time was 25.4 minutes and the ureterovescical anastomosis time was 17.4 minutes. Mean intraoperative renal surface temperature was 20.3 C. The average incision length was 6.1 cm. The kidney graft was retroperitonealized for the final position. The 6-month outcome of 67 patients who underwent RAKT was recently reported.16 There were no instances of graft vascular thrombosis, stenosis, and leak. There was no delayed graft function. The mean 6-month serum creatinine was 1.2 mg/dL. The patient survival at latest follow-up was 96.3% and the graft survival (death-censored) at latest follow-up was 100%. There was no surgical site infection. During the same year, Tsai et al. from Taiwan17 reported their experience with 10 patients. The kidney was placed in the retro-peritoneum through the Gibson incision (7.7 6 1.04 cm) in the iliac fossa. The robot was docked from behind the patient’s back and the assistant surgeon stood between the two legs of the patient. The robotic arms were attached to the robotic ports and set to lift the abdominal wall about 3 cm higher. A 30-degree endoscope was placed over the Gibson incision. The kidney was placed into the abdomen through the Gibson incision. Vascular anastomosis was carried out by the robot using two half continuous sutures of 6.0 polytetrafluoroethylene. The ureteral implantation was done through the Gibson incision using the Gregoir-Lich extravesical technique. Their mean BMI was 22.8 6 3.5 kg/m2 (range: 18.9 28.2). All of the patients with robotic surgery resumed oral intake and ambulation within 24 hours after operations. Overall, the average posttransplant hospital stay was 13.6 6 3.5 days.

10.4 DISCUSSION Minimally invasive surgical approaches, including open with mini incision, laparoscopic and robotic kidney transplantation, are feasible and safe. The da Vinci robotic surgical system has the clear advantages of threedimensional vision and control of the camera by the surgeon. Articulated instruments with a wide range of movements allow for ease of suturing. Tracks of the surgeon’s movements 1300 times/s eliminates the tremor and increases the precision, essential for performing a reliable vascular anastomosis. However, vascular anastomosis through robotic arms can be technically demanding due to a lack of tactile feedback.18 This issue can be overcome using expanded polytetrafluoroethylene suture which is more resistant to grasp and less likely to break. Our experience demonstrates the benefit of RAKT in obese recipients,10 also in terms of graft survival compared to open technique. It is known that obese patients who are able to avoid SSI have the same kidney transplant success rate as patients with a normal BMI.6 We strongly believe that RAKT offers a real alternative to dialysis for obese renal failure patients, and may help to reduce health disparities due to end-stage renal disease in populations with a higher prevalence of obesity. Furthermore, considering the low success rate of medical weight loss before transplantation,19 as an evolution of our idea for the treatment of the obese patients with

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ESRD, we proposed to well selected patients a combined approach with robotic sleeve gastrectomy and RAKT. We hypothesized that the weight loss and comorbidity management may further improve the outcomes after transplantation. In the literature, different techniques have been proposed. Both transperitoneal and extraperitoneal approaches to kidney transplantation are feasible. Also the incisions made for the graft placement are at different anatomical locations. Every technique has its advantages, described by the authors. Hand assistance, easier through an upper mid-line incision, could facilitate some operative steps, such as handling the graft during performance of the vascular anastomoses, and improving exposure especially in obese recipients. In case of sudden hemorrhage, the hand-assisting surgeon could save unnecessary blood loss. Incision in the upper abdomen also avoids the highly colonized pubic and groin areas. However, this type of incision puts the patient at a slightly higher risk of developing incisional hernia. In the rare case of conversion this incision should be extended to gain full access to iliac vessels. Moreover, in our view, working transperitoneally avoids the traditional disadvantages of retroperitoneoscopy, such as limited working space, easy collapse during suctioning, and blurred vision, while still maintaining the option of the graft placement in a retroperitoneal pocket. In our series, the graft remained in an intraperitoneal location and we have been satisfied with this approach, although some complications such as a para-transplant hernia20 and renal pedicle torsion21 are possible. Furthermore, the intraperitoneal location of the graft increases the risk for complications related to standard ultrasonography-guided percutaneous kidney graft biopsy. For this reason, we have chosen to perform kidney graft biopsies under laparoscopic guidance. Intraoperative regional hypothermia proposed by Menon et al. is a reasonable technique. We believe that this approach can be useful for surgeons with limited experience in robotic suturing as a protection if a longer time to perform the anastomosis is needed. If the anastomosis time is kept to the 30 40 minutes range, intraoperative cooling of the graft may not be necessary. For achievement of immediate function of the graft we consider it important to decrease the level of the pneumoperitoneum to below 10 mmHg. This maneuver seems to decrease the potential negative effect to the microcirculation of the graft from the higher intraperitoneal pressure.

10.5 CONCLUSION Robotic-assisted kidney transplantation is an emerging modality of minimally invasive surgery and an increasing number of surgeons worldwide are trying to apply different techniques. Despite the enthusiasm for RAKT, the current high cost is the most prohibitive factor for its widespread use. By achieving adequate kidney graft function and minimizing surgical complications, robotic-assisted renal transplantation gives an opportunity for the disadvantaged group of obese patients with ESRD to have more realistic access to transplantation with good outcomes. We believe that in the long term this will compensate for the initial cost and will prove effective even from a financial viewpoint. However, robotic surgery in organ transplantation is a very advanced application of this technology and a high level of expertise is needed for any surgeon who considers this approach.

References 1. Trinh QD, Sammon J, Sun M, et al. Perioperative outcomes of robot-assisted radical prostatectomy compared with open radical prostatectomy: results from the nationwide inpatient sample. Eur Urol 2012;61(4):679 85. 2. Sood A, Jeong W, Peabody JO, Hemal AK, Menon M. Robot-assisted radical prostatectomy: inching toward gold standard. Urol Clin North Am 2014;41(4):473 84. 3. Hockstein NG, Gourin CG, Faust RA, Terris DJ. A history of robots: from science fiction to surgical robotics. J Robot Surg 2007;1(2):113 18. 4. Hoznek A, Zaki SK, Samadi DB, et al. Robotic assisted kidney transplantation: an initial experience. J Urol 2002;167(4):1604 6. 5. Segev DL, Simpkins CE, Thompson RE, Locke JE, Warren DS, Montgomery RA. Obesity impacts access to kidney transplantation. J Am Soc Nephrol 2008;19(2):349 55. 6. Lynch RJ, Ranney DN, Shijie C, Lee DS, Samala N, Englesbe MJ. Obesity, surgical site infection, and outcome following renal transplantation. Ann Surg 2009;250(6):1014 20. 7. Meier-Kriesche HU, Vaghela M, Thambuganipalle R, Friedman G, Jacobs M, Kaplan B. The effect of body mass index on long-term renal allograft survival. Transplantation 1999;68(9):1294 7. 8. Meier-Kriesche HU, Arndorfer JA, Kaplan B. The impact of body mass index on renal transplant outcomes: a significant independent risk factor for graft failure and patient death. Transplantation 2002;73(1):70 4. 9. Giulianotti P, Gorodner V, Sbrana F, et al. Robotic transabdominal kidney transplantation in a morbidly obese patient. Am J Transplant 2010;10(6):1478 82.

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10. Oberholzer J, Giulianotti P, Danielson KK, et al. Minimally invasive robotic kidney transplantation for obese patients previously denied access to transplantation. Am J Transplant 2013;13(3):721 8. 11. Ayloo SM, D’Amico G, West-Thielke P, et al. Combined robot-assisted kidney transplantation and sleeve gastrectomy in a morbidly obese recipient. Transplantation 2015;99(7):1495 8. 12. Boggi U, Vistoli F, Signori S, et al. Robotic renal transplantation: first European case. Transpl Int 2011;24(2):213 18. 13. Abaza R, Ghani KR, Sood A, et al. Robotic kidney transplantation with intraoperative regional hypothermia. BJU Int 2014;113(4):679 81. 14. Menon M, Sood A, Bhandari M, et al. Robotic kidney transplantation with regional hypothermia: a step-by-step description of the Vattikuti Urology Institute-Medanta technique (IDEAL phase 2a). Eur Urol 2014;65(5):991 1000. 15. Menon M, Abaza R, Sood A, et al. Robotic kidney transplantation with regional hypothermia: evolution of a novel procedure utilizing the IDEAL guidelines (IDEAL phase 0 and 1). Eur Urol 2014;65(5):1001 9. 16. Sood A, Ghosh P, Jeong W, et al. Minimally invasive kidney transplantation: perioperative considerations and key 6-month outcomes. Transplantation 2015;99(2):316 23. 17. Tsai MK, Lee CY, Yang CY, Yeh CC, Hu RH, Lai HS. Robot-assisted renal transplantation in the retroperitoneum. Transpl Int 2014;27(5): 452 7. 18. Patel M, Porter J. Robotic retroperitoneal surgery: a contemporary review. Curr Opin Urol 2013;23(1):51 6. 19. Wu T, Gao X, Chen M, van Dam RM. Long-term effectiveness of diet-plus-exercise interventions vs. diet-only interventions for weight loss: a meta-analysis. Obes Rev 2009;10(3):313 23. 20. Gao Z, Zhao J, Sun D, Yang D, Wang L, Shi L. Renal paratransplant hernia: a surgical complication of kidney transplantation. Langenbecks Arch Surg 2011;396(3):403 6. 21. Roza AM, Johnson CP, Adams M. Acute torsion of the renal transplant after combined kidney-pancreas transplant. Transplantation 1999;67(3):486 8.

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C H A P T E R

11 Orthotopic Kidney Transplantation Bulang He1,2 1

Sir Charles Gairdner Hospital, Perth, Western Australia 2The University of Western Australia, Perth, Australia

11.1 INTRODUCTION Kidney transplantation is a definitive treatment for patients with end stage kidney disease. The standard surgical technique for kidney transplantation has been well established since the 1950s1; in it, the kidney graft is placed in the extraperitoneal space of the lower abdomen with renal vein anastomosed to the external iliac vein and renal artery anastomosed to the internal or external iliac artery. However, in certain circumstances, such as occlusion of the iliac vessels or severe adhesions due to repeated previous pelvic surgery, it may not be possible to place the kidney graft at the lower abdomen. In such situations, the kidney graft can be transplanted at the original location or immediately next to the native kidney. This is called an orthotopic kidney transplant (OKT). OKT was first described by Gil-Vernet et al. in 1978.2 A large series study of OKT has demonstrated equivalent patient and graft long-term survival in comparison with heterotopic kidney transplant at the iliac fossa.3

11.2 INDICATIONS OKT is not commonly practiced in most transplant centers as it is technically more challenging. However it should be considered in the following conditions: (1) poor quality or occlusive iliac vessel disease; (2) multiple previous pelvic surgeries and expected severe extensive adhesions; (3) bilateral retained kidney grafts from previous transplants; (4) pediatric kidney transplant in small children; (5) demand in contact sports such as football players.

11.3 SURGICAL TECHNIQUES 11.3.1 Orthotopic Kidney Transplant by Open Surgery In open surgery, OKT can be performed either at the left or right side through a flank or midline incision. However, the left side is the preferred approach in the literature.3,4 Nephrectomy is performed first with preservation of longer renal vein and renal artery. The dissection is usually extended to the renal hilum at the level of segmental arteries. The ureter is divided at a level superior to the pelvis ureter junction. Following nephrectomy, the splenic artery is dissected and prepared for renal artery anastomosis if the renal artery is deemed not suitable for reanastomosis. The kidney graft is placed at the orthotopic region with the renal vein anastomosed to the recipient renal vein in an end-to-end fashion. To avoid renal vein anastomotic stricture, interrupted stitches are preferred. Alternatively, a growth factor is left in situ allowing the anastomosis extension if running sutures are used. The renal artery is often anastomosed to the splenic artery in end-to-end fashion as the renal artery is often of small caliber or poor quality. The renal artery can be anastomosed directly onto the aorta if the aorta is of good quality. In cases of severe aortoiliac occlusive disease, a bifurcated Dacron vascular graft may be used with one end anastomosed to the aorta and one branch end for renal artery anastomosis while another branch Kidney Transplantation, Bioengineering, and Regeneration. DOI: http://dx.doi.org/10.1016/B978-0-12-801734-0.00011-4

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end is sutured closed.5 The employment of a vascular graft will allow caliber-matched anastomosis to avoid technical failure and graft loss. After completion of vessel anastomosis, the kidney graft is reperfused and hemostasis is checked. The urinary tract is reconstructed by renal pelvis to pelvis (pyelo pyelic) anastomosis or ureter-toureter anastomosis with a ureteric stent placed in situ. 5/0 or 6/0 reabsorbable stitches are used in running suture or interrupted suture fashion. In the case of pediatric kidney transplant in a small child, the kidney graft is placed at the flank area of the orthotopic region.6 The renal vein is anastomosed to the inferior vena cava and the renal artery is anastomosed to the aorta in an end-to-side fashion. The kidney is reperfused following completion of vessel anastomosis. The ureter is usually anastomosed to the bladder using the Lich Gregoir technique with a stent placed in situ. 11.3.1.1 Outcomes of Orthotopic Kidney Transplant by Open Surgery From a large series study by Musquera,3 the long-term patient and graft survival are comparable between OKT and heterotopic kidney transplant (62.5% vs 65.9% and 34.5% vs 29.2% respectively at 20 years). Complications included renal artery stenosis in 3.6%, renal artery thrombosis in 4.8%, and renal vein thrombosis , 1%. Urinary fistula occurred in 9.5% and urinary tract obstruction is seen in 2.3%.

11.3.2 Orthotopic Kidney Transplant by Laparoscopic Surgery Kidney transplant by laparoscopic surgery has been explored over the last decade; there are increasing reports of kidney transplant by laparoscopic surgery7,8 or robotic surgery.9 11 The benefits would be similar to other minimally invasive surgeries including a smaller incision, less analgesia use, quicker recovery, and better cosmesis. To date, there has been no clinical case report of OKT by laparoscopic surgery. In a large live animal model, He et al.12 have reported successful OKT by laparoscopic surgery. This technique may be employed in the human OKT in the future. In the animal pig model, the technique is described as follows: 11.3.2.1 Laparoscopic Donor Nephrectomy on Left Side The pig is placed on right lateral recumbency after intubation for general anesthesia. The camera port is first inserted via a small incision (1.5 cm) at left of midline and cranial to the umbilicus (Fig. 11.1). Pneumoperitoneum is established with pressure set at 12 mmHg. The other ports are placed under vision forming a “V” shape (Fig. 11.1). The left kidney is identified and the bowel dissected medially to expose the renal hilum. The renal artery, vein, and ureter are subsequently dissected. The renal artery is dissected to the origin of aorta and the renal vein to the origin of vena cava to maximize vessel length and facilitate transplantation (Fig. 11.2). The kidney is mobilized free from its attachments. The ureter is divided first at the level of the lower pole of the kidney (Fig. 11.3). A small lower midline abdominal incision (6 cm) (Fig. 11.1) is made. The muscular layer is incised through the left paramidline. The peritoneal layer remained intact at this stage prior to dividing of renal artery and vein. The renal artery and vein are clamped by endoscopic Bulldog (B Brown, Germany) and then divided (Fig. 11.4). The kidney is delivered by opening the peritoneum layer through the precreated midline incision. FIGURE 11.1 (1) Camera port, (2) For fan retractor, (3) For left hand, (4) For right hand, (5) For irrigation and suction, (6) A small midline incision.

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FIGURE 11.2 Laparoscopic donor nephrectomy. The renal artery and vein are dissected to the aorta and vena cava respectively.

FIGURE 11.3 The ureter is divided.

FIGURE 11.4

Endoscopic Bulldog placed over the renal artery (RA) and renal vein (RV) respectively just beyond aorta and vena cava.

11.3.2.2 Laparoscopic Kidney Transplant in an Orthotopic Region The kidney graft is prepared on a back table. The mark suture is placed at upper and lower corner of the renal vein (Fig. 11.5). The kidney graft is wrapped in a tailored surgical pack for easy handling during the transplantation and is delivered to the orthotopic region through the same midline incision. The renal artery is anastomosed in end-to-end fashion to the renal artery stump (Fig. 11.6) and the renal vein is anastomosed in end-to-end fashion to the renal vein stump (Fig. 11.7) by 5/0 Prolene sutures using laparoscopic technique. For venous anastomosis, the posterior side can be anastomosed with a continuous suture pattern and the anterior side by interrupted sutures. Alternatively, two separate running sutures can be used for posterior side and anterior side anastomosis then two sutures are tied with a growth factor to prevent anastomotic stricture (Fig. 11.7B). For

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FIGURE 11.5 The kidney graft is wrapped in tailored surgical pack. A mark suture is placed at superior and inferior corner. RA, renal artery; RV, renal vein.

FIGURE 11.6

Renal artery anastomosis in end-to-end fashion by 5/0 Prolene interrupted suture.

FIGURE 11.7 (A B), Renal vein anastomosis in end-to-end fashion by using running 5/0 Prolene suture, one for posterior side and one for anterior side. The two sutures are tied with a “growth factor.”

arterial anastomosis, an interrupted suture pattern is preferred (Fig. 11.6). Alternatively, two separate continuous sutures are used for posterior side and anterior side anastomosis. The two sutures are tied after removing the proximal vascular clamp, allowing the blood flow through the anastomosis and distension of the anastomosis. The kidney is reperfused following completion of vessel anastomosis (Fig. 11.8). The ureter is anastomosed in an end-to-end fashion by interrupted 5/0 polydioxanone sutures (Fig. 11.9). Lastly, the kidney graft is fixed by three separate 4/0 polydioxanone sutures at the site of upper pole, lateral side, and lower pole (Fig. 11.10). Hemostasis is checked again and the wound and port incision are closed in layers. 11.3.2.3 Outcomes of Orthotopic Kidney Transplant by Laparoscopic Surgery In large live animal pig model, laparoscopic kidney transplant at the orthotopic region has been demonstrated feasible and safe.12 The estimated blood loss from surgery was minimal. The hemoglobin was stable on blood test at pre- and postsurgery. The kidney graft functioned immediately after reperfusion as long as the vessel anastomosis

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FIGURE 11.8 The kidney is reperfused following completion of the renal vein (RV) and renal artery (RA) anastomosis.

FIGURE 11.9

FIGURE 11.10

The ureter anastomosis is in an end-to-end fashion by interrupted 5/0 PDS sutures.

The kidney graft is fixed at the orthotopic region by a suture (4/0 PDS) at upper pole, interpolar, and lower pole.

is satisfactory. Doppler ultrasound showed normal perfusion waveforms of kidney parenchyma. The animal survived on the transplant kidney graft with creatinine level peaked at about 200 µmol/L on day 3 and recovered and normalized at the level of 120 µmol/L from day 7 posttransplant without need of renal replacement therapy over the 4-week study period. There was no urine leakage. However, there was mild narrowing of ureter anastomosis as a result of mucosa edema. Histopathology of kidney graft revealed normal kidney cortex or mild changes secondary to mild hydronephrosis. Understandably, at this early phase, the vessel anastomotic time is longer than OKT. The anastomotic time may improve in the future with increased proficiency in laparoscopic suture skill.

11.4 CONCLUSION OKT is an alternative procedure where heterotopic kidney transplant is not feasible due to the medical conditions of the patient or the demands of sport. The retroperitoneal approach with flank incision is a preferred technique. The splenic artery is often used for renal artery end-to-end anastomosis. The renal vein is most always

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used for renal vein end-to-end anastomosis. The pyelo pyeli anastomosis is usually performed with a ureteric stent placed in situ. The patient and graft survival is comparable to the conventional heterotopic kidney transplant. The vascular and urological complications are within the acceptable range. Recently, minimally invasive laparoscopic surgery has been employed successfully in OKT in the large animal pig model. This technique may be translated to the clinical OKT in the near future.

References 1. Murray JE, Merrill JP, Harrison JH. Kidney transplantation between seven pairs of identical twins. Ann Surg 1958;148(3):343 59. 2. Gil-Vernet JM, Caralps A, Ruano D. New approach to the splenic vessels. J Urol 1978;119(3):313 15. 3. Musquera M, Peri LL, Alvarez-Vijande R, Oppenheimer F, Gil-Vernet JM, Alcaraz A. Orthotopic kidney transplantation: an alternative surgical technique in selected patients. Europ Urol 2010;58(6):927 33. 4. Gil-Vernet JM, Gil-Vernet A, Caralps A, Carretero P, Talbot-Wright R, Andreu J, et al. Orthotopic renal transplant and results in 139 consecutive cases. J Urol 1989;142(2 Pt 1):248 52. 5. Rodrigues P, D’Imperio M, Campagnari M, Azevedo LA, Campagnari JC, van Bellen B. Alternative grafting technique for patients unsuited to heterotopic transplantation due to diseased pelvic conditions. Urol Int 2004;73(4):316 19. 6. Mackie FE, Kennedy SE, Rosenberg AR, Kainer G, Frawley JE, Haghighi KS. Safety and adequacy of percutaneous biopsies in pediatric orthotopic kidney transplantation. Int J Organ Transplant Med 2012;3(1):26 31. 7. Modi P, Pal B, Modi J, Singla S, Patel C, Patel R, et al. Retroperitoneoscopic living-donor nephrectomy and laparoscopic kidney transplantation: experience of initial 72 cases. Transplantation 2013;95(1):100 5. 8. He B, Mou L, Sharpe K, Swaminathan R, Hamdorf J, Delriviere L. Laparoscopic kidney transplant by extra peritoneal approach: the safe transition from laboratory to the clinic. Am J Transplant 2014;14(8):1931 6. 9. Menon M, Sood A, Bhandari M, Kher V, Ghosh P, Abaza R, et al. Robotic kidney transplantation with regional hypothermia: a step-bystep description of the Vattikuti Urology Institute-Medanta technique (IDEAL phase 2a). Europ Urol 2014;65(5):991 1000. 10. Abaza R, Ghani KR, Sood A, Ahlawat R, Kumar RK, Jeong W, et al. Robotic kidney transplantation with intraoperative regional hypothermia. BJU Int 2014;113(4):679 81. 11. Tzvetanov I, D’Amico G, Benedetti E. Robotic-assisted kidney transplantation: our experience and literature review. Curr Transplant Rep 2015;2(2):122 6. 12. He B, Musk GC, Mou L, De Boer B, Delriviere L, Hamdorf J. Laparoscopic surgery for orthotopic kidney transplant in the pig model. J Surg Res 2013;184(2):1096 101.

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12 Kidney Transplantation Combined With Other Organs Junichiro Sageshima1, Linda Chen2, Gaetano Ciancio2, Alberto Pugliese3 and George W. Burke III2,4 1

University of California, Davis, Department of Surgery, Division of Transplantation, Sacramento CA, United States 2 University of Miami Miller School of Medicine, Department of Surgery, Miami Transplant Institute, Miami, FL, United States 3Diabetes Research Institute, Department of Medicine, Division of Diabetes, Endocrinology and Metabolism, Department of Microbiology and Immunology, Miami, FL, United States 4University of Miami Miller School of Medicine, Diabetes Research Institute, Miami, FL, United States

12.1 PREAMBLE It was posited that a chapter on the role of kidney transplantation following a previous transplant of a different organ would be less than relevant for a book on kidney transplantation, bioengineering, and regeneration—perhaps an afterthought. Taking the liberty of pursuing a metaphysical conceit (with apologies to John Donne) and as a prism through which to analyze this further, it may be inferred, as a metric of organ importance, that the shorter the distance (length of the arterial tree) from the heart to the organ, the greater the importance of that organ. For those intraabdominal organs, including liver, spleen, and tail of the pancreas, the arterial supply is from the celiac axis, making them a higher priority than the head of the pancreas and small bowel (arterial supply derived from the superior mesenteric artery (SMA)). The renal arteries come off laterally just inferior to the SMA, putting the kidneys next in line. However, the nearness (or distance) of the kidneys to the heart needs to be tempered with the need for the kidneys to be connected to the bladder, some 20 cm distant. This anatomical directive, which required the development of ureters to bridge the transabdominal distance between the kidneys and bladder, forces one to rethink the original premise of organ importance, and distance from the heart. In fact, for the kidneys this represents a critical balance between cardiac proximity (for perfusion of the kidneys) and bladder proximity (for transit of urine through the ureters). This critical balance actually emphasizes the importance of the role of the kidneys, which would likely be located much closer to the heart were it not for the need to transport urine into the bladder. Thus the kidneys could be judged as the jewels of the abdomen and while their importance does not carry the immediacy of a transplant of the heart or liver, it does have a considerable impact on the longevity of the recipient.1,2 The kidneys play a central role relative to their location. Perhaps arterial distance from the kidneys, rather than arterial distance from the heart, should be the new metric for organ priority, following the compass analogy of John Donne.3 This compass was initially centered on the heart (in our conceit); perhaps it should be repositioned to trace circles (orbs and distance) from the kidneys, to recreate a new, revitalized order of organ importance. The following is a description detailing the benefit that many other organ transplants—including pancreas, liver, and heart—experience from the addition of a kidney transplant.

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12.2 KIDNEY COMBINED WITH PANCREAS TRANSPLANTATION This section describes simultaneous pancreas kidney (SPK), pancreas after kidney (PAK), kidney after pancreas (KAP) transplantation, and the kidney transplant alone (KTA). Pancreas transplantation is performed to restore euglycemia and long-term insulin independence. It has the potential to improve patient survival when combined with a kidney transplant. This has become the optimal therapy for patients with type I diabetes (T1D) and end-stage renal disease (ESRD).4,5 The importance of glycemic control was established in a series of papers published by the Diabetes Control and Complications Trial (DCCT), the landmark paper of which was published in the New England Journal of Medicine in 1993.6 Since that time glycemic control has been shown to be important in limiting the progression of diabetic complications, particularly retinopathy, nephropathy, and neuropathy.6,7 More recently the importance of glycemic control has been demonstrated to have long-term beneficial effects on the risk of cardiovascular disease.8 11 Together this series of reports emphasizes the importance of long-term glycemic control in the health of patients with T1D, although this effect may take decades to become evident. Patients with T1D and ESRD who underwent SPK transplantation were (somewhat surprisingly) identified, in a large proportion of instances, to have evidence of hypercoagulability. This was reported in a publication looking at thromboelastograms that were performed intraoperatively at the time of transplantation and that demonstrated a hypercoagulable pattern in a remarkably high percentage of patients.12 In addition, many patients have a clinical history of clotting (dialysis access, deep venous thrombosis, etc.), or laboratory data suggesting a tendency towards hypercoagulability that includes low PT/INR, elevated fibrinogen, and/or platelet count.12 Furthermore, the pancreas transplant venous system is positioned to develop venous stasis since the portal vein, splenic vein, and superior mesenteric vein are high-capacitance vessels (returning venous blood from the spleen and small bowel), which are only receiving venous return from the pancreas itself when transplanted. The reduced venous flow through these large veins within the pancreas contributes to venous stasis. When combined with endothelial cell damage that occurs in every solid organ transplant, this establishes the three criteria for Virchow’s triad of venous thrombosis (hypercoagulability, venous stasis, and endothelial cell damage), and explains why the incidence of thrombosis of pancreas transplants is the highest of any solid organ. In addition, the pancreas transplant recipient often presents technical challenges since patients with T1D and renal failure often have comorbidities that include atherosclerosis, which may involve the heart as well as peripheral arteries. The pancreas transplant exocrine component is drained either into the small bowel or into the bladder. At the Miami Transplant Institute, bladder drainage has been performed for over 25 years. This allows the drainage of the duodenum and pancreas exocrine secretions into the bladder and is a very safe way to handle the exocrine enzymes. The pH of the urine generally is between 7 and 9, which is protective, keeping the pancreatic exocrine enzymes from becoming activated. The alternative is enteric drainage, which is more physiologic, avoiding the metabolic acidosis that can be a consequence of bladder exocrine drainage. Enteric drainage is performed by most US pancreas transplant programs. Ultimately, since the goal of pancreas transplantation is long-term restoration of euglycemia, the specific technique of exocrine drainage, bladder or enteric, is center-dependent, where the team can determine if their technique associates favorably with long-term pancreas transplant survival. The venous drainage of the pancreas transplant is generally through the systemic system either through the external iliac vein or the inferior vena cava. Other approaches include venous drainage through the mesenteric tree or the portal vein, which ultimately allows the more physiologic first pass of insulin through the liver. However, in studies over time, results have not been shown to be different between venous drainage through mesenteric or systemic tributaries.13 The result of venous drainage through the systemic venous tree, bypassing the liver, is hyper C-peptidemia, generally in the context of euglycemia. It is theoretically possible that the high levels of c-peptide may contribute to maintaining normal HbA1c, in the context of immunosuppressive agents that are associated with insulin resistance (e.g., calcineurin inhibitors (CNI) (tacrolimus and cyclosporine A), steroids, and rapamycin). The arterial in-flow approaches for the pancreas transplant include the creation of a Y-graft that utilizes the donor external iliac artery, which is generally anastomosed to the SMA of the pancreas transplant and the internal iliac artery, which is anastomosed to the splenic artery of the pancreas transplant. The common iliac artery of the donor Y-graft is then anastomosed to the arterial tree of the recipient, generally to the external iliac or common iliac artery. Many variations of this arterial anastomosis have been applied depending on the degree of

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143

atherosclerotic changes within the recipient arterial tree.14 16 In some centers, both kidney and pancreas transplants are placed on the ipsilateral side.17 Immunosuppression in patients who receive an SPK transplant has evolved over the recent decades. A number of antibodies have been used at the time of transplantation for induction therapy.18 These have included anti-CD 25 monoclonal antibodies19,20 (currently the only anti-CD 25 formulation available is basiliximab). At the University at the Miami Transplant Institute, anti-CD 25 monoclonal antibody has been used in combination with the polyclonal, rabbit-derived antibody mixture thymoglobulin for induction therapy.21,22 Other centers have used alemtuzumab for induction.23,24 Maintenance immunosuppression generally includes tacrolimus and mycophenolate mofetil with or without steroids.19,25 In our experience at the Miami Transplant Institute, in a prospective, randomized 10-year study comparing mycophenolate mofetil to rapamycin, the results with rapamycin were significantly better from the standpoint of reduced rate of acute rejection over time.21 Furthermore rapamycin was better tolerated specifically from the standpoint of gastrointestinal function, whereas mycophenolate mofetil generally exacerbated the underlying gastroparesis that occurs in many patients with T1D. The death-censored kidney graft survival was not different between the two groups (Fig. 12.1A), 74% in the rapamycin group versus 70% in the mycophenolate mofetil group. Death-censored pancreas 10-year graft survival (Fig. 12.1B) was 98% in the rapamycin group, whereas for mycophenolate mofetil the 10-year death-censored pancreas transplant survival rate was 84% (P 5 12). This was not statistically significant, however, the rate of acute rejection was statistically significantly better in the rapamycin than the mycophenolate mofetil group.21 With longer followup, the reduced rate of rejection for pancreas transplant recipients treated with rapamycin may result in greater graft survival, and hence longer periods of euglycemia, which may translate to prolonged patient survival.9 From a metabolic standpoint there was also evidence of rapamycin-related insulin resistance. Thus, HbA1c was higher throughout the 10 years of followup in the rapamycin versus mycophenolate mofetil group, with equivalent c-peptide secretion. Nonetheless, the HbA1c in the rapamycin group was normal at all time points over the 10 years.21 Over the course of time there are suggestions that pancreas transplantation may have a positive impact on the complications of T1D. For example, similar to results of the DCCT studies over time, reversal of diabetes changes requires clearly more than 5 years of normoglycemia and oftentimes 10 20 years.26,27 There is evidence for improvement in renal function after pancreas transplantation documented by reduction of proteinuria and stable creatinine clearance.28,29 For those patients without ESRD, a higher percentage of patients experienced improved or stabilized retinopathy after pancreas transplantation alone than intensive insulin therapy.30 There has been some question regarding the optimal form of transplantation involving the kidney and pancreas transplant.31 Much data, in particular recently, suggest that the combination of SPK transplant offers the best outcome for both kidney and pancreas.32,33 However, a living donor kidney offers excellent kidney transplant survival, and, if this is then followed by a pancreas transplant, this can also result in very good kidney and pancreas transplant survival.34,35 The loss of either pancreas or kidney graft has a significant impact on patient survival with kidney loss being more important. The loss of kidney transplant function is predicted to have over five- or sixfold greater importance than the loss of a pancreas transplant.1,2 However, long-term normoglycemia and kidney function is certainly possible with either approach to transplantation.32,36 In most analyses that compare SPK to kidney transplant alone, the incremental improvement in patient survival related to pancreas transplantation survival is modest. Overall survival outcomes are superior for SPK recipients compared with recipients of deceased donor kidneys, but not for recipients of living donor kidney transplants. It should also be mentioned that pancreas transplantation with enteric drainage, pancreas after kidney (PAK) transplantation, is a risk factor for duodenal leak. This may be related to the long-term immunosuppression necessary to preserve renal function in the kidney transplant recipient.37 If the pancreas transplant is performed within 1 year of the kidney transplant, there is a smaller risk of immunosuppression-related complications.

12.2.1 Recurrence of Autoimmunity in the Pancreas Transplant Challenges for pancreas transplantation in the future include recognizing and addressing the recurrence of autoimmunity in the pancreas transplant after transplantation.38 In our experience generally this occurs somewhere between five and up to 20 years after SPK transplantation.39 This has been identified in patients who return with significant hyperglycemia (oftentimes frank diabetic ketoacidosis) in the context of unchanged creatinine and stable measurements of urine amylase, i.e., exocrine pancreas transplant function. This is then

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(A) 100

% Kidney graft survival

90

80 74% 70 70% 60 Rapa (N=84, 10 events)

50

P = .16 MMF (N=86, 18 events)

0 0

12

24

36

48 60 72 84 Months since transplant

96

108

120

(B) 98%

100

90 % Pancreas graft survival

84% 80

70

60

Rapa (N=84, 2 events)

50

P = .12

MMF (N=86, 7 events) 0 0

12

24

36

48 60 72 84 Months since transplant

96

108

120

FIGURE 12.1 Simultaneous pancreas kidney transplant recipient treated with rapamycin versus mycophenolate mofetil. (A) Percent death-censored kidney transplant survival. (B) Percent death-censored pancreas transplant survival.

confirmed by biopsy of the pancreas as well as the kidney transplant, in order to show the autoimmune process in the pancreas (as well as the lack of rejection in the kidney and pancreas) as the explanation for the hyperglycemia. In fact, these biopsies generally demonstrate the presence of insulitis with T-cell infiltrates and variable insulin staining. The T-cell infiltrates generally include CD4 and CD8 T cells. In the most severe case, where beta cell insulin staining is absent, we have demonstrated the presence of insulin staining in the exocrine ductal cells, along with markers of cell proliferation (KI-67).40 We also have demonstrated the importance of measuring autoantibodies, specifically GAD65, IA-2, and ZnT8, which tend to rise between 3 months and several years prior to the development of clinical hyperglycemia (Fig. 12.2). Autoreactive T cells have also been identified in the peripheral blood as well as the pancreas transplant and the peripancreas transplant tissues in those patients who develop recurrence of autoimmunity.41,42 This approach, specifically the potential to identify T-cell subsets and/or biomarkers in the pancreas transplant biopsy, may allow insight into the pathogenesis of T1D.

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145

FIGURE 12.2 Kaplan Meier analysis of T1DR-free survival according to autoantibodies on followup among 193 SPK recipients followed at the University of Miami who either developed T1DR or remained normoglycemic. (A) Autoantibody positivity: positive versus negative, hazard ration (HR)=14.53, P=.0005. (B) Number of autoantibodies: overall, P=.0001; 1 versus 0, HR=3.53, P=.2672; 2 versus 0, HR=14.60, P=.0005*; 3 versus 0, HR=53.88, P , .0001*; 2 versus 1, HR=5.65, P=.0131**; 3 versus 1, HR=17.04, P , .0001*; 3 versus 2, HR=3.00, P=.0261**; P value *was or **was not significant after correction for multiple comparisons. (C) Autoantibody conversion: overall, P , .0001; persistence versus negative, HR=1.88, P=.6486; conversion versus negative, HR=27.11, P , .0001*; conversion versus persistence, HR=15.57, P=.0003*; *significant P values after correction for multiple comparisons. Modified from Vendrame F, Hopfner Y-Y, Diamantopoulos S, et al. Risk factors for type 1 diabetes recurrence in immunosuppressed recipients of simultaneous pancreas-kidney transplants. Am J Transplant 2015. doi:10.1111/ajt.13426, American Journal of Transplantation, John Wiley and Sons Publisher, 1 Copyright 2015 The American Society of Transplantation and the American Society of Transplant Surgeons.

The particular importance of this finding relates to the principle of this book (as applied to the pancreas, not the kidney), which is the potential for bioengineering concepts to lead to replacement of chronically failed organs, and providing a cure for specific, organ-related diseases. From our work, it has become clear that in order to effectively treat T1D, attention must also be directed to treatment of the autoimmune process. It will not be enough to replace the pancreas physically. The issue of recurrence of autoimmunity after pancreas transplantation may help to lead to interventions that might benefit T1D primarily, or contribute to effective pancreas regeneration approaches that incorporate avoidance of recurrent autoimmunity.

12.2.2 Conclusion The challenges for SPK transplantation in the future will be finding the balance between immunosuppression that protects from rejection with resultant long-term graft function and the potential side effects of this

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immunosuppression, which include cancer and infections. Immunosuppression has evolved over the past 20 years so low rates of acute rejection with excellent graft survival and low rates of viral sequelae and lymphoproliferative disorders have been demonstrated. Improved monitoring for both rejection (alloimmunity) and the development of autoimmunity are goals for the future. Ideally, the Holy Grail of transplantation is the development of tolerogenic protocols that may allow lifelong organ function with freedom from chronic immunosuppression.

12.3 KIDNEY COMBINED WITH LIVER TRANSPLANTATION Since its first report in 1984,43 simultaneous liver and kidney transplantation (SLK) has been performed for numerous medical conditions.44 46 First, chronic kidney disease (CKD) may develop during the course of various end-stage liver diseases47; e.g., membranous nephropathy in hepatitis B virus cirrhosis48 and membranoproliferative, fibrillary, and other type of glomerulonephritis in hepatitis C virus cirrhosis49 51 (Table 12.1). Second, liver disease may develop and progress to end-stage liver disease in patients with end-stage kidney disease52; e.g., hepatitis C.53 55 Third, both liver and kidney may be affected by structural diseases; e.g., polycystic liver and kidney disease.56,57 Fourth, metabolic disorders may affect the kidney function as they progress over time58; e.g., primary hyperoxaluria,59 61 methylmalonic academia,62,63 and atypical hemolytic uremic syndrome.64 66 Lastly, advanced liver disease may be accompanied by acute kidney injury (AKI), including hepatorenal syndrome.67,68 This secondary kidney dysfunction is more prevalent than primary kidney disease in adult patients with advanced cirrhosis. Extrarenal vasodilatation in such patients may lead to systemic circulatory dysfunction, intrarenal vasoconstriction, and eventual kidney failure that is refractory to plasma volume expansion. While hepatorenal syndrome accounts for only one-third of AKI in hospitalized patients with cirrhosis,69 the overall incidence of AKI in this population is 20% and the mortality rate reaches 58% at 1 month—a sevenfold increase as compared with patients without kidney injury.70 For these patients with poor prognosis, SLK potentially provides complete recovery of combined organ failure and likely offers survival benefit over liver transplantation alone.71 75 However, not all AKIs in liver transplant candidates become permanent,76 78 nor do all parenchymal kidney diseases progress to CKD requiring renal replacement therapy.79,80 Indeed, the recovery of native kidney function after SLK has been reported.81,82 Furthermore, some of the patients with liver and kidney failure may be too sick to undergo SLK with unacceptably high mortality and morbidity rates.83,84 Of note, the patient and graft survival of SLK recipients is inferior to that of kidney alone or kidney pancreas recipients of contralateral kidney allografts owing to a higher mortality rate during the first year posttransplant.85 Thus the fair and just allocation scheme is desired for kidney, liver, and kidney liver transplant candidates. The model for end-stage liver disease (MELD) scoring system, currently used for adult liver allocation, was validated in the different patient populations with advanced liver disease. It has been accepted as an objective TABLE 12.1

Common Kidney Diseases Associated With Liver Disease

Kidney disease

Liver disease

Membranoproliferative GN

HCV, HBV, PSC, A1AT deficiency

Membranous glomerulopathy

HBV, HCV, PBC, PSC

Diabetic nephropathy

HCV

IgA nephropathy

Alcoholic cirrhosis

Fibrillary GN

HCV

ANCA-associated vasculitis

PBC, PSC

Anti-GBM disease

A1AT deficiency

Autoimmune complex GN

Autoimmune hepatitis

Tubulointerstitial nephritis

PBC, PSC, HCV

A1AT, alpha-1 antitrypsin; ANCA, antineutrophil cytoplasmic autoantibody; GBM, glomerular basement membrane; GN, glomerulonephritis; HBV, hepatitis B; HCV, hepatitis C; PBC, primary biliary cirrhosis; PSC, primary sclerosing cholangitis.

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scale of disease severity and predictor of liver waitlist mortality.86 Since its introduction to liver graft allocation in 2002, the number of adult SLK has increased substantially in the United States, mainly because of prioritization of liver transplant candidates with kidney dysfunction (Fig. 12.3). The percentage of SLK, however, varies significantly among the Organ Procurement and Transplantation Network (OPTN) regions from 3% to 12% of all liver transplantation and from 0% to 44% at a center level.87,88 This nonuniformity raised the concern about over- or underutilization of kidney grafts due to variable selection criteria and different philosophies among transplant centers, because the preferential use of kidneys in liver recipients will increase wait time for solitary kidney transplant candidates. To standardize the evaluation and selection of SLK candidates, the expert panels held several consensus conferences and published recommendations (Table 12.2).88 91 Although the details of these recommendations differ, there are a few key components in common.

12.3.1 The Reversibility of AKI Estimated by the Duration of AKI The initial SLK guideline excluded patients with AKI who are not on dialysis89; most AKI that required dialysis was also considered possibly reversible.76,77,93 SLK was not recommended if the duration of dialysis is short (i.e., for # 6 weeks)89 because a single-center study demonstrated no benefit of SLK over liver transplantation in patients requiring pretransplant dialysis for short time.94 Subsequent SLK guidelines, however, included AKI patients even without requiring dialysis using various definitions of AKI.88,90,91 The difference among the guidelines may be in part attributed to recent increase in waiting time for liver transplant candidates with prolonged kidney insult while waiting. The duration of kidney dysfunction—even without dialysis—was shown to predict posttransplant kidney function,95,96 and a certain proportion of patients with pretransplant kidney dysfunction will progress to consideration for kidney transplantation.96,97 There is no universal consensus as to the duration of AKI beyond which no kidney recovery is possible. It is difficult to set single thresholds for recoverability prediction because the AKI reversibility may also be affected by etiology of AKI and other coexisting conditions.96 99 For example, hepatitis C virus infection and diabetes mellitus are associated with post liver transplant kidney failure.79,100 Thus it is probably reasonable to use a longer AKI threshold (e.g., 8 12 weeks) to allocate a kidney graft for patients without other comorbid conditions and a shorter AKI threshold (e.g., 4 6 weeks) for patients with comorbidities that can chronically affect kidney function.

600

10

8 7

400

6 300

5 4

200

3 2

100

% Liver–kidney transplant

Number of liver–kidney transplant

9 500

1 0

20

00 20 01 20 02 20 03 20 04 20 05 20 06 20 07 20 08 20 09 20 10 20 11 20 12 20 13 20 14

0

Year of transplant

FIGURE 12.3 Total number and percentage of simultaneous liver kidney transplantation in the United States. Columns indicate the number of liver kidney transplants; a solid line indicates percentage of liver kidney transplants of total deceased-donor liver transplants; and a broken line indicates percentage of liver kidney transplants of total deceased-donor kidney transplants. Organ Procurement and Transplantation Network (OPTN, http://optn.transplant.hrsa.gov) accessed on July 7, 2015. Note the model for end-stage liver disease (MELD) prioritization for liver transplantation was implemented in February 2002.

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TABLE 12.2

Published Guidelines on Simultaneous Liver Kidney Transplantation

References

Recommendations for clinical practice

89

Davis et al.

Eason et al.88

a. b. c. d. a. b. c. d. e.

Charlton et al.90

f. a. b. c. d.

Nadim et al.91

e. f. a.

b.

Martin et al.92

a. b. c.

Patients with CKD with a measured CCr of # 30 mL/min Patients with AKI and/or HRS on dialysis for $ 6 weeks SLK not recommended in patients with AKI not requiring dialysis Patients with prolonged AKI with kidney biopsy showing fixed renal damage Patients with ESRD (with cirrhosis and symptomatic portal hypertension or hepatic vein wedge pressure gradient $ 10 mmHg) Patients with CKD with GFR # 30 mL/min Patients with AKI or HRS with creatinine $ 2 mg/dL and dialysis $ 8 weeks Patients with CKD and kidney biopsy demonstrating .30% glomerulosclerosis or 30% fibrosis Other criteria to be considered in granting exceptions are the presence of comorbidities such as diabetes, hypertension, or other preexisting renal disease, along with proteinuria, renal size, and duration of elevated creatinine $ 2 mg/dL Net benefit should be considered when evaluating patients .65 year old, given the unfavorable outcomes in the elderly Patients with ESRD on dialysis Patients with CKD not on dialysis but with GFR ,30 mL/min (by MDRD-6 equation or direct measurement) and proteinuria ( . 3 g/day with 24-h measurement or urine protein/creatinine ratio . 3) Patients with sustained AKI requiring dialysis for .6 weeks (at least 2 times per week) Patients with sustained AKI not requiring dialysis but with GFR ,25 mL/min (by MDRD-6 equation or direct measurement) for .6 weeks Patients with categories (c) or (d) above for total of .6 weeks Patients with metabolic or genetic diseases Patients with persistent AKI for $ 4 weeks with one of the following: i. Stage 3 AKI as defined by modified RIFLE, i.e., a threefold increase in creatinine from baseline, creatinine $ 4 mg/dL with an acute increase of $ 0.5 mg/dL or on dialysis ii. eGFR # 35 mL/min (MDRD-6 equation) or GFR # 25 mL/min (iothalamate clearance) Patients with CKD as defined by the National Kidney Foundation for 3 months with one of the following: i. eGFR # 40 mL/min (MDRD-6 equation) or GFR # 30 mL/min (iothalamate clearance) ii. Proteinuria $ 2 g/day iii. Kidney biopsy showing .30% global glomerulosclerosis or .30% interstitial fibrosis iv. Metabolic disease Patients with CKD with GFR ,30 mL/min Patients with AKI with dialysis .8 weeks Patients with extensive glomerulosclerosis

AKI, acute kidney injury; CCr, creatinine clearance; CKD, chronic kidney disease; ESLD, end-stage liver disease; ESRD, end-stage renal disease; GFR, glomerular filtration rate; HRS, hepatorenal syndrome; MDRD, modification of diet in renal disease; RIFLE, risk, injury, failure, loss of kidney function, and end-stage kidney disease; Scr, serum creatinine; SLK, simultaneous liver kidney.

12.3.2 The Severity of Chronic Kidney Disease Defined by the Estimated Glomerular Filtration Rate While patients with CKD stage V (estimated glomerular filtration rate (eGFR) , 15 mL/minute/1.73 m2 for at least 3 months) require an assessment whether to start dialysis or to receive kidney transplantation,101 clinical guidelines do not recommend preemptive solitary kidney transplantation when eGFR is above 20 mL/minute.101,102 This recommendation is supported by registry data and multicenter data analyses.103,104 Nevertheless, the SLK guidelines include patients with higher pretransplant eGFR (i.e., 30 mL/minute or higher) in the SLK candidacy.88,90 92 One reason to include such patients is inaccuracy to estimate kidney function in patients with advanced liver disease using conventional methods. Even a small increase in serum creatinine levels may indicate significant deterioration of kidney function; the MDRD and Cockcroft Gault formulae were shown to overestimate the kidney function in this population.105 107 Another reason that justifies the use of higher eGFR cutoff is the impact of nephrotoxic medication, which will be used after transplantation; e.g., calcineurin inhibitors (CNI: cyclosporine, tacrolimus).90 Although it is uncommon,88,108 patients with marginal pretransplant kidney function may progress to CKD and end-stage kidney disease after liver transplantation,79,96,97,109 which translate into poor patient survival.99,100 A recent registry data analysis demonstrated significantly inferior survival during the first 4 months after liver transplantation in patients who were listed for SLK but received an isolated liver graft.74 While several risk scores were proposed to predict posttransplant kidney function,80,110 no single method is universally accepted thus far.

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12.3 KIDNEY COMBINED WITH LIVER TRANSPLANTATION

12.3.2.1 Histological Changes of Native Kidneys In addition to the standard kidney imaging studies such as Doppler ultrasonography and kidney nuclear scan, kidney biopsy may help assess the chronicity and reversibility of the kidney disease. Besides conventional percutaneous needle biopsy, transjugular biopsy has been shown to be safe and efficacious to evaluate injury to the kidney in liver transplant candidates.111,112 Open wedge or needle biopsy performed during liver transplantation may help decide if a patient also requires a kidney transplant. We have, in selected cases, intentionally delayed kidney transplantation after liver transplantation,113 while preserving the kidney graft on a perfusion machine.114 This will allow us to hemodynamically stabilize a patient and evaluate permanent kidney biopsy sections if the reversibility of kidney dysfunction is uncertain. This strategy did not adversely affect the patient survival (Fig. 12.4). Often pathological findings of kidney biopsy correlate with impaired kidney function; however, the amount of histological changes (e.g., interstitial fibrosis, glomerulosclerosis) required to cause irreversible kidney failure is controversial. Studies on age-related glomerulosclerosis have demonstrated significant glomerular changes in healthy subjects older than 50 years.115,116 Although the degree of interstitial fibrosis and glomerulosclerosis may be associated with the impaired kidney function,117,118 a single cutoff point (i.e., 30% for both interstitial fibrosis and glomerulosclerosis used in the guidelines88,91) may not adequately classify the severity and irreversibility of functional damage, implying the need for a composite histological scoring system similar to donor kidney biopsy grading.119,120 SLK likely benefits many of the patients who have end-stage liver and kidney disease on dialysis as compared with receiving isolated liver transplantation and remaining on dialysis.71 73 However, the overall outcome of SLK has declined since the introduction of MELD.121 The etiologies of kidney dysfunction and liver disease have a significant impact on the transplant outcome.122,123 For example, hepatitis C virus infection—the leading cause of liver disease requiring liver transplantation—has been identified as a poor prognostic factor of the SLK outcome.113,123 125 For a patient with hepatitis C and other comorbidities, the transplant risk may be exceptionally high and the benefit of SLK (or the additional kidney to the liver transplant) may be minimal. If kidney grafts that are lost early in SLK recipients (i.e., death with functioning graft) were allocated to patients waiting for a solitary kidney graft, the net societal benefit could be greater.126 Until the newer treatments for hepatitis C totally shift the paradigm,127,128 it is important to take these factors into consideration. Similarly, the donor factors affect the SLK outcomes, raising the debate as to whether marginal donors such as donors after circulatory death should be used for SLK.129,130

100

% Patient survival

90

80

70

60

Delta CIT > 12 h (N=28, 6 events)

50

Delta CIT ≤ 12 h (N=38, 12 events) 0 0

4

8

12 16 Months since transplant

20

24

FIGURE 12.4 The patient survival after combined liver and kidney transplantation at the University of Miami/Jackson Memorial Hospital, stratified by the delta cold ischemia time (delta CIT: kidney cold ischemia time minus liver cold ischemia time). When a patient is hemodynamically unstable after liver transplantation or the reversibility of kidney dysfunction is uncertain, kidney transplantation from the same donor was intentionally delayed, while preserving the kidney graft on a perfusion machine. Intraoperative kidney biopsy was performed in selected cases and permanent sections were reviewed to determine the need for kidney transplantation. Additional ischemia time to the kidney did not alter the patient outcome.

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12. KIDNEY WITH OTHER ORGAN TRANSPLANT

Modifiable factors, such as immunosuppressive treatment, need to be optimized to maximize the outcome. Because infection is the major cause of morbidity and mortality, it may be important to avoid overimmunosuppression caused by depleting antibody induction.131,132 The proper management of CNI and other immunosuppressive medication is also the key to maintaining kidney function after SLK or liver transplantation.90,133,134 While the introduction of CNIs significantly improved overall survival in virtually all types of transplantation, their well-known side effect—nephrotoxicity—is the dominant causative factor for kidney disorders in nonrenal transplant recipients.135 The early posttransplant exposure to CNI can render healing from AKI difficult. Kidney function declines to 60% of pretransplant after liver transplantation in patients with normal kidney function.76,79 While mechanisms of functional and structural nephrotoxicity by CNIs have been well studied,136 methods to predict or prevent nephrotoxicity have not been established. Theoretically, CNIs and other classes of immunosuppressive or antiinflammatory agents may stabilize primary kidney diseases of liver transplant patients; however, the effect of CNI in this setting is still unclear.47 Conversely, renal vasoconstriction causing ischemic kidney dysfunction associated with severe liver failure will be exacerbated by CNIs. Optimization of immunosuppression using a nonnephrotoxic combination may decrease the need for renal replacement therapy in combination with or after the liver transplantation. However, when pretransplant kidney failure persists or new onset kidney failure develops after liver transplantation, kidney transplantation provides a better survival rate than remaining on dialysis.109,137 In contrast to the increasing number of SLK in adult patients, SLK is still uncommon in children and adolescents.46,138 Metabolic liver diseases causing end-stage kidney disease, especially primary hyperoxaluria, are the most common indication. Primary hyperoxaluria type 1 is an autosomal recessive disorder by gene mutations encoding liver-specific peroxisomal enzyme. Overproduced oxalate is excreted in the urine, where it forms oxalate stone, nephrocalcinosis, and eventual kidney failure. Unless preemptive liver transplantation is performed before the development of kidney disease,139 SLK (or sequential) transplantation is often required.59 61,140,141 Autosomal recessive polycystic kidney disease—the second most common indication—is caused by fibrocystin gene mutations and leads to end-stage kidney and liver disease.56 Because the disease manifestations can vary in kidney and liver, an individualized assessment and management is required whether to transplant solitary kidney or SLK.142 The SLK outcome in children depends on the cause of liver and kidney disease; generally children with a metabolic disease have a good prognosis. However, transplantation of a reduced-size graft with an adult kidney into a small child can still present a technical challenge.138,143

12.4 KIDNEY COMBINED WITH HEART TRANSPLANTATION Patients with concomitant end-stage heart failure and irreversible renal insufficiency are candidates for simultaneous heart and kidney transplantation (SHK). While SHK is relatively uncommon—around 100 cases annually, accounting for only 1% of deceased donor kidney transplants in the United States—the actual number of SHK is increasing (Fig. 12.5) in response to the increasing number of waitlisted patients.144 Similar to other nonrenal transplant candidates, heart transplant candidates with renal insufficiency have higher waitlist mortality. In US registry data analyses, the hazard ratios (HR) of waitlist mortality or becoming too sick to transplant were 1.6 for CKD stage III and 3.2 for CKD stage IV/V or dialysis-dependent patients.145 The 3-month and 1-year waitlist mortality rates of SHK waitlisted patients were 7% and 19% for nondialysis-dependent patients and 21% and 31% for dialysis-dependent patients, respectively.144 In contrast to dismal waitlist mortality, multiple studies demonstrated that the posttransplant survival of SHK recipients was identical to isolated heart transplant recipients, reaching 87% at 1 year and 75% at 5 years; SHK seems to mitigate the negative effect of renal insufficiency without conferring significant procedural risks.144,146 148 In a time-varying Cox proportional hazard model, SHK was shown to have greater mortality reduction (HR: 0.25) than isolated heart transplantation (HR: 0.67) from time of SHK listing.144 Although the impact of SHK is striking in patients who are on dialysis, the long-term impact of SHK is modest in patients who are not on dialysis.147 For example, 1-year and 5-year survival rates were significantly higher in SHK recipients than propensity-matched heart-alone recipients with dialysis dependence (84% vs 69% and 73% vs 51%, respectively), whereas the differences were small between SHK and heartalone recipients without dialysis dependence (91% vs 81% and 80% vs 69%, respectively).144 Therefore, despite potential benefits of SHK, the routine use of kidney grafts in heart transplant candidates with moderate renal insufficiency is not recommended, ensuring the fair utilization of the limited resource of kidneys for kidneyalone transplant candidates. When SHK candidates (eGFR , 33 mL/minute) are stratified by recipients’ risk factors, only low-risk patients were shown to clearly benefit from SHK over heart transplant alone, underscoring the importance of patient selection.149 Additionally, some of the renal insufficiency is considered reversible after

I. KIDNEY TRANSPLANTATION

151

12.5 PROTECTION OF KIDNEY BY OTHER ORGANS

120

10

8 7

80

6 60

5 4

40

3 2

20

% Heart–kidney transplant

Number of heart–kidney transplant

9 100

1 0

20 00 20 01 20 02 20 03 20 04 20 05 20 06 20 07 20 08 20 09 20 10 20 11 20 12 20 13 20 14

0

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FIGURE 12.5 Total number and percentage of simultaneous heart kidney transplantation in the United States. Columns indicate the number of heart kidney transplants; a solid line indicates percentage of heart kidney transplants of total heart transplants; and a broken line indicates percentage of heart kidney transplants of total deceased-donor kidney transplants. Organ Procurement and Transplantation Network (OPTN, http://optn.transplant.hrsa.gov) accessed on July 7, 2015.

isolated heart transplantation (i.e., certain forms of cardiorenal syndrome).150 While no universal criteria for SHK indications have been established,151,152 a thorough assessment of chronicity using kidney imaging studies and biopsies may reduce unnecessary kidney transplantation. We implemented staged SHK from a single donor using machine preservation of the kidney in order to hemodynamically stabilize the patient after heart transplantation and to increase the time to assess the kidney status.153 The efficacy of other strategies, such as using a ventricular assist device, to maintain or enhance kidney function has yet to be evaluated.154 As is the case with kidney failure after liver transplantation, kidney failure after heart transplantation is a negative predictor of posttransplant survival, and a kidney transplant after heart transplantation offers significant survival benefit.137,155,156 Because dialysis time prior to kidney transplantation affects graft survival,157 it may be reasonable to prioritize kidney transplantation in a heart transplant recipient whose marginal pretransplant kidney function is unlikely to recover after heart transplantation. Interestingly, there seems to be no difference in kidney graft survival by the quality of the kidney donor in prior heart transplant recipients; living donor, standard criteria deceased donor, and even extended criteria deceased donor transplants seem to provide similar advantages.157 Thus in a prior heart transplant recipient, shorter waiting time by accepting extended criteria donors may be beneficial, when no living donor is available157,158.

12.5 PROTECTION OF KIDNEY BY OTHER ORGANS When a kidney is transplanted with another organ, a lower rejection rate and prolonged graft survival have been observed. Traditionally, this observation was seen when the kidney was transplanted with the liver, establishing its reputation as an immunoprotective organ.159 161 Even in patients with positive pretransplant crossmatch, hyperacute rejection may not occur and the crossmatch may become negative.162 164 The kidney grafts last longer in liver transplant recipients from the same donor,85,165 and kidney graft rejection rates are lower when a liver allograft is placed concurrently,161 offering immunological advantage in SLK patients. This phenomenon was also observed in pediatric patients.166,167 Olausson et al. transplanted a partial auxiliary liver merely to facilitate the kidney transplantation in highly sensitized patients together with a kidney from the same crossmatch-positive donor.168 In five of seven cases, crossmatch turned negative with excellent graft function and no rejection, whereas two remained crossmatch positive with one primary nonfunction and one early graft failure. In contrast, others reported that liver is not always protective against kidney rejection.169 172 While the liver may help improve kidney graft survival in sensitized patients,173,174 Dar et al. demonstrated persistence of preexisting HLA class II antibodies and antibody-mediated rejections in SLK patients.175 Preexisting or de novo class II

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donor specific antibodies were also shown to be an independent predictor of patient death and liver allograft loss.176 Interestingly, the protection is also observed with nonliver organs. Opelz et al. demonstrated prolongation of long-term kidney graft survival not only in SLK recipients but also in SHK recipients.177 Heart seems to protect kidney and vice versa.146,178,179 Using the US registry data, Rana et al. also demonstrated the protective effect of a liver, heart, and kidney graft for the other cotransplanted organ, but failed to demonstrate the protective effect of pancreas or intestine grafts.180 Several mechanisms have been proposed to explain how one organ protects the other, but the exact mechanism remains to be explored further.181 184

Acknowledgements The authors would like to express their appreciation to Ms. Zulimi Pastor Clark for her persistence and patience in the preparation of this manuscript.

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126. Kiberd B, Skedgel C, Alwayn I, Peltekian K. Simultaneous liver kidney transplantation: a medical decision analysis. Transplantation 2011;91(1):121 7. 127. Coilly A, Roche B, Duclos-Vallee JC, Samuel D. Management of post-transplant hepatitis C in the direct antiviral agents era. Hepatol Int 2015;9(2):192 201. 128. Price JC, Terrault NA. Treatment of hepatitis C in liver transplant patients: interferon out, direct antiviral combos in. Liver Transpl 2015;21(4):423 34. 129. Wadei HM, Bulatao IG, Gonwa TA, et al. Inferior long-term outcomes of liver-kidney transplantation using donation after cardiac death donors: single-center and organ procurement and transplantation network analyses. Liver Transpl 2014;20(6):728 35. 130. Alhamad T, Spatz C, Uemura T, Lehman E, Farooq U. The outcomes of simultaneous liver and kidney transplantation using donation after cardiac death organs. Transplantation 2014;98(11):1190 8. 131. Hibi T, Nishida S, Sageshima J, et al. Excessive immunosuppression as a potential cause of poor survival in simultaneous liver/kidney transplantation for hepatitis C. Transpl Int 2014;27(6):606 16. 132. Uemura T, Schaefer E, Hollenbeak CS, Khan A, Kadry Z. Outcome of induction immunosuppression for liver transplantation comparing anti-thymocyte globulin, daclizumab, and corticosteroid. Transpl Int 2011;24(7):640 50. 133. McKenna GJ, Trotter JF, Klintmalm E, et al. Limiting hepatitis C virus progression in liver transplant recipients using sirolimus-based immunosuppression. Am J Transplant 2011;11(11):2379 87. 134. Manzia TM, Angelico R, Toti L, et al. Long-term, maintenance MMF monotherapy improves the fibrosis progression in liver transplant recipients with recurrent hepatitis C. Transpl Int 2011;24(5):461 8. 135. Ojo AO. Renal disease in recipients of nonrenal solid organ transplantation. Semin Nephrol 2007;27(4):498 507. 136. Campistol JM, Sacks SH. Mechanisms of nephrotoxicity. Transplantation 2000;69(12 Suppl):SS5 10. 137. Cassuto JR, Reese PP, Sonnad S, et al. Wait list death and survival benefit of kidney transplantation among nonrenal transplant recipients. Am J Transplant 2010;10(11):2502 11. 138. Jalanko H, Pakarinen M. Combined liver and kidney transplantation in children. Pediatr Nephrol 2014;29(5):805 14 quiz 12. 139. Kemper MJ. The role of preemptive liver transplantation in primary hyperoxaluria type 1. Urol Res 2005;33(5):376 9. 140. Hoppe B. An update on primary hyperoxaluria. Nat Rev Nephrol 2012;8(8):467 75. 141. Malla I, Lysy PA, Godefroid N, et al. Two-step transplantation for primary hyperoxaluria: cadaveric liver followed by living donor related kidney transplantation. Pediatr Transplant 2009;13(6):782 4. 142. Telega G, Cronin D, Avner ED. New approaches to the autosomal recessive polycystic kidney disease patient with dual kidney-liver complications. Pediatr Transplant 2013;17(4):328 35. 143. Sakamoto S, Kasahara M, Fukuda A, et al. Pediatric liver-kidney transplantation for hepatorenal fibrocystic disease from a living donor. Pediatr Transplant 2012;16(1):99 102. 144. Schaffer JM, Chiu P, Singh SK, Oyer PE, Reitz BA, Mallidi HR. Heart and combined heart-kidney transplantation in patients with concomitant renal insufficiency and end-stage heart failure. Am J Transplant 2014;14(2):384 96. 145. Singh TP, Almond CS, Taylor DO, Graham DA. Decline in heart transplant wait list mortality in the United States following broader regional sharing of donor hearts. Circ Heart Fail 2012;5(2):249 58. 146. Narula J, Bennett LE, DiSalvo T, Hosenpud JD, Semigran MJ, Dec GW. Outcomes in recipients of combined heart-kidney transplantation: multiorgan, same-donor transplant study of the International Society of Heart and Lung Transplantation/United Network for Organ Sharing Scientific Registry. Transplantation 1997;63(6):861 7. 147. Gill J, Shah T, Hristea I, et al. Outcomes of simultaneous heart-kidney transplant in the US: a retrospective analysis using OPTN/UNOS data. Am J Transplant 2009;9(4):844 52. 148. Karamlou T, Welke KF, McMullan DM, et al. Combined heart-kidney transplant improves post-transplant survival compared with isolated heart transplant in recipients with reduced glomerular filtration rate: analysis of 593 combined heart-kidney transplants from the United Network Organ Sharing Database. J Thorac Cardiovasc Surg 2014;147(1):456 61 e1. 149. Russo MJ, Rana A, Chen JM, et al. Pretransplantation patient characteristics and survival following combined heart and kidney transplantation: an analysis of the United Network for Organ Sharing Database. Arch Surg 2009;144(3):241 6. 150. Cruz DN, Bagshaw SM. Heart-kidney interaction: epidemiology of cardiorenal syndromes. Int J Nephrol 2010;2011:351291. 151. Lewis RM, Verani RR, Vo C, et al. Evaluation of chronic renal disease in heart transplant recipients: importance of pretransplantation native kidney histologic evaluation. J Heart Lung Transplant 1994;13(3):376 80. 152. Labban B, Arora N, Restaino S, Markowitz G, Valeri A, Radhakrishnan J. The role of kidney biopsy in heart transplant candidates with kidney disease. Transplantation 2010;89(7):887 93. 153. El Hinnawi A, Sageshima J, Ciancio G, et al. Renal graft outcome after single-donor staged heart-kidney transplantation using pulsatile machine perfusion of the renal grafts. Am J Transplant 2013;13:48 [Abstract]. 154. Singh M, Shullo M, Kormos RL, et al. Impact of renal function before mechanical circulatory support on posttransplant renal outcomes. Ann Thorac Surg 2011;91(5):1348 54. 155. Bourge RC, Naftel DC, Costanzo-Nordin MR, et al. Pretransplantation risk factors for death after heart transplantation: a multiinstitutional study. The Transplant Cardiologists Research Database Group. J Heart Lung Transplant 1993;12(4):549 62. 156. Lonze BE, Warren DS, Stewart ZA, et al. Kidney transplantation in previous heart or lung recipients. Am J Transplant 2009;9(3):578 85. 157. Cassuto JR, Reese PP, Bloom RD, et al. Kidney transplantation in patients with a prior heart transplant. Transplantation 2010;89(4):427 33. 158. Srinivas TR, Stephany BR, Budev M, et al. An emerging population: kidney transplant candidates who are placed on the waiting list after liver, heart, and lung transplantation. Clin J Am Soc Nephrol 2010;5(10):1881 6. 159. Calne RY, Sells RA, Pena JR, et al. Induction of immunological tolerance by porcine liver allografts. Nature 1969;223(5205):472 6. 160. Fung J, Makowka L, Tzakis A, et al. Combined liver-kidney transplantation: analysis of patients with preformed lymphocytotoxic antibodies. Transplant Proc 1988;20(Suppl. 1):88 91. 161. Rasmussen A, Davies HF, Jamieson NV, Evans DB, Calne RY. Combined transplantation of liver and kidney from the same donor protects the kidney from rejection and improves kidney graft survival. Transplantation 1995;59(6):919 21.

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162. Morrissey PE, Gordon F, Shaffer D, et al. Combined liver-kidney transplantation in patients with cirrhosis and renal failure: effect of a positive cross-match and benefits of combined transplantation. Liver Transpl Surg 1998;4(5):363 9. 163. Neumann UP, Lang M, Moldenhauer A, et al. Significance of a T-lymphocytotoxic crossmatch in liver and combined liver-kidney transplantation. Transplantation 2001;71(8):1163 8. 164. Creput C, Durrbach A, Samuel D, et al. Incidence of renal and liver rejection and patient survival rate following combined liver and kidney transplantation. Am J Transplant 2003;3(3):348 56. 165. Simpson N, Cho YW, Cicciarelli JC, Selby RR, Fong TL. Comparison of renal allograft outcomes in combined liver-kidney transplantation versus subsequent kidney transplantation in liver transplant recipients: analysis of UNOS Database. Transplantation 2006; 82(10):1298 303. 166. de la Cerda F, Jimenez WA, Gjertson DW, Venick R, Tsai E, Ettenger R. Renal graft outcome after combined liver and kidney transplantation in children: UCLA and UNOS experience. Pediatr Transplant 2010;14(4):459 64. 167. Rogers J, Bueno J, Shapiro R, et al. Results of simultaneous and sequential pediatric liver and kidney transplantation. Transplantation 2001;72(10):1666 70. 168. Olausson M, Mjornstedt L, Norden G, et al. Successful combined partial auxiliary liver and kidney transplantation in highly sensitized cross-match positive recipients. Am J Transplant 2007;7(1):130 6. 169. Eid A, Moore SB, Wiesner RH, DeGoey SR, Nielson A, Krom RA. Evidence that the liver does not always protect the kidney from hyperacute rejection in combined liver-kidney transplantation across a positive lymphocyte crossmatch. Transplantation 1990;50(2):331 4. 170. Katznelson S, Cecka JM. The liver neither protects the kidney from rejection nor improves kidney graft survival after combined liver and kidney transplantation from the same donor. Transplantation 1996;61(9):1403 5. 171. Askar M, Schold JD, Eghtesad B, et al. Combined liver-kidney transplants: allosensitization and recipient outcomes. Transplantation 2011;91(11):1286 92. 172. Nilles KM, Krupp J, Lapin B, Sustento-Reodica N, Gallon L, Levitsky J. Incidence and impact of rejection following simultaneous liverkidney transplantation. J Hepatol 2015;62(2):340 5. 173. Hanish SI, Samaniego M, Mezrich JD, et al. Outcomes of simultaneous liver/kidney transplants are equivalent to kidney transplant alone: a preliminary report. Transplantation 2010;90(1):52 60. 174. Leca N, Warner P, Bakthavatsalam R, et al. Outcomes of simultaneous liver and kidney transplantation in relation to a high level of preformed donor-specific antibodies. Transplantation 2013;96(10):914 18. 175. Dar W, Agarwal A, Watkins C, et al. Donor-directed MHC class I antibody is preferentially cleared from sensitized recipients of combined liver/kidney transplants. Am J Transplant 2011;11(4):841 7. 176. O’Leary JG, Gebel HM, Ruiz R, et al. Class II alloantibody and mortality in simultaneous liver-kidney transplantation. Am J Transplant 2013;13(4):954 60. 177. Opelz G, Margreiter R, Dohler B. Prolongation of long-term kidney graft survival by a simultaneous liver transplant: the liver does it, and the heart does it too. Transplantation 2002;74(10):1390 4 discussion 70 71. 178. Luckraz H, Parameshwar J, Charman SC, Firth J, Wallwork J, Large S. Short- and long-term outcomes of combined cardiac and renal transplantation with allografts from a single donor. J Heart Lung Transplant 2003;22(12):1318 22. 179. Pinderski LJ, Kirklin JK, McGiffin D, et al. Multi-organ transplantation: is there a protective effect against acute and chronic rejection? J Heart Lung Transplant 2005;24(11):1828 33. 180. Rana A, Robles S, Russo MJ, et al. The combined organ effect: protection against rejection? Ann Surg 2008;248(5):871 9. 181. Sumimoto R, Kamada N. Specific suppression of allograft rejection by soluble class I antigen and complexes with monoclonal antibody. Transplantation 1990;50(4):678 82. 182. Murase N, Starzl TE, Tanabe M, et al. Variable chimerism, graft-versus-host disease, and tolerance after different kinds of cell and whole organ transplantation from Lewis to brown Norway rats. Transplantation 1995;60(2):158 71. 183. Cre´put C, Durrbach A, Menier C, et al. Human leukocyte antigen-G (HLA-G) expression in biliary epithelial cells is associated with allograft acceptance in liver-kidney transplantation. J Hepatol 2003;39(4):587 94. 184. Ingelsten M, Gustafsson K, Olausson M, Haraldsson B, Karlsson-Parra A, Nystrom J. Rapid increase of interleukin-10 plasma levels after combined auxiliary liver-kidney transplantation in presensitized patients. Transplantation 2014;98(2):208 15.

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C H A P T E R

13 Pediatric Renal Transplantation Ashton Chen, Jen-Jar Lin and Andrew M. South Wake Forest Baptist Health, Winston-Salem, NC, United States

ABBREVIATIONS AAMR ACR AMR AR ATN BKVN BP CAMR CAN CCR CMV CNI CVD CVP DD DM DSA EBV ELISA ESRD FSGS GI GSR HUS IL-2 IVIG LD MMF MPGN mTOR NAPRTCS OPTN PRA PSR PTLD

acute antibody-mediated rejection acute cellular rejection antibody-mediated rejection acute rejection acute tubular necrosis BK virus nephropathy blood pressure chronic antibody-mediated rejection chronic allograft nephropathy chronic cellular rejection cytomegalovirus calcineurin inhibitor cardiovascular disease central venous pressure deceased-donor diabetes mellitus donor-specific antibodies Epstein Barr virus enzyme-linked immunosorbent assay end-stage renal disease focal segmental glomerulosclerosis gastrointestinal graft survival rate hemolytic uremic syndrome interleukin-2 intravenous immunoglobulin living-donor mycophenolate mofetil membranoproliferative glomerulonephritis mammalian target of the rapamycin North American Pediatric Renal Trials and Collaborative Studies Organ Procurement and Transplantation Network panel-reactive antibodies patient survival rate posttransplant lymphoproliferative disorder

Kidney Transplantation, Bioengineering, and Regeneration. DOI: http://dx.doi.org/10.1016/B978-0-12-801734-0.00013-8

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© 2017 Elsevier Inc. All rights reserved.

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Scientific Registry of Transplant Recipients United Network for Organ Sharing United States Renal Data System urinary tract infection vesicoureteral reflux

13.1 INTRODUCTION There are many benefits to transplant compared to dialysis in children, including improved quality of life and survival and decreased risk of cardiovascular disease, metabolic bone disease, and growth delay associated with chronic renal failure and dialysis (see the following paragraphs). The precise selection criteria for pediatric kidney transplantation vary from center to center, but generally include up-to-date immunizations, particularly with live vaccine (varicella and MMR), stable home environment and social support network, absence of active infection or malignancy, and a urinary bladder with adequate capacity, high compliance, and efficient emptying by spontaneous voiding or intermittent catheterization. The incidence of end-stage renal disease (ESRD) in children is 16 per 1 million in Whites, 15 per 1 million in African Americans, and 18 per 1 million in other races.1 There is an increased incidence of ESRD with increasing age, with the highest rate in adolescents age 15 19 (21 per 1 million). Cystic, hereditary, and congenital kidney diseases are the most common causes of pediatric ESRD in the United States (34%) while primary glomerular disease accounts for 22% and secondary glomerulonephritis/vasculitis accounts for 11% of cases.1 The etiology of ESRD differs between White and African American children. The most common causes of ESRD in Whites are renal aplasia/hypoplasia/dysplasia (16%), obstructive uropathy (15%), and focal segmental glomerulosclerosis (FSGS) (12%). In African American recipients, FSGS is the most common cause (23%), followed by obstructive uropathy (15%) and renal aplasia/hypoplasia/dysplasia (13%). Causes of ESRD are similar between Hispanic and White children.2 The survival of pediatric renal grafts in earlier years was meager due to complications from allograft thrombosis and limited immunosuppression,3 but it has improved since the 1980s, as indicated in the 1987 2007 data from the North American Pediatric Renal Trials and Collaborative Studies (NAPRTCS), which demonstrated an improvement in 10-year patient survival rate and graft survival rate (GSR) in both living-donor (LD) and deceased-donor (DD) transplantation.4 Similarly, the short- and long-term survival of pediatric renal grafts has improved in the past 25 years according to the 1987 2012 data from Scientific Registry of Transplant Recipients.5 Furthermore, high-risk pediatric recipients based on peak panel-reactive antibodies (PRA), previous transplant history, and human leukocyte antigen (HLA) mismatch have had a similar improvement in death-censored graft survival as low-risk groups, while highly sensitized pediatric recipients experienced a better improvement in PSR.5 The implementation of Share-35 policy in 2005 by American Organ Procurement and Transplantation Network has changed the donor characteristics of pediatric recipients. With this policy, the priority for kidneys from deceased donors less than 35 years old was assigned to recipients less than 18 years old, zero mismatch transplants, recipients with a PRA greater than 80%, or candidates receiving a kidney and a nonrenal organ.6 It has increased HLA-mismatching and DD grafts for pediatric recipients.7,8 According to a 2010 NAPRTCS report, the age distribution of recipients remained unchanged with 13 17 years being the most common age at transplantation (39.2%), followed by 6 12 years (32.8%), 2 5 years (14.7%), and less than 1 year (5.3%). Recipients were 59% male, 59% Whites, 17% Hispanics, 17% African Americans, and 7% other races. Adolescents were more likely to receive a DD graft. Children less than 5 years old were more likely to receive a LD graft. LD transplant increased from 43% in 1987 to 61% in 2001 but it decreased to its lowest point in 2007 at 37%, likely related to Share-35 policy. About 24% primary transplants were preemptive. Among them, Whites were 31%, African Americans 13%, and Hispanics 16%.4 Many studies have demonstrated racial and ethnic minority pediatric patients in the United States and other countries have a longer waiting time for transplantation and lower rates of preemptive and LD transplantation.9,10 Main causes of death in pediatric recipients are infection (28.5%), cardiopulmonary events (14.7%), malignancy (11.3%), and dialysis-related complications (3.1%).4 The primary causes of graft failure are chronic rejection (35%) and acute rejection (13%). Risk factors for a decreased GSR include DD kidney, African Americans, prior dialysis, and prior transplant.4 A lower GSR in adolescents,5,11 13 female recipients,5 pretransplant dialysis,5,14 and recipients with FSGS5,12,15 was also reported in other studies. Compared with Whites, African Americans have a lower 3- and 5-year GSR.16 Analysis of nearly 14,000 pediatric kidney transplant recipients from the United States Renal

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Data System demonstrated approximately a twofold higher rate of graft loss in African Americans compared with Whites.17 Reasons for racial disparities in transplant outcomes are unclear and require further study.

13.2 SURGICAL ASPECT OF PEDIATRIC RENAL TRANSPLANTATION 13.2.1 Native Nephrectomy Prior to Transplantation Data in pediatric pretransplant native nephrectomy are limited. Polyuria is not an indication for native nephrectomy since it may interfere with fluid management and blood pressure control during dialysis. However, there are circumstances in which native kidneys are removed because they may cause short- or long-term risks to the recipients or grafts, such as intractable hypertension, recurrent kidney infections, heavy proteinuria, large polycystic kidneys, and risk of malignancy.18 20 Heavy proteinuria and hypoalbuminemia increase the risk of thromboembolic events during surgery.19,21 Removal of nephrotic native kidneys reduces the risk of acute graft thrombosis, intravascular volume depletion, nutritional deficit, and delayed wound healing.22 An improved serum protein level during surgery improves posttransplant fluid management23 and is associated with a favorable posttransplant graft function in children.20

13.2.2 Transplant Surgery The kidney graft can be placed intra- or extraperitoneally, depending on the child’s body shape, height, and the sizes of blood vessels. The minimum weight and/or age limit for kidney transplantation varies among pediatric centers, ranging from 6 months old/5 6 kg weight to 12 months old/10 kg.24 The best option is a LD,25 followed by an adult-sized DD.26 The last choice is a pediatric donor, preferably .6 years of age.22,27 29 The exception is a recipient with a thrombosed inferior vena cava who will require a smaller kidney with a graft vein implanted in patent pelvic vein.30,31 Grafts of less than 24 hours of cold ischemia time are favored since those greater than 24 hours are 1.5 times more likely to fail regardless of donor age.27 In general, a midline abdominal incision is usually made for recipients less than 10 kg. The graft is placed within the right side of the abdomen by anastomosing the donor artery to the aorta below the inferior mesenteric artery and the donor vein to the inferior vena cava below the left renal vein using end-to-side technique.32 The donor’s ureter is implanted into the recipient’s bladder using either a Ledbetter Politano procedure or one of its modifications.33 For infants weighing 10 20 kg, the graft is placed extraperitoneally when possible34,35 with the renal artery and vein anastomosed to the common or external iliac artery and vein, respectively. For those recipients greater than 30 kg, the surgical technique is the same as those for adult recipients.36,37 The extraperitoneal approach allows earlier oral feeding, avoids postoperative intestinal adhesion, prevents twisting or kinking of the graft, and reduces motility of ureteral anastomosis.33,38 Recently, some centers have successfully performed extraperitoneal renal transplantation on infants less than 10 kg using a J-shaped external pararectal incision extending cranially to a point near the costal border.31,38,39 The graft is placed in a position similar to that of the native kidney. This incision avoids muscle splitting leading to less postoperative pain, permits good exposure of the great vessels, prevents handling the intestine with subsequent ileus, enables the whole length of the graft ureter to remain extraperitoneally, preserves peritoneal dialysis access, and keeps possible surgical complications within the retroperitoneum.39,40 As a result, surgical success in small children has improved, equivalent to those achieved in older children and adolescents.31,41,42 Immediate postoperative complications include lymphocele, renal artery or venous thrombosis, renal artery stenosis, urinary leakage from anastomosis, ureteral stenosis, and ureteral necrosis.39,43 Prophylactic placement of an indwelling double-J ureteral stent does not reduce the risk of urinary leakage or obstruction. Stent migration and the need of general anesthesia to remove the stents are common.44 In addition, an association between ureteral stent and BK nephropathy has been reported.45 In general, the ureteral stent is not recommended except for selected cases in small children.33

13.2.3 En Bloc Pediatric Kidney Transplant To expand the donor pool, en bloc kidney transplantation from young pediatric donors to adult recipients has been advocated but in an earlier report it suffered a high incidence of renal artery stenosis, venous thrombosis, and ureteral complication.46 With improved surgical technique, its complications have been reduced, similar to that in adult single allografts.47

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13.2.4 Perioperative Management Factors contributing to early graft loss in small children include inadequate surgical technique, postoperative vessel angulation, and the increased hemodynamic need of an adult graft in a small child.48 Recipients less than 2 years old are at risk of vascular thrombosis due to the smaller size of blood vessels and inadequate renal perfusion.41,49 Other risk factors for vascular thrombosis are cold ischemia time greater than 24 hours and acute tubular necrosis.50 An adult kidney sequesters 250 300 mL of blood so an infant’s cardiac output needs to be doubled to perfuse the adult graft.51 Therefore, intraoperative and immediate postoperative volume management is critical to maintain anastomosis patency and avoid hypoperfusion of grafts in small pediatric recipients.38,48 Usually central venous pressure is kept greater than 10 cm H2O and mean arterial pressure greater than 60 mmHg with crystalloid, colloid, and sometimes mannitol prior to the release of vascular clamping.52 Postoperatively, the intravenous fluid is usually given as insensible loss plus full replacement of urine output with Ringer’s lactate or saline plus bicarbonate solution to maintain CVP greater than 10 cm H2O during the first 24 48 hours. Inotropic agents such as dopamine can be added to maintain urine output or mean BP above 60 mmHg. When there is an unexplained decrease in urine output, sonogram of the graft should be performed to rule out vascular thrombosis. Arterial hypertension is usually managed with calcium channel blockers. Perioperative antibiotics such as cephalexin are used to prevent wound infection, followed by cotrimoxazole to prevent urinary tract infection (UTI) and Pneumocystis jiroveci pneumonia.53

13.2.5 Posttransplant Urologic Complications Bladder dysfunction including abnormal bladder capacity (26%), abnormal urinary flow (50%), and residual urine (32%) occurs in pediatric recipients but does not seem to affect graft function.54 Patients with augmented cystoplasty have a higher incidence of UTI after transplantation. This is related to vesicoureteral reflux (VUR) or noncompliance with clean intermittent catheterization.55 VUR into a graft is more frequent in children than in adult recipients even with the same surgical team using the same techniques.56 Its incidence was 36%57 to 58%58 before 1992 but has been reduced to 14.5% after 1998 due to improvement in surgical technique.59 The incidence of UTI was not different between pediatric recipients with and without,57,59 but those with VUR had a higher incidence of acute pyelonephritis57,58 which could be prevented by antibiotic prophylaxis.58 Correction of VUR in pediatric recipients reduced the incidence of UTI in a small series but it was associated with obstructive complications.60 Whether antireflux surgery can reduce the risk of febrile UTI in a large pediatric cohort is unclear. VUR was associated with worse graft function and survival in two pediatric studies61,62 but not in others.59,63 It has been linked to the pathologic findings of interstitial fibrosis and tubular atrophy in pediatric recipients.64 In this aspect, persistent inflammation in areas of interstitial fibrosis and tubular atrophy were negative prognostic indicators for long-term graft function.65,66 Therefore, it remains a controversy whether the presence or degree of VUR in pediatric recipients affects graft outcome. A similar controversy exists in the association of UTI with graft function in pediatric recipients. Despite acute graft dysfunction during febrile UTI, long-term renal function was not different between pediatric patients with and without infection in some reports59,67,68 but others reported lower graft function in those with recurrent UTI.58,69 In this regard, analysis of 1996 2000 USRDS data that included 28,942 Medicare adult and pediatric recipients showed that UTIs occurring after 6 months posttransplant were associated with an increased risk of death and graft loss.70

13.3 DESENSITIZATION 13.3.1 ABO Incompatibility Blood group antigens A and B are targets of natural antibodies of IgM and/or IgG types that can cause renal allograft rejection.71 In contrast, the presence of antibodies against non-A/B blood group antigens such as RH, Kell, Duffy, etc., does not seem to affect kidney allograft survival since they are not expressed on the endothelium.72 Plasmapheresis or immunoadsorption, depletion of B-cells prior to transplantation, and maintenance immunosuppression after transplantation are strategies to reduce the risk of acute rejection in ABO-incompatible recipients. Plasmapheresis indiscriminately removes all plasma proteins including anti-A or anti-B antibodies. Immunoadsorption using columns coated with protein A (binding most of IgG) or sheep antihuman

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immunoglobulin (binding most antibody isotypes) removes only antibodies but is not antigen-specific. Antigenspecific immunoadsorption73 is coated with Staphylococcus A protein on which either isoagglutinin A (anti-A column) or B (anti-B column) is implanted to adsorb anti-A or anti-B immunoglobulins, respectively. In general, a 1/4 or 1/32 reduction in anti-A or -B immunoglobulins prior to transplantation is accepted by many centers but a titer of 1/256 or less has been advocated.74 Due to a preparative regimen of several days, immunoadsorption and plasmapheresis are largely restricted to LD transplant. Splenectomy or cyclophosphamide to deplete B-cells is now replaced by rituximab, which is usually given within a month before transplantation to reduce the risk of antibody rebound.75 The typical posttransplant maintenance immunosuppression after ABO desensitization includes tacrolimus, mycophenolate mofetil (MMF), and corticosteroids. Plasmapheresis or immunoadsorption is used in cases with a significant rise of antibody titers.76 Grafts after ABO-incompatible kidney transplantation in pediatric patients have a good outcome. Tyden et al. showed 100% and 90% GSR at 1 year and 3 years, respectively, in ABO-incompatible group compared with 96% and 92% in ABO-compatible group.77 Shishido et al. reported 94% and 86% GSR at 1 year and 10 years, respectively, in ABO-incompatible group compared with 95% and 88% in ABO-compatible group.78 It is interesting to see that the 10-year graft survival in ABO-incompatible group in this pediatric study was higher than 72.9% survival in the United States and 76.1% survival in the Japanese study.79

13.3.2 Antibody Sensitization PRA is a complement-fixating assay to test the ability of recipient’s serum to lyse a panel of T-cells from a group of potential donors.80 For a PRA of 20% 80%, there is 50% chance to be transplanted compared to nonsensitized patients while the chance is only 5% for PRA greater than 80%. Approximately 3% of sensitized children on the waiting list have a PRA above 80%.81 PRA detects only anti-HLA I antibodies and does not reflect all donors. A positive T-cell cross-match has a high-risk for hyper-acute rejection and is considered an absolute contraindication to kidney transplantation. A positive B-cell cross-match indicates the presence of anti-HLA I and/or II antibodies. The presence of anti-HLA II alloantibodies is associated with a high-risk of hyperacute rejection. In this aspect, solid phase assays in which purified HLA I or II antigen attaches to flow cytometry beads or enzymelinked immunosorbent assay plates can identify specific anti-HLA I or II antibodies.82 Therefore, combined solid phase and cross-match assays allow for specification and quantitation of anti-HLA antibodies. Donor-specific antibodies (DSA) are produced when nonimmunosuppressed recipients are exposed to foreign antigens such as HLA-I and -II, ABO antigens, and possibly endothelial monocyte antigens. These antibodies are the result of persistence of donor proteins in the recipient follicular dendritic cells by chimerism.83 Sensitization may occur after blood transfusion but the main cause is previous transplantation. Prevention of sensitization from multiple transfusions in transplant candidates is important. Cyclosporine coverage during transfusion has been suggested84 but it is not widely used because of hyperkalemia. MMF exposure in a 4-year-old sensitized recipient reduced anti-HLA antibodies.85 The use of leukocyte-poor blood in transplant candidates on sensitization rate is unknown. Plasmapheresis, intravenous immunoglobulin (IVIG), and rituximab in combination are commonly used for antibody desensitization. Splenectomy at the time of transplantation, in combination of rituximab, is reserved for recipients with a high-risk profile.86 The goal of plasmapheresis is the removal of DSA to achieve a negative cross-match assay before transplantation. In addition to blocking anti-HLA antibodies, IVIG has immunomodulatory effects including downregulation of B-cell differentiation and antibody production, inhibition of T-cell proliferation and complement system, suppression of dendritic cells, and anticytokine activity.87,88 Desensitization using plasmapheresis and low-dose IVIG before LD transplantation improves the 8-year survival in adult recipients89 but there is no consensus in pediatric desensitization protocols. Tyan et al. reported the successful use of IVIG in a 13-year-old boy.90 Three doses of 2 g/kg IVIG every 4 weeks did not reduce PRA in an 11-year-old child. Subsequently, plasmapheresis every other day for 2 weeks reduced PRA with a negative cross-match assay.91 In contrast, 500 mg/kg IVIG weekly for 3 consecutive weeks every 12 weeks after 40 months reduced PRA to zero in a 7-year-old girl with a failed graft.92 These reports suggest IVIG’s effect on pediatric desensitization is unpredictable. Rituximab is often given after plasmapheresis to prevent B-cell differentiation and antibody rebound. This regimen can be used in conjunction with IVIG, or alone if patients fail IVIG.91 Since rituximab does not inhibit antibody production by plasma cells, bortezomib has been used alone in anti-HLA-sensitized adult recipients but results were disappointing.93,94 The combination of bortezomib and plasmapheresis seems to be a better desensitizing treatment than IVIG plus plasmapheresis in adults.95 There is no report regarding the use of bortezomib on sensitized pediatric kidney transplant candidates although its successful use in combination

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with rituximab and plasmapheresis has been reported in a heart transplant recipient.96 An alternate strategy for antibody desensitization is a paired living-donor program. After transplantation, triple agents including MMF, steroids, and tacrolimus are usually employed as maintenance immunosuppression. Most patients require additional plasmapheresis and IVIG after transplantation, depending on DSA titer and graft function. Future multicenter controlled pediatric clinical trials are needed to determine the most efficacious protocols.97

13.4 TRANSPLANT IMMUNOSUPPRESSION While adult data guide pediatric practice, there has been significant progress in developing pediatric protocols and obtaining outcome data. In pediatrics special attention has been focused on minimizing or avoiding steroids or calcineurin inhibitor (CNI). As of 2017, there is no evidence that the various induction agents improve longterm outcomes, though survival may be increased in LD recipients.4,98

13.4.1 Induction Therapy The goal of induction therapy is to prevent T-cell activation. The approach to induction therapy varies widely among transplant centers.4,49 The immunosuppressive effects of antithymocyte globulins is mediated through the depletion of circulating T-cells by complement-mediated lysis as well as blockage of T-cell proliferation.99 Polyclonal antithymocyte globulins can be derived from horse (Atgam) or rabbit (thymoglobulin). In a singlecenter pediatric study, the incidence of acute rejection was lower in Thymoglobulin-treated recipients when compared to the historical Atgam-treated recipients.100 Thymoglobulin reduced the incidence of AR in pediatric recipients.101 It is usually given as 1.5 2 mg/kg/dose when the CD3 count exceeds 20 cells/mm.3,102 Alemtuzumab (Campath-1H) is a humanized anti-CD52 antibody. It depletes T- and B-cells, monocytes, and natural killer cells and its effects last for months. In 42 pediatric LD recipients who received pretransplant alemtuzumab and posttransplant tacrolimus monotherapy, the 4-year GSR and PSR were comparable to those reported for LD recipients in the US-transplant SRTR database.103 In 101 pediatric LD recipients who received the first dose of alemtuzumab 12 29 days prior to transplantation and the second dose intraoperatively, along with a low-dose CNI, their 3-year GSR and AR episodes were comparable to NAPRTCS data. However, the incidences of subclinical Epstein Barr virus (EBV) and cytomegalovirus (CMV) infections were high.104 In a retrospective study, alemtuzumab was given intraoperatively at 0.5 mg/kg to 25 pediatric DD recipients. Steroids were discontinued 4 days later. Tacrolimus monotherapy was started the day after transplantation while MMF was added only in cases of high immunological risk or prolonged delayed graft function. Over a mean follow-up of 2 years, tacrolimus monotherapy was maintained in 48% of children and steroids were avoided in 80%. Incidence of AR was 12% within the first year and 16% at 2 years but the incidence of BK or CMV infection was 16%.105 In another retrospective study on 21 pediatric DD recipients who received alemtuzumab intraoperatively and maintained on tacrolimus and MMF, PSR was 100% and GSR 95% after a mean follow-up of 33 months (range 1 6.5 years). None of the patients had CMV infection, posttransplant lymphoproliferative disorder (PTLD), or BK virus nephropathy.106 These studies suggest that alemtuzumab can be a candidate as induction agent for steroid avoidance but prospective studies are needed to confirm these findings. Basiliximab (Simulect) and daclizumab (Zenapax) act on the inducible alpha-H chain of the interleukin-2 (IL-2) receptors on the surface of the activated lymphocytes. They saturate the IL-2 receptors, acting as a competitive antagonism of IL-2-dependent T-cell proliferation. Data from NAPRTCS demonstrated their use was associated with a decreased risk of thrombosis.107 Both antibodies have been studied extensively in children and have been shown to be safe and effective but neither confers increased long-term graft or patient survival.108

13.4.2 Maintenance Therapy Pediatric maintenance agents usually comprise of CNI (tacrolimus, cyclosporine), cell cycle inhibitors (azathioprine, MMF), and steroids. The most common maintenance protocol consists of tacrolimus, MMF, and steroids, followed by a steroid-free protocol with tacrolimus and MMF, though this differs internationally.4,109 Tacrolimus is used more commonly than cyclosporine.49,110 The combination of CNI, rapamycin, and corticosteroids should be avoided due to the risk of PTLD.11

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Steroids have multiple adverse effects on children including growth retardation, cushingoid habitus, infection, impaired wound healing, aseptic bone necrosis, cataracts, glucose intolerance, hypertension, hyperlipidemia, gastric ulcer disease, obesity, behavioral changes, and acne. In addition, the negative cosmetic effect may play a role in poor adherence. Therefore there have been attempts to withdraw steroids earlier after transplantation. Alternate-day steroid therapy improved linear growth and did not increase rejection episodes or affect renal function in retrospective111 and prospective112 pediatric studies. In a single-center retrospective study of 60 children, a steroid-free protocol using Thymoglobulin induction and maintenance tacrolimus and MMF had a 93% PSR and 63% GSR at 10 years with improved linear growth.113 Analysis of United Network for Organ Sharing database from 2002 to 2009 showed pediatric recipients who received only tacrolimus and MMF immunosuppression had a lower risk of AR 6 months after renal transplantation and a better 5-year GSR compared to steroidbased protocols. However, this data analysis was limited by steroid withdrawal patients having less delayed graft function and pretransplant dialysis, less likely to be African American, and more frequently receiving a LD graft.114 The cooperative clinical trials in pediatric transplantation study of corticosteroid withdrawal in pediatric kidney transplantation was halted at 1 year due to an increased incidence of PTLD. However, those children whose corticosteroids were successfully withdrawn did not have a higher rate of later rejection, and their longterm graft survival was equivalent to the control group.115 In a randomized, multicenter trial, 130 children were randomized to steroid-free or steroid-based immunosuppression, with concomitant tacrolimus, MMF, and standard-dose daclizumab (Steroid-based group) or extended-dose daclizumab (Steroid-free group). At 3-year follow-up, both groups had comparable graft and patient survival but the Steroid-free group had better linear growth, lower systolic BP, and lower cholesterol levels.116 There is no evidence to date of a higher risk of DSA in steroid-free protocols after renal transplantation. Further studies are needed to determine the long-term benefits of steroid-free protocols. The dose of cyclosporine is higher in children due to a more rapid metabolism.117 Among the side effects of cyclosporine, hirsutism, facial dimorphisms, and gingival hyperplasia are the main concerns in children since they can affect compliance.118 Neoral is a microemulsion form of cyclosporine that has a more consistent intestinal absorption with fewer dose adjustments compared to Sandimmune.119 Tacrolimus shares similar side effects with cyclosporine except for cosmetic effects but it has more frequent neurologic symptoms.120 Posttransplant diabetes mellitus associated with tacrolimus occurs via diminished insulin secretion complicated by insulin resistance from concomitant steroid use.121 Tacrolimus is not associated with a higher risk of PTLD, likely due to the lower dose.122 Extended-release tacrolimus is a once-daily formulation with comparable efficacy to twice-daily tacrolimus. Long-term renal function is greater than with cyclosporine123 125 but there remains a concern with how it compares with the twice-daily tacrolimus in terms of dosage monitoring, benefits in compliance, and rejection episodes in children.126 There are numerous drug and food interactions with cyclosporine and tacrolimus through cytochrome p450 system 3a4. Grapefruit not only contains substrates that inhibit CYP3A4 but it also increases membrane transporter p-glycoprotein and interferes with intestinal absorption and tissue distribution of cyclosporine, tacrolimus, and possible sirolimus.127 Diarrhea can lead to an increased tacrolimus level.128 A higher dose of tacrolimus is needed for African Americans due to the fact that CYP3A*51 alleles are expressed in 90% of African Americans but only 5% of Whites.129 The clearance of tacrolimus can be affected by food, medications such as corticosteroids and antibiotics, hypoalbuminemia, anemia, and hepatic dysfunction.130 Azathioprine inhibits purine synthesis, preventing gene replication and cell division. This in turn blocks cellmediated immunity, inhibits primary antibody synthesis, and decreases circulating monocytes and granulocytes.131 The main side effect of azathioprine is myelosuppression. Other side effects include viral infection, hepatotoxicity, pancreatitis, alopecia, and neoplasia, most notably skin cancer.132 To date, azathioprine is no longer the first-line immunosuppression in pediatric recipients but is still useful in select patients such as patients with MMF-associated colitis. MMF is rapidly metabolized to mycophenolic acid, which blocks conversion of inosine IMP to guanosine IMP in purine biosynthesis pathway. The net result is a decrease in the number of functional B- and T-cells and the inhibition of their response to antigen challenges.131 Hematological and gastric symptoms are the main side effects. Neutropenia and thrombocytopenia can be reversed with the reduction in MMF dose but frequent reduction or withholding of the MMF dose can lead to impaired graft survival due to rejection.133 Frank esophagitis and gastritis with occasional GI hemorrhage occur in approximately 5% of patients.134 Colonoscopic findings of chronic diarrhea from MMF include nonspecific, inflammatory bowel disease-like, graft-versus-host disease-like, and ischemic-like colitis.135 The enteric coated mycophenolic acid, myfortic, has fewer gastrointestinal side effects and a faster intestinal absorption.133 MMF in combination with tacrolimus results in a higher blood level of

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MMF.136 The use of therapeutic drug target monitoring of MMF has been suggested in patients with high immunologic risk; noncompliance; renal, hepatic, or bowel dysfunction; reduced dose or withdrawal of CNI; or drug interaction137 but its role in long-term graft outcome remains controversial.137,138 The pharmacokinetics of MMF are different between adults and pediatric patients and there is a weak negative association between its clearance and age in pediatric patients.139 The mammalian target of the rapamycin (mTOR) is a cytosolic serine/threonine enzyme that exists in two distinct multiprotein complexes, mTORC1 and mTORC2. It triggers the differentiation and proliferation of lymphocytes after activation by cytokines from the antigen-activated lymphocytes. Sirolimus or everolimus complexes with the immunophilin FK-binding protein 12, which inhibits mTORC1. mTOR inhibitors also promote T-cell apoptosis140 and inhibit proliferation of vascular muscle cells.141 This dual immunosuppressive and antiproliferative effect can be beneficial to long-term graft outcome. mTOR inhibitors do not possess nephrotoxicity.142 They have fewer GI side effects than MMF.143 Everolimus is the 40-O-(2-hydroxyethyl) derivative of sirolimus with improved GI absorption but a shorter elimination half-life,144 making it a twice-daily dosing versus the daily dosing of sirolimus in adults. In younger children, however, sirolimus is usually given twice a day due to higher clearance.145 There is a competitive interaction between sirolimus and CNI for being a substrate for p-glycoprotein and cytochrome p450 isoenzymes,146 147 allowing for a smaller CNI dose and fewer side effects. In a retrospective pediatric study, addition of sirolimus to cyclosporine on a steroid-withdrawal protocol led to stable graft function at 1 year without steroid-related side effects.148 In pediatric patients with chronic allograft nephropathy (CAN) or high immunologic risk, conversion of CNI to sirolimus preserved short-term149 151 or long-term152 153 graft function while decreased short-term graft function was reported in another study.154 However, there were high incidences of proteinuria, hypercholesterolemia, oral ulcers, myalgia, infection, and leukopenia from sirolimus149 153 resulting in a high withdrawal rate.149,153,154 A prospective, single-arm trial of CNI-free immunosuppression with sirolimus and MMF in pediatric recipients had biopsy-proven AR episodes occurring in almost one in three patients and a high rate of sirolimus-related patient withdrawal.155 Another pediatric prospective study using basiliximab induction and sirolimus, cyclosporine, and steroids for maintenance immunosuppression was discontinued at 1 year due to a high incidence of PTLD.115 A prospective combination of everolimus, corticosteroids, and a 50% dose of cyclosporine in 19 pediatric recipients had good 3-year patient and graft survival but there was a high incidence of biopsy-proven AR, hyperlipidemia, and bacterial infection.156 This increased incidence of AR was not observed in 13 LD recipients in the same trial at 1 year157 or in another study with additional basiliximab induction.155 Therefore, evidence from studies of sirolimus replacing CNI as maintenance immunosuppression in pediatric recipients has not been encouraging and the use of everolimus to reduce CNI exposure awaits further controlled studies. Furthermore, long-term side effects of mTOR inhibitors on spermatogenesis or growth retardation have not been examined in pediatric recipients. CTLA-4 signaling pathway is critical for T-cell costimulation and the induction of acquired tolerance in vivo.158 Belatacept is a recombinant protein that contains the extracellular domain of CTLA-4 fused to an IgG heavy chain tail. In a randomized adult BENEFIT trial, 666 patients who received basiliximab induction and MMF plus corticosteroid immunosuppression were randomized to low- or high-dose belatacept or cyclosporine. Renal function was higher and the incidence of chronic rejection was lower at 12 months in the belatacept group compared to the cyclosporine group.159 At 5 years, the benefit on renal function was sustained and the concern for PTLD in EBV-negative patients in the earlier study was not observed.160 A pediatric study on belatacept is currently under investigation.

13.4.3 Acute Rejection The rejection process is initiated and maintained by interaction among CD4 and CD8 T-cells, B-cells, and macrophages. This interaction mediates activation and expression of proinflammatory molecules on intragraft endothelial cells, which in turn augments recruitment and activation of leukocytes. The binding of alloantibodies to the cytokine-activated endothelial cells is associated with complement C4d deposition, resulting in regional vasculitis and endothelial cell death.161 AR is characterized histologically by the necrosis of renal parenchyma with lymphocyte and macrophage infiltrates. Its time course includes hyperacute rejection (immediately after engraftment), accelerated AR (within 1 week after transplantation), early AR (within 6 months after transplantation), and late AR (more than 12 months after transplantation). Hyperacute rejection is characterized by rapid thrombotic occlusion of the graft within minutes of vascular anastomosis. The only treatment is surgical removal. Accelerated AR carries a poor prognosis and its risk factors include delayed graft function

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and presence of de novo DSA.162 Both hyperacute rejection and accelerated AR are rare now with the widespread use of cross-match assay. The incidence of AR is declining in pediatric recipients163 but it remains higher than that in adult recipients.4,164 AR can be acute cellular rejection (ACR) or acute antibody-mediated rejection but mixed rejection can occur. Continuous and diffuse C4d staining along peritubular capillaries is present in 30% of rejection biopsies, and DSA are present in 90% of C4d 1 cases.165 Surveillance biopsies may be useful in diagnosing subclinical AR but data on long-term outcome is lacking.166,167 AR is a risk factor for graft dysfunction and chronic rejection. The incidences of graft failure and patient death after AR are 5% 7%.4 Graft loss from AR at 5 years after transplantation is approximately 13%,109 with infants and young children having the best outcome among age groups.168 Risk factors for AR in LD and DD recipients are similar, including years of transplant, adolescent age, and African American race.49 An elevated percentage of circulating CD25 level169 or CD69 level170 or a five-gene set on peripheral blood171 has been proposed as a marker for AR in pediatric recipients. 13.4.3.1 Acute Cellular Rejection ACR is mainly a T-cell-mediated rejection that involves tubular, interstitial, and intimal infiltration of inflammatory cells. Tubulitis is essential for its diagnosis, while glomerular changes are limited to mesangial expansion. ACR is associated with de novo DSA production and the risk for future AMR and graft loss.172 Standard treatment is intravenous methylprednisolone 10 25 mg/kg pulse therapy for 3 days, followed typically by Thymoglobulin at 1.5 2 mg/kg/dose for 10 14 days. High-dose oral prednisone in children has an equivalent result to intravenous therapy in terms of the rate of rejection reversal.173 Alemtuzumab has been used in adult patients with ACR who were previously treated with OKT3 or Thymoglobulin174 or who were resistant to steroids or Thymoglobulin.175 NAPRTCS 2001 07 data showed that 53% LD and 47% DD recipients had complete reversal of rejection, 43% LD and 47% DD recipients had a partially reversed rejection, and 4% LD and 6% DD recipients lost the graft.49 Despite the decreasing AR frequency, the reversibility rate of AR in pediatric recipients has not improved since the 1990s. 13.4.3.2 Acute Antibody-Mediated Rejection The incidence of AAMR in children is not known. In adults, the incidence was less than 5% in unsensitized patients but between 40% and 90% in sensitized patients.176 The risk for AAMR in children includes HLA mismatches, AR, nonadherence to treatment, insufficient immune suppression, previous transplantation, and blood transfusion.177 179 De novo DSA has been detected in up to 24% of children with renal transplants, equally found in those with steroid-free or steroid-based regimen.180 They are usually anti-HLA II antibodies,181 which are associated with a worse prognosis than are anti-HLA I antibodies.182 In AAMR, alloantibodies preferentially attack the peritubular and glomerular capillaries183,184 through activation of classical complement pathway, resulting in C4d binding to tissue and depositing in peritubular capillaries.185 The 2007 Banff criteria for AAMR included circulating DSA, diffuse deposition of C4d in more than 50% peritubular capillaries, acute tissue injury, and clinical evidence of graft dysfunction. The acute tissue injury includes acute tubular injury; neutrophils or monocytes in the peritubular capillaries or glomeruli; and intimal, intramural, or transmural inflammation or fibrinoid necrosis of the arteries.186 AAMR has been described in C4d-negative grafts. In such cases microcirculatory injury and the presence of Class II DSA indicate a worse outcome. For this reason, the updated 2013 Banff AMR criteria did not preclude a negative C4d staining.187 188 Since not all patients with anti-HLA antibodies have AR or graft loss, assays for C1q-binding DSA have been developed and C1q-positive DSA are associated with short- or long-term graft loss.172 189 There is evidence that non-HLA antibodies are also associated with the development of AAMR since it was reported in identical HLA recipients. These non-HLA antigens include polymorphic antigens of endothelial cells, the major histocompatibility complex class one-related chain A (MICA) on endothelial surface, the angiotensin II type 1 receptor, and vimentin.190 AAMR in general has a worse prognosis than ACR in adults.179 There are no agreed-upon pediatric protocols to treat AMR but commonly employed treatments alone or in combination include IVIG, plasmapheresis/immunoadsorption, rituximab, and an enhancement of existing immunosuppression with or without steroids.191 193 Other treatment has been reported in pediatric cases. Three pediatric recipients with AAMR who failed IVIG, rituximab, plasmapheresis, and steroid pulse therapy were treated with one dose of 900 or 1200 mg eculizumab but only one patient responded.194 Successful treatment with plasmapheresis, IVIG, and eculizumab was reported in a 7-year-old child who developed AMR at day 4 after transplantation.195 A recent retrospective study in 4 children with AAMR who received 4 doses of bortezomib (1.3 mg/m2) at days 1, 4, 8, and 11 and various combinations of rituximab, steroids, plasmapheresis, and IVIG suggested bortezomib therapy is an effective and

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safe method for rapid reduction in DSA levels.196 Because of the prolonged need for increased immunosuppression treatment, patients have increased risk of infectious and malignant complications.

13.4.4 Chronic Renal Allograft Injury Chronic renal allograft injury is characterized clinically by a progressive decrease in allograft function, usually accompanied by proteinuria and hypertension, and pathologically by interstitial fibrosis and tubular atrophy with or without mononuclear infiltration. It is the end result from a variety of etiologies including nonimmune causes such as CNI toxicity, arterial hypertension, chronic urinary obstruction, viral and bacterial infection, and immune causes such as chronic antibody-mediated rejection and chronic cellular rejection.197 CAMR is a chronic injury to the glomerular and tubular basement membrane as a result of capillary injury.198 Clinically it presents as proteinuria, hypertension, and declining graft function over time. The glomerular lesion is characterized by reduplication and lamination of glomerular basement membrane (transplant glomerulopathy). There are tubular basement membrane multilayers with margination of mononuclear leukocytes. Eventually these lesions lead to tubular atrophy and interstitial fibrosis.199 C4d deposition may or may not be present and DSA levels usually fluctuate, suggesting additional complement-independent mechanisms mediating the injury from antibodies against endothelial HLA antigens. Patients with positive DSA and untreated or inadequately treated AMR have a higher risk for CAMR.200,201 Surveillance biopsies may facilitate early detection of CAMR, which could lead to more prompt treatment and improved outcomes.200 Twenty pediatric recipients with CAMR were prospectively treated with 4 weekly IVIG (1 g/kg) and 1 dose of rituximab (375 mg/m2) at 1 week after the last IVIG infusion. Response to treatment was defined as a reduction of the rate of functional loss by at least 30% within 6 months compared with the period 6 months prior to treatment. After 2 years of follow-up, all 9 patients without transplant glomerulopathy (100%) and 5 of 11 patients with transplant glomerulopathy (45%) responded.177 CCR is incompletely described in pediatric transplant recipients and is usually comprised of intimal inflammation with intercurrent chronic allograft arteriopathy.202 A low-dose CNI in combination with a low-dose of sirolimus or everolimus in prospective pediatric studies improves graft function.203,204

13.5 METABOLIC COMPLICATIONS AFTER TRANSPLANTATION Despite the improvement in patient survival since the advent of pediatric renal transplantation, there remain significant deleterious effects on a patient’s metabolism, leading to poor cardiovascular, endocrine, musculoskeletal, and developmental outcomes. Contributing factors include medication side effects, complications from prior procedures, sequel from the underlying disease, and declining allograft function.

13.5.1 Cardiovascular Disease Children with ESRD have cardiovascular disease and while transplantation attenuates the risk factors, CVD remains a significant contributor to morbidity and mortality.4,205 207 Risk factors for CVD include hypertension, obesity, hyperglycemia, dyslipidemia, and anemia, and early interventions should focus on treatment of these conditions. Hypertension is a common posttransplant complication, especially with steroid and CNI use.208,209 Steroid avoidance improves BP.116 Appropriate lifestyle and pharmacologic management is critical. Adequate BP control can improve or stabilize end-organ damage such as left ventricular remodeling and carotid intima-media thickness.210,211 There is evidence angiotensin-converting enzyme inhibitors or angiotensin II receptor blockers may protect and improve pediatric graft function over time.212 214

13.5.2 Endocrine Disorders Dyslipidemia, glucose intolerance, and DM are less common in pediatric recipients compared to adults but remain significant posttransplant complications. Posttransplant dyslipidemia improves over time and steroid avoidance improves cholesterol levels.116,206 Pediatric patients often have abnormalities in glucose metabolism. Glucocorticoids reduce peripheral insulin sensitivity by decreasing the binding of insulin to insulin receptors and by decreased glucose utilization. CNI and mTOR inhibitors reduce insulin secretion by pancreatic β-cells.215 Impaired glycemic control is associated with higher BP and being overweight and obese.216 In this aspect, the presence of metabolic syndrome in transplant patients has increased in the past few years.217 Insufficient or

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deficient 25-vitamin D levels have been reported in 76%218 to 92%219 of pediatric recipients. Vitamin D deficiency was associated with short stature and hyperparathyroidism219 but its association with posttransplantation proteinuria remains to be determined.

13.5.3 Growth Growth impairment occurs in patients with ESRD and in transplant recipients. Pretransplant growth deficits, posttransplant growth patterns, and final adult height improve dramatically across age groups after transplantation.4,220 Pubertal growth is maintained but catch-up growth during puberty typically does not occur. Steroid avoidance improves growth and graft function predicts growth.116 While improved growth is a desired outcome, obesity remains a significant issue. Most patients experience increases in BMI posttransplant, which in turn increases the risk of cardiovascular and metabolic disease.216,221

13.5.4 Metabolic Bone Disease There is improvement in metabolic bone disease posttransplant but the physiology does not return to normal.206,222 224 Pediatric recipients have increased loss of bone mass and strength and increased rates of fractures and avascular necrosis.225 228 Persistent posttransplant metabolic bone disease is a modifiable cardiovascular risk factor.229 Key interventions include maintaining normal graft function; targeting normal vitamin D, calcium, and phosphorus levels; and ensuring adequate nutrition and physical activity. Growth hormone may have a role in improving bone structure and final adult height.230 232

13.5.5 Development The metabolic complications of kidney disease and renal transplantation affect pediatric neurocognitive development. Patients with chronic kidney disease and ESRD have worse neurocognitive outcomes but this improves following renal transplantation.233 However, many patients have persistent developmental abnormalities and require some degree of educational and social support.234 Overall IQ, education levels, and social outcomes are normal to near-normal.235 237

13.6 RECURRENCE Disease recurrence accounts for 6.6% of primary allograft failure and 9.6% of subsequent allograft failure according to NAPRTCS 2010 data. The primary cause for disease recurrence in pediatric recipients is glomerular disease.

13.6.1 Focal Segmental Glomerulosclerosis FSGS recurs in 20% 50% of primary pediatric transplants with equally high incidence of graft loss.4,238 Risk factors for its recurrence include early onset of nephrotic syndrome, rapid progression to ESRD (less than 3 years), resistant to treatment, White or Asian race, and recurrence in previous transplant.239,240 For unclear reasons, familiar FSGS does not seem to recur in a graft. The recurrence can occur within the first 24 hours after the graft is reperfused or several months later. The presence of circulating permeability factors has been suggested in recurrent FSGS.241 There is no benefit to LD transplantation in children with recurrent FSGS. Treatment includes plasmapheresis242 and high-dose CNI,243 but there are mixed results with rituximab.244

13.6.2 Alport Syndrome In individuals with X-linked Alport syndrome or hereditary nephritis, antiglomerular basement membrane disease may develop after transplantation leading to allograft failure in 3% 5% of male cases,245 especially when there is an associated vascular rejection.246 The risk is higher in subsequent transplants and does not typically respond to treatment.

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13.6.3 Membranoproliferative Glomerulonephritis Membranoproliferative glomerulonephritis is characterized by subendothelial and mesangial deposits of immune complexes. It has been recently reclassified into immunoglobulin-positive MPGN caused by chronic infection or autoimmune disease and immunoglobulin-negative MPGN, which consists of dense deposit disease and C3 glomerulopathy.247 The latter is more common in pediatric patients, although still a relatively rare cause of glomerular disease in children. MPGN can recur in 20% 30% of cases in what is classically termed MPGN type 1. In cases of dense deposit disease (classically referred to as MPGN type 2), it can recur in up to 90% of cases.248 In a study based on NAPRTCS data, recurrence of MPGN type 2 resulted in allograft failure in 15% of children by 5 years after kidney transplantation249 and there is no evidence of effective treatment.250

13.6.4 IgA Nephropathy IgA nephropathy can recur but graft survival is not different from that in other glomerular diseases.238 However, there were case reports that it can present with crescentic lesions leading to deterioration of graft function.250

13.6.5 Hemolytic Uremic Syndrome Shiga toxin associated hemolytic uremic syndrome is unlikely to recur after kidney transplantation. Atypical HUS is a genetic disorder of complement pathway such as Factor H, B, or I deficiency, a genetic mutation resulting in deficiency of von Willebrand factor cleaving protease, ADAMTS13, and other metabolic disorders.251 Atypical HUS is highly likely to recur after transplantation, between 20% and 80%.251 ADAMTS13 deficiency is associated with high mortality and few patients undergo transplantation, with those that do having high rates of allograft failure from vascular thrombosis or disease recurrence. In patients with Factor H, I, or membrane cofactor protein mutations, recurrence is 67% 76%.252 Plasmapheresis is the main treatment and prophylactic plasmapheresis has some benefit compared to plasmapheresis after recurrence.253 There have been several successful simultaneous liver kidney transplants for patients with Factor H deficiency using presurgery plasmapheresis plus fresh frozen plasma infusion pre- and intraoperatively.252 Recently, weekly eculizumab starting 1 month prior to transplantation in a pediatric LD recipient with Factor H deficiency maintained graft function after 1 year of follow-up.254

13.6.6 Membranous Nephropathy Unlike in adult kidney transplant recipients, recurrence of primary membranous nephropathy is rare in children and does not impact graft function. De novo membranous nephropathy can occur but does not usually affect graft function or outcome.

13.6.7 Systemic Lupus Erythematosus The risk of recurrence in patients with lupus nephritis is low, likely from adequate maintenance immunosuppression.255 Patients with antiphospholipid syndrome have a higher risk of thrombotic complications. Recurrence in pediatric patients is not clinically significant and does not affect allograft outcome.238 In pediatric patients, allograft survival is not different from that in nonlupus renal disease, but patient survival is worse.256

13.6.8 ANCA-Associated Vasculitis/Pauci-Immune Glomerulonephritis Recurrence of ANCA-associated vasculitis and pauci-immune glomerulonephritis is rare due to adequate maintenance immunosuppression. If it recurs, it can be managed with cyclophosphamide, corticosteroids, and sometimes plasmapheresis.257 It is preferable that patients maintain remission without systemic signs or active nephritis for at least 6 months prior to transplantation.

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13.7 METABOLIC DISEASES Primary hyperoxaluria type I is secondary to a deficiency of hepatic alanine glyoxylate aminotransferase leading to excess production of oxalate and deposition in kidneys with associated renal injury. As kidney function declines there is reduced oxalate excretion and calcium oxalate is deposited in other tissue and organs. The recurrence is nearly 100% shortly after kidney transplantation. Treatment with intense plasmapheresis after kidney transplantation is needed to reduce the body load of oxalates. Therefore, a preemptive combined liver kidney transplant is preferred.258,259 For patients with long-term ESRD, sequential liver kidney transplantation with hemodialysis after liver transplantation may be beneficial to reduce total body oxalate levels.260 Patients with the Gly170Arg AGXT mutation may respond to pyridoxine, which allows them to receive an isolated kidney transplant with long-term pyridoxine supplementation.260 Patients with methylmalonic acidemia develop ESRD due to interstitial nephritis. It may be partially ameliorated by kidney transplantation, but methylmalonic acid continues to be produced after kidney transplantation so combined liver kidney transplantation has been suggested in selected cases.261 Kidney transplantation and cysteine-depleting therapy with cysteamine extends the life expectancy to the fifth decade and beyond for children with nephropathic cystinosis.262,263 Patients need to take cysteamine posttransplant since transplant does not correct or prevent nonrenal morbidity such as visual impairment, hypothyroidism, myopathy, central nervous system disease, and pancreatic insufficiency.262,264 Although cystine may accumulate in the interstitium of the renal graft, it does not cause graft failure. There is no risk of recurrence of primary disease in the renal graft.

13.8 CANCER AFTER KIDNEY TRANSPLANTATION Cancer accounts for 11% of mortality in pediatric recipients.4 It occurs in 2% 9% of pediatric recipients265 267 and its incidence increases with years of transplantation.267 PTLD accounts for 82% of pediatric cancer cases.2 The second most common cancer in pediatric recipients is skin cancer, which includes squamous cell carcinoma, basal cell carcinoma, lip cancer, and melanoma.268 Skin cancer usually develops 12 15 years after transplantation269 and so only a few cases are reported during childhood. Treatment of skin cancer relies on surgical excision with adequate margins, reduction of immunosuppression, and a change to mTOR inhibitors.270 Anogenital cancers involving the vulva, scrotum, penis, perianal skin, and anus are the third most frequent malignancy (4%) in pediatric recipients.271 They occur on average 12 years after transplantation268 and are associated with human papilloma virus infection. Kaposi’s sarcoma accounts for 3.2% of cancer in pediatric recipients271 and is associated with human herpes virus-8 infection. Its treatment includes surgical excision, chemotherapy, and a change to mTOR inhibitors.272 Renal carcinoma, mostly renal cell carcinoma in native kidneys, accounts for 0.6% of cancer in pediatric recipients.267,273 Surgery is the main therapy.274

13.8.1 Epstein Barr Virus and Posttransplant Lymphoproliferative Disorder Pediatric recipients are at increased risk of primary EBV infection due to the fact that up to 50% are EBV seronegative at time of transplant.275 EBV-related PTLD can be the result of reactivation or primary infection. Young children are at higher risk compared to their adult counterparts, especially in the setting of an EBV-positive donor. The incidence of PTLD in pediatric kidney transplant recipients is 1% 6%,4,276 below the reported 33% incidence in combined liver kidney, 15% in heart, and 10% in liver transplants.277 It follows a bimodal distribution, with the first peak occurring during the first year and the second within the third year after transplantation.278 EBV infection and, to a lesser extent, CMV infection, are involved in the pathogenesis of the majority of cases.279 The development of PTLD after EBV infection relies on the equilibrium between EBV-infected B-cells and EBVspecific, CD8-positive cytotoxic T-cells.280 Hence, the degree of immunosuppression,266 HLA mismatching,281 and the absence of EBV-specific cytotoxic T cells282 are major risk factors for PTLD. Recent large-scale data analysis does not support earlier reports that the use of Thymoglobulin283 or prior therapy of recombinant growth hormone284 increases the risk of PTLD. EBV-negative PTLD can be associated with T-cell, B-cell, or plasma-cell clonality.285 The World Health Organization (WHO) has classified PTLD into four categories: early lesion,

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polymorphic, monomorphic, and classical Hodgkin’s lymphoma. Pediatric EBV-related PTLD are mostly of the early or polymorphic type.286 PTLD can be insidious and patients often present with nonspecific symptoms such as fever, malaise, graft dysfunction, peripheral adenopathy, hepatosplenomegaly, and diarrhea. Thus, diagnosis requires a strong clinical suspicion. Routine monitoring of quantitative EBV blood PCR has been utilized as a screening tool. Its role in predicting and monitoring PTLD remains controversial. An increased EBV viral load has been shown to correlate with the development of PTLD or EBV-associated symptoms in pediatric solid organ transplant PTLD277,287 or renal transplant.288 290 However, not every case of EBV-related PTLD is associated with elevated EBV viral load.265 A chronically elevated viral load is not predictive of PTLD289,291 or EBV-associated symptoms.292 This controversy may be explained by variations in PCR assay methods, cut-offs of positivity, blood samples being used (peripheral blood lymphocytes, whole blood, or plasma), different primer sets and target genes, report of PCR results (genome copies/105 PBL, genome copies/mL, or genome copies/μg DNA), definition of viral loads, study design (prospective vs retrospective), and EBV serostatus at the time of transplantation.293 Therefore, vigilant monitoring, aggressive radiologic testing, and tissue biopsy are key steps in following renal transplant children with EBV infection.294 Consensus is lacking on treatment of PTLD, but the first step is usually reduction in immunosuppression, whether it is associated with EBV infection or not.295 How to reduce immunosuppression is center-specific. However, it has been demonstrated that different immunosuppression agents have different effects on different viruses.296 MMF inhibits the proliferation of newly EBV-infected B-cells in vitro297 and potentiates the effect of anti herpes virus drugs.298 A higher MPA concentration is associated with a lower risk of EBV infection in pediatric renal transplant.292 Therefore, studies examining the steps of immunosuppression are needed in pediatric renal transplantation. Early lesion in children usually responds to reduction of immunosuppression but only 50% of pediatric polymorphic PTLD respond.299 Those who failed reduction of immunosuppression carry a mortality rate of 50% 90%.300 Rituximab is routinely used for CD20-positive PTLD. A combination therapy with rituximab, low-dose cyclophosphamide, and prednisolone improved the 2-year PTLD-free GSR to 80%.301 Additional radiotherapy and surgery are used for those who failed combined rituximab and chemotherapy and those with central nervous system involvement, T-cell non-Hodgkin’s lymphoma, or Hodgkin’s disease.286 Therapy with the EBVspecific cytotoxic T-lymphocytes from immunocompetent donors who are challenged with EBV-transformed Blymphoblastoid cell lines is currently under investigation.302

13.9 INFECTION There is increased infection risk in pediatric recipients due to chronic immunosuppression. The infection risk is highest in the immediate posttransplant period due to higher degree of immunosuppression. Pediatric recipients are at risk for common community-acquired infections and UTI. They are also at risk for opportunistic infections such as Pneumocystis jiroveci and fungal infections. Prophylaxis against P. jiroveci with trimethoprimsulfamethoxazole or pentamidine in individuals with sulfa allergy is a common practice among most pediatric transplant centers. The most common opportunistic viral infections in pediatric transplant are BK virus, CMV, and EBV.

13.9.1 BK Virus BK virus belongs to the family Polyomaviridae of small nonenveloped DNA viruses. BK virus subtype I is the most commonly identified subtype in BKV nephropathy (BKVN), although types II and III have been reported.303 BK virus infection usually presents as a nonspecific viral illness in those with intact immunity and occurs mainly during childhood, with greater than 90% of adults being seropositive. After primary infection, renal tubular epithelial cells and the uroepithelial cell layer are the main sites of viral latency or replication.304 Immunosuppression, age, White race, male sex, HLA mismatch, seroconversion, and history of allograft rejection are risk factors for the development of BKVN with a high graft loss rate.305 308 The incidence of BKVN was 4.6%, with a rate of graft loss of 24% within 2 years after diagnosis, in pediatric transplant recipients according to 2007 NAPRTCS registry.309 In pediatric patients, primary BKV infection from seropositive kidney graft is common.306 There is still a debate as to the screening method for BKVN. Urine screening has a high negative predictive value and a lower inhibitory effect on PCR method and it can be assessed for decoy cells. However, a positive urine

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screening requires the confirmation of viremia. BKVN with a negative viremia has been reported.310 Therefore, the diagnosis of BKVN requires the demonstration of polyomavirus cytopathic changes and interstitial nephritis.311 In pediatric centers with an established BK virus screening protocol, the incidence of viremia ranges 16% 26% during the first 3 years after transplantation.312 314 Reduction of immunosuppression in patients without AR resulted in the clearance of viremia in 20%314 to as high as 83% 100%.312,313 For patients not responding to reduction of immunosuppression, cidofovir, a cytosine nucleoside analog, has had mixed results in uncontrolled pediatric reports315 317 but its direct tubular toxicity is a major concern.318 As reported in 2014, CMX001, a lipophilic nucleotide analog formed by covalently linking 3-(hexasecycloxy)propan-L-ol to cidofovir, successfully treated a young child with BKVN.319 Leflunomide is an inhibitor of pyrimidine synthesis with case reports of its success in treating BKVN without nephrotoxicity.320,321 A 2-month ciprofloxacin treatment, followed by leflunomide if there was no significant decrease in BK viremia, and a small reduction in immunosuppression reduced the incidence of BKVN to 1% in 19 pediatric transplant patients.322

13.9.2 Cytomegalovirus CMV infection can lead to symptoms of fever, hepatitis, leukopenia, thrombocytopenia, pneumonia, and gastrointestinal disease. CMV can lead to opportunistic infections such as P. jiroveci, fungal infections, and bacterial infections. CMV can also increase risk of allograft rejection and PTLD due to dysregulation of the immune system including altered T-cell function and upregulation of cytokines and proinflammatory markers. It is the practice of most pediatric transplant centers to implement viral prophylaxis with valgancyclovir or ganciclovir for a period of time (usually 6 months) posttransplant, particularly for CMV seronegative recipients in the setting of a CMV seropositive donor. There are no prospective, randomized, controlled trials providing clear indications as to whether preemptive or prophylactic treatment is the optimal approach. It is still recommended in pediatric patients to use intravenous ganciclovir for treatment of active CMV infection due to the lack of efficiency data on oral therapy. Viral load is a good predictor of response to treatment.323

13.10 TRANSITION The rate of improvement in pediatric graft and patient survival over the past 25 years was attenuated in recipients transplanted at 13 17 years old.5 The American OPTN 1987 2010 data indicated recipients who received their first kidney transplant at age 14 16 years were at the highest risk of graft loss, with inferior outcomes starting at 1 and amplifying at 3, 5, and 10 years after transplantation.324 Other studies also reported a higher risk of graft loss seen during late adolescence and early adulthood.325,326 These findings have been attributed to poor adherence to immunosuppressive drugs,327 331 alterations in health insurance coverage,332,333 or difficulties with transition between pediatric and adult posttransplant care.334 337 It should be noted that during early adulthood, notably between 18 and 25 years, patients become independent while facing a transition from childhood to adulthood. This age period denotes a developmental stage that has been conceptually termed “emerging adulthood.”338 Adherence to immunosuppressive medication is associated with a longer graft survival in pediatric renal transplant recipients.339 In pediatric solid organ transplant recipients, the rate of poor adherence among adolescents was three times higher than younger children327 and 60% higher than adults between 24 and 44 years old.331 Greater family dysfunction,327 risk-taking behavior,340,341 brain immaturity,342 344 neurocognitive impairment from chronic renal failure345 or young age at onset of ESRD,346 developmental delays at young age,347 and lower attention and executive function348 are associated with nonadherence during adolescence and emerging adulthood. In addition, adherence to general care such as clinic appointments and routine blood monitoring deteriorates during adolescence and emerging adulthood in renal transplant recipients.349 Greater frequency of visiting in young liver transplant recipients350 or a strong relationship with providers in adult renal transplant recipients351 improved the adherence but more studies are needed in this area. The transfer from pediatric to adult-oriented care is associated with adverse outcomes in pediatric renal transplant recipients. The graft failure rate was 35% within 3 years of transfer to adult service.352 The risk of graft failure was highest immediately following transfer.335 In 2008, through the initiative of the Pediatric Committee of the American Society of Transplantation, a jointly sponsored international consensus conference on Adolescent Transition to Adult Care in Solid Organ Transplantation recommended a transition process incorporating the developmental aspect of adolescents with solid organ transplant.353 In 2011 the International Society of Nephrology and

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International Pediatric Nephrology Institution released a consensus statement on the transition of children with renal disease and suggested interventions at all levels of adherence.354 However, these recommendations did not address the key factor, the self-management skill, in the readiness for transition.355,356 Setup of a multidisciplinary transition clinic improved graft survival357,358 or adherence and renal function.359 In addition, since those who were transferred before 21 years old had a higher graft failure rate than those who were transferred after 21 years old,337 it has been suggested to extend traditional pediatric care to the emergent adulthood group.360

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Long term results of liver-kidney transplantation in children with primary hyperoxaluria. Pediatr Nephrol 2001;16(12):946 50. 259. Ellis SR, Hulton SA, McKiernan PJ, et al. Combined liver-kidney transplantation for primary hyperoxaluria type 1 in young children. Nephrol Dial Transpl 2001;16(2):348 54. 260. Cochat P, Liutkus A, Fargue S, et al. Primary hyperoxaluria type 1: still challenging!. Pediatr Nephrol 2006;21(8):1075 81. 261. Van Calcar SC, Harding CO, Lyne P, et al. Renal transplantation in a patient with methylmalonic acidaemia. J Inherit Metab Dis 1998; 21(7):729 37. 262. Nesterova G, Gahl W. Nephropathic cystinosis: late complications of a multisystemic disease. Pediatr Nephrol 2008;23(6):863 78. 263. Langlois V, Geary D, Murray L, et al. Polyuria and proteinuria in cystinosis have no impact on renal transplantation. A report of the North American Pediatric Renal Transplant Cooperative Study. Pediatr Nephrol 2000;15(1 2):7 10. 264. Ueda M, O’Brien K, Rosing DR, et al. Coronary artery and other vascular calcifications in patients with cystinosis after kidney transplantation. Clin J Am Soc Nephrol 2006;1(3):555 62. 265. Axelrod DA, Holmes R, Thomas SE, et al. Limitations of EBV-PCR monitoring to detect EBV associated post-transplant lymphoproliferative disorder. Pediatr Transplant 2003;7(3):223 7. 266. Dharnidharka VR, Sullivan EK, Stablein DM, et al. North American pediatric renal transplant cooperative study, risk factors for posttransplant lymphoproliferative disorder (PTLD) in pediatric kidney transplantation: a report of the north american pediatric renal transplant cooperative study (NAPRTCS). Transplantation 2001;71(8):1065 8. 267. Koukourgianni F, Harambat J, Ranchin B, et al. Malignancy incidence after renal transplantation in children: a 20-year single-centre experience. Nephrol Dial Transplant 2010;25(2):611 16. 268. Euvrard S, Kanitakis J, Cochat P, et al. Skin cancers following pediatric organ transplantation. Dermatol Surg 2004;30(4 Pt 2):616 21. 269. Coutinho HM, Groothoff JW, Offringa M, et al. De novo malignancy after paediatric renal replacement therapy. Arch Dis Child 2001; 85(6):478 83.

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270. Euvrard S, Morelon E, Rostaing L, et al. Sirolimus and secondary skin-cancer prevention in kidney transplantation. N Engl J Med 2012;367(4):329 39. 271. Penn I. De novo malignances in pediatric organ transplant recipients. Pediatr Transplant 1998;2(1):56 63. 272. Riva G, Luppi M, Barozzi P, et al. How I treat HHV8/KSHV-related diseases in posttransplant patients. Blood 2012;120(20):4150 9. 273. Simard JF, Baecklund E, Kinch A, et al. Pediatric organ transplantation and risk of premalignant and malignant tumors in Sweden. Am J Transplant 2011;11(1):146 51. 274. Tsaur I, Obermuller N, Jonas D, et al. De novo renal cell carcinoma of native and graft kidneys in renal transplant recipients. BJU Int 2011;108(2):229 34. 275. Matas AJ, Smith JM, Skeans MA, et al. OPTN/SRTR 2011 Annual Data Report: kidney. Am J Transplant 2013;13(Suppl. 1):11 46. 276. Collins MH, Montone KT, Leahey AM, et al. Post-transplant lymphoproliferative disease in children. Pediatr Transplant 2001;5(4):250 7. 277. Holmes RD, Sokol RJ. Epstein-Barr virus and post-transplant lymphoproliferative disease. Pediatr Transplant 2002;6(6):456 64. 278. Schober T, Framke T, Kreipe H, et al. Characteristics of early and late PTLD development in pediatric solid organ transplant recipients. Transplantation 2013;95(1):240 6. 279. Opelz G, Daniel V, Naujokat C, et al. Epidemiology of pretransplant EBV and CMV serostatus in relation to posttransplant non-Hodgkin lymphoma. Transplantation 2009;88(8):962 7. 280. Merlo A, Turrini R, Dolcetti R, et al. The interplay between Epstein-Barr virus and the immune system: a rationale for adoptive cell therapy of EBV-related disorders. Haematologica 2010;95(10):1769 77. 281. Opelz G, Dohler B. Pediatric kidney transplantation: analysis of donor age, HLA match, and posttransplant non-Hodgkin lymphoma: a collaborative transplant study report. Transplantation 2010;90(3):292 7. 282. Dharnidharka VR, Harmon WE. Management of pediatric postrenal transplantation infections. Sem Nephrol 2001;21(5):521 31. 283. Hertig A, Zuckermann A. Rabbit antithymocyte globulin induction and risk of post-transplant lymphoproliferative disease in adult and pediatric solid organ transplantation: an update. Transplant Immunol 2015;32(3):179 87. 284. Longmore DK, Conwell LS, Burke JR, et al. Post-transplant lymphoproliferative disorder: no relationship to recombinant human growth hormone use in Australian and New Zealand pediatric kidney transplant recipients. Pediatr Transplant 2013;17(8):731 6. 285. Nelson BP, Nalesnik MA, Bahler DW, et al. Epstein-Barr virus-negative post-transplant lymphoproliferative disorders: a distinct entity? Am J Surg Pathol 2000;24(3):375 85. 286. Mucha K, Foroncewicz B, Ziarkiewicz-Wroblewska B, et al. Post-transplant lymphoproliferative disorder in view of the new WHO classification: a more rational approach to a protean disease? Nephrol Dial Transplant 2010;25(7):2089 98. 287. Rowe DT, Qu L, Reyes J, et al. Use of quantitative competitive PCR to measure Epstein-Barr virus genome load in the peripheral blood of pediatric transplant patients with lymphoproliferative disorders. J Clin Microbiol 1997;35(6):1612 15. 288. Shroff R, Trompeter R, Cubitt D, et al. Epstein-Barr virus monitoring in paediatric renal transplant recipients. Pediatr Nephrol 2002; 17(9):770 5. 289. Ishihara M, Tanaka E, Sato T, et al. Epstein-Barr virus load for early detection of lymphoproliferative disorder in pediatric renal transplant recipients. Clin Nephrol 2011;76(1):40 8. 290. Wagner HJ, Wessel M, Jabs W, et al. Patients at risk for development of posttransplant lymphoproliferative disorder: plasma versus peripheral blood mononuclear cells as material for quantification of Epstein-Barr viral load by using real-time quantitative polymerase chain reaction. Transplantation 2001;72(6):1012 19. 291. Tanaka E, Sato T, Ishihara M, et al. Asymptomatic high Epstein-Barr viral load carriage in pediatric renal transplant recipients. Pediatr Transplant 2011;15(3):306 13. 292. Hocker B, Fickenscher H, Delecluse HJ, et al. Epidemiology and morbidity of Epstein-Barr virus infection in pediatric renal transplant recipients: a multicenter, prospective study. Clin Infect Dis 2013;56(1):84 92. 293. Green M, Webber SA. EBV viral load monitoring: unanswered questions. Am J Transplant 2002;2(10):894 5. 294. Comoli P, Ginevri F. Monitoring and managing viral infections in pediatric renal transplant recipients. Pediatr Nephrol 2012;27(5):705 17. 295. Frey NV, Tsai DE. The management of posttransplant lymphoproliferative disorder. Med Oncol 2007;24(2):125 36. 296. Brennan DC, Aguado JM, Potena L, et al. Effect of maintenance immunosuppressive drugs on virus pathobiology: evidence and potential mechanisms. Rev Med Virol 2013;23(2):97 125. 297. Alfieri C, Allison AC, Kieff E. Effect of mycophenolic acid on Epstein-Barr virus infection of human B lymphocytes. Antimicrob Agents Chemother 1994;38(1):126 9. 298. Neyts J, Andrei G, De Clercq E. The novel immunosuppressive agent mycophenolate mofetil markedly potentiates the antiherpesvirus activities of acyclovir, ganciclovir, and penciclovir in vitro and in vivo. Antimicrob Agents Chemother 1998;42(2):216 22. 299. Tsao L, Hsi ED. The clinicopathologic spectrum of posttransplantation lymphoproliferative disorders. Arch Pathol Lab Med 2007; 131(8):1209 18. 300. Taylor AL, Marcus R, Bradley JA. Post-transplant lymphoproliferative disorders (PTLD) after solid organ transplantation. Crit Rev Oncol Hematol 2005;56(1):155 67. 301. Gross TG, Orjuela MA, Perkins SL, et al. Low-dose chemotherapy and rituximab for posttransplant lymphoproliferative disease (PTLD): a Children’s Oncology Group Report. Am J Transplant 2012;12(11):3069 75. 302. Bollard CM, Rooney CM, Heslop HE. T-cell therapy in the treatment of post-transplant lymphoproliferative disease. Nat Rev Clin Oncol 2012;9(9):510 19. 303. Kapusinszky B, Chen SF, Sahoo MK, et al. BK polyomavirus subtype III in a pediatric renal transplant patient with nephropathy. J Clin Microbiol 2013;51(12):4255 8. 304. Hirsch HH, Steiger J, Polyomavirus BK. Lancet Infect Dis 2003;3(10):611 23. 305. Ali AM, Gibson IW, Birk P, et al. Pretransplant serologic testing to identify the risk of polyoma BK viremia in pediatric kidney transplant recipients. Pediatr Transplant 2011;15(8):827 34. 306. Smith JM, McDonald RA, Finn LS, et al. Polyomavirus nephropathy in pediatric kidney transplant recipients. Am J Transplant 2004; 4(12):2109 17.

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307. Ramos E, Drachenberg CB, Wali R, et al. The decade of polyomavirus BK-associated nephropathy: state of affairs. Transplantation 2009; 87(5):621 30. 308. Vasudev B, Hariharan S, Hussain SA, et al. BK virus nephritis: risk factors, timing, and outcome in renal transplant recipients. Kidney Int 2005;68(4):1834 9. 309. Smith JM, Dharnidharka VR, Talley L, et al. BK virus nephropathy in pediatric renal transplant recipients: an analysis of the North American Pediatric Renal Trials and Collaborative Studies (NAPRTCS) registry. Clin J Am Soc Nephrol 2007;2(5):1037 42. 310. Vats A, Shapiro R, Singh Randhawa P, et al. Quantitative viral load monitoring and cidofovir therapy for the management of BK virusassociated nephropathy in children and adults. Transplantation 2003;75(1):105 12. 311. Hirsch HH, Brennan DC, Drachenberg CB, et al. Polyomavirus-associated nephropathy in renal transplantation: interdisciplinary analyses and recommendations. Transplantation 2005;79(10):1277 86. 312. Zarauza Santovena A, Garcia Meseguer C, Martinez Mejia S, et al. BK virus infection in pediatric renal transplantation. Transplant Proc 2015;47(1):62 6. 313. Ginevri F, Azzi A, Hirsch HH, et al. Prospective monitoring of polyomavirus BK replication and impact of pre-emptive intervention in pediatric kidney recipients. Am J Transplant 2007;7(12):2727 35. 314. Hymes LC, Warshaw BL. Polyomavirus (BK) in pediatric renal transplants: evaluation of viremic patients with and without BK associated nephritis. Pediatr Transplant 2006;10(8):920 2. 315. Pallet N, Burgard M, Quamouss O, et al. Cidofovir may be deleterious in BK virus-associated nephropathy. Transplantation 2010; 89(12):1542 4. 316. Hymes L. BK virus nephropathy: a pediatric nephrologist’s perspective. Transplant Rev 2010;24(1):28 31. 317. Araya CE, Lew JF, Fennell RS, et al. Intermediate dose cidofovir does not cause additive nephrotoxicity in BK virus allograft nephropathy. Pediatr Transplant 2008;12(7):790 5. 318. Izzedine H, Launay-Vacher V, Deray G. Antiviral drug-induced nephrotoxicity. Am J Kidney Dis 2005;45(5):804 17. 319. Reisman L, Habib S, McClure GB, et al. Treatment of BK virus-associated nephropathy with CMX001 after kidney transplantation in a young child. Pediatr Transplant 2014;18(7):E227 31. 320. Jung YH, Moon KC, Ha JW, et al. Leflunomide therapy for BK virus allograft nephropathy after pediatric kidney transplantation. Pediatr Transplant 2013;17(2):E50 4. 321. Araya CE, Garin EH, Neiberger RE, et al. Leflunomide therapy for BK virus allograft nephropathy in pediatric and young adult kidney transplant recipients. Pediatr Transplant 2010;14(1):145 50. 322. Zaman RA, Ettenger RB, Cheam H, et al. A novel treatment regimen for BK viremia. Transplantation 2014;97(11):1166 71. 323. Sanghavi SK, Abu-Elmagd K, Keightley MC, et al. Relationship of cytomegalovirus load assessed by real-time PCR to pp65 antigenemia in organ transplant recipients. J Clin Viro 2008;42(4):335 42. 324. Andreoni KA, Forbes R, Andreoni RM, et al. Age-related kidney transplant outcomes: health disparities amplified in adolescence. JAMA Int Med 2013;173(16):1524 32. 325. Foster BJ, Dahhou M, Zhang X, et al. Association between age and graft failure rates in young kidney transplant recipients. Transplantation 2011;92(11):1237 43. 326. Van Arendonk KJ, James NT, Boyarsky BJ, et al. Age at graft loss after pediatric kidney transplantation: exploring the high-risk age window. Clin J Am Soc Nephrol 2013;8(6):1019 26. 327. Dew MA, Dabbs AD, Myaskovsky L, et al. Meta-analysis of medical regimen adherence outcomes in pediatric solid organ transplantation. Transplantation 2009;88(5):736 46. 328. Dobbels F, Ruppar T, De Geest S, et al. Adherence to the immunosuppressive regimen in pediatric kidney transplant recipients: a systematic review. Pediatr Transplant 2010;14(5):603 13. 329. Rianthavorn P, Ettenger RB. Medication non-adherence in the adolescent renal transplant recipient: a clinician’s viewpoint. Pediatr Transplant 2005;9(3):398 407. 330. Shaw RJ, Palmer L, Blasey C, et al. A typology of non-adherence in pediatric renal transplant recipients. Pediatr Transplant 2003;7(6):489 93. 331. Pinsky BW, Takemoto SK, Lentine KL, et al. Transplant outcomes and economic costs associated with patient noncompliance to immunosuppression. Am J Transplant 2009;9(11):2597 606. 332. Willoughby LM, Fukami S, Bunnapradist S, et al. Health insurance considerations for adolescent transplant recipients as they transition to adulthood. Pediatr Transplant 2007;11(2):127 31. 333. White PH. Access to health care: health insurance considerations for young adults with special health care needs/disabilities. Pediatrics 2002;110(6 Pt 2):1328 35. 334. Chaturvedi S, Jones CL, Walker RG, et al. The transition of kidney transplant recipients: a work in progress. Pediatr Nephrol 2009; 24(5):1055 60. 335. Samuel SM, Nettel-Aguirre A, Hemmelgarn BR. Pediatric Renal Outcomes Canada Group, Graft failure and adaptation period to adult healthcare centers in pediatric renal transplant patients. Transplantation 2011;91(12):1380 5. 336. Watson AR, Harden P, Ferris M, et al. Transition from pediatric to adult renal services: a consensus statement by the International Society of Nephrology (ISN) and the International Pediatric Nephrology Association (IPNA). Pediatr Nephrol 2011;26(10):1753 7. 337. Foster BJ, Platt RW, Dahhou M, et al. The impact of age at transfer from pediatric to adult-oriented care on renal allograft survival. Pediatr Transplant 2011;15(7):750 9. 338. Arnett JJ. Emerging adulthood. A theory of development from the late teens through the twenties. Am Psychol 2000;55(5):469 80. 339. Chisholm-Burns MA, Spivey CA, Rehfeld R, et al. Immunosuppressant therapy adherence and graft failure among pediatric renal transplant recipients. Am J Transplant 2009;9(11):2497 504. 340. Reyna VF, Farley F. Risk and rationality in adolescent decision making: implications for theory, practice, and public policy. Psychol Sci Public Interest 2006;7(1):1 44. 341. Casey BJ, Getz S, Galvan A. The adolescent brain. Devel Rev 2008;28(1):62 77.

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342. Casey BJ, Tottenham N, Liston C, et al. Imaging the developing brain: what have we learned about cognitive development? Trends Cogn Sci 2005;9(3):104 10. 343. Rubia K, Smith AB, Woolley J, et al. Progressive increase of frontostriatal brain activation from childhood to adulthood during eventrelated tasks of cognitive control. Hum Brain Mapp 2006;27(12):973 93. 344. Casey BJ, Jones RM, Hare TA. The adolescent brain. Ann N York Acad Sci 2008;1124:111 26. 345. Gipson DS, Hooper SR, Duquette PJ, et al. Memory and executive functions in pediatric chronic kidney disease. Child Neuropsychol 2006;12(6):391 405. 346. Johnson RJ, Warady BA. Long-term neurocognitive outcomes of patients with end-stage renal disease during infancy. Pediatr Nephrol 2013;28(8):1283 91. 347. Gipson DS, Duquette PJ, Icard PF, et al. The central nervous system in childhood chronic kidney disease. Pediatric nephrology 2007; 22(10):1703 10. 348. Hooper SR, Gerson AC, Butler RW, et al. Neurocognitive functioning of children and adolescents with mild-to-moderate chronic kidney disease. Clin J Am Soc Nephrol 2011;6(8):1824 30. 349. Zelikovsky N, Schast AP, Palmer J, et al. Perceived barriers to adherence among adolescent renal transplant candidates. Pediatr Transplant 2008;12(3):300 8. 350. Shemesh E, Annunziato RA, Shneider BL, et al. Improving adherence to medications in pediatric liver transplant recipients. Pediatr Transplant 2008;12(3):316 23. 351. Chisholm MA, Lance CE, Mulloy LL. Patient factors associated with adherence to immunosuppressant therapy in renal transplant recipients. Am J Health Syst Pharm 2005;62(17):1775 81. 352. Watson AR. Non-compliance and transfer from paediatric to adult transplant unit. Pediatr Nephrol 2000;14(6):469 72. 353. Bell LE, Bartosh SM, Davis CL, et al. Adolescent transition to adult care in solid organ transplantation: a consensus conference report. Am J Transplant 2008;8(11):2230 42. 354. Watson AR, Harden PN, Ferris ME, et al. Transition from pediatric to adult renal services: a consensus statement by the International Society of Nephrology (ISN) and the International Pediatric Nephrology Association (IPNA). Kidney Int 2011;80(7):704 7. 355. Sawyer SM, Aroni RA. Self-management in adolescents with chronic illness. What does it mean and how can it be achieved? Med J Aust 2005;183(8):405 9. 356. Dobbels F, Van Damme-Lombaert R, Vanhaecke J, et al. Growing pains: non-adherence with the immunosuppressive regimen in adolescent transplant recipients. Pediatr Transplant 2005;9(3):381 90. 357. Prestidge C, Romann A, Djurdjev O, et al. Utility and cost of a renal transplant transition clinic. Pediatr Nephrol 2012;27(2):295 302. 358. Harden PN, Walsh G, Bandler N, et al. Bridging the gap: an integrated paediatric to adult clinical service for young adults with kidney failure. BMJ 2012;344:e3718. 359. McQuillan RF, Toulany A, Kaufman M, et al. Benefits of a transfer clinic in adolescent and young adult kidney transplant patients. Canad J Kidney Health Dis 2015;2:45. Available from: http://dx.doi.org/10.1186/s40697-015-0081-6. 360. Foster BJ. Heightened graft failure risk during emerging adulthood and transition to adult care. Pediatr Nephrol 2015;30(4):567 76.

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C H A P T E R

14 Indications for Renal Transplantation: Evaluation of Transplant Candidates Opas Traitanon1,2 and Lorenzo Gallon1 1

Northwestern University, Chicago, IL, United States 2Thammasart University Hospital, Pathumthani, Thailand

14.1 INTRODUCTION Kidney transplantation, at this time, provides the best treatment option for end stage renal disease (ESRD) patients. However, while the incidence of treated ESRD has almost doubled over the last decade, the number of potential donor kidneys has not shown the same type of substantial growth. Consequently, the median waiting time for patients receiving a kidney allograft has steadily increased (Fig. 14.1). Data from the United States Renal Data System (USRDS) in 2014 showed that the average wait time for newly listed transplant candidates currently ranges from 3 to 5 years (Fig. 14.2). In order to ensure that the limited and vital resources we have are used optimally, all transplant candidates should undergo pretransplant evaluation to ensure that they are fit for anesthesia and operation. All the candidates in the transplant waiting list should be capable of surviving the current waiting time for transplant, and should also have a reasonable life expectancy as well as an expected greater quality of life after the procedure.

Transplant rate 10

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FIGURE 14.1

Percent of dialysis patients waitlisted versus unadjusted transplant rates.

Kidney Transplantation, Bioengineering, and Regeneration. DOI: http://dx.doi.org/10.1016/B978-0-12-801734-0.00014-X

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© 2017 Elsevier Inc. All rights reserved.

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Counts: subsequent listings Wait time: subsequent listings

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FIGURE 14.2 Kidney waiting list counts versus waiting time.

14.2 INDICATIONS AND CONTRAINDICATIONS 14.2.1 Potential Indications for Kidney Transplantation Renal transplantation is the treatment of choice in most patients with ESRD. In the past decade, significant improvements in transplant management now allows most patients with ESRD to be considered for kidney transplantation, unless there is an apparent contraindication. Although there is increased risk of death in the first few months following transplant, the long-term survival and quality of life are significantly higher in patients having had a kidney transplantation compared to those patients on dialysis.1 5 Potential kidney transplant candidates should be referred to a transplant program for evaluation when their glomerular filtration rate falls below 20 30 mL/minute or when initial renal replacement therapy is expected in the following 12 months.6 8 For patients already on chronic dialysis, the transplant option should be offered and discussed with them when all other medical conditions have stabilized.

14.2.2 Contraindications for Kidney Transplantation There are certain contraindications to renal transplantation: • • • • •

Advanced stage or untreated cancer with short life expectancy Untreated active infection Severe psychiatric disease Unresolvable psychosocial problems Persistent substance abuse

The following conditions require careful evaluation or possibly correction before proceeding to transplantation. Some conditions might be a contraindication in one transplant center but not in another. • Unreconstructable coronary artery disease or refractory congestive heart failure (may be a candidate for combined organ transplantation). • History of malignancy. The cancer-free interval required is varied depending on the stage and type of cancer (the detailed discussions follow).

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All

0–17

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Waiting list or transplantation among incident ESRD patients by age (0 74 years) (USRDS 2014).

• Substance abuse history. Patients must present evidence of involvement in at least 12 months of drug-free rehabilitation. This includes written documentation of participation in rehabilitation including negative random toxicologic screens. • Active hepatitis or chronic liver disease. Candidates with chronic hepatitis B or C or persistently abnormal liver function testing should be evaluated by a hepatologist. Patients may be candidates for combined liver and kidney transplantation. • Structural genitourinary abnormality or recurrent urinary tract infection. Urologic consultation is required prior to transplantation. • Treated and controlled active infection. • Proven noncompliance patients. • Severe malnutrition. • Severe hyperparathyroidism. • Past psychosocial abnormality that might interfere with posttransplant compliance. • Cerebrovascular disease with significant disabilities. • Aortoiliac disease. Patients with abnormal femoral pulses or disabling claudication, rest pain, or gangrene should be evaluated by a vascular surgeon prior to consideration. Patients with significant aortoiliac occlusive disease may require angioplasty or aortoiliac grafting prior to transplantation. Advanced age alone should not be considered a contraindication if the patients are medically and surgically suitable for kidney transplantation, although it is still unclear whether the benefits of transplantation are sufficient enough to consider transplantation over chronic dialysis. Data from USRDS 2014 showed a gradual increase of patients over age 65 in the transplant waiting list in the past decade (Fig. 14.3), meaning there is an increasing number of patients over 65 years of age who require chronic dialysis and kidney transplantation. Most published studies show better survival of elderly patients with kidney transplantation in comparison to patients with chronic dialysis.1,9,10 However, the recipients in the studies could be a sample of cases that do not perfectly represent the population of all elderly patients. Elderly candidates should have a reasonable expectancy beyond the current waiting time for transplantation and should have an expected improved quality of life after transplantation.

14.3 PRETRANSPLANT EVALUATION FOR TRANSPLANT CANDIDATES 14.3.1 Initial Evaluation The first step for pretransplant evaluation is to inform the patients and/or family members about the ESRD treatment options available and the risks and benefits of kidney transplantation compared to the patients’ current treatment. If the patients decide to proceed with kidney transplantation, the physician should carefully review the medical history, underlying renal diseases, previous nonrenal illnesses, and other comorbidities of the

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14. PRETRANSPLANT RECIPIENT EVALUATION

Initial Diagnostic Studies for Kidney Transplant Candidates

• Complete blood count, Blood group, PT/PTT/INR • Human leukocyte antigen (HLA typing) and panel reactive antibody testing (PRA) • Blood chemistry & metabolic profile: BUN, creatinine, uric acid, electrolytes, calcium, phosphate, liver function test, albumin, globulin, parathyroid hormone (PTH), lipid profile • Urinalysis • Serology: hepatitis B and C, human immunodeficiency virus (HIV), cytomegalovirus (CMV), Epstein Barr virus (EBV), varicella virus, herpes simplex virus, rapid plasma regain (RPR) • Pregnancy test for fertile women • Chest X-ray • Electrocardiogram • Prostatic specific antigen in elderly men • Pap smear (all women), mammography (elderly women) • Screening colonoscopy (elderly patients)

patient. A detailed history of cardiovascular disease including family history should be obtained. Physical examination should include checking for signs of congestive heart failure, carotid artery disease, or peripheral vascular disease. Elderly patients should also be examined for common malignancy such as prostate, breast, and cervical cancer. The initial testing is summarized in Table 14.1.

14.3.2 Cardiovascular Disease Cardiovascular disease is the greatest cause of death of both dialysis and transplant patients. Data from USRDS 2014 showed the rate of death from cardiovascular disease is 3.6 per 1000 patients per year, almost double compared to the rate of death by infection and malignancy; cardiovascular disease is also the leading cause of death with a functioning graft. Transplant candidates should be assessed for cardiovascular risk to ensure that they are fit to undergo the anesthesia and transplant surgery. However, extensive cardiovascular evaluation in the transplant candidates still remains controversial since many patients may be subjected to numerous unnecessary screening procedures and sometimes receive false-positive results from such screenings. There is little evidence that screening asymptomatic transplant candidates for cardiac diseases will improve posttransplant outcomes. Screening could also lead to rejection of a transplant candidate who otherwise might benefit from transplantation, or could delay the transplant procedure, which could lead to increased costs and complications.11 Furthermore, recent metaanalysis also showed that noninvasive cardiac tests (myocardial perfusion scintigraphy, dobutamine stress echocardiography) are as effective as coronary angiography at predicting future adverse cardiovascular events in cases of advanced chronic kidney disease.12 Although there has been no consensus about the best way to perform cardiac evaluation in the transplant candidates, most transplant centers still do the screening for cardiac diseases. The goal of this screening is to identify those patients who might benefit from cardiac intervention and risk factor modification before undergoing kidney transplantation. For most transplant candidates, examining the medical history carefully, conducting a physical examination, a chest X-ray and a 12-lead electrocardiogram (EKG) are the standard initial noninvasive tests. Echocardiograms may be performed to assess the structural abnormalities, left ventricular functions, and valvular abnormalities. In asymptomatic low cardiovascular risk patients, the initial evaluation (medical history, physical examination, basic EKG, chest X-ray 6 echocardiography) has a very high negative predictive value13 and should be appropriate. Patients with negative test results may be placed on the transplant waitlist. In cases of patients with high cardiovascular risk (elderly patients, diabetes mellitus, history of cardiovascular disease, family history of cardiovascular disease), additional stress testing should be performed. These additional stress tests, such as the dobutamine stress echocardiography, myocardial perfusion scintigraphy, or exercise stress electrocardiography, can be performed in select patients. Although exercise stress electrocardiography is more physiologic and does not require any injection, it may not be suitable for some dialysis patients who might have low exercise tolerance and motor disability. Dobutamine stress echocardiography and myocardial perfusion scintigraphy were shown to have moderate sensitivity and specificity in detecting coronary artery stenosis in patients who are kidney transplant candidates (pooled sensitivity 0.79, pooled specificity 0.89 for dobutamine stress echocardiography; pooled sensitivity 0.74, pooled specificity 0.70 for myocardial perfusion scintigraphy).14 However, there is no clear evidence for superiority of one method of stress testing over the other.13 The selection of stress test should be based on the expertise of each center

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191

itself. High-risk patients with positive stress testing should undergo coronary angiography for further evaluation. Patients with significant coronary lesions should undergo a revascularization procedure. Patients with successful revascularization may proceed to be placed on the transplant waitlist after careful evaluation and should be reevaluated on a regular basis. Coronary CT angiography is also an emerging option to evaluate the obstructive coronary lesions in the transplant candidates.15 On the other hand, there is evidence that noninvasive cardiac tests may not be sufficient to detect the coronary artery disease especially in the diabetes patients with advanced chronic kidney disease.16 18 One study showed that myocardial perfusion scintigraphy could not detect half of the patients who had significant coronary lesions on angiography18; many transplant centers have a lower threshold for coronary angiography in high-risk diabetic patients. The decision tree for cardiovascular evaluation is given in Fig. 14.4. Patients with known ischemic heart disease should also be eligible for transplantation if they are low-risk asymptomatic patients with negative noninvasive tests. Patients who have undergone successful revascularization or patients who have noncritical diseases and are on appropriate medical therapy may also be candidates for kidney transplantation with careful evaluation.

14.3.3 Cerebrovascular Disease There is an increased incidence of atherosclerotic cerebrovascular disease in dialysis patients as well as in transplant patients.19 22 The mortality rate following stroke after transplantation is quite high so transplant

FIGURE 14.4

Suggested decision tree for cardiovascular evaluation. Source: Modified from ERBP Guideline.

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candidates should be carefully evaluated for evidence of carotid stenosis, especially elderly patients with high risk for cerebrovascular diseases (hypertension, smoking, dyslipidemia, history of cerebrovascular disease). Patients with a history of transient ischemic attack are considered at high risk and should be evaluated by a neurologist. CT scan or magnetic resonance imaging (MRI) and carotid Doppler or magnetic resonance angiography may be performed in these patients. It is also important to screen for cerebral aneurysms in high-risk patients with autosomal dominant polycystic kidney disease (ADPKD) although there is not enough evidence to support all ADPKD patients undergoing transplantation.6

14.3.4 Peripheral Vascular Disease Peripheral vascular disease is also an important part of pretransplant evaluation and can cause allograft ischemia in some transplant cases. Peripheral vascular disease is also associated with higher mortality posttransplantation.23 Diabetes, hypertension, dyslipidemia, smoking, and a history of vascular disease are among the risk factors for peripheral vascular disease. Patients with history of claudication, diabetes, and poor peripheral pulse by physical examination may be candidates for Doppler vascular screening.7 CT scan and CT angiography could also help in the evaluation of anatomical lesions and assess the flow. Patients with large uncorrectable abdominal aneurysms, severe occlusive common iliac disease, active gangrene, or recent atheroembolic events are not suitable candidates for kidney transplantation.6

14.3.5 Malignancy All transplant candidates should be carefully evaluated for the potential of occult malignancy. Chronic dialysis increases risk of some malignancies.24 High risks were observed for malignancy of the kidney, bladder, thyroid, and other endocrine organs although in cancers of the lung, colorectum, prostate, breast, and stomach the risks were not consistently increased.24 Younger patients were found to have higher risk than older patients on chronic dialysis and may benefit the most from cancer screening. Posttransplant immunosuppression may enhance the growth of malignant cells. Furthermore, chronic immunosuppression after transplantation has an impact on both viral and tumor surveillance of the recipient immune system and has been linked to the higher incidence of malignancy after transplantation. From the 2014 USRDS report, malignancy was the cause of death in 1.8 patients per 1000 patients per year, which is about the same rate as that of infection, and about half of that of cardiovascular disease. Active malignancy is generally an absolute contraindication for transplantation. However, patients who were successfully treated for malignancy can be suitable candidates for kidney transplantation. There is limited data available regarding the rate of recurrence after transplantation.25,26 One study showed that 53% of malignancy recurrence occurred within 2 years of successful treatment, 33% of recurrence occurred in patients treated between 2 and 5 years before transplantation, and only 13% of cases recurred if successfully treated for more than 5 years.25 Based on the available data, most published clinical guidelines recommend a waiting time of at least 2 5 years to minimize the risk of malignancy recurrence after transplantation.6,27,28 Breast carcinomas, symptomatic renal carcinomas, sarcomas, bladder carcinomas, nonmelanoma skin cancers and multiple myeloma are among the malignancies that have highest recurrence rates after transplantation. Some malignances such as basal cell carcinoma of the skin and superficial lesions of bladder carcinoma may not require the wait period after successful treatment of the lesions.27 However, patients with advanced breast cancer (stages III and IV) and patients with multiple myeloma should not undergo renal transplantation due to the very high risk of recurrence.6 The recommended wait times for some common cancers are summarized in Table 14.2. There are many clinical tools available to screen the transplant candidates for malignancies. Various transplant centers may have their own screening program, which may differ from one another based on the risk factor of patients and incidence of common malignancies in the local population. The evaluation should be individually considered based on patient’s age, sex, family history, and environmental exposures. Patients with high risk for renal and bladder cancer may be screened by renal ultrasound,29 urinalysis, urine cytology, and cystoscopy. All female candidates should undergo pelvic examination and cervical cytology to exclude occult cervical cancer. Breast examination should be performed as well as a screening mammogram in women over age 40. Male patients over age 50 should have digital rectal examination and prostatic specific antigen tested. Fecal occult blood, colonoscopy or double contrast barium enema X-ray may be performed in individuals with high risk of colorectal cancer. Patients with hepatitis B or C virus

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TABLE 14.2

193

Recommended Wait Times for Common Malignancies (Canadian Guidelines)

Type

Recommended wait time

Cervival cancer • localized cervical cancer • in situ cervical lesions • invasive cervical lesion Breast cancer

• 2 years • 2 years • No firm recommendation 5 years (2 years for early in situ lesions)

Bladder cancer

2 years

Renal cell carcinoma

2 years

Colorectal cancer

5 years

Hodgkin’s disease, non-Hodgkin’s lymphoma, posttransplant lymphoproliferative disorder or leukemia

2 years

Lung cancer

2 years

Melanoma

5 years

Prostate cancer

2 years

Thyroid cancer

2 years

Testicular cancer

2 years

infection should be screened for hepatocellular carcinoma using alpha-fetoprotein, liver ultrasonography, CT scan, or MRI.

14.3.6 Infection All transplant candidates should be free from active infection and should receive immunizations for prevalent or life-threatening infections. The pretransplant vaccinations should include hepatitis B (if nonimmune), influenza (annually), pneumococcal vaccine (every 5 years), and the varicella vaccine (if negative for varicella-zoster antibodies).6,13 Vaccination should be administered early in the course of disease since the response rate may be reduced in patients with worse kidney function.30 Serologic status of hepatitis B and C virus, cytomegalovirus (CMV) and Epstein Barr virus (EBV) should be obtained in all transplant candidates. Patients with evidence of chronic hepatitis B or C infection require consultation with a hepatologist for possible treatment options prior to transplantation. All patients should be assessed for a history of tuberculosis exposure (direct contact from friends or family and travel history) especially in the endemic area such as south/southeast Asia and Africa. Purified protein derivatives (PPD) skin test or quantiferon assay may be useful in cases with questionable chest X-ray’s although the PPD skin test might give negative results in ESRD patients. Patients with active or latent tuberculosis should be treated before undergoing transplantation.31 HIV infection is not a contraindication for kidney transplantation. The rapid advancement of highly active antiretroviral therapy has led to markedly improved survival and quality of life of HIV infected patients.32 The estimated prevalence of HIV infection in advance stage kidney disease is around 0.5% 1.5% and that number is expected to gradually increase in the future.33,34 Many transplant centers now routinely perform kidney transplantation in well-controlled HIV infected patients. Recent data has shown that the long-term outcomes of HIV patients who received kidney transplantation are similar to non-HIV-infected patients and significantly better than HCV-infected or HIV/HCV coinfected recipients.35,36 Kidney transplantation from HIV positive donors to HIV positive recipients is also a suitable option. Data on carefully selected patients show that the patient and graft survival are good for up to 5 years posttransplant, with more than 80% graft survival and more than 70% patient survival at 5 years.37 The HIV-infected patients should be carefully selected and evaluated before transplantation. The patients should have undetectable HIV viral load with CD4 counts more than 200/µL and have been stable for at least 3 months. The patients should be free from any opportunistic infections for at least 6 months and show no signs of possible progressive multifocal leukoencephalopathy, chronic intestinal cryptosporidiosis, or lymphoma.13

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14.4 PRETRANSPLANT RENAL AND SYSTEMIC DISEASES 14.4.1 Glomerulonephritis Glomerulonephritis is the primary cause of ESRD in about 30% 50% of kidney transplant recipients.38 Recurrent glomerular disease after transplantation is quite common (5% 20% of recipients)8,27,39 and is the third most leading cause of posttransplant graft loss after chronic rejection and death with functioning graft.38,40 Despite the risk of recurrent glomerular disease, there is no contraindication to the first kidney transplant in patients with ESRD due to primary glomerulonephritis. The most common form of recurrent disease is IgA nephropathy although it is not the dominant glomerulopathy responsible for graft loss. Primary focal segmental glomerulosclerosis (FSGS) frequently recurs after kidney transplantation (30% 50%),38,41 44 and graft loss occurs in 30% 50% of patients with recurrence. If the patients lose their graft due to FSGS recurrence, the risk of recurrence in the second transplant is very high.45 Recurrence usually occurs early after transplantation, even as early as the first day after transplantation. Late recurrence can occur but tends to be a slower process with less proteinuria.40 The patients and the donors (in living donation) should be informed about the risk of recurrence and graft loss. Secondary FSGS, however, does not recur after transplantation. Membranoproliferative glomerulonephritis (MPGN) type 2 also has a high recurrent rate (more than 80%) after transplant and the risk of graft loss is also high. The risk of recurrence and risk of graft loss after recurrence in primary glomerulonephritis are summarized in Table 14.3. To date there is no evidence that any specific immunosuppressive regime or induction agent affects the risk of recurrent glomerular disease, and therefore, preoperative counseling regarding the risks of graft loss is critical.

14.4.2 Autoimmune Diseases Although significant improvement has been made in the past decades, some patients still experience ESRD from autoimmune diseases. Patients with clinically active systemic lupus erythematosus (SLE) should wait until the disease is quiescent for at least 6 months off cytotoxic agents.46 SLE recurs in 10% 30% of patients but rarely leads to graft failure.27,47,48 Scleroderma patients should wait for at least 6 months off cytotoxic agents and have limited extrarenal diseases.46 Patients with vasculitis (Wegener’s granulomatosis, microscopic polyangiitis, pauciimmune necrotizing glomerulonephritis, and Henoch Schonlein purpura) develop recurrent diseases in about 20% of cases posttransplantation, which rarely leads to graft loss.27,38 Patients with vasculitis should have quiescent disease for at least 12 months off immunosuppressive agents.46

14.4.3 Primary Hyperoxaluria Primary hyperoxaluria is a rare condition characterized by the overproduction of oxalate due to the shortage of an enzyme that normally prevents oxalate accumulation. Deposits of calcium oxalate can lead to kidney damage, kidney failure, and injury to other organs.49 Screening for primary hyperoxaluria should be considered for all young patients with ESRD secondary to stones. Patients with kidney failure from primary hyperoxaluria type 1 should not undergo kidney transplantation alone due to the very high risk of recurrence.50,51 Combined liver and kidney transplantation is the treatment of choice.52 54 Sequential transplantation (liver transplantation followed by kidney transplantation) may allow the clearance of oxalate from the patients and protect the new kidney that will be transplanted later.55 57 TABLE 14.3

Risk of Recurrence and Risk of Graft Loss After Recurrence in Primary Glomerulonephritis

Type

Risk of recurrent

Risk of graft loss

Primary FSGS

30% 50%

High

MPGN Type 1

20% 50%

Moderate

MPGN Type 2

80% 100%

High

Membranous nephropathy

10% 40%

Moderate

IgA nephropathy

Up to 100%

Low

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14.4.4 Hemolytic Uremic Syndrome Transplant candidates with proven shiga-toxin Escherichia coli-associated hemolytic uremic syndrome have very low risk of recurrence after transplantation and can proceed with either deceased or living donor kidney transplantation.11,58 60 Atypical hemolytic uremic syndrome (aHUS) is a rare condition characterized by dysregulation of the alternate complement pathway. aHUS can present as familial (,20%) but most often the cases are of the sporadic type. Most common genetic abnormalities include the mutation of complement factor H (CFH, 20% 30%), membrane cofactor protein (MCP, 10% 15%), complement factor I (CFI, 5% 10%), complement C3 (2% 10%), thrombomodulin (THBD, 3% 5%), and complement factor B (CFB, 1% 4%).61,62 About 10% of the patients may have combined mutations. Antibodies to CFH have been identified in about 6% of patients with sporadic aHUS.61 aHUS with CFH, CFI, and CFB mutations have high recurrent rates (70% 100%) after kidney transplantation, and graft failure after recurrence is high (80% 90%).61,63 65 However, aHUS with MCP mutation has less than 20% recurrent rate (unless associated with other mutations).61 65 The recurrence of aHUS usually occurs within the first year posttransplant. Ischemia-reperfusion injury, immunosuppressive drugs, and infections are among the triggering factors of posttransplant aHUS recurrence. In the past, kidney transplantation was not recommended for the patients with aHUS, however, with the improvement in aHUS management over the past decades, isolated kidney transplantation can be safely done in aHUS patients in centers with experience managing this condition. Eculizumab, a humanized monoclonal anti-C5 antibody recently approved for aHUS treatment, has been used as a prophylactic treatment in aHUS patients who have undergone kidney transplantation with promising results.66 68 Combined liver and kidney transplantation can correct the genetic defects and offer a curative treatment for aHUS, but earlier reports were disappointing.69,70 Nevertheless, recent reports with the use of preoperative eculizumab or plasma therapy with combined liver and kidney transplantation have demonstrated good results, with 80% success rate,71 the newer therapy has allowed isolated kidney transplantation as a more preferable option.

14.5 REEVALUATION AFTER WAITLISTING The growing number of patients on the transplant waiting list and the shortage of kidneys available for transplantation have consequently led to a steady increase in the current wait time for the patients on the list. The transplant candidates on the waiting list should be reevaluated by the transplant nephrologist in order to keep their health status up to date. The timing and frequency of such evaluations should depend on the age and comorbid conditions of the patients. Patients with advanced age, high risk for cardiovascular disease, and diabetes should be periodically reassessed for cardiovascular status. Patients who experience a major comorbidity (such as major cardiovascular events) while on the transplant waiting list should be carefully reassessed after the conditions are successfully treated.

References 1. Wolfe RA, Ashby VB, Milford EL, Ojo AO, Ettenger RE, Agodoa LY, et al. Comparison of mortality in all patients on dialysis, patients on dialysis awaiting transplantation, and recipients of a first cadaveric transplant. New Engl J Med 1999;341(23):1725 30. 2. Rabbat CG, Thorpe KE, Russell JD, Churchill DN. Comparison of mortality risk for dialysis patients and cadaveric first renal transplant recipients in Ontario, Canada. J Am Soc Nephrol 2000;11(5):917 22. 3. Meier-Kriesche HU, Ojo AO, Port FK, Arndorfer JA, Cibrik DM, Kaplan B. Survival improvement among patients with end-stage renal disease: trends over time for transplant recipients and wait-listed patients. J Am Soc Nephrol 2001;12(6):1293 6. 4. Schnuelle P, Lorenz D, Trede M, Van Der, Woude FJ. Impact of renal cadaveric transplantation on survival in end-stage renal failure: evidence for reduced mortality risk compared with hemodialysis during long-term follow-up. J Am Soc Nephrol 1998;9(11):2135 41. 5. Oniscu GC, Brown H, Forsythe JL. Impact of cadaveric renal transplantation on survival in patients listed for transplantation. J Am Soc Nephrol 2005;16(6):1859 65. 6. Knoll G, Cockfield S, Blydt-Hansen T, Baran D, Kiberd B, Landsberg D, et al. Canadian Society of Transplantation: consensus guidelines on eligibility for kidney transplantation. CMAJ 2005;173(10):S1 25. 7. Bunnapradist S, Danovitch GM. Evaluation of adult kidney transplant candidates. Am J Kidney Dis 2007;50(5):890 8. 8. European Best Practice Guidelines for Renal Transplantation (part 1). Nephrol Dial Transpl 2000;15(Suppl. 7):1 85. 9. Rao PS, Merion RM, Ashby VB, Port FK, Wolfe RA, Kayler LK. Renal transplantation in elderly patients older than 70 years of age: results from the Scientific Registry of Transplant Recipients. Transplantation 2007;83(8):1069 74. 10. Oniscu GC, Brown H, Forsythe JL. How great is the survival advantage of transplantation over dialysis in elderly patients? Nephrol Dial Transpl 2004;19(4):945 51.

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11. European Renal Best Practice Transplantation Guideline Development G. ERBP guideline on the management and evaluation of the kidney donor and recipient. Nephrol Dial Transpl 2013;28(Suppl. 2):ii1 71. 12. Wang LW, Masson P, Turner RM, Lord SW, Baines LA, Craig JC, et al. Prognostic value of cardiac tests in potential kidney transplant recipients: a systematic review. Transplantation 2015;99(4):731 45. 13. Abramowicz D, Cochat P, Van Biesen W, Vanholder R. ERBP guideline on the management and evaluation of the kidney donor and recipient. Nephrol Ther 2014;10(6):427 32. 14. Wang LW, Fahim MA, Hayen A, Mitchell RL, Lord SW, Baines LA, et al. Cardiac testing for coronary artery disease in potential kidney transplant recipients: a systematic review of test accuracy studies. Am J Kidney Dis 2011;57(3):476 87. 15. Winther S, Svensson M, Jorgensen HS, Bouchelouche K, Gormsen LC, Pedersen BB, et al. Diagnostic performance of coronary CT angiography and myocardial perfusion imaging in kidney transplantation candidates. J Am Coll Cardiol Img 2015;8(5):553 62. 16. Pilmore H. Cardiac assessment for renal transplantation. Am J Transpl 2006;6(4):659 65. 17. Holley JL, Fenton RA, Arthur RS. Thallium stress testing does not predict cardiovascular risk in diabetic patients with end-stage renal odisease undergoing cadaveric renal transplantation. Am J Med 1991;90(5):563 70. 18. Welsh RC, Cockfield SM, Campbell P, Hervas-Malo M, Gyenes G, Dzavik V. Cardiovascular assessment of diabetic end-stage renal disease patients before renal transplantation. Transplantation 2011;91(2):213 18. 19. Seliger SL, Gillen DL, Longstreth Jr. WT, Kestenbaum B, Stehman-Breen CO. Elevated risk of stroke among patients with end-stage renal disease. Kidney Int 2003;64(2):603 9. 20. Oliveras A, Roquer J, Puig JM, Rodriguez A, Mir M, Orfila MA, et al. Stroke in renal transplant recipients: epidemiology, predictive risk factors and outcome. Clin Transplant 2003;17(1):1 8. 21. Kawamura M, Fijimoto S, Hisanaga S, Yamamoto Y, Eto T. Incidence, outcome, and risk factors of cerebrovascular events in patients undergoing maintenance hemodialysis. Am J Kidney Dis 1998;31(6):991 6. 22. Adams Jr HP, Dawson G, Coffman TJ, Corry RJ. Stroke in renal transplant recipients. Arch Neurol 1986;43(2):113 15. 23. Snyder JJ, Kasiske BL, Maclean R. Peripheral arterial disease and renal transplantation. J Am Soc Nephrol 2006;17(7):2056 68. 24. Maisonneuve P, Agodoa L, Gellert R, Stewart JH, Buccianti G, Lowenfels AB, et al. Cancer in patients on dialysis for end-stage renal disease: an international collaborative study. Lancet 1999;354(9173):93 9. 25. Penn I. Evaluation of transplant candidates with pre-existing malignancies. Ann Transplant 1997;2(4):14 17. 26. Chapman JR, Sheil AG, Disney AP. Recurrence of cancer after renal transplantation. Transplant Proc 2001;33(1 2):1830 1. 27. Kasiske BL, Cangro CB, Hariharan S, Hricik DE, Kerman RH, Roth D, et al. The evaluation of renal transplantation candidates: clinical practice guidelines. Am J Transplant 2001;1(Suppl. 2):3 95. 28. Kasiske BL, Ramos EL, Gaston RS, Bia MJ, Danovitch GM, Bowen PA, et al. The evaluation of renal transplant candidates: clinical practice guidelines. Patient Care and Education Committee of the American Society of Transplant Physicians. J Am Soc Nephrol 1995;6(1):1 34. 29. Gulanikar AC, Daily PP, Kilambi NK, Hamrick-Turner JE, Butkus DE. Prospective pretransplant ultrasound screening in 206 patients for acquired renal cysts and renal cell carcinoma. Transplantation 1998;66(12):1669 72. 30. Soni R, Horowitz B, Unruh M. Immunization in end-stage renal disease: opportunity to improve outcomes. Semin Dialysis 2013;26(4):416 26. 31. British Thoracic Society Standards of Care C, Joint Tuberculosis C, Milburn H, Ashman N, Davies P, Doffman S, et al. Guidelines for the prevention and management of Mycobacterium tuberculosis infection and disease in adult patients with chronic kidney disease. Thorax 2010;65(6):557 70. 32. Mills EJ, Barnighausen T, Negin J. HIV and aging--preparing for the challenges ahead. New Engl J Med 2012;366(14):1270 3. 33. Trullas JC, Cofan F, Tuset M, Ricart MJ, Brunet M, Cervera C, et al. Renal transplantation in HIV-infected patients: 2010 update. Kidney Int 2011;79(8):825 42. 34. Gomez V, Fernandez A, Galeano C, Oliva J, Diez V, Bueno C, et al. Renal transplantation in HIV-infected patients: experience at a tertiary hospital in Spain and review of the literature. Transplant Proc 2013;45(3):1255 9. 35. Locke JE, Mehta S, Reed RD, MacLennan P, Massie A, Nellore A, et al. A National Study of Outcomes among HIV-Infected Kidney Transplant Recipients. J Am Soc Nephrol 2015. Mar 19. pii: ASN.2014070726. 36. Sawinski D, Forde KA, Eddinger K, Troxel AB, Blumberg E, Tebas P, et al. Superior outcomes in HIV-positive kidney transplant patients compared with HCV-infected or HIV/HCV-coinfected recipients. Kidney Int 2015. Available from: http://dx.doi.org/10.1038/ki.2015.74. 37. Muller E, Barday Z, Mendelson M, Kahn D. HIV-positive-to-HIV-positive kidney transplantation--results at 3 to 5 years. New Engl J Med 2015;372(7):613 20. 38. Briganti EM, Russ GR, McNeil JJ, Atkins RC, Chadban SJ. Risk of renal allograft loss from recurrent glomerulonephritis. New Engl J Med 2002;347(2):103 9. 39. Cosio FG, Alamir A, Yim S, Pesavento TE, Falkenhain ME, Henry ML, et al. Patient survival after renal transplantation: I. The impact of dialysis pretransplant. Kidney Int 1998;53(3):767 72. 40. Fairhead T, Knoll G. Recurrent glomerular disease after kidney transplantation. Curr Opin Nephrol Hypertens 2010;19(6):578 85. 41. Canaud G, Dion D, Zuber J, Gubler MC, Sberro R, Thervet E, et al. Recurrence of nephrotic syndrome after transplantation in a mixed population of children and adults: course of glomerular lesions and value of the Columbia classification of histological variants of focal and segmental glomerulosclerosis (FSGS). Nephrol Dial Transpl 2010;25(4):1321 8. 42. Fuentes GM, Meseguer CG, Carrion AP, Hijosa MM, Garcia-Pose A, Melgar AA, et al. Long-term outcome of focal segmental glomerulosclerosis after pediatric renal transplantation. Pediatr Nephrol 2010;25(3):529 34. 43. Hickson LJ, Gera M, Amer H, Iqbal CW, Moore TB, Milliner DS, et al. Kidney transplantation for primary focal segmental glomerulosclerosis: outcomes and response to therapy for recurrence. Transplantation 2009;87(8):1232 9. 44. Pardon A, Audard V, Caillard S, Moulin B, Desvaux D, Bentaarit B, et al. Risk factors and outcome of focal and segmental glomerulosclerosis recurrence in adult renal transplant recipients. Nephrol Dial Transpl 2006;21(4):1053 9. 45. Ponticelli C. Recurrence of focal segmental glomerular sclerosis (FSGS) after renal transplantation. Nephrol Dial Transpl 2010;25(1):25 31. 46. Knoll G, Cockfield S, Blydt-Hansen T, Baran D, Kiberd B, Landsberg D, et al. Canadian Society of Transplantation consensus guidelines on eligibility for kidney transplantation. CMAJ 2005;173(10):1181 4.

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47. Lionaki S, Skalioti C, Boletis JN. Kidney transplantation in patients with systemic lupus erythematosus. World J Transpl 2014;4(3):176 82. 48. Goral S, Ynares C, Shappell SB, Snyder S, Feurer ID, Kazancioglu R, et al. Recurrent lupus nephritis in renal transplant recipients revisited: it is not rare. Transplantation 2003;75(5):651 6. 49. Hoppe B. An update on primary hyperoxaluria. Nat Rev Nephrol 2012;8(8):467 75. 50. Harambat J, van Stralen KJ, Espinosa L, Groothoff JW, Hulton SA, Cerkauskiene R, et al. Characteristics and outcomes of children with primary oxalosis requiring renal replacement therapy. Clin J Am Soc Nephrol 2012;7(3):458 65. 51. Cochat P, Rumsby G. Primary hyperoxaluria. New Engl J Med 2013;369(7):649 58. 52. Rumsby G, Cochat P. Primary hyperoxaluria. New Engl J Med 2013;369(22):2163. 53. Bergstralh EJ, Monico CG, Lieske JC, Herges RM, Langman CB, Hoppe B, et al. Transplantation outcomes in primary hyperoxaluria. Am J Transpl 2010;10(11):2493 501. 54. Lorenzo V, Alvarez A, Torres A, Torregrosa V, Hernandez D, Salido E. Presentation and role of transplantation in adult patients with type 1 primary hyperoxaluria and the I244T AGXT mutation: single-center experience. Kidney Int 2006;70(6):1115 19. 55. Malla I, Lysy PA, Godefroid N, Smets F, Malaise J, Reding R, et al. Two-step transplantation for primary hyperoxaluria: cadaveric liver followed by living donor related kidney transplantation. Pediatric Transpl 2009;13(6):782 4. 56. Sasaki K, Sakamoto S, Uchida H, Shigeta T, Matsunami M, Kanazawa H, et al. Two-step transplantation for primary hyperoxaluria: a winning strategy to prevent progression of systemic oxalosis in early onset renal insufficiency cases. Pediatric Transpl 2015;19(1):E1 6. 57. Mor E, Nesher E, Ben-Ari Z, Weissman I, Shaharabani E, Eizner S, et al. Sequential liver and kidney transplantation from a single living donor in two young adults with primary hyperoxaluria type 1. Liver Transpl 2013;19(6):646 8. 58. Artz MA, Steenbergen EJ, Hoitsma AJ, Monnens LA, Wetzels JF. Renal transplantation in patients with hemolytic uremic syndrome: high rate of recurrence and increased incidence of acute rejections. Transplantation 2003;76(5):821 6. 59. Ferraris JR, Ramirez JA, Ruiz S, Caletti MG, Vallejo G, Piantanida JJ, et al. Shiga toxin-associated hemolytic uremic syndrome: absence of recurrence after renal transplantation. Pediatr Nephrol 2002;17(10):809 14. 60. Loirat C, Niaudet P. The risk of recurrence of hemolytic uremic syndrome after renal transplantation in children. Pediatr Nephrol 2003; 18(11):1095 101. 61. Alasfar S, Alachkar N. Atypical hemolytic uremic syndrome post-kidney transplantation: two case reports and review of the literature. Front Med 2014;1:52. 62. Bresin E, Daina E, Noris M, Castelletti F, Stefanov R, Hill P, et al. Outcome of renal transplantation in patients with non-Shiga toxin-associated hemolytic uremic syndrome: prognostic significance of genetic background. Clin J Am Soc Nephrol 2006;1(1):88 99. 63. Noris M, Caprioli J, Bresin E, Mossali C, Pianetti G, Gamba S, et al. Relative role of genetic complement abnormalities in sporadic and familial aHUS and their impact on clinical phenotype. Clin J Am Soc Nephrol 2010;5(10):1844 59. 64. Lahlou A, Lang P, Charpentier B, Barrou B, Glotz D, Baron C, et al. Hemolytic uremic syndrome. Recurrence after renal transplantation. Groupe Cooperatif de l’Ile-de-France (GCIF). Med 2000;79(2):90 102. 65. Zuber J, Le Quintrec M, Sberro-Soussan R, Loirat C, Fremeaux-Bacchi V, Legendre C. New insights into postrenal transplant hemolytic uremic syndrome. Nat Rev Nephrol 2011;7(1):23 35. 66. Weitz M, Amon O, Bassler D, Koenigsrainer A, Nadalin S. Prophylactic eculizumab prior to kidney transplantation for atypical hemolytic uremic syndrome. Pediatr Nephrol 2011;26(8):1325 9. 67. Zuber J, Le Quintrec M, Krid S, Bertoye C, Gueutin V, Lahoche A, et al. Eculizumab for atypical hemolytic uremic syndrome recurrence in renal transplantation. Am J Transpl 2012;12(12):3337 54. 68. Matar D, Naqvi F, Racusen LC, Carter-Monroe N, Montgomery RA, Alachkar N. Atypical hemolytic uremic syndrome recurrence after kidney transplantation. Transplantation 2014;98(11):1205 12. 69. Loirat C, Saland J, Bitzan M. Management of hemolytic uremic syndrome. Presse Med 2012;41(3 Pt 2):e115 35. 70. Remuzzi G, Ruggenenti P, Colledan M, Gridelli B, Bertani A, Bettinaglio P, et al. Hemolytic uremic syndrome: a fatal outcome after kidney and liver transplantation performed to correct factor h gene mutation. Am J Transpl 2005;5(5):1146 50. 71. Saland J. Liver-kidney transplantation to cure atypical HUS: still an option post-eculizumab? Pediatr Nephrol 2014;29(3):329 32.

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15 Early Postoperative ICU Care of the Kidney Transplant Recipient Xavier Wittebole, Diego Castanares-Zapatero, Michel Mourad, Virginie Montiel, Christine Collienne and Pierre-Franc¸ois Laterre St Luc university Hospital, Universite´ Catholique de Louvain (UCL), Brussels, Belgium

15.1 INTRODUCTION In 2009, the Kidney Disease Improving Global Outcomes (known as KDIGO) published clinical practice guidelines on the posttransplant care of kidney transplant recipients.1,2 Those extensive guidelines (more than 150 pages, more than 900 references) address immunosuppression therapy (induction, initial and long-term maintenance of immunosuppressive drugs, acute and chronic rejection), graft monitoring and infections (kidney function monitoring, graft biopsy, vaccination, viral and other infections), associated cardiovascular risk factors and diseases, malignancies, and other chronic disorders such as transplant bone disease, hematological complications, and metabolic and endocrine disorders such as gout. However, there is very little information on how to manage the kidney transplant recipient in the first hours or days after surgery. The European Best Practice Guideline Group (EBPG) published guidelines on the evaluation and selection of kidney transplant donor and recipient, as well as posttransplant care, in 2000 and 2002. While this group endorsed the KDIGO guidelines, they recently updated their own guidelines, mainly on patient and donor selection, and perioperative care, in order to avoid redundancies with the KDIGO work.3,4 In those guidelines, a whole chapter is dedicated to the perioperative care of patients undergoing kidney transplantation. The role of preoperative dialysis, the use of central venous pressure (CVP) monitoring, the type of fluid as well as the use of dopaminergic agents and antithrombotic agents, and timing of bladder catheter removal are discussed. We will review and further discuss some of those recommendations.

15.2 FLUID THERAPY Patients undergoing kidney transplantation should be properly hydrated in order to avoid intravascular volume deficit and hypovolemia as precipitating factors for postoperative kidney injury. Hence, fluid therapy to maintain adequate organ perfusion is mandatory. Fluid therapy may also be required as maintenance solution, specifically given to cover the patient’s daily basal requirements of water and electrolytes, or as replacement solutions to correct or prevent deficits that cannot be compensated by oral intakes.5 There are increasing data, mainly coming from the critical care area and septic patients studies, demonstrating that the type of fluid may be of importance in avoiding worsening acute kidney injury. The mechanisms proposed to explain fluid-induced kidney injury include activation of the tubuloglomerular feedback, increased intravascular oncotic pressure, osmotic nephrosis, and kidney parenchymal edema and/or venous congestion.6 Two major debates have taken place since the 1990s: colloid versus crystalloid solutions, and balanced versus

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unbalanced solutions. Both debates mainly focus on the use of resuscitation fluids for correction of the intravascular volume status in various clinical settings, including perioperative and postoperative care.

15.2.1 Colloids Colloid solutions include albumin solutions, available in two different preparations (the isooncotic 4% or 5% and the concentrated 20% 25% solutions) and synthetic solutions such as hydroxyethyl starches (HES) derived from maize or potato, gelatins derived from bovine collagen, and dextrans (polysaccharide) solutions. Despite major modifications in their structure (molecular weight, degree of molar substitution that is the number of hydroxyethyl residues per glucose unit) over time, leading to faster clearance from the body,7 starches should certainly be avoided as fluid therapy. Indeed, there are at least 4 large randomized controlled trials that specifically confirmed an increased risk for acute kidney injury and need for renal replacement therapy with starch solutions when compared to balanced or unbalanced crystalloid solutions in either severe sepsis patients or general ICU patients. In a French study involving 129 patients with severe sepsis, the use of HES was found, in a multivariate analysis, to be an independent risk factor for renal failure when compared to a gelatin solution.8 In the German VISEP study, the use of HES was associated with a significantly higher rate of acute renal failure (34.9% vs 22.8%, P 5 .002), a significantly higher number of days on renal replacement therapies (18.3% vs 9.2%) and a trend toward increased mortality at day 90 compared to Ringer lactate solution in patients with severe sepsis.9 A dose effect was clearly demonstrated in this study, with higher dose of HES leading to a significant increase in mortality when compared to lower dose. Likewise, in the Scandinavian 6S study, the primary outcome of death or need for dialysis at day 90 was significantly higher in the group of septic patients receiving HES as compared to Ringer lactate solution.10 Similarly to the German study, the use of HES was also associated with an increased need for renal replacement therapy during the ICU stay. Finally, the Australian and New Zealand CHEST study confirmed, in a large general ICU population (about 7000 patients), a significantly increased need for renal replacement therapy in those patients who received HES as compared to those treated with normal saline solution (NS).11 However, there was no difference in mortality in that particular study. Those data on deleterious side effects on kidney function were confirmed in various metaanalyses, and some authors proposed alternate volume replacement should be used in place of HES products.12 With those data in mind, both the United States Food and Drug Administration and the European Medicines Agency issued, in 2013, warnings on the use of HES in septic patients, leaving the debate open for the perioperative setting. This led to various publications with some conflicting results. In the particular setting of kidney transplantation, Legendre et al. already raised concerns, back in the early 1990s, on the use of HES in the management of brain dead kidney donors, as it could induce osmotic nephrosis-like lesions.13 To confirm the impact of these histopathological findings, the same group prospectively studied 47 kidney recipients.14 They demonstrated a potential for nephrotoxicity with HES as there were significantly more dialysis requirements in those patients receiving a kidney from an HES-treated donor and the evolution of the creatinine value after transplantation was better in those patients receiving a kidney from a gelatin-treated donor. However, the long-term effect at 5 years was not different between groups in terms of serum creatinine value.15 Other authors did not confirm the relationship between osmosis-like lesions in the kidney recipient and the use of HES as other confounding factors could have influenced the results.16 Some authors also did not confirm any difference in outcome between patients receiving a kidney from a donor treated with HES or another type of solution such as crystalloid17,18 while others even observed an improved outcome with 3rd-generation HES solutions on renal function of kidney recipients.19 However, those studies were retrospective and the latest one19 compared two different types of HES solutions without any other control solution. Finally, a large prospective observational study to assess the effect of HES, given to the kidney donor, on graft function in the recipient was conducted in Los Angeles.20 Data were completed for 986 kidneys transplanted from 529 donors. In this study, HES use was associated with a 41% increased risk of delayed graft failure in the kidney transplant recipient. Those latest data certainly reinforce the statement by the EBPG, which already raised caution on the use of starches in kidney donor management and kidney recipient patients, 2 years before this latest study was published.3,4 Other synthetic colloid solutions were certainly not studied to the extent of starch. Gelatins exist in three different solutions (succinilated, urea-linked, or oxypolygelatin), are eliminated by glomerular filtration, and are known to induce allergic reactions. In a well-known animal model of peritonitis (cecal ligature and punction or CLP) gelatins were shown to increase markers of renal failure (urea and creatinine) and kidney damage (NGAL), and induce structural changes as seen with starches.21 In humans, data on the use of gelatins in adequately powered randomized trials are scarce. Such a trial is planned in Germany (the GENIUS study for gelatin in ICU and

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sepsis; EudraCT 2015-000057-20), but this study will evaluate the safety of gelatins in critically ill septic patients, as compared to a crystalloid solution. In a study comparing the use of crystalloids and colloids, there was a trend toward increased kidney injury (as assessed by the RIFLE score R), a significant increase in the use of renal replacement therapy, and a prolonged ICU stay with gelatins,22 confirming to a large extent a previous trial.23 Likewise, in a similar large before-and-after study, gelatins were shown to increase the need for renal replacement therapy as compared to crystalloids without achieving any better hemodynamic outcome in cardiac surgery patients.24 Hence, the use of gelatins at the present time remains questionable. Dextrans are polysaccharides that are mainly eliminated by glomerular filtration and the digestive tract system, while cellular uptake and storage has also been described. In a canine model of kidney transplantation, the use of dextran was associated with decreased renal function and histopathological features of osmotic nephrosis-like lesions.25 Some case reports, reviewed elsewhere26 also describe such lesions associated with kidney failure in humans. On top of those potential kidney side effects, dextrans may also induce severe allergic reactions and compromise hemostasis, which makes their use difficult to justify in the ICU setting or in kidney transplant patients. From a theoretical point of view, albumin contained in natural colloid albumin solutions would be of great interest and show major advantages such as the major determinant of plasma colloid pressure, an ability to bind and transport various molecules including drugs, free radicals scavenging, and the protection of the endothelial surface layer. However, they are not equivalent in terms of sodium and chloride content; indeed the 4% or 5% solution displays sodium and chloride concentrations at 140 160 mmol/L and 100 130 mmol/L respectively while the 20% and 25% solutions contain 100 125 mmol/L sodium and 20 25 mmol/L chloride. Since this difference could account for different effects on the kidney (see the balanced unbalanced solution debate later in the chapter), results from a particular study would probably not extend to other studies. In the critical care setting, albumin was not demonstrated to be superior in term of renal failure, need for renal replacement therapy, or mortality when compared to crystalloids.27,28 In the Saline versus Fluid Evaluation or SAFE study,27 4% albumin was compared to 0.9% saline and did not result in any better outcome, including for the kidney. In the Albumin Italian Outcome Study or ALBIOS study,28 a 20% albumin solution was prescribed to target a plasma albumin level of 30 g/L. Again, the various outcomes including the incidence of acute kidney injury were not affected by the treatment. Various factors such as the high chloride content of the albumin solution in the SAFE study or the high oncotic pressure in the ALBIOS study could explain the nonpositive results since those two characteristics could lead to renal failure (see previous). Furthermore, the cost of albumin solutions (in the absence of clear benefit in the general ICU population) prevents its generalized use. The situation is slightly different for kidney transplant patients. Indeed, in this context, observational studies showed albumin solution was associated with increased urine output, graft renal function and 1-year graft survival.29 31 In a large series of 438 patients transplanted with kidneys from cadaveric donors, there was even a linear dose effect with albumin, the higher dose (1.2 1.6 g/kg body weight) achieving the better outcomes in term of urine volume, serum creatinine and glomerular filtration rate, delayed graft failure, and graft function at 1 year.32 Because of the design of those studies, other factors (such as concomitant medications and the use of mannitol) may have affected the results. In a recent retrospective study on about 2000 kidney-transplanted patients over a 20-year period, the use of albumin was found to be an independent factor of protection for acute rejection and chronic graft dysfunction.33 Beyond the fact this study was retrospective, the importance of the results are weakened by the very limited number of patients (91) treated with albumin. Controlled clinical data investigating the effect of albumin in kidney transplant patients were not available until recently. A first study on 44 patients assessed the outcome of patients receiving intraoperative 20% albumin and 0.9% saline versus 0.9% saline alone to target a CVP between 10 and 15 mmHg.34 There was no difference between groups at baseline. The volume infused during surgery was equivalent (roughly 4 L). Neither urine output nor the various creatinine values at days 1, 3, and 5 were different between groups. Another study analyzed intraoperative 20% albumin versus 0.9% saline, to target a CVP of 12 15 mmHg, on the outcome of kidney graft function in 80 patients.35 Groups were well balanced apart from a trend towards an increased kidney donor age in the albumin group (49 vs 45; P 5 .051). None of the outcomes (hemodynamic parameters, creatinine serum value and urine output) was affected by the treatment group. Hence, the authors concluded albumin solution should be used rationally, considering its risk benefit and cost benefit ratios.

15.2.2 Crystalloids As demonstrated in the various studies in the section on colloids, crystalloids perform better than, or at the worst are equivalent to, colloid solutions. However, there is a major debate on which type of crystalloid solution

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should be used: Unbalanced (with a high sodium and chloride content) or balanced (with a composition that mimics more the plasma content). Beyond its well-known effects on metabolic acidosis,36,37 chloride-rich solutions induce the so-called glomerulo-tubulo-feedback mechanism: the increased delivery of chloride to the macula densa cells located in the distal part of the nephron induces the release of various vasoactive mediators, which in turn promote vasoconstriction of the afferent arteriole and finally a reduced glomerular filtration rate. This is supported by both animal and human studies.38,39 There are many observational and retrospective studies assessing the effect of chloride-poor solution on renal function. While not always consistent, most of those studies report on decreased acute kidney injury, decreased need for renal replacement therapy and improved urine output with chloride-poor solutions. The largest retrospective study, on 53,448 patients treated for sepsis in an intensive care unit, demonstrated, in a propensitymatched cohort, that those who received balanced solutions (3396 patients or 6.4% of the total cohort) had a decreased mortality.40 In this study, renal failure, ICU, and hospital length of stay were not affected by the type of crystalloid solution. There are also some prospective randomized controlled trials on that topic. In a prospective, before-andafter study, the implementation of a chloride-restrictive policy led to a significant decrease in the incidence of acute kidney injury and need for renal replacement therapy.36 Likewise, in this study involving 1644 ICU admissions in 1533 patients, the chloride-restrictive strategy was associated with a significantly lower increase in serum creatinine, while mortality and need for RRT at 3 months were not affected. A large prospective randomized controlled trial comparing 0.9% saline and a balanced solution was started on April 1, 2014.41,42 The SPLIT study (Saline vs Plasma-Lyte 148 for Intensive care fluid Therapy) planned to recruit 2300 patients in four New Zealand tertiary ICUs. Results of this particular study are supposed to be published by the end of 2015. While awaiting those results, a panel of experts proposed balanced solutions may be a reasonable default choice.43 In the particular setting of kidney transplantation, there are few studies evaluating the effect of “normosaline” solution (NS) versus balanced solutions. In a study involving 74 patients during living donation kidney transplantation, the use of Ringer’s lactate solution resulted in lower metabolic acidosis and hyperkalemia than the use of NS.44 This recent study confirmed the results of another trial that was stopped prematurely, after the safety analysis of 51 patients, because of increased incidence of metabolic acidosis and hyperkalemia with the use of NS.45 Kidney function was not affected by the type of solution. The study by Kim et al. evaluated the effect of NS versus Plasmalyte in 60 patients undergoing living donor kidney transplantation.46 While postoperative serum creatinine, urine output, and incidence of graft failure were unaffected by the type of crystalloid, Plasmalyte better maintained the acid base and electrolyte balances. The better metabolic profile with Plasmalyte as compared to NS or even Ringer’s lactate solution was confirmed in a large recent metaanalysis.47 In summary, and awaiting some new data, it seems reasonable to avoid synthetic colloid when fluid therapy is required in kidney recipient patients. Concentrated 20% albumin (with low chloride content) and balanced crystalloids are probably preferable to normal saline because of its high chloride content, which may be deleterious for the kidney function as well as the metabolic status since it induces acidosis as confirmed by a recent metaanalysis.48 Furthermore, the high cost of albumin solutions makes its cost benefit ratio below that of balanced crystalloid solution.

15.3 CENTRAL VENOUS PRESSURE MONITORING CVP is the measurement of pressures within the thorax in the superior vena cava; it normally provides some estimates of the volume status of the systemic circulation, as it is a surrogate for the right atrial pressure. It does not provide any information on cardiac output nor on the volume status of the pulmonary circulation in the presence of left ventricular dysfunction. For this purpose, pulmonary artery catheters were frequently placed in patients, until large studies and metaanalysis were unable to confirm a benefit on mortality with its use.49 51 Likewise, apart from cardiac surgery patients,52 data on how CVP monitoring affects outcome are lacking. Furthermore, a single value of CVP does not preclude if a patient will respond to fluid challenge and increase its cardiac output.53 Hence, many studies questioned the interest in CVP and many other parameters such as stroke volume variation, pulse pressure variation, and inferior vena cava diameter assessed by ultrasound were proposed in critically ill patients as well as in the operating room. The EBPG recommend to measure and correct CVP values in order to decrease the incidence of hypovolemia and delayed graft function (recommendation grade 2D).3,4 They base their recommendation on small trials. In a

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retrospective analysis assessing potential risk factors for graft failure, a CVP value below 8 mmHg at awakening was significantly associated with delayed graft failure.54 Othman and colleagues demonstrate in 40 patients that perioperative infusion of sodium chloride targeting a CVP above 15 mmHg was associated with earlier onset of diuresis and better graft outcome.55 Some new studies, published after the ERBP guidelines, confirmed the positive impact of CVP monitoring.56 As already mentioned, some authors questioned the role of routine CVP monitoring and proposed some other tools to assess the volume status of the patient.57,58 However, the PVC monitoring may easily be maintained after surgery (as opposed to transesophageal Doppler) and it may help in diagnosing conditions known to worsen kidney function such as renal venous hypertension59 and the abdominal compartment syndrome (ACS) (see the following).

15.4 DOPAMINE AND OTHER MEDICATIONS The use of dopamine to improve renal function after various insults was a matter of debate for more than 20 years. Dopamine is the immediate precursor of norepinephrine and its effects are dose-dependent: small doses (0.3 up to 3 5 mcg/kg/minute) activate dopamine and beta-receptors, while at higher doses, alpha-receptorrelated effects are observed. In summary, the various effects of dopamine on the kidney could be described as increase in renal blood flow, increase in natriuresis through direct effects on renal tubules, and increase in free water clearance through a stimulation of the E2 prostaglandin synthesis, which antagonizes the antidiuretic hormone.60 However, dopamine effects on renal blood flow could potentially also be detrimental, since it induces vasodilation in the cortex and inner medulla while it decreases blood flow from the outer medulla, shunting blood away from it. Some old physiological studies performed in healthy volunteers demonstrated that low-dose dopamine (LDD) increased renal blood flow and promoted natriuresis and diuresis.61,62 Based on its potential beneficial effects, dopamine has been extensively used at the so-called “renal dose” for years in critical care in order to improve renal function. While LDD improved hemodynamic and renal parameters in patients with septic shock,63 others were not able to demonstrate any benefit from LDD apart from its effect on diuresis.64 A large randomized controlled trial did not confirm positive effect of such treatment in 324 patients with signs of systemic inflammatory response syndrome and early renal dysfunction.65 None of the evaluated outcomes (peak value and increase from baseline value of serum creatinine concentration, number of patients with a creatinine value above 300 micromol/L, need for renal replacement therapy, ICU and hospital length of stay) were affected by treatment.65 Observational66 and prospective randomized67 studies in shock patients even raised some concern on the use of dopamine as vasopressor. The many studies performed in this area were evaluated in various metaanalyses. The renal protective effect was not confirmed in critically ill patients68 and, as mentioned, dopamine was even found deleterious when used as a vasopressor in septic shock patients because of increased side-effect events.69 Dopamine has also extensively been studied in kidney transplantation as an agent that might improve graft function when given to the kidney donor, during surgery or in the kidney recipient. A retrospective observational analysis of a 152 consecutive kidney transplantation database found that donor catecholamine treatment was associated with improved graft survival.70 This was later confirmed in a larger database.71 This was further studied in a rodent model of brain death.72 The use of dopamine in the donor was associated with better serum creatinine value in the recipient. Interestingly, 10 days after transplantation, dopamine treatment also decreased monocyte infiltration and cytokine mRNA expression, confirming a potential role of dopamine as an immunemodulatory agent.73 Finally, small-dose dopamine (4 mcg/kg/minute) given for a median time of 344 minutes (IQR: 215 minutes) to heart-beating donors was further evaluated in a randomized controlled trial. The need for dialysis was evaluated in the 487 subsequent recipients.74 The main outcome of dialysis was significantly reduced in patients receiving a dopamine-treated graft. In the subsequent multivariate analysis, the use of dopamine remained statistically significant; other risk factors for graft failure included cold ischemic time and recipient body weight. To enhance urine output and prevent arterial vasospasm and acute tubular necrosis, LDD has frequently been used in kidney recipients. Despite its general use, results from clinical studies were conflicting. Some studies reported on some beneficial effects such as slightly better creatinine clearance and improved urine output.75,76 In a short-course treatment study (3 hours infusion) with LDD, Dalton demonstrated that during the period of time the kidneys were exposed to LDD, urine flow rate, effective renal plasma flow, creatinine clearance, and urinary sodium excretion were improved.77 Clinical consequences were not evaluated. Others could

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not confirm those positive results. DeLosAngeles was not able to demonstrate that a combined infusion of dopamine (3 mcg/kg/minute) with furosemide improved creatinine clearance in 20 patients as compared to 20 others not receiving the treatment.78 Cardiac arrhythmias, however, were increased in the dopamine group. Resistive index, renal blood flow velocity, and vascular resistance assessed by Doppler ultrasound were evaluated in a prospective study.79 None of these indices were modulated by LDD infusion. The authors hypothesized that the denervated kidney might not respond the same way as normal kidneys in healthy persons. In a retrospective study of 105 patients, Ciapetti and colleagues compared 57 patients kidney recipients receiving dopamine (0.5 2.5 mcg/kg/minute) to 48 controlled patients.80 While kidney donor characteristics, intraoperative procedure, and kidney recipient characteristics were similar, patients treated with LDD had an increased heart rate and ICU length of stay (20.4 vs 29 hours, P , .05). Kidney function parameters and urine output as well as graft rejection at day 28 were equivalent in both groups. Mortality at 6 months was also increased in the dopamine-treated patients, but the small number of patients analyzed in the study limit the interpretation of this data. Taking all those data together, the use of dopamine as a renoprotective agent in kidney recipient patients is certainly not supported outside of prospective randomized trial. This is actually confirmed by the ERBP guidelines.3 Fenoldopam is a synthetic benzazepine derivate that acts as a selective dopaminergic 1 receptor agonist, causing vasodilation of both systemic and renal vessels. It also promotes sodium excretion.81 A metaanalysis, gathering data on 1290 critically ill patients, suggested fenoldopam reduced the risk for acute kidney injury and mortality while the risk for hypotension was clinically acceptable.82 However, a later randomized controlled study in cardiac surgery patients was stopped early because of futility.83 While need for renal replacement therapy and mortality were equivalent between both groups, hypotension was significantly more frequent in treated patients. Fenoldopam was evaluated in kidney- transplanted recipients. Oral treatment in 6 patients evaluated 3 6 months after transplantation was assessed using a loading dose of 100 mg and after 3 weeks of treatment, 100 mg thrice daily.84 Despite those patients being on oral cyclosporine (a potent renal vasoconstrictor), the administration of fenoldopam improved mean effective renal plasma flow. However, the other variables such as glomerular filtration rate, urine output, and fractional excretion of sodium were not significantly ameliorated. Curiously, despite the conflicting results with dopamine (see previous), there are two studies comparing, in kidney recipient, the effect of fenoldopam with dopamine.85,86 In the first study, a continuous infusion of fenoldopam at a dose of 0.05 mcg/kg/minute for 48 hours did not significantly improve urine output, the first postoperative day creatinine value, or the renal vascular resistive index assessed by Doppler ultrasound.85 In the second study, Sorbello and colleagues showed that fenoldopam for a 2-hour continuous infusion treatment at a dose of 0.1 mcg/kg/minute increased urine sodium concentration and decreased urine potassium concentration.86 There was no observed effect on urine output. A recent study in liver transplant recipients showed however some promise on the use of fenoldopam87 but data are certainly too weak in the kidney transplant literature to recommend the use of fenoldopam as a nephroprotective agent. Medications aimed at increasing the urine output such as diuretics or mannitol are often used in patients during or after kidney transplantation. For instance mannitol was reported to be used, as an antioxidant or diuretic, or both, in about 65% of centers performing living donor nephrectomy.88 Positive effects are indeed reported in the literature with mannitol prescribed to the donor89 or recipient90 or even used in the storage solution91 but those studies involved a very limited number of patients and drawing any conclusion is therefore of limited value. A recent systematic review and metaanalysis concluded mannitol does not convey any additional benefit in preventing acute kidney injury.92 The authors proposed that a large well-conducted randomized controlled trial should be undertaken in kidney recipients. Likewise, the use of diuretics resulted in conflicting data with some studies unable to demonstrate any positive effect.93 Actually, it is clearly reported that diuretics are ineffective in preventing acute kidney injury and might even be detrimental.94,95 Therefore the prescription of diuretics should be restricted to those patients with limited diuresis and clear evidence of volume overload. Furthermore, the use of diuretics (or mannitol) should not induce hypovolemia since it could worsen kidney injury. One exception might be spironolactone. Indeed there are some very interesting experimental data showing that spironolactone was of great interest in models of ischemia-reperfusion kidney injury.96,97 This, however, has to be translated into clinical studies and practice. Many other medications aimed at decreasing the oxidative stress induced by the transplant procedure related ischemia-reperfusion have been evaluated in experimental models as well as in clinical practice. For instance, N-acetyl-cysteine was studied as a renal protective agent when given to the donor98 or the recipient.99 Results of these two studies, which did not show any positive impact on the various parameters of kidney

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function, did not confirm the positive results observed in another study that suggested NAC reduced oxidative stress assessed by levels of thiobarbituric acid reactive substances and improved kidney function up to 1 year after transplantation.100 Other molecules studied in experimental models or clinical practice included calcium channel blockers, atrial natriuretic peptide, brain natriuretic peptide, vitamin E, and nitric oxide generators. However, strong clinical evidence is still lacking and none of these molecules should be recommended at the present time.101 Future research should certainly focus on the role of the innate system activation in ischemia-reperfusion injury as inhibition of toll-like receptor was demonstrated of benefit in a model of ischemic heart injury.102

15.5 ABDOMINAL COMPARTMENT SYNDROME AND RENAL ALLOGRAFT COMPARTMENT SYNDROME Increased pressure within the abdominal cavity leads to intraabdominal hypertension (IAH) when intraabdominal pressure is above 12 mmHg and ACS when the rise in pressure constantly exceeds 20 mmHg, and is accompanied by a new onset of organ failure within or outside of the abdominal cavity.103 Many conditions, such as decreased abdominal wall compliance, increased abdominal content, capillary leak and others are associated with the development of this syndrome.103 The relationship between increased intraabdominal pressure and kidney function was already described by Wendt in 1876. The different mechanisms involved in the pathophysiology of ACS-induced renal failure were reviewed in 2011.104 Beyond the effects of ACS on hemodynamics, which lead to decreased cardiac output, local arterial inflow to the kidney is also impaired. Likewise, the IAH-induced increase in venous pressure decreases venous blood flow. While compression of the ureter does not seem relevant to postrenal kidney injury, direct compression of the kidney parenchyma, as it was demonstrated in ischemia-reperfusion models,105 certainly is an important contributor to renal failure. In a surgical population, the level of IAH was demonstrated to be an independent risk factor with worsening renal failure associated with increased IAH values.106 This was confirmed in liver-transplanted patients; higher value of IAH ( . 25 mmHg) was associated with higher creatinine value, greater need for diuretics, increased incidence of renal failure, and worse outcome.107 Initially described by Humar,108 the terminology of renal allograft compartment syndrome (RACS) was proposed by Ball and colleagues to define this subtype of secondary ACS.109 Actually two different scenarios may occur: Renal failure due to classical intraabdominal hypertension with increased pressure within the whole abdominal cavity and retroperitoneal space, and, renal failure related to compression of the parenchyma within the kidney capsule, as met in the ischemia-reperfusion model described previously.105 Indeed, transplantation induces ischemia-reperfusion injury and inflammation in the kidney with activation of the innate immune system, leading to swelling within a closed space (capsule).101 Risk factors for RACS include weight discrepancy between the donor and the recipient, child recipient, and a noncompliant retroperitoneal space, which is limited laterally by the pelvic sidewall, posteriorly and medially by the peritoneum and its contents, and anteriorly by the abdominal wall musculature.109 Allograft width and length were also proposed as potential risk factor.110 Diagnosis is made upon clinical assumption and ultrasound. Reversed diastolic blood flow in interlobar and segmental renal arteries, in conjunction with minimized venous flow, defines the sonographic criteria for RACS.109 RACS is rarely reported in the literature. Pertek described three cases of posttransplant renal failure due to occult bleeding related retroperitoneal hematoma; evolution was excellent after abdominal decompression.111 Fontana reported on a case where RACS was induced by the presence of a large amount of ascites.112 While intraabdominal pressure was moderately elevated (14 mmHg), paracenthesis allowed recovery of kidney function. Decoster observed RACS in a patient who underwent simultaneous pancreas kidney transplantation.113 Again, in this case report, the IAP was only moderately increased; surgical decompression resulted in immediate recovery of kidney function. Pediatric cases were also reported.114 After early recognition, initial management includes the treatment of the precipitating factor and surgical decompression is usually required; this allows dramatic improvement of graft function.109 A particular technique of mesh hood fascial closure may apply to prevent and treat RACS.115 Various types of mesh are used to close the abdominal wall (porcine collagen graft, prosthetic mesh (PTFE), polypropylene assisted mesh). Interestingly this procedure does not preclude biopsy procedure or imaging of the renal graft postoperatively.116 Subcutaneous placement of the kidney graft and intraperitoneal placement of the graft117 have also been proposed.

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15.6 OTHER CONSIDERATIONS The ERBP Guidelines also recommend on dialysis session prior to transplantation, the use of antithrombotic agents in the perioperative period, the placement of a ureteral catheter (JJ stent), and the optimal timing of bladder catheter removal.3,4 Routine hemodialysis sessions should not be performed before the surgical procedure as there are no demonstrated benefits (recommendation 1 C). It could even delay the surgical procedure and increase the cold ischemia time. Actually, this procedure should be restricted to those patients with severe metabolic disorders such as hyperkalemia. Likewise, hemofiltration should be performed only if fluid overload is evident. Antithrombotic agents are not recommended for the prevention of graft thrombosis and bladder catheter should be removed as soon as possible, balancing the risk between infection and urinary leak. However, the authors propose to monitor adverse rate in each transplant center in order to help in the decision of catheter removal. Curiously, the guidelines do not approach the need for specific biologic monitoring or noninvasive techniques to assess graft functioning. Usual kidney function tests (urea or BUN, creatinine serum values) should be performed routinely after kidney transplantation and urine output monitoring is certainly warranted. The use of other kidney function markers such as neutrophil gelatinase-associated lipocalin (NGAL), KIM-1, or others might be reliable markers of early and delayed graft failure.118 However, their routine use at the present time does not add to the usual blood test as it was demonstrated for NGAL, for instance, that it was not able to compete with markers used in clinical practice.119 Postoperative graft function may also be assessed with ultrasound or nuclear imaging techniques. Ultrasound may help diagnose vascular complications (arterial stenosis, arterial and venous thrombosis, pseudoaneurysm formation), urological complications (urinary obstruction and leaks), and collections.120,121 Renal scintigraphy has also been proposed by some authors to assess kidney function as well as posttransplant complications such as vascular compromise, acute tubular necrosis, and urine leak.122 Finally, some specificities for the management of children after kidney transplantation are also described in the literature.123

15.7 CONCLUSIONS Above usual postoperative care, management of the kidney-transplanted patient warrants particular attention to avoid anything that could compromise the kidney function in this particular ischemia-reperfusion period. On top of avoiding usual nephrotoxic agents, patient care should include adequate maintenance of volume and hemodynamic status with fluid therapy with balanced solutions. There are no specific medications at the present time that might help graft function recovery, and, in case of persistent renal failure, adequate work-up with ultrasound, scintigraphic imaging, and measurement of the intraabdominal pressure may help the physician. Finally, care of the donor is of importance as it was demonstrated in 2015 that mild hypothermia in the donor might improve graft function after kidney transplantation.124 This is however beyond the scope of this chapter.

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C H A P T E R

16 Approaches to Minimize Delayed Graft Function in Renal Transplantation Maria Francesca Egidi and Domenico Giannese University of Pisa, Pisa, Italy

ABBREVIATIONS ACR AKI AMR ATN BMI CIT CNI DBD DCD DGF ECD GFR GI HD HMP IRI IS NGAL PD SCD s.creat. Thymo US

acute cellular rejection acute kidney injury antibody-mediated rejection acute tubular necrosis body mass index cold ischemia time calcineurin inhibitor donation after brain death donation after cardiac death delayed graft function extended criteria donor glomerular filtration rate graft index hemodialysis hypothermic machine perfusion ischemia-reperfusion injury immunosuppressive regimens neutrophil gelatinase-associated lipocalin peritoneal dialysis standard criteria donor serum creatininemia Thymoglobulin ultrasonography

16.1 INTRODUCTION In the present chapter the authors attempt to give a comprehensive picture of delayed graft function (DGF) in relation to different definitions, causes, and the impact on graft outcomes. DGF is a complication in the immediate posttransplant (TX) period that not only increases morbidity, length of hospitalization, and health care costs, but more importantly enhances immunogenicity, chronic allograft damage, and decreased long-term patient and graft survival.1,2 Therefore DGF is a relevant prognostic indicator. Risk factors for DGF derive from the donor and the recipient.3 Although the incidence of DGF has remained unchanged over the past decades, it is crucial to stress that in the current era this post-TX complication requires major endeavor with its prevention and Kidney Transplantation, Bioengineering, and Regeneration. DOI: http://dx.doi.org/10.1016/B978-0-12-801734-0.00016-3

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management. In particular, the imbalance between organ demand and supply has induced the increasing use of “suboptimal donors,” defined as extended criteria donor (ECD) and donor after cardiac death (DCD) whose grafts will suffer more from DGF impact when compared to standard criteria donors (SCD).4 6 There is no effective treatment for DGF; however, early diagnosis and therapeutic interventions may improve short and long-term results. In order to prevent or reverse the initial kidney injury and the deleterious consequences several biomarkers and new immunosuppressive regimens (IS) are under investigation.

16.2 DEFINITIONS DGF can affect 20% 50% of recipients of cadaveric TX and 4% 10% of living donors. This large variability is related to the various definitions of DGF, most of them based on dialysis requirement or serum creatinine (s. creat.) values.7 9 This lack of consensus complicates the interpretation of the results on patient and graft outcomes and also generates discrepancies from the literature data. One systematic review by Yarlagadda reported 18 definitions and at least 10 different techniques for DGF diagnosis, based on need for dialysis, histological findings, urinary output and s.creat.7 The need for dialysis in the first week after TX is the most commonly used definition for DGF and has shown high specificity and sensitivity for predicting graft outcome.10 However, there is an important selection bias: The indication for dialysis can be subjective, being a clinician decision, and depends on single-center experience and policy. Furthermore, patients with a residual urinary output and glomerular filtration rate (GFR), such as the candidates for a preemptive TX, may not require post-TX dialysis despite a suboptimal graft function. These circumstances are reasons to underestimate the real DGF incidence.7 Aitken et al.10 assumed that DGF is a syndrome with different variety of etiologies and after a retrospective analysis of data collected in 762 recipients in the first 30 days post-TX, described 4 distinct patterns of DGF: 1. A prolonged hemodialysis: a prolonged period of hemodialysis, then a fall in s.creat. 2. A single hemodialysis: a single session of hemodialysis required, then be s.creat. fell. 3. A slow decline: immediate reduction in s.creat; however, s.creat. took a long time to reach baseline and .1 week for the s.creat to half. 4. A slow rise, then a gradual decline: s.creat. initially rose, then declined slowly with .1 week for s.creat. to half. No dialysis required. Although the number of patients who suffered DGF was limited (24.9%), this classification is relevant and could reflect the potential injuries incurred by the kidneys including the type and the length of the ischemia. In fact, cold ischemia time (CIT) or acute kidney injury (AKI) correlate with the need for dialysis, whereas the rate of decline of s.creat. may be influenced by warm ischemia and ischemia-reperfusion injury (IRI). Recipients of marginal kidneys could be consistent with this observation, as ECD kidneys are more likely to require dialysis than DCD kidneys, which conversely show a slower decline in s.creat. to the ultimate baseline. This classification focuses on the renal function recovering rate and could be a reliable predictor of medium-long term graft outcomes.9 The DGF definitions do not allow the differentiation from the other concomitant causes of nonfunctioning graft. It has been clearly documented that IRI activates an immunological cascade but its relationship with acute cellular rejection (ACR) or antibody-mediated rejection (AMR) can be proven only by renal biopsy. Besides irreversible thrombotic events, other possible etiologies such as calcineurin inhibitors (CNI) acute toxicity and/or thrombotic microangiopathy should be taken under consideration, not only for diagnostic approach but for pharmacological management. The induction strategies, in particular with the antithymocyte globulin Thymoglobulin (Thymo), have been designed not only to minimize CNI nephrotoxicity but also to ensure an immunological protection of the graft during DGF.11,12

16.3 PATHOPHYSIOLOGY The etiology of DGF is multifactorial and involves different risk factors including CIT and AKI, which perhaps play the major role in the inflammatory and immunological cascade. The development of immune response and the presentation of intra- or extracellular antigens result in poor long-term outcome of the graft.13,14 After the organ procurement, the cellular components of the kidney, despite storage in hypothermic solution, change their metabolic pathways from aerobic to anaerobic. This hypoxic status during the ischemic period causes the loss of osmotic equilibrium and increases the permeability of cellular membranes, with

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consequent cell necrosis and further irreversible damage. The endothelial and tubular epithelial cells, because of the great number of metabolic processes, are extremely vulnerable to the toxic effects of the ischemia. The following reperfusion phase induces the restoration of the metabolic processes but concomitantly triggers the expression of inflammatory cytokines and adhesion molecules that play an integral role in DGF development.15 18 These events stimulate interactions among leukocytes, platelets, and erythrocytes that plug the capillaries and create a low-flow condition with tubular obstruction and detrimental intraluminal pressure.19 The immune system activation and DGF coexist, interact, and lead to a complex interaction that involves different mediators, dendritic cells, and complement components. Acute tubular necrosis (ATN) is the corresponding histological picture of DGF that can also be associated to ACR or AMR. ATN shows different degrees of severity that range from the dilatation and loss of tubular epithelial cells to the significant damage of the microvascular epithelium. The tubular repair process is often associated with multiple mitosis. On the other side, the persisting damage induces an early and irreversible tubule-interstitial fibrosis with poor outcome of the graft.13 The incidence of ATN is increased by CIT longer than 24 hours, the presence of preformed antibodies, the type of pre-TX dialysis and the poor quality of the donor.20,21 Associations among gene polymorphism, DGF, and ACR or AMR are under investigation. Some genes appear to play a minor role in DGF pathogenesis but can be strongly related to ACR.22 Recent studies have shown that CC genotype (CX3CR1 gene V2491) appear to be an independent and significant predictor of higher risk for DGF.23 It has not yet been determined what, if any, are the potential repair mechanisms possibly linked to the genetic asset of the recipient.

16.4 IMAGING As described in the previous section, the definition of DGF is based on clinical features, but imaging could be a worthwhile aid. Ultrasonography (US) is the noninvasive approach used in every TX phase and is the primary imaging technique for the evaluation of an early diagnosis of DGF. Several reports agree that indexes of perfusion and elevated resistive index (RI) differ in patients with DGF when compared to a control group.24 An US evaluation performed in 79 patients in the early post-TX period showed higher RI of upper and middle segments in cases of DGF. Also a multivariate analysis evidenced that older donors and high final s.creat. correlate with the risk for DGF.25 In agreement with those data are the findings of a contrast-enhanced US study where the inflow was significantly delayed, and lower, in patients with DGF. The renal biopsies performed in this latter group showed a picture of ACR or ATN respectively. As expected, the inflow time to the cortex and the pyramids was longer in ACR than in ATN.26 Two studies performed after 2 4 weeks post-TX identified particular features in patients with increase of RI and pulsatility. Both investigations evidenced that graft and recipient characteristics such as age, diabetes, and DGF had significant impact in abnormal US findings. However, the second study reported that donor features such as hypotension, use of catecholamines, HLA mismatch, and impaired central venous pressure were also relevant.27 Scintigraphy with 99 m Tc-DTPA is another imaging technique useful for diagnosis of DGF and appears to be more accurate and sensitive than US. Yazici28,29 described a formula defined as the graft index. In 179 patients who underwent a renal scintigraphy after 2 days from TX, this index was the most accurate parameter for DGF identification.30 The obvious limitations of this imaging are the exposure of the patient to radionuclides and the involved costs. Magnetic resonance imaging is free from radioactive risk, has proven reliability in detecting perfusion impairment during DGF, and correlates well with GFR, RI, and CIT.31 Noninvasive tests are more and more sophisticated and well equipped in terms of sensitivity. Therefore, radiology imaging should be considered an important and complementary tool for the detection of renal dysfunction, including DGF.

16.5 NEW BIOMARKERS AND PREDICTIVE MODELS In recent years, new markers of kidney injury have acquired an important role. There are emerging and promising markers able to predict DGF, but the complexity of AKI pathophysiology has low probability to detect one specific biomarker rather than a full panel. Biomarkers can be found not only in urine but in serum as well. This is relevant as DGF induces oligo-anuria in most cases. As of 2017, studies are based on a small number of patients due to the limitations for the high costs of production and validation of the laboratory through standardized techniques. Basically, the new biomarkers correspond to the different functional components of the kidney.

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Neutrophil gelatinase-associated lipocalin (NGAL), a small protein expressed in various epithelial cells (e.g., kidney, gastrointestinal tract, lungs), filtered through the glomerulus and secreted in tubular cells is greatly increased in the serum and urine of patients with AKI.32 34 Different studies have shown that high urine and serum NGAL levels shortly after TX are predictors of DGF and are associated with reduced graft function at 1 year.35 38 Recent reports have shown that clusterin and interleukin 18 can predict DGF.39 Leptin, an adipocytokine whose circulating levels are increased in chronic kidney diseases, appears to be a good marker for DGF. In recipients with DGF, leptin values, just after 1 day, were approximately two times higher than in patients with immediate graft function. After correcting for gender and body mass index (BMI), day-1 leptin predicted DGF with a performance slightly better than s.creat.40 Assessments conducted on gene expression during DGF show a correlation with some potential inflammatory and protective molecules. For instance genes for IL-1R1, IL10, IFNA1 during DGF seem to promote and upregulate inflammation, whereas the gene for TGF-beta is downregulated.41 As a consequence, the absence of the immunomodulatory effect of TGF-beta activates immunity and triggers inflammation that result in chronic damage.42 The interleukin 2 (IL2), is under investigation for a possible link with DGF because of an established role in the immunological response. Nevertheless, the correlation between rs6822844 gene polymorphism in the IL2-IL21 region and graft function did not show any statistically significant difference between the patients who developed DGF and the group with rapid recovery.43 In a swine model of IRI, Castellano,44 after the discovery of a remarkable decrease of Klotho produced by the tubular cells, analyzed kidney biopsies from cadaveric donors at the implantation time. If compared with patients with immediate graft function, patients with DGF showed downregulation of Klotho. Interestingly in this latter group, Klotho levels continued to be lower up to 2 years after TX. Taken together these data support the hypothesis that a persistent deficiency of Klotho, with its antisenescence and antifibrotic effects, may contribute to chronic allograft dysfunction in DGF patients. In order to prevent or reduce the severe consequences and complications of DGF, several predictive models have been proposed. Irish and coworkers45,46 defined a model in 2003 and later revised in 2010, based on 18 parameters that included: recipient’s ethnicity, gender, BMI, diabetes, previous TX, preformed antibodies, pre-TX transfusions, HLA mismatches, duration of dialysis, and donor’s age, s.creat., weight, age, hypertension, causes of death, DCD, CIT, and warm ischemia time. Jeldres and Chapal47,48 proposed a model based on 5 6 parameters. In 2015, Zaza49 identified a preoperative predictive model for DGF by the analysis of pre-TX variables detected in 2755 patients transplanted between 1984 and 2012. Recipients’ characteristics at the time of TX such as age, body weight, modality, and duration of dialysis were statistically associated with DGF development. Finally, Decruyenaere et al.50 combined into a single metaanalysis model based upon the four existing predictive models and studied parameters not included in previous analyses. The main purpose of the study was to achieve a better degree of calibration without overestimating the risk of DGF. The study resulted in the identification of two relevant parameters not included in the previous models: cardiac function and preoperative blood pressure.

16.6 PROGNOSTIC IMPACT DGF has short- and long-term consequences with significant reduction of patient and graft survival.51,52 The impact on the immediate post-TX period not only prolongs hospitalization but increases morbidity and health care costs. It is well documented that DGF can induce ACR/AMR and chronic allograft dysfunction but there is not complete agreement about the role of DGF alone on graft survival because of possible unrecognized rejection or poor intrinsic graft quality.53 55 The diagnosis of rejection, often based on clinical findings rather than graft biopsies, might have led to its underestimation. Data from 2012 based on biopsy-proven ACR during DGF condition have documented a clear relationship between these complications.56 The link between DGF and ACR/AMR may have changed over time due to new IS, including the induction with Thymo, a strategy for protecting the kidney from the immunological cascade and for minimizing the nephrotoxic effects of CNI.57 As reported by several studies, there are three different pictures based on the presence or absence of DGF and rejection: DGF-ACR-; DGF 1 ACR-; and DGF 1 ACR 1 . Patient survival up to 10 years was similar in all the groups with the exception that in case of DGF 1 ACR 1 , graft survival, even at 1 year post-TX, was always significantly lower.13,51 Finally, since biopsies are performed infrequently in the early post-TX phases, it is difficult to establish the scenario of possible ACR/AMR without DGF because the majority of rejections have ATN, a basic component of DGF. DGF is particularly frequent in the ECD group, and results in higher number of premature deaths due to severe complications. In an attempt to limit the onset of DGF or ACR triggered by DGF, these recipients might receive more IS with an increased risk for lethal infections.6,10,57

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16.7 INTERVENTIONS AND APPROACHES TO THE PATIENT WITH DGF In recent years several attempts have been made to limit the incidence of DGF, especially in grafts of donors with more risk factors such as ECD, DCD, major trauma, etc. During the final phases of establishing brain death, the donor has a nonphysiological status with metabolic and inflammatory abnormalities that impact the quality of retrievable organs and compromise the recipient and graft survival. The first step for avoiding DGF is to optimize the donor’s care. Ethical and legal issues do not allow specific donor pretreatment strategies. The approaches for organ protection can be attempted only after the death and the consent for donation. Interventions have been directed to improve perfusion, to reduce CIT, to fight the oxidative damage caused by IRI, and, finally, to ameliorate the repair process. The first point of concern is that CIT is probably the most important determinant for the severity of IRI; therefore, the use of cold preservation solutions containing antioxidants and a careful hemodynamics during surgery are crucial.58 In the ECD era, an important aid comes from the possibility to use hypothermic machine perfusion (HMP). This device allows more stabilized conditions than the cold storage solution. In an animal model, after 1-hour vascular clamping, canine kidneys were stored for 24 hours and then biopsied. The kidneys stored in cold solution were available to 40% according to established biopsy criteria, as opposed to those saved in HPM, which were available to 100%.59 In humans, the incidence of DGF is less if the kidney is kept in these devices.60 The organs preserved in HMP reduce the duration of DGF with a faster drop in s.creat. during the recovery phase.61,62 The use of HMP increased 1-year ECD graft survival whereas DCD grafts did not show a statistically significant difference. The best results occur with pulsatile rather than continuous kidney perfusion flow.59 HMP allows the evaluation of the different components of the perfusion fluid and monitoring of kidney status during the time between organ procurement and TX procedure. Concerning the economic impact, the use of HMP per se entails an increase in the costs but its use saves resources by reducing DGF, primary nonfunctioning grafts, and length of hospitalization.63 Three main factors should be addressed for preventing or at least minimizing IRI: The preservation of the microcirculatory fluidity, the inhibition of oxygen radicals with antioxidants and the block of inflammatory cells.64,65 Drug regimens consisting of prostaglandin E, dopamine, and vasodilator agents able to increase nitric oxide synthesis, in addition to isovolemic hemodilution and hypertonic solutions, have been shown to also be beneficial for edema prevention.66,67 During the implantation, the incidence of DGF can be reduced by administration of mannitol, which minimizes CIT effects.68 Different antioxidant agents have been unsuccessfully tested, including N-acetylcysteine, alone or in association with vitamin C.69,70 During the TX procedure, the administration of high doses of erythropoietin showed a slight tendency to DGF reduction but this event was not statistically significant.71,72 In a recent paper, the authors analyzed by logistic regression the classic risk factors for DGF along with two novel ones, the recipient’s residual diuresis and the perioperative saline load. These two strategies resulted in an efficacious IRI prevention. The simplistic but intriguing conclusion from the study, retrospectively performed on 1784 kidney TX recipients, was that the susceptibility to rejection enhanced by IRI decreased and impacted positively patient and graft outcomes.73 Prolonged cold storage and rewarming induce stress and nutrient deprivation mainly in the renal tubular epithelium and may induce apoptosis and autophagy. However attempts focused on autophagy blockage did not seem to be protective for the development of DGF.74,75 Monoclonal antibodies against ICAM-1 or CTLA4 designed for inhibiting the interaction between neutrophils and endothelial cells and T-cell activation, have been explored with discordant results in animal models.76 78 Few preclinical studies have shown that thiazolidinediones, although designed with the purpose of enhancing glucose sensitivity and improving free fatty acid metabolism, have instead shown efficacy in reducing IRI because of downregulation of specific antiinflammatory genes in endothelial and myeloid cells. Experimental and clinical data suggest that Thymo might ameliorate IRI, and consequently reduce the incidence of DGF. The mechanisms of the effects of Thymo are multiple such as a direct block of cell cell interaction, reduction of leukocyte rolling and adhering along endothelial surface, downmodulation of adhesion molecules, reduction of inflammatory mediations and chemokine receptors.79,80,85 During the repair phases, the tubular regeneration is well documented whereas the potential regrowth of the endothelial cells is probably limited. This has generated interest in the hematopoietic progenitor cells and their ability to differentiate into mature cells of the microvascular system.87 Mesenchymal stromal cells and growth factors are still under deep investigation. However, the need for a mixture of growth elements rather than a single one and the association with tumorogenesis have limited this approach.88 There are no effective treatments for DGF; however, early diagnosis and some possible therapeutic interventions may improve the TX outcomes. Markers for detecting AKI and DGF are crucial and in continuous development but so far the only reliable diagnostic approach is sustained by the clinical findings of

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anuria, oliguria, renal dysfunction, increased RI, and the patient’s conditions. The main reason to avoid excessive ultrafiltration is to allow some degree of diuresis that will be further impaired by intravascular fluid depletion. This last condition can induce hypotension, a condition that generates low perfusion of the graft and worsens the lesions secondary to ATN. Different IS regimens have been proposed during DGF phases. In order to prevent further vasoconstriction, CNI minimization or avoidance in the early post-TX period have been studied in several trials.89 Most of these studies have reached similar conclusions and aimed Thymo or anti-IL-2 receptor antibody induction (basilixamab or the no longer available daclizumab). The literature reports very few experiences with the initial use of m-TOR inhibitors (Sirolimus and Everolimus) that despite the lack of nephrotoxicity, have been avoided because of their antifibroblastic property and consequently impaired wound healing. However, with particular interest for ECD kidneys, the m-TOR inhibitors have been revisited and utilized in combination with a short Thymo course.90

16.7.1 Center Features According to the literature, the risk for AKI increases 3% for each hour of CIT.91 Thirty-six hours is an acceptable time to prevent long-term injury.13 Some TX centers have established a policy to discard organs from marginal donors and prolonged CIT.5 The impact of CIT is more noticeable in DCD kidneys who show higher percentage of DGF.6,8,9 For these reasons, the introduction of CNI in DCD grafts should be delayed and eventually introduced at low doses after induction therapy with Thymo. The need of activating health personnel during the nighttime prolongs CIT. This situation does not increase surgical complications, but it can increase CIT and the possibility of developing DGF.92 The minimally invasive and robotic surgery reduce surgical complications and hospitalization and improve short- and long-term graft survival.93

16.7.2 Recipient Features There are some recipient features that increase the incidence of DGF3,94: these include pre-TX anuria, mannitol, hemodynamic instability and absence of diuresis during surgery, low quality of reperfusion, old age of the recipients, duration of pre-TX dialysis and obesity. This last feature is strongly linked to DGF after correction for recipient’s age, gender and years of dialysis.4 In fact a BMI greater than 30 predisposes to the development of DGF.3,57,94 Increased risks for DGF up to 8.7% are generated by each point of body BMI and also by increased triceps skinfold thickness.94,95 This link between obesity and DGF is related to a difficult and prolonged surgery. The obesity induces the rise of prothrombotic events with endothelial damage. Post-TX micro- thrombotic complications are known to increase the risk for DGF.94 Finally, in obese recipients there is an over activation of the sympathetic system and a greater renal vasoconstriction, conditions that can further increase the CNI related ischemic damage and the risk for DGF.96

16.7.3 Donor Features Donor features that increase the incidence of DGF are: Age over 60 years, s.creat. above 1.36 mg/dL, long time of CIT, causes of death, ECD and DCD, AKI, hypertension, diabetes mellitus, and arteriosclerosis.3,7,41,51,57,94 Today the use of ECD is raised by the need to find an increasing number of organs for TX. In order to minimize the incidence of DGF, the graft of donor of .60 years old requires more invasive interventions that unfortunately may result in an early post-TX mortality.57 The grafts from AKI donors have a chance to develop DGF almost double compared to SCD (68% vs 36%). However, the data of mortality and long-term graft survival collected are similar to SCD transplants. This reassures about the safety of this type of donor.51 The comparison between SCD and DCD favors the former as shown by biopsies performed pre-TX: in DCD there are more hypoxic injuries and complement activated coagulation cascade that will enhance DGF and immune response.97

16.8 CONCLUSIONS DGF is a common post-TX complication induced by immunologic and nonimmunologic events mainly related to IRI. The real incidence of DGF has to be determined because of different definitions and diagnostic criteria. Risk factors for DGF are associated with recipient’s and donor’s characteristics. In particular, for the urgent

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demand for organs, more and more marginal donors are used with a consequent impact on renal function in short- and long-term follow up. There are no effective and standardized treatments for DGF; however, an early diagnosis with novel biomarkers, procurement, and preservation techniques offer promising results.

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Aitken E, Cooper C, Dempster N, et al. Delayed graft function is a syndrome rather than a diagnosis. Exp Clin Transplant 2015;13:19 25. 11. Wang CJ, Tuffaha A, Shang D, et al. A CD3 1 count-based thymoglobulin induction regimen permits delayed introduction of calcineurin inhibitors in kidney transplantation. Clin Transplant 2012;26:900 9. 12. Murad G, Morelon E, Noel C, et al. The role of thymoglobulin induction in kidney transplantation: an update. Clin Transplant 2012;26:e450 64. 13. Ponticelli CE. The impact of cold ischemia time on renal transplant outcome. Kidney Int 2015;87:272 5. 14. Malyszko J, Lukaszyk E, Glowinska I, et al. Biomarkers of delayed graft function as a form of acute kidney injury in kidney transplantation. Sci Rep 2015;5:11684. 15. Lang P, Pardon A, Audard V. Long-term benefit of mycophenolate mofetil in renal transplantation. Transplantation 2005;79:S47 8. 16. Laskowski I, Pratschke J, Wilhelm MJ, et al. 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Perit Dial Int 2009;29 (Suppl. 2):117 22. 22. Israni AK, Li N, Cizman BB, et al. Association of donor inflammation and apoptosis-related genotypes and delayed allograft function after kidney transplantation. Am J Kidney Dis 2008;52:331 9. 23. Dabrowska-Zamojcin E, Dziedziejko V, Sadranow K, et al. Association between the CX3CR1 gene V249I polymorphism and delayed kidney allograft function. Transpl Immunol 2015;32:172 4. 24. Liang WX, Cai MJ, Jiang L, et al. Ultrasonic imaging characteristics of transplanted kidney with delayed graft function. Genet Mol Res 2014;13:6878 84. 25. Contti MM, Garcia PD, Kojima CA, et al. Quantified power Doppler as a predictor of delayed graft function after renal transplantation. Int Urol Nephrol 2015;47:405 12. 26. Grzelak P, Szymczyk K, Strzelczyk J, et al. Perfusion of kidney graft pyramids and cortex in contrast-enhanced ultrasonography in the determination of the cause of delayed graft function. Ann Transplant 2011;16:48 53. 27. Rodrigo E, Lopez-Rasines G, Ruiz JC, et al. Determinants of resistive index shortly after transplantation: independent relationship with delayed graft function. Nephron Clin Pract 2010;1113:178 86. 28. Yazici B, Yazici A, Oral A, et al. Comparison of renal transplant scintigraphy with renal resistance index for prediction of early graft dysfunction and evaluation of acute tubular necrosis and acute rejection. Clin Nucl Med 2013;38:931 5. 29. Yazici B, Oral A, Gokalp C, et al. A new quantitive index for baseline renal transplant scintigraphy with 99m Tc-DTPA in evaluation of delayed graft function and prediction of 1-year graft function. Clin Nucl Med 2015;40:548 52. 30. Obeidat MA, Luyckx VA, Grebe SO, et al. Post-transplant nuclear scans correlate with renal injury biomarkers and early allograft outcomes. Nephrol Dial Transplant 2011;26:3038 45. 31. Hueper K, Gueler F, Brasen JH, et al. Functional MRI detects perfusion impairment in renal allografts with delayed graft function. 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32. Mishra J, Dent C, Tarabishi R, et al. Neutrophil gelatinase-associated lipocalin (NGAL) as a biomarker for acute renal injury after cardiac surgery. Lancet 2005;365:1231 8. 33. Makris K, Markou N, Evodia E, et al. Urinary neutrophil gelatinase-associated lipocalin (NGAL) as an early marker of acute kidney injury in critically ill multiple trauma patients. Clin Chem Lab Med 2009;47:79 82. 34. Bennett M, Dent CL, Ma Q, et al. Urine NGAL predicts severity of acute kidney injury after cardiac surgery: a prospective study. Clin J Am Soc Nephrol 2008;3:665 73. 35. Hollmen ME, Kyllo¨nen LE, Merenmies J. Serum neutrophil gelatinase-associated lipocalin and recovery of kidney graft function after transplantation. BMC Nephrol 2014;15:123. 36. Bataille A, Abbas S, Semoun O, et al. Plasma neutrophil gelatinase-associated lipocalin in kidney transplantation and early renal function prediction. Transplantation 2011;92(9):1024 30. 37. Buemi A, Musuamba F, Frederic S, et al. Is plasma and urine neutrophil gelatinase-associated lipocalin (NGAL) determination in donors and recipients predictive of renal function after kidney transplantation? Clin Biochem 2014;47:68 72. 38. Van den Akker EK, Hesselink DA, Manintveld OC, et al. Neutrophil gelatinase-associated Lipocalin, but not kidney injury marker 1, correlates with duration of delayed graft function. Eur Surg Res 2015;55:319 27. 39. Hall IE, Yarlagadda SG, Coca SG, et al. IL-18 and urinary NGAL predict dialysis and graft recovery after kidney transplantation. J Am Soc Nephrol 2010;21:189 97. 40. Fonseca I, Oliveira JC, Santos J, et al. Leptin and adiponectin during the first week after kidney transplantation: biomarkers of graft dysfunction? Metabolism 2015;64:202 7. 41. Pribylova-Hribova P, Kotsch K, Lodererova A, et al. TGF-β1 mRNA upregulation influences chronic renal allograft dysfunction. Kidney Int 2006;69:1872 9. 42. Guerrieri D, Re L, Petroni J, et al. Gene expression profile in delay graft function: inflammatory markers are associated with recipient and donor risk factor. Mediators Inflamm 2014;167361. 43. Kwiatkowska E, Domanski L, Kloda K, et al. IL2-IL21 gene cluster polymorphism is not associated with allograft function after kidney transplantation. Nephrology 2014;46:2415 20. 44. Castellano G, Intini A, Stasi A, et al. Complement modulation of anti-aging factor Klotho in ischemia/reperfusion injury and delayed graft function. Am J Transplant 2015. Available from: http://dx.doi.org/10.1111/ajt.13415. 45. Irish WD, McCollum DA, Tesi RJ, et al. Normogram for predicting the likelihood of delayed graft function in adult cadaveric renal transplant recipients. J Am Soc Nephrol 2003;14(11):2967 74. 46. Irish WD, Ilsley JN, Schnitzler MA, et al. A risk prediction model for delayed graft function in the current era of deceased donor renal transplantation. Am J Transplant 2010;10(10):2279 86. 47. Jeldres C, Cardinal H, Duclos A, et al. Prediction of delayed graft function after renal transplantation. Can Urol Assoc J 2009;3(5):377 82. 48. Chapal M, Le Borgne F, Legendre C, et al. A useful scoring system for the prediction and management of delayed graft function following kidney transplantation from cadaveric donors. Kidney Int 2014;86(6):1130 9. 49. Zaza G, Ferraro PM, Tessari G, et al. Predictive model for delayed graft function based on easily available pre-renal transplant variables. Intern Emerg Med 2015;10(2):135 41. 50. Decruyenaere A, Decruyenaere P, Peeters P, et al. Validation in a single-center cohort of existing predictive models for delayed graft function after kidney transplantation. Ann Transplant 2015;20:544 52. 51. Molina M, Apaza J, Gonzalez ME, et al. Results of kidney transplantation from deceased donors with acute kidney injury. Transplant Proc 2015;47:42 4. 52. Almond PS, Troppmann C, Escobar F, et al. Economic impact of delayed graft function in cadaveric renal transplantation. Transplant Proc 1991;23:1304. 53. Rosenthal JT, Danatovich GM, Wilkinson A, et al. The high costs of delayed graft function in cadaveric renal transplantation. Transplantation 1991;51:1115 18. 54. Raimundo M, Guerra J, Teixeira C, et al. Intermediate early graft function is associated with increased incidence of graft loss and worse long-term graft function in kidney transplantation. Transplant Proc 2013;45:1070 2. 55. Wu WK, Famure O, Li Y, et al. Delayed graft function and the risk of acute rejection in the modern era of kidney transplantation. Kidney Int 2015;88:851 8. 56. Requia˜o-Moura LR, Ferraz E, Matos AC, et al. Comparison of long-term effect of thymoglobulin treatment in patients with a high risk of delayed graft function. Transplant Proc 2012;44:2428 33. 57. Tanrisev M, Ho¸sco¸skun C, A¸sc¸ G, et al. Long-term outcome of kidney transplantation from elderly living and expanded criteria deceased donor. Ren Fail 2015;37:249 53. 58. Andrews PM, Cooper M, Verbesey J, et al. Mannitol infusion within 15 min of cross-clamp improves living donor kidney preservation. Transplantation 2014;98:893 7. 59. Lindell LS, Muir H, Brassil J, et al. Hypothermic machine perfusion preservation of the DCD kidney: machine effects. J Transplant 2013;802618. 60. Forde JC, Shields WP, Azhar M, et al. Single centre experience of hypothermic machine perfusion of kidneys from extended criteria deceased heart-beating donors: a comparative study. Ir J Med Sci 2014;14(4):109 12. 61. Burgos Revilla FJ, Hevia V, Diex V, et al. Machine perfusion: initial results in an expanded criteria donor kidney transplant program. Transpl Proc 2015;47:19 22. 62. Oliver E, Lopez-Delgado JC, Gil-Vernet S, et al. Machine perfusion for preservation of kidneys from expanded criteria donors after brain death improves transplantation results. Org Tiss Cells 2013;16:99 105. 63. Go´mez V, Galeano C, Diez V, et al. Economic impact of the introduction of machine perfusion preservation in a kidney transplantation program in the expanded donor era: cost-effectiveness assessment. Transplant Proc 2012;44:2521 4. 64. Aydin Z, van Zonneveld AJ, de Fijter JW, et al. New horizons in prevention and treatment of ischaemic injury to kidney transplants. Nephrol Dial Transplant 2007;22:342 6.

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65. Yates PJ, Hosgood SA, Nicholson ML. A biphasic response to nitric oxide donation in an ex vivo model of donation after cardiac death renal transplantation. J Surg Res 2012;175:316 21. 66. Brauer RB, Marx T, Ulm K, et al. Effect of perioperative administration of a drug regimen on the primary function of human renal allografts. Transplant Proc 2010;42:1523 5. 67. McCune TR, Wombolt DG, Whelan TV, et al. Vasodilatation vs. immunotherapy to prevent delayed graft function: delayed graft function as an indication of immune activation. Int Immunopharmacol 2005;5:85 92. 68. Andrews PM, Cooper M, Verbesey J, et al. Mannitol infusion within 15 min of cross-clamp improves living donor kidney preservation. Transplantation 2015;98:893 7. 69. Orban JC, Quintard H, Cassuto E, et al. Effect of N-acetylcysteine pretreatment of deceased organ donors on renal allograft function: a randomized control trial. Transplantation 2015;99:746 53. 70. Sahraei Z, Salamzadeh J, Nafar M. Effect of N-acetylcysteine and vitamin C on kidney allograft function biomarkers interleukin-18 and neutrophil gelatinase-associated lipocalin. Transplantation 2015;9:56 62. 71. Coupes B, de Freitas DG, Roberts SA, et al. rhErythropoietin-b as a tissue protective agent in kidney transplantation: a pilot randomized controlled trial. BMC Res Notes 2015;8:21. 72. Xin H, Ge YZ, Wu R, et al. Effect of high-dose erythropoietin on graft function after kidney transplantation: a meta-analysis of randomized controlled trials. Biomed Pharmacother 2015;96:29 33. 73. Chaumont M, Racape´ J, Broeders N, et al. Delayed graft function in kidney transplants: time evolution, role of acute rejection, risk factors, and impact on patient and graft outcome. J Transplant 2015;163757. 74. Jain S, Keys D, Nydam T, et al. Inhibition of autophagy increases apoptosis during re-warming after cold storage in renal tubular epithelial cells. Transpl Int 2014;28:214 23. 75. Salahudeen AK, Joshi M, Jenkins JK, et al. Apoptosis versus necrosis during cold storage and rewarming of human renal proximal tubular cells. Transplantation 2001;72(5):798 804. 76. Kelly KJ, Williams Jr. WW, Colvin RB, et al. Intercellular adhesion molecule-1-deficient mice are protected against ischemic renal injury. J Clin Invest 1996;97(4):1056 63. 77. Ichikawa H, Flores S, Kvietys PR, et al. Molecular mechanisms of anoxia/reoxygenation-induced neutrophil adherence to cultured endothelial cells. Circul Res 1997;81:922 31. 78. Aydin Z, van Zonneveld AJ, de Fijter JW, et al. New horizons in prevention and treatment of ischaemic injury to kidney transplants. Nephrol Dial Transplant 2007;22(2):342 6. 79. Pre´ville X, Flacher M, LeMauff B, et al. Mechanisms involved in antithymocyte globulin immunosuppressive activity in a nonhuman primate model. Transplantation 2001;71:460 8. 80. Beiras-Fernandez A, Thein E, Chappel D, et al. Polyclonal anti-thymocyte globulins influence apoptosis in reperfused tissues after ischaemia in a non-human primate model. Transpl Int 2004;17:453 7. 81. Goggins WC, Pascual MA, Powelson JA, et al. A prospective, randomized, clinical trial of intraoperative versus postoperative Thymoglobulin in adult cadaveric renal transplant recipients. Transplantation 2003;76:798 802. 82. Cravedi P, Codreanu I, Satta A, et al. Cyclosporine prolongs delayed graft function in kidney transplantation: are rabbit anti-human thymocyte globulins the answer? Nephron Clin Pract 2005;101:c65 71. 83. Chappell D, Beiras-Fernandez A, Hammer C, et al. In vivo visualization of the effect of polyclonal antithymocyte globulins on the microcirculation after ischemia/reperfusion in a primate model. Transplantation 2006;81:552 8. 84. Aydin Z, van Zonneveld AJ, de Fijter JW, et al. New horizons in prevention and treatment of ischaemic injury to kidney transplants. Nephrol Dial Transplant 2007;22:342 6. 85. Mehrabi A, Mood Zh A, Sadeghi M, et al. Thymoglobulin and ischemia reperfusion injury in kidney and liver transplantation. Nephrol Dial Transplant 2007;22(Suppl. 8):viii54 60. 86. Patel SJ, Duhart Jr BT, Krauss AG, et al. Risk factors and consequences of delayed graft function in deceased donor renal transplant patients receiving antithymocyte globulin induction. Transplantation 2008;86:313 20. 87. Rabelink TJ, de Boer HC, de Koning EJ, et al. Endothelial progenitor cells: more than an inflammatory response. Arterioscler Thromb Vasc Biol 2004;24(5):834 8. 88. Devarajan P. Has HGF met other partners? Met-independent epithelial morphogenesis induced by HGF. focus on “Hepatocyte growth factor induces MDCK cell morphogenesis without causing loss of tight junction functional integrity.” Am J Physiol Cell Physiol 2004;286(3): C475 7. 89. House AA, Nguan CY, Luke PP, et al. Sirolimus use in recipients of expanded criteria donor kidneys. Drugs 2008;68(Suppl. 1):41 9. 90. Tahir W, Hakeem A, Dawrant M, et al. Early sirolimus conversion as rescue therapy in kidneys with prolonged delayed graft function in deceased donor renal transplant. Transplant Proc 2015;47(6):1610 15. 91. Frei U, Noeldeke J, Machold-Fabrizii V, et al. Prospective age-matching in elderly kidney transplant recipients--a 5-year analysis of the Eurotransplant Senior Program. Am J Transplant 2008;8(1):50 7. 92. Fockens MM, Alberts VP, Bemelman FJ, et al. Renal transplantation at night. Ned Tijdschr Geneeskd 2014;158:a7779. 93. Sood A, Ghosh P, Jeong W, et al. Minimally invasive kidney transplantation: perioperative considerations and key 6-month outcomes. Transplantation 2015;99(2):316 23. 94. Tsirigoti L, Kontogianni MD, Darema M, et al. Exploring associations between anthropometric indices and graft function in patients receiving renal transplant. J Hum Nutr Diet 2014. Available from: http://dx.doi.org/10.1111/jhn.12289. 95. Weissenbacher A, Jara M, Ulmer H, et al. Recipient and donor body mass index as important risk factors for delayed graft function. Transplantation 2012;93:524 9. 96. Lambert E, Sari CI, Dawood T, et al. Sympathetic nervous system activity is associated with obesity-induced subclinical organ damage in young adults. Am J Kidney Dis 2010;54:1043 51. 97. Damman J, Bloks VW, Daha MR, et al. Hypoxia and complement-and-coagulation pathways in the deceased organ donor as the major target for intervention to improve renal allograft outcome. Transplantation 2014;99(6):1293 300.

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C H A P T E R

17 Histocompatibility Testing in the Transplant Setting Michael D. Gautreaux Wake Forest School of Medicine, Winston-Salem, NC, United States

17.1 INTRODUCTION There are two basic systems of defense that animals use against pathogens or invaders: innate and adaptive. Innate defenses, as the name suggests, are defenses one is born with. Innate immune responses are not specific to a particular pathogen. Innate immunity includes external barriers (e.g., intact skin, mucus membranes), secretory components (e.g., enzymes, histamine, oxygen radicals, etc.), and certain leukocytes (such as phagocytes, NK cells, platelets). A hallmark of innate immunity is that there is no increase in strength after exposure. Innate immune responses have been found among both vertebrates and invertebrates, as well as in plants, and the basic mechanisms that regulate them are conserved.1 Adaptive immunity, also known as acquired immunity, is generally referred to as the “immune system.”2 The adaptive immune system is composed of highly specialized cells and processes that eliminate or prevent pathogen growth. In addition, the adaptive immune system can potentiate the effects of certain parts of the innate immune system. The adaptive immune system performs three basic functions: (1) to discriminate between “self” and “nonself”; (2) generate a specific response to what is perceived as nonself antigens and facilitate the clearance of such antigens; and (3) develop specific memory to such antigens to facilitate a quicker and greater response to such antigens in the future. The pioneering work of Medawar3 demonstrated that rejection to transplants is a form of adaptive immunity. Two factors play a strong role in the speed in which rejection occurs: The degree of presensitization and the magnitude of the HLA mismatch between a donor and recipient. Mostly for historical reasons, graft rejection is usually classified on the basis of histopathology or time after transplant, rather than immune effector mechanisms. Subsequent research and clinical experience have shown three patterns of rejection based on clinical presentation and histological criteria. These types of rejection are hyperacute, acute, and chronic; each will be discussed separately in the following sections.

17.2 TYPES OF REJECTION 17.2.1 Hyperacute Rejection Hyperacute rejection is characterized by rapid endothelial cell disruption, platelet margination, complement activation, and thrombotic occlusion of the graft vasculature within minutes of vascular anastomosis. Hyperacute rejection is the only form of rejection that is exclusively mediated by a humoral mechanism, the remaining forms of rejection appear to have both cellular and humoral components. Preformed antibodies to histocompatibility antigens bind to the graft’s endothelial cells and activate complement causing endothelial cell injury.4 This

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process exposes subendothelial basement membrane proteins that activate platelets. As a result of these processes the organ suffers irreversible ischemic damage. Hyperacute rejection has been virtually eliminated in kidney transplantation because of the prospective crossmatch test. Prospective crossmatches are not the norm outside of the renal transplant setting because of the short ideal preservation time for hearts, lungs, and livers. Most crossmatches for these organs are done retrospectively. As a result, there are cases in which a transplant occurs across a positive crossmatch. Hyperacute rejection of a heart is extremely rare; however, it has been documented.5 More common, however, is significantly lower 1-year survival of a heart transplanted across a positive crossmatch.5,6

17.2.2 Acute Rejection Accelerated rejection and acute rejection are different names for the same basic process. The only difference between the two is the time of onset after the transplant has occurred. Accelerated rejection is said to occur within a week or two after transplant (sometimes as long as 1 month), and acute rejection is said to occur 2 weeks to several months after transplant. For the sake of simplicity, both types will be referred to as acute rejection. Acute rejection, from an effector standpoint, can actually happen at any time after a transplant and is considered an active immune response to the graft. Acute rejection can have humoral and/or cellular mechanisms of action.7,8 Acute humoral rejection is characterized by necrosis of graft endothelial cells. The histologic pattern is one of vasculitis rather than massive vascular thrombosis seen with hyperacute rejection. A more accurate name for this process is acute vascular rejection because the vascular endothelium is destroyed, primarily by antibody-mediated mechanisms. Lymphocytes (especially donor-specific CD8 1 T-cells) also appear to directly kill endothelial cells and, in addition, provide cytokines to stimulate inflammatory responses (primarily CD-4 1 T-cell mediated). Acute cellular rejection is mediated by lymphocytes and macrophages infiltrating into the parenchyma and lysing the parenchymal cells. The infiltrating lymphocytes are T-cells and NK cells. The recognition of donor HLA markers by alloreactive CD8 1 T-cells appears to be the most important mechanism in acute cellular rejection. This conclusion is supported by the finding that CD8 1 T-cells predominate in cellular infiltrates and by the knowledge that most graft vascular and parenchymal cells express class I HLA, which is recognized by CD8 1 T-cells; however, they are usually resistant to killing by NK cells and macrophages. Animal studies have demonstrated that adoptive transfer of cytotoxic T-cells (CD8 1 ) can eliminate tumor grafts.9 The realization that antibodies and CTLs are the primary effectors in the acute graft rejection response has led to the idea that this process is similar to the immune response to viruses. This is in contrast to the idea that chronic rejection (discussed in the next section) represents a variant of the wound healing process. The potential mechanisms by which the immune system can recognize foreign HLA will be discussed in the following.

17.2.3 Chronic Rejection Chronic rejection is a much more insidious process than acute rejection. The damage from chronic rejection slowly compromises organ function through two pathways: Parenchymal remodeling with fibrosis and graft vascular disease. Patchy interstitial inflammation can give way to fibrosis and parenchymal injury. The fibrosis seen with chronic rejection may represent wound healing following cellular necrosis due to acute rejection. Indeed, the incidence of chronic rejection is increased following severe or persistent acute rejection, perhaps due to insufficient or ineffective immunosuppression.10 The most common histologic manifestation of chronic rejection in heart and kidneys is a progressive narrowing of the graft’s arteries. This is referred to as obliterative arteriopathy (OA). OA can be likened to an accelerated form of atherosclerosis. In this form of chronic rejection, the vascular lumen is replaced by an accumulation of smooth muscle cells and connective tissue.11,12 This predisposes the graft to chronic ischemic damage and infarction. In lung allografts, destruction of the small bronchioles, termed obliterative bronchiolitis (OB), is the major manifestation of chronic rejection, while vascular disease is thought to be of lesser importance.13 Chronic rejection can begin within weeks to months after transplantation. Indeed, chronic and acute rejection can be occurring within the same graft at the same time. In general, the incidence of chronic rejection increases with time after rejection and, with the notable exception of the liver,14 eventually afflicts a majority of solid organ allografts. By 5 years posttransplant, chronic rejection affects up to 80% of lung transplants,15,16 60% of heart transplants,17 50% of kidney transplants,18 but only about 10% of liver allografts.19

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17.3 HISTOCOMPATIBILITY TESTING The goal of histocompatibility testing is to ensure that the transplanted organ remains viable in the recipient for the longest period of time. Three sets of tests are performed to accomplish this goal. The first is determining the human leukocyte antigen (HLA) types of the patient and donor. The second is determining the presence and identity of any anti-HLA antibodies by testing against a reference cell panel, otherwise known as panel-reactive antibody or panel-reactive antibody (PRA) testing. Lastly, donor-specific antibody response is measured in the crossmatch assay.

17.3.1 HLA Typing For transplantation, it is recommended that HLA typing and, more importantly, antibody testing be performed on all patients prior to transplant. Time constraints make a pretransplant crossmatch impractical for most heart and liver cases. The maximum preservation time for a kidney is 24 36 hours; whereas, the ideal preservation time for a heart is 4 6 hours, a liver 8 12 hours, and pancreas 12 18 hours.20 Unless there are sufficient reasons to believe a patient may have a positive crossmatch, such as a high PRA or identified antibody specificity, the crossmatch is usually performed retrospectively. These considerations will be addressed below. HLA matching is one of the major considerations for allocating kidneys from deceased donors. The stringent time constraints and lack of computerized algorithms from UNOS mean that HLA matching is not usually a consideration for either heart or liver allocation. However, several publications suggest that there may be sufficient benefits of HLA matching to warrant its consideration.21 26 17.3.1.1 HLA Typing Methods 17.3.1.1.1 Complement-Dependent Cytotoxicity Complement-dependent cytotoxicity (CDC) had been the predominant form of HLA testing until it was supplanted by molecular or DNA typing (discussed in the following). CDC or serological typing relies on HLAspecific antibodies, from characterized alloantisera or monoclonal antibodies, binding to the HLA molecules expressed on the surface of patient or donor cells. Complement is added to the reactions and if specific antibodies are bound on the cells the complement cascade is initiated and cell death occurs. Special stains are added to identify dead cells from live cells and the cells are visualized microscopically. The percentage of cell death in each reaction is noted and each well given a score. Multiple reactions are analyzed to determine a patient or donor’s phenotype. Indeed, for one complete HLA typing, 300 individual reactions or more may be analyzed in order to assign an HLA phenotype.27 The CDC typing test relies on allospecific antisera or monoclonal antibodies, which permit identification of HLA-A, HLA-B, and HLA-DR antigens recognized by the World Health Organization. If possible, private (split) specificities should be differentiated from public (broad) specificities. By way of example, A9 is considered a broad antigen. As better-defined antisera became available, it was realized that two separate antigens were recognized by the original anti-A9 antisera. These new specificities, A23 and A24, are considered private specificities. Fig. 17.1 gives an overview of the CDC tests. Peripheral blood lymphocytes are separated into T-cell and Bcell-enriched fractions. In the case of the figure, superparamagnetic beads with pertinent monoclonal antibodies to either T-cell antigens or B-cell antigens are incubated with the buffy coat of a peripheral blood prep or a single-cell suspension derived from a peripheral lymph node or a piece of spleen. Once magnetically separated into either T-cells or B-cells, the cells are incubated with antisera with precharacterized antisera to various HLA specificities. Depending on the laboratory protocol, the cells/sera are then washed with buffer and then incubated with rabbit complement. A vital dye preparation is added (in the case of the figure acridine orange for green, denoting viable cells, and ethidium bromide to stain dead cells red). Each well is visualized and independently scored utilizing the ASHI scoring system (Fig. 17.2).28 The percentages of cell death are estimated and a score given appropriately. An example of a scoring sheet is given in Table 17.1. 17.3.1.1.2 Molecular Typing Molecular or DNA-based typing allows for the identification of HLA types independent of expression on the cell surface. Most molecular HLA typing procedures rely, in whole or part, on the polymerase chain reaction (PCR) process. The three most common forms of molecular HLA typing are sequence-specific primer (SSP) test, the sequence-specific oligonucleotide probe (SSOP) process, and the sequence-based typing (SBT) test.

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FIGURE 17.1 Schematic of a CDC-based HLA typing utilizing magnetic beads.

FIGURE 17.2 Diagram of the ASHI scoring system for CDC-based testing.

DNA-based typing has several benefits when compared to serological based typing: Less blood is required, viable cells are not essential, there is less cost, and the typing process can be performed faster because of greater reliability. An excellent primer for molecular typing for HLA typing purposes is “DNA Methods for HLA Typing: A Workbook for Beginners.”29 SSP relies on amplification of specific regions of the HLA molecule after binding of a specific piece of DNA as an initiator of DNA replication (Fig. 17.3).30 Once the DNA is amplified, an agarose gel is run with a special stain, such as ethidium bromide, in order to determine if a specific primer amplified the patient’s input DNA. If amplification has occurred, then the patient’s DNA reacted with the specific primers in a particular well. The reactivity of a particular set of primers in a well is scored as positive, negative, or no reaction (Fig. 17.4). The reaction pattern of the various sets of primers is then interpreted to provide the final typing. Recently, a modified form of SSP has gained a considerable following. Real-time or quantitative PCR, utilizes specific primers to amplify DNA with specific sequences. However, instead of relying upon gel electrophoresis for readout, fluorescent dyes are intercalated into the amplified DNA. Upon completion of amplification, a specialized melting curve is generated and the release of the fluorescent dye at a specific temperature, under controlled conditions, indicates the presence or absence of specific amplification.31

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TABLE 17.1 Scoring Sheet for CDC-Based HLA Testing Well

Specificity

Reaction

1A

Positive control

8

1B

Negative control

1

1C

A1

1

1D

A1,36

1

1E

A1,36,80

1

1F

A2

8

2F

A2

8

2E

A3

8

2D

A3

8

2C

A23,24

1

2B

A23

1

2A

A24

1

3A

A25,26,34,11

1

3B

A25,26

1

3C

A26

1

3D

A11

1

3E

A68,69

4

3F

A68,69,33,34

2

4F

A29

1

4E

A30

1

4D

A30,31

1

4C

A30,31

1

4B

A32

1

4A

A32,25,26,34,43

1

5A

A33,34

1

5B

A34

1

5C

B51,52

1

5D

B51,7801

1

5E

B7,8101

1

5F

B7,8101

1

6F

B8

1

6E

B8

1

6D

B44,45

1

6C

B44

1

6B

B45

1

7B

B64,65

1

7C

B64,65

1 (Continued)

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TABLE 17.1 (Continued) Well

Specificity

Reaction

7D

B62,63,75

8

7E

B62,63,75

8

7F

B62

8

8F

B38,39

1

8E

B38

1

8D

B39

1

8C

B57,58

1

8B

B57

1

8A

B18

8

9A

B18

8

9B

B49,50

1

9C

B49

1

9D

B54,55,56,42

1

9E

B55,42

1

9F

B56

1

10F

B27

1

10E

B27

2

10D

B35

1

10C

B35,53

1

10B

B51,52,53

1

10A

B37

1

11A

B60,61,48

1

11B

B60

1

11C

B41,42

1

11D

B42

1

11E

B70,62,50,35

1

11F

B70,35,50,75,56

1

12F

Bw4

1

12E

Bw6

8

12D

Cw1

1

12C

Cw2

8

12B

Cw3

8

12A

Cw4

8

SSOP relies on a specific piece of DNA that is labeled to act as a specific detector or probe for a particular HLA sequence.32 Similar to SSP, sequences of DNA specific to particular HLA types are utilized. However, instead of utilizing the specific sequence to initiate DNA amplification, the specific DNA is labeled with a particular compound and then allowed to bind to the DNA. The DNA sequence is used to probe the patient’s DNA. The pattern of reactivity for a series of probes is then interpreted to provide the HLA typing.

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FIGURE 17.3

Schematic of PCR-based HLA typing utilizing DNA primers.

FIGURE 17.4

A ethidium-bromide stained gel along with a legend to show how individual wells are to be considered negative or

positive.

SBT relies on DNA sequencing technology to determine the unique sequence of base pairs in a particular HLA allele.33 The input DNA is amplified with locus-specific primers. Then that DNA is used as a template for sequencing reactions with dye labeled dideoxynucleotides. The termination of the elongating DNA chain by the specific dideoxynucleotide provides the sequence by size separation through a capillary gel electrophoresis. The sequence from the reaction is then compared with an HLA sequence database in order to assign the HLA type. The level of typing resolution is not usually necessary for solid organ transplantation. However, the potential for allele-specific antibodies could necessitate knowing a patient and/or donor’s high-resolution HLA typing. The demand for high throughput and low cost has fueled a new method of sequencing DNA sequences, called next generation sequencing,34 which holds promise for making high-resolution HLA typing become the norm for all solid organ patients. Outside the cost of acquiring the next generation sequencers, the costs of actually performing the HLA typing are poised to drop to previously unimaginable levels.

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17.3.1.2 Specimen Requirements Individual laboratories may have their own preferred set of requirements for serological or molecular HLA typing and it is best to check an institution’s HLA laboratory to determine its requirements. The suggestions in this section reflect the general consensus of the HLA community. Complete serological HLA typing (HLA-A, B, and DR) generally requires 30 mL of anticoagulated blood. The anticoagulants of choice are acid citrate dextrose (ACD; yellow-top Vacutainer tubes) or sodium heparin (green-top Vacutainer tubes). The use of lithium heparin is strongly discouraged because of problems with cell viability and purity. Samples for serological typing must be kept at room temperature and should be delivered to the HLA laboratory as rapidly as possible. In general, these samples maintain useful viability for 12 24 hours with sodium heparin and 24 72 hours with ACD. HLA typing on donors can be performed on whole blood, lymph nodes, or spleen sections. Occasionally, whole blood may not be the optimal specimen due to the adverse effects of steroids and blood transfusions. For lymph nodes and spleens, it is imperative that these tissues not be placed in any type of fixative. It is recommended that 2 or 3 lymph nodes and a piece of spleen at least 2 cm2 be provided to the HLA lab for testing. More lymph nodes and spleen would be beneficial if the organ is exported to another center to allow for confirmation typing and crossmatching at that location. Donor tissue specimens should be placed in a complete tissue culture medium such as RPMI 1640. Saline and Ringer’s lactate are not complete media. Check with an HLA lab to determine its recommendation. Molecular HLA typing usually requires much less volume of sample than serological typing. Generally, a single 10-mL tube of anticoagulated blood is sufficient. The anticoagulants of choice for DNA typing are ACD (yellow-top Vacutainer tube) and EDTA (lavender-top Vacutainer tube). Once again, this is a guideline. 17.3.1.3 Interpretation HLA types are assigned based on the reactivities of the specific antisera in the CDC method or specific DNA sequences in the molecular assay. Since HLA antigens are codominantly expressed, the goal is to detect two antigens or alleles each for HLA-A, HLA-B, and HLA-DR. If only one antigen or allele is identified for a particular locus, it is designated as a blank. Blanks usually represent homozygosity at the particular locus but such homozygosity can only be proven by performing typings on additional family members to determine haplotype inheritance patterns. A blank can also indicate expression of a particular allele that is not identifiable by current technology. Current guidelines recommend that a patient be retyped every 5 years if any of their HLA loci have a blank locus or if they possess an antigen that can be split with more current tissue typing procedures.

17.3.2 Serum Screening for HLA Antibodies The purpose of PRA screening is twofold. The first consideration is to determine the likelihood of a patient having a positive crossmatch with a random donor, i.e., a predictor of crossmatch compatibility. This is the reason that a percent score is given with antibody screening, e.g., a patient with a 5% PRA will likely have a positive crossmatch with 5% of the potential donors. The second, and more important, consideration for antibody screening is determination of the specificity of any alloreactive antibodies directed against particular HLA antigens. Federal regulations require a prospective crossmatch, whenever medically possible, for presensitized transplant recipients regardless of organ.35 Since serum screening is strongly recommended with the initial patient typing, the results of the serum screen may necessitate a prospective crossmatch in a cardiac transplant setting. The American Society of Histocompatibility and Immunogenetics (ASHI), the accrediting body for HLA laboratories, sets the definition of sensitized patient as having a greater than 15% PRA or having an identified allospecific antibody.36 UNOS standards reflect ASHI standards in the area of histocompatibility although transplant centers may have a certain amount of latitude in the definition of a “sensitized” patient. 17.3.2.1 Serum Screening Methods 17.3.2.1.1 Standard Panel Testing The patient’s sera are tested against a panel of lymphocytes from individuals whose HLA phenotypes have been predetermined. Ideally, the panel provides a spectrum of HLA antigens that are representative of the local population. Panels may be made by an individual laboratory from freshly isolated lymphocytes, frozen lymphocytes, or purchased from commercial sources. The use of frozen cells or commercially prepared panels eliminates the great concentration of personnel resources required to utilize fresh lymphocytes within the limited span of

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viability of these cells. Whatever the source of the panel lymphocytes, the actual assay is performed by the CDC process, in a manner very similar to the complement-based HLA typing described previously. 17.3.2.1.2 Solid-Phase Microarrays Solid-phase microarrays, better known as Luminex, serum screening utilizes solubilized and purified HLA antigens bound to a solid support, a microsphere. The purified HLA proteins serve as targets for HLA-specific antibodies that may exist in a patient’s serum. The presence of anti-HLA antibodies is detected by antihuman IgG conjugated to the fluorochrome FITC. A colorless substrate for the enzyme is added and a colored product develops if the antibody/enzyme complex is present. The absorbance of the colored product is then measured spectrophotometrically. The microspheres themselves have two dyes, a red dye and an infrared dye, at varying concentrations that allow for discrimination of the individual beads. Usually, a result is considered positive if the MFI of the FITC reading is above a cut-off value determined by the laboratory’s parameters. Solid-phase microarray serum screening offers several advantages over cell panel screening. First, the solidphase microarray is not dependent on viability of lymphocytes. Fresh lymphocytes maintain viability up to 72 hours and frozen lymphocytes may have lower viability because of the freezing and thawing process. The second and perhaps most important advantage is that only antibodies against HLA antigens are detected. Autolymphocytotoxic antibodies, which cause false positive reactions in the cell panel test and could subsequently confound the interpretation of such tests, do not react in the solid-phase microarrays system. 17.3.2.2 Specimen Requirements Serum derived from clotted whole blood is the specimen of choice for all antibody screening methods. It is vitally important that the specimen be collected in a plain tube with no additives (plain red-top Vacutainer tube). Specimen tubes with clot activators and serum separators are unacceptable because the additives can interfere with the serum screen assay. The serum from a full-draw 10-mL tube of clotted blood is sufficient for the serum screen assays. If a patient has a potential sensitizing event, such as a blood transfusion, it is best to wait 2 weeks from such an event prior to drawing a sample to allow for detection of any new anti-HLA specificities. 17.3.2.3 Interpretation A negative PRA test is indicative of a lack of anti-HLA antibodies. A positive result may indicate the presence of anti-HLA antibodies. Studies have demonstrated that sensitized patients have poorer outcomes in transplant when compared to unsensitized patients. In one study of 120 heart transplant patients, a PRA of 25% or greater was associated with a poorer outcome even in the presence of a negative crossmatch.33 In another study, patients with a T-cell PRA or a B-cell PRA greater than 11% had significantly poorer 3-year survival than patients with a PRA of less than 11%.37 Care must be taken in standard panel testing to determine if the antibodies present are anti-HLA in nature. Certain patients can develop autolymphocytotoxic antibodies that are usually of the IgM isotype. These react most often with B-cells, and are considered harmless to most kidney grafts.38 The best way to determine if a patient has an autoantibody is to perform an autocrossmatch. Some labs prefer to perform this at the time of the patient’s initial typing workup, while other labs perform autocrossmatches at the time of final crossmatch. Autoantibodies are not a problem with ELISA and the microarray serum screenings because these assays only have purified HLA antigens with which the serum may react. IgM antibodies directed against HLA have been demonstrated and are suspected of being harmful to a graft but there is little direct evidence to substantiate that fact.39 The serum screen assay allows the transplant team to determine if a prospective crossmatch is necessary. The crossmatch assay will be discussed in the next section. A PRA in excess of 15% indicates that a patient may have developed an anti-HLA antibody and is therefore considered sensitized. The serum screen may also determine if a patient has developed an antibody to a particular HLA antigen. If the medical situation allows, the safest course seems to be avoiding a transplant with a heart bearing a particular HLA antigen to which the potential recipient has been sensitized.

17.3.3 Crossmatching Because of the time constraints involved with both liver and cardiac transplantation, it is often necessary to perform such a transplant before the results of a crossmatch are known. As mentioned previously, a sensitized patient must have a prospective crossmatch prior to receiving any transplant, if medical circumstances permit.

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Crossmatch testing detects the presence of antibodies in the recipient that react specifically with the tissue of the donor. A positive crossmatch does not appear to directly lead to hyperacute rejection in the cardiac and liver transplant settings as it does in the renal setting, although cases of hyperacute rejection have been documented in both.40 42 More commonly, the presence of preformed donor-specific antibodies appears to lead to a dramatically lessened 1-year survival. Several studies have shown that a positive T-cell crossmatch can lead to a dramatically poorer clinical outcome. One study reported a 1-year survival rate of 73% when the crossmatch was negative but when the B-cell crossmatch was positive the survival rate was 62%. More significantly, when the T-cell crossmatch was positive the 1-year survival rate was 28%.43 Another study touched on the potential importance of positive B-cell crossmatches. In this study, about 9% of the patients with a negative T-cell crossmatch and a positive B-cell crossmatch were found to have an increased frequency of severe rejection when compared to crossmatch negative controls.44 The study did not focus on whether the antibodies were directed against HLA class I or HLA class II. In another report, the frequency of positive crossmatches in patients undergoing retransplantation was eightfold higher than the controls.45 It is important to determine the isotype of the antibody in the case of positive crossmatches since positive crossmatches caused by IgM antibodies have not been found to alter the outcome of cardiac transplantations.46 17.3.3.1 Crossmatch Methods 17.3.3.1.1 Complement-Dependent Cytotoxicity Crossmatch The standard crossmatch procedure is a CDC assay that detects the presence of antibodies in the recipient to antigens in donor lymphocyte preparations. There are many variations of this test depending on the target lymphocyte preparation, extension of incubation times, inclusion of wash steps, or utilization of antiglobulin reagents. The basic cytotoxicity crossmatch, called the NIH crossmatch because of its description by the NIH, consists of a 30-minute incubation with the serum followed by the addition of complement and an additional 60minute incubation. This is considered to be the least sensitive of the CDC crossmatch techniques. ASHI and UNOS guidelines require a crossmatch test that is more sensitive than the basic NIH crossmatch. Several modifications of the CDC crossmatch allow for greater sensitivity. Perhaps the simplest modification is the NIH extended technique, which calls for extended incubation time (120 minutes vs 60 minutes) after the addition of complement. The Amos modified crossmatch calls for the introduction of a wash step or steps prior to the addition of complement to wash away anticomplementary factors that may be present in the patient’s serum. Antiglobulin techniques call for several wash steps after the serum incubation followed by the addition of antihuman globulin (AHG; usually antikappa light chain antibody) and complement. Antiglobulin crossmatches are useful for identifying weak antibodies or anti-HLA antibodies of an isotype that does not fix complement. Another important consideration is the lymphocyte preparation that is used for the target of the crossmatch. The lymphocytes used as targets for the crossmatch can be an unseparated lymphocyte preparation containing both T-cells and B-cells or separate T-cell-enriched or B-cell-enriched preparations. Use of a separated T-cell or B-cell crossmatch may be more informative in determining the significance of a possible positive crossmatch. 17.3.3.1.2 Flow-Cytometric Crossmatch As mentioned in the serum screen section, flow-cytometric crossmatching is now considered to be the most sensitive type of crossmatch available. The flow-cytometric crossmatch relies on indirect immunofluorescence to detect the presence of antidonor antibodies in the serum of a potential recipient. With appropriate reagents, the flow-cytometric crossmatch can detect the presence of antibodies directed against donor T-cells or B-cells and the isotype of the reacting antibody.47 17.3.3.2 Specimen Requirements 17.3.3.2.1 Recipient Specimens Serum derived from clotted whole blood is the specimen of choice for all crossmatch methods. The serum should have been collected within the last 30 days prior to transplant with serum collected within 48 hours of transplant being ideal. Individual labs and transplant programs may have their own definition of what is considered to be a “current” sample. It is vitally important that the specimen be collected in a collection tube with no additives (plain red-top Vacutainer tube). Specimen tubes with clot activators and serum separators are unacceptable because the additives can interfere with the crossmatch assay. The serum from a full-draw 10-mL tube of clotted blood is

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sufficient for crossmatches. If a patient has a potential sensitizing event, such as a blood transfusion, it is best to wait, if possible, 2 weeks from such an event to allow for detection of any new anti-HLA specificities. In sensitized patients, previous sera representing peaks of reactivity (as determined by PRA testing) from within the last 6 24 months should also be included in a crossmatch. In regraft patients, it is also recommended that serum from the time of rejection of the previous allograft be included in a crossmatch. 17.3.3.2.2 Donor Specimens Donor lymphocytes for crossmatching can be isolated from whole blood, lymph nodes, or spleen sections. As mentioned previously, whole blood may not be optimal due to the adverse effects of steroids and blood transfusions on peripheral blood lymphocytes. If whole blood is to be used, 50 mL of anticoagulated blood is usually sufficient. The anticoagulant of choice is acid citrate dextrose (ACD; yellow-top Vacutainer tubes). As mentioned earlier, tissue specimens, such as lymph nodes and spleens, should not be placed into any type of fixative. 17.3.3.3 Interpretation A negative crossmatch is one predictor of a favorable transplant outcome. Data from several studies indicate that a positive crossmatch, especially a positive T-cell crossmatch, is an indicator of a poor clinical outcome. However, it must be recognized positive crossmatches in different crossmatch methodologies can indicate different levels of risk that certain transplant programs may feel more comfortable accepting.

17.4 CONCLUSION A great deal of insight has been gathered regarding the role of the immune system in acute and chronic rejection. Central to these advances has been the elucidation of the role of HLA as a target for antibodies and T-cells through both the direct and indirect pathways of allorecognition. Regardless of the direction that any of these areas of research may take, one thing that increases optimism is the near certainty that all of the great advances of the 2000s in defining the mechanisms of rejection will be outstripped by the advances in understanding that will come in the next decades.

References 1. Alberts B, Johnson A, Lewis J, et al. Molecular biology of the cell. Innate immunity. 4th edition New York: Garland Science; 2002. Available from: http://www.ncbi.nlm.nih.gov/books/NBK26846/. 2. Alberts B, Johnson A, Lewis J, et al. Molecular biology of the cell. Chapter 24, the adaptive immune system. 4th edition New York: Garland Science; 2002. Available from: http://www.ncbi.nlm.nih.gov/books/NBK21070/. 3. Medawar PB. The behaviour and fate of skin autografts and skin homografts in rabbits. J Anat 1944;78:176 99. 4. Kissmeyer-Nielson F, Olsen S, Peterson VP, Fjeldborg O. Hyperacute rejection of kidney allografts associated with pre-existing humoral antibody against donor cells. Lancet 1966;2:662 5. 5. Ratkovec RM, Hammond EH, O’Connell JB, et al. Outcome of cardiac transplant recipients with a positive donor-specific crossmatch-preliminary results with plasmapheresis. Transplantation 1992;54:651 5. 6. Zerbe TR, Arena VC, Kormos RL, Griffith BP, Hardesty RL, Duquesnoy RJ. Histocompatibility and other risk factors for histological rejection of human cardiac allografts during the first three months following transplantation. Transplantation 1991;52:485 90. 7. Abbas AK, Lichtman AH, Pober JS. Chapter 16: Immunity to tissue transplants. In: Abbas AK, Lichtman AH, Pober JS, editors. Cellular and Molecular Immunology. Philadelphia, PA: W.B. Saunders Co; 1991. 8. Mason DW, Morris PJ. Effector mechanisms in allograft rejection. Ann Rev Immunol 1986;4:119 45. 9. Engers HD, Glasebrook AL, Sorenson GD. Allogeneic tumor rejection induced by the intravenous injection of Lyt-2 1 cytotoxic T lymphocyte clones. J Exp Med 1982;156:1280 5. 10. Humar A, Kerr S, Gillingham KJ, Matas AJ. Features of acute rejection that increase risk for chronic rejection. Transplantation 1999;68:1200 3. 11. Demetris AJ, Murase N, Lee RG, et al. Chronic rejection. A general overview of histopathology and pathophysiology with emphasis on liver, heart and intestinal allografts. Ann Transplant 1997;2:27 44. 12. Pethig K, Heublein B, Wahlers T, Haverich A. Mechanism of luminal narrowing in cardiac allograft vasculopathy: inadequate vascular remodeling rather than intimal hyperplasia is the major predictor of coronary artery stenosis. Working Group on Cardiac Allograft Vasculopathy. Am Heart J 1998;135:628 33. 13. Griffith BP, Hardesty RL, Armitage JM, et al. A decade of lung transplantation. Ann Surg 1993;218:310 18. 14. Wiesner RH, Batts KP, Krom RA. Evolving concepts in the diagnosis, pathogenesis, and treatment of chronic hepatic allograft rejection. Liver Transpl Surg 1999;5:388 400. 15. Sundaresan S. Bronchiolitis obliterans. Semin Thorac Cardiovasc Surg 1998;10:221 6. 16. Haverich A. Experience with lung transplantation. Ann Thorac Surg 1999;67:305 12.

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17. Billingham ME. Pathology of graft vascular disease after heart and heart-lung transplantation and its relationship to obliterative bronchiolitis. Trans Proc 1995;27:2013 16. 18. Cecka M. Clinical outcome of renal transplantation: factors influencing patient and graft survival. Surg Clin North Amer 1998;78:133 48. 19. Wiesner RH, Batts KP, Krom RA. Evolving concepts in the diagnosis, pathogenesis and treatment of chronic hepatic allograft rejection. Liver Transpl Surg 1999;5:388 400. 20. http://optn.transplant.hrsa.gov/learn/about-transplantation/how-organ-allocation-works/. 21. Hosenpud JD, Edwards EB, Lin HM, Daily OP. Influence of HLA matching on thoracic transplant outcomes. Analysis from the UNOS/ ISHLT Thoracic Registry. Circulation 1996;94:170 4. 22. Leivestad T, Foerster A, Simonsen S, Bratlie A, Thasby E, Geiran O. HLA-DR matching reduces rejection rate in heart transplantation. Transpl Int 1996;9(Suppl. 1):S230 3. 23. Sheldon S, Yonan NA, Aziz TN, et al. The influence of histocompatibility on graft rejection and graft survival within a single center population of heart transplant recipients. Transplantation 1999;68:515 19. 24. Dyer PA, Claas FH. A future for HLA matching in clinical transplantation. Eur J Immunogenet 1997;24:17 28. 25. Smith J, Ellis H, Hunt D, Laylor R, Danskine A, Rose M, et al. Prospective HLA matching is feasible in thoracic organ transplantation. Transpl Proc 1997;29:1467 8. 26. Ketheesan N, Tay GK, Witt CS, Christiansen FT, Taylor RR, Dawkins RL. The significance of HLA matching in cardiac transplantation. J Heart Lung Transplant 1999;18:556 1230. 27. Amos D, Bashir H, Boyle W, MacQueen M, Tiilikainen A. A simple micro cytotoxicity test. Transplantation 1969;7:220 3. 28. Hopkins KA. The Basic Lymphocyte Microcytotoxicity Tests: Standard and AHG Enhancement. ASHI Laboratory Manual 4th Edition., 2000. 29. Hurley CK. DNA Methods for HLA Typing: A Workbook for Beginners. Available at www.ashi-hla.org/images/uploads/ DNAMethodsforHLATyping-Workbook.pdf, 2008. 30. Bunce M, Passey B. HLA typing by sequence-specific primersdoi:10.1007/978-1-62703-493-7_1 In: Zachary AA, Leffell MS, editors. Transplantation immunology: methods and protocols, second edition, methods in molecular biology, vol. 1034. Springer Science 1 Business Media, LLC; 2013. 31. Antovich ZR, Russnak NL, Quinto K, Frome M, Grumet FC. LinkSeq and SureTyper a new agile system for HLA typing. Tissue antigens 2011;77:473 4. 32. Trajanoski D, Fidler S. Immunogenetics: methods and applications in clinical practicedoi:10.1007/978-1-61779-842-9_1 In: Christiansen FT, Tait BD, editors. Methods in molecular biology, vol. 882. New York: Springer Science 1 Business Media; 2012. 33. Lazaro A, Tu B, Yang R, et al. Human leukocyte antigen (HLA) typing by DNA sequencing. Available from: http://dx.doi.org/10.1007/ 978-1-62703-493-7_1doi: In: Zachary AA, Leffell MS, editors. Transplantation immunology: methods and protocols, second edition, methods in molecular biology, vol. 1034. Springer Science 1 Business Media, LLC; 2013. 34. Trachtenberg EA, Holcomb CL. Next-generation HLA sequencing using the 454 GS FLX system. In: Zachary AA, Leffell MS, editors. Transplantation immunology: methods and protocols, second edition, methods in molecular biology, vol. 1034. Springer Science 1 Business Media, LLC; 2013. . p. 2013. Available from: http://dx.doi.org/10.1007/978-1-62703-493-7_1. 35. Department of Health and Human Services, Health Care Financing Administration. Medicare, Medicaid and CLIA Programs: Revision of Laboratory Regulations. Federal Register 1990; 55:9538 9610. 36. American Society for Histocompatibility and Immunogenetics. ASHI Standards for Histocompatibility Testing Revised October 2014. Accessed at www.ashi-hla.org/images/uploads/2014ASHIStandardsandGuidance.pdf. 37. Kobashigawa JA, Sabad A, Drinkwater D, et al. Pretransplant panel reactive antibody screens. Are they truly a marker for poor outcome after cardiac transplantation? Circulation 1996;94:294 7. 38. Ting A. The lymphocyte crossmatch test in clinical renal transplantation. Transplantation 1983;35:403 6. 39. Braun WE. Laboratory and clinical management of the highly sensitized organ transplant recipient. Hum Immunol 1989;26:245 60. 40. Loh E, Bergin JD, Couper GS, Mudge GH. Role of panel-reactive antibody cross-reactivity in predicting survival after orthotopic heart transplantation. J Heart Lung Transplant 1994;13:194 201. 41. Ratkovec RM, Hammond EH, O’Connell JB, et al. Outcome of cardiac transplant recipients with a positive donor-specific crossmatch-preliminary results with plasmapheresis. Transplantation 1992;54:651 5. 42. Zerbe TR, Arena VC, Kormos RL, Griffith BP, Hardesty RL, Duquesnoy RJ. Histocompatibility and other risk factors for histological rejection of human cardiac allografts during the first three months following transplantation. Transplantation 1991;52:485 90. 43. Smith JD, Danskine AJ, Laylor RM, Rose ML, Yacoub MH. The effect of panel reactive antibodies and the donor specific crossmatch on graft survival after heart and heart-lung transplantation. Transpl Immunol 1993;1:60 5. 44. Bunke M, Ganzel B, Klein JB, Oldfather J. The effect of a positive B-cell crossmatch on early rejection in cardiac transplant recipients. Transplantation 1993;56:1595 7. 45. Ensley RD, Hunt S, Taylor DO, et al. Predictors of survival after repeat heart transplantation. The Registry of the International Society for Heart and Lung Transplantation and Contributing Investigators. J Heart Lung Transpl 1992;11:S142 58. 46. Kerman RH, Kimball P, Scheinen S, et al. The relationship among donor-recipient HLA mismatches, rejection and death from coronary artery disease in cardiac transplant recipients. Transplantation 1994;57:884 8. 47. Bray RA. Lymphocyte crossmatching by flow cytometry. In: Zachary AA, Leffell MS, editors. Transplantation immunology: methods and protocols, second edition, methods in molecular biology, vol. 1034. Springer Science 1 Business Media, LLC; 2013. Available from: http://dx.doi. org/10.1007/978-1-62703-493-7_1In: Zachary AA, Leffell MS, editors. Transplantation immunology: methods and protocols, second edition, methods in molecular biology, vol. 1034. Springer Science 1 Business Media, LLC; 2013.

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18 The Immune Response to the Allograft Fiona Carty and Karen English National University of Ireland, Maynooth, Ireland

18.1 INTRODUCTION Despite the progress made in solid organ transplantation since the 1950s, alloimmunity remains the greatest barrier to successful graft survival. Tolerance of the transplanted organ by the recipient immune system is the goal in clinical transplantation; however, manipulation of the recipient immune response is crucial for successful clinical outcomes.1 3 Although 1-year survival rates for renal allografts have increased since the 1990s due to improved immunosuppression regimens, long-term outcomes have not significantly improved, suggesting that ameliorating acute rejection does not necessarily guarantee successful graft survival. In fact, these findings suggest that chronic rejection is still a major obstacle to successful renal transplantation.4 6 Herein we review current knowledge on the immune response to the allograft, and discuss the homeostatic proliferation following lymphopenia that contributes to allograft rejection.

18.2 ISCHEMIA REPERFUSION Ischemia reperfusion injury (IRI) is an unavoidable side effect of solid organ transplantation; however, the degree to which IRI occurs is a key factor affecting the clinical outcome of the transplant. Approximately onethird of transplants are significantly affected by IRI, and this increases to one half when donor organs are isolated after circulatory arrest of the donor.7,8 Twenty percent of kidney transplant patients suffer from acute kidney injury as a direct result of IRI, leading to delayed graft function (DGF), requiring dialysis within a week of transplantation. Others suffer from slow graft function (SGF) and both of these complications increase the likelihood of graft loss and acute rejection, while DGF increases the likelihood of chronic allograft dysfunction.9 IRI occurs when blood supply to tissue is interrupted, and the organ is subsequently exposed to hypoxic conditions. This causes mitochondrial damage, ATP depletion, necrosis, and vascular damage within the donor tissue. Reperfusion then triggers the release of free oxygen radicals causing further damage to the donor tissue, and also carries lymphocytes to the graft.10,11 This ischemic injury initiates the production of danger associated molecular patterns (DAMPs), activating both the complement system and innate immune cells through pattern recognition receptors (PRRs). DAMPs in this context are typically associated with cell damage and death. They include necrotic cells, cellular debris, heat shock proteins, tissue factor (TF), and high-mobility group protein box 1 (HMGB1). The importance of DAMPs in graft survival is apparent in islet transplantation, where it has been shown that the clinical outcome of graft survival directly correlates to TF expression.12,13 DAMPs activate PRRs on innate immune cells such as toll like receptors (TLRs), nucleotide-binding oligomerization domain (NOD) and NOD-like receptors, C type lectin receptors, receptors for advanced glycation end products (RAGE), and retinoic acid inducible gene 1 receptors. Signaling through these receptors activates the inflammasome and complement system, amplifying the inflammatory response. Proinflammatory cytokines, precoagulants, and chemoattractants are produced, subsequently causing recruitment of innate immune cells, and eventually recruitment of adaptive immune cells.1,14 Kidney Transplantation, Bioengineering, and Regeneration. DOI: http://dx.doi.org/10.1016/B978-0-12-801734-0.00018-7

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The complement system is activated upon tissue damage, and has a significant role in IRI. C3a and C5a are important initiators and regulators of the inflammatory response, and have been shown to induce production of proinflammatory cytokines by tubular epithelial cells (TECs) and macrophages, and activate endothelial cells in renal IR. In addition, complement mediates epithelial cell damage, leukocyte infiltration, and chemotaxis of interleukin (IL)-17 producing innate immune cells.15 The membrane attack complex (MAC) formed by C5b-C9 has also been shown to contribute to renal IRI. MAC forms pores in cell membranes, causing cell activation, inducing the expression of proinflammatory cytokines, adhesion molecules, and TF, further contributing to acute inflammation and renal IRI.7,16 Inflammasome activation, complement, and chemotactic factors guide neutrophils and γδ T cells to the ischemic tissue. Neutrophils and γδ T cells are among the first populations of cells to infiltrate the graft following IRI. These cells release significant amounts of IL-17, resulting in natural-killer T (NKT) cell activation, leukocyte recruitment, inflammatory cytokine production, chemokine production, enhanced tissue damage and allograft rejection.17 19 Activated NK cells subsequently migrate to the areas of ischemic damage and further contribute to tissue damage in the ischemic kidney. This is thought to be associated with NK cell mediated death of TECs.20 Elevated levels of stromal derived factor-1 (SDF-1) in the ischemic kidney induce the infiltration of macrophages that accumulate in early stages of IRI.21 TLR4 stimulation via hypoxia, glucose deprivation, and HMGB-1 activates macrophages, stimulating the production of IL-6, IL-23, IL-17, and TNF-α.22 24 Despite the significant contribution of macrophages to tissue damage and inflammatory responses in early IRI, macrophages can also play a reparative role in the late stages. Antiinflammatory macrophages mediate proreparative effects in IRI through growth factors and antiinflammatory cytokines.25,26 Infiltration of activated, effector memory CD41 and CD81 T lymphocytes via chemotaxis is observed following moderate and severe IRI. These activated T cells contribute to tissue damage, mediated by inflammatory cytokines and Fas/Fas ligand (FasL) interactions.8,14,27,28 Inhibition of NF-κB in T cells led to decreased CD41 T cell infiltration and leukocyte accumulation in a murine IRI kidney model. NF-κB inhibition in T cells also resulted in decreased levels of IL-1 and TNF-α, and improved survival.29,30 While systemic NF-κB inhibition has also been shown to attenuate IRI, it was not as successful as T cell specific NF-κB inhibition. This is consistent with studies demonstrating that NF-κB can also play a protective role in IRI, and suggests that the impaired T cell activation due to NF-κB inhibition is responsible for attenuation of IRI.30,31 It is widely accepted that regulatory T cells have protective effects in IRI.32 36 Many of these studies have shown that depleting Tregs exacerbates IRI, while adoptive transfer or recruitment of Tregs to damaged tissue ameliorates IRI. The protective effects of Tregs include impairing migration of innate immune cells to IRI areas and suppression of inflammatory cytokine production. These effects are mediated by Treg-derived IL-10 and PDL molecules.32,37

18.3 T CELLS AND MECHANISMS OF ALLORECOGNITION Heterodimeric T-cell receptors (TCR) expressed on the surface of T cells, consist of either the αβ or γδ chains. TCR diversity results from variations and diversity in these chains, which allow T cells to recognize a wide range of peptides expressed on major histocompatibility complex (MHC) by APCs. T cells go through a rigorous “education” process of positive and negative selection in the thymus, determined by the strength of interaction between TCR and self-peptide MHC complexes. The T cells that are unable to interact with self MHC complexes sufficiently are eliminated by apoptosis. T cells that interact with MHC-I complexes become CD81 T cells, while T cells that interact with MHC class II complexes become CD41 T cells. T cells displaying high levels of recognition with self-peptides on MHC complexes are eradicated to ensure that potentially self-reactive T cells do not survive. Following antigen recognition, naı¨ve CD41 and CD81 T cells are activated and undergo clonal expansion. During activation, costimulatory molecules and cytokines expressed by antigen presenting cells (APCs) dictate the phenotypic and functional fate of the T cell.38 Upon reperfusion of the transplanted organ, recipient leukocytes infiltrate the graft, while donor DCs migrate to the lymph nodes and spleen via CCL19 and CCL21. While the early innate immune response to the allograft associated with IRI is largely unspecific, and occurs even in syngeneic transplants, allorecognition by the adaptive immune response is considerably more specific.2,39 The first step in the adaptive immune response to the allograft is mediated by T cells. MHC complexes expressed by allogeneic tissue are the predominant target of the recipient immune system, though minor antigens

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FIG. 18.1 Pathways of allorecognition. The direct pathway (A) occurs when recipient CD41 T cells recognize foreign MHC II molecules presented by donor APCs. This is thought to be via the “high determinant density” model or the “multiple binary complex” model. In the indirect pathway (B) recipient APCs take up donor MHC molecules and present them as foreign peptides to recipient CD41 or CD81 T cells. In the semidirect pathway (C) intact donor MHC molecules are transferred to recipient APCs. Intact MHC I molecules can be presented to CD81 T cells, while at the same time, processed MHC molecules can be presented by recipient MHC II molecules to CD41 T cells.

such as ABO glycolipids expressed by red blood cells and endothelial cells also play a role. Grafts from HLA-identical siblings have significantly better clinical outcomes than those from HLA-nonidentical donors. Recipient T cells recognize these MHC molecules as foreign through the direct, indirect, and semidirect pathways (Fig. 18.1).40,41 The direct pathway (Fig. 18.1A) occurs when T cells recognize the MHC II molecules on donor cells within the transplanted organ, particularly APCs but also on nonimmune cells such as endothelial cells.41,42 While the direct pathway is a predominant factor in initiating the adaptive immune response, due to the limited number of donor lymphocytes within the graft, activity along this pathway subsides eventually, with only structural donor cells such as endothelial and tubular epithelial cells remaining in the graft to activate the direct pathway. Studies from as early as the 1950s have shown that alloantigen presented by donor DCs are recognized by recipient CD41 memory T cells.42 Two models for the allorecognition of foreign cells have been proposed, the “high determinant density” model and the “multiple binary complex” model. The “high determinant density model,” also coined the “antigen density model,” suggests that alloreactive T cells can recognize amino acid polymorphisms exposed on foreign MHC molecules regardless of the peptide being presented. In this model, each foreign APC is expressing an abundance of polymorphisms in each MHC molecule, therefore activating a vast number of T cells. The “multiple binary complex” model on the other hand is peptide dependent. This hypothesis suggests that T cells recognize selfpeptide presented by a foreign MHC molecule due to the difference in orientation of the self-peptide in the foreign MHC molecule.41,43 45 It remains to be elucidated which model is most likely to cause the alloresponse; however, it is widely believed that both models may contribute. The recognition of nonself MHC by recipient T cells, despite MHC restriction, is understood to be mediated by cross reactivity. This phenomenon is associated with T cell ability to recognize peptides on foreign MHC following previous priming to recognize self MHC. The mechanisms by which T cells can recognize these noncognate peptides have been explained by Yin & Mariuzza46 and are facilitated by structural changes of TCR binding site, differential TCR docking to the MHC peptide complex,47 structural degeneracy, molecular mimicry, and antigen-dependent tuning of peptide MHC flexibility. Importantly the MHC mismatch stimulates diverse T cell responses and drives the alloreactive T cell response.2,38,41 Indirect pathway recognition (Fig. 18.1B) occurs when T cells recognize processed MHC molecules on the surface of recipient APCs in a self-restrictive fashion. The indirect pathway can be activated as long as the graft persists, and thus is the predominant source of antigen presentation causing allorecognition in the long term. Recipient DCs trafficking through the graft take up soluble MHC alloantigens and apoptotic/necrotic donor cells.

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They process and present these antigens on MHC II molecules to CD41 T cells in lymphoid tissues. CD81 T cells are activated in a phenomena known as cross-priming, where APCs present alloantigens on their MHC I complexes. B cells also play a role in the indirect pathway, through capture of alloantigen via the B cell receptor (BCR).48,49 Following B cell presentation of alloantigen to T cells, B cells produce high affinity alloantibody.50 While deletion of the direct pathway delays allograft rejection, the indirect pathway eventually compensates, suggesting an essential role in long-term rejection of the graft.41 The third and final pathway for allorecognition is the “semidirect” pathway (Fig. 18.1C), which exploits the ability of immune cells to exchange surface molecules. The model hypothesizes that MHC peptide complexes are acquired by recipient APCs from other cells, via cell cell contact or release and uptake of the MHC between exosomes.51 53 The now-chimeric recipient APCs present intact donor MHC I complexes to CD81 T cells, while presenting processed donor MHC complexes as peptides to CD41 T cells.41,43 While there is no compelling in vivo evidence for this model as of yet, it would explain observations in other studies, i.e., that graft rejection still occurred despite the absence of both the direct and indirect pathways.54 Interactions between the MHC peptide complex on APCs and the TCR on T cells induces a signal through the CD3 complex. T cells then require costimulation, through accessory molecules such as CD28 and OX40. The outcome of APC: T cell interaction depends on the activity of costimulatory molecules. CD28 activation increases expression of IL-2 by T cells, promoting T cell proliferation. In contrast to this, activated T cells express CTLA-4, which binds the same ligands as CD28, but with greater affinity. Therefore, CTLA-4 acts as a negative regulator through competing with CD28 for costimulatory ligands. The third signal that contributes to naive T cell activation is the production of cytokines such as IL-12, triggering T cells to undergo clonal expansion and differentiation.55 The T cell populations mediating the immune response are determined by the cytokine environment in which the antigen presentation occurs. It is widely believed that acute rejection is primarily mediated by Th1 cells, which are induced in the context of IFN-γ and IL-12 costimulation, usually following the direct pathway of APC presentation. Th1 cells release IFN-γ, TNF-α, and IL-2 amplifying the immune response and activating cell death signals by promoting the proliferation of cytotoxic CD81 T cells and interacting with donor cells via Fas/FasL interactions.40,56 Alloreactive Th1 cells can also cause a delayed type hypersensitivity (DTH) by macrophages through production of nonspecific mediators such as nitric oxide and reactive oxygen species and activation of B cell production of alloreactive antibodies.2,40 This Th1 response is thought to be ameliorated by Th2 cells, which release IL-4, IL-10, and IL-13 among other cytokines, suggesting that Th2 cells can delay or inhibit allograft rejection.57 59 However, there is much debate on the role of Th2 cells in allograft rejection. While Th2 cytokines have been linked to tolerance,60 Barbara et al. demonstrated that Th2 cells can cause allograft rejection to the same extent as Th1 cells.61 Studies have convincingly linked Th2 cytokine expression with chronic rejection in renal and cardiac allografts.57,62 The presence of IL-23 in the inflammatory environment induces Th17 differentiation, while the addition of IL-1β results in the production of Th1/17 cells.63,64 Th17 cells are present during acute rejection; however, the role of Th17 in chronic rejection remains unclear.56 Chronic rejection (associated with the indirect pathway) has been linked to donor-specific Th1/17 cell populations producing IL-17 and IFN-γ.65 In the context of allograft rejection, it is thought that the outcome of the immune reaction may depend on the ratio of alloreactive T cells to Tregs. Nguyen et al. have shown that the suppressive capacity of Tregs in transplant recipients prior to transplantation correlates with the success of immediate graft function.66 Therefore, Treg function could potentially be used as an indication of DGF and SGF. Higher levels of CD45RO expression and reduced levels of CD27 expression on regulatory T cells are associated with better graft outcome, suggesting that the effector memory Treg subpopulation in particular may contribute to the immunosuppressive microenvironment.67 Tregs mediate their protective effects in a number of ways: They express CTLA4, acting as a negative regulator for T cell activation. Furthermore, signaling through CTLA4 induces production of indoleamine 2, 3-dioxygenase (IDO), which inhibits T cell proliferation. Treg-derived IL-10 also dampens inflammation by inhibiting APC activity, and by inducing the conversion of T cells to T regulatory type 1 (TR1) cells.3

18.4 B CELLS B cell derived alloantibodies may not be considered the predominant mediator of allograft rejection, but they certainly contribute to the rejection process, particularly in the context of chronic rejection. Alloantibodies bind to the graft, disrupting the function of graft endothelial cells and enhancing fixation of complement cellular uptake by APCs.68,69

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Alloantibodies specific for MHC molecules, ABO blood group antigens, or antigens expressed by the graft endothelium can be present in the serum of those who have previously rejected transplants, received blood transfusions, or have been pregnant. Studies investigating the roles of these antibodies have generated ambiguous results. While alloantibodies can cause hyperacute rejection immediately after graft reperfusion,70 72 transferred alloantibodies have been shown to support graft survival in vivo.73 In hyperacute rejection these preformed antibodies bind to donor tissue, activating the complement cascade. Antibody mediated hyperacute rejection generally occurs in patients with existing antibodies against human leukocyte antigens (HLA) antigens. However, this type of humoral hyperacute response is rare due to extensive antibody screening prior to transplantation.69,74,75 Patients who have not been sensitized can develop de novo antibodies against HLA following transplantation. These antibodies can be produced from 6 months posttransplantation and have been shown to contribute to chronic rejection and allograft failure in renal transplants.76 Donor-specific antibodies (DSAs) are produced by two types of memory B cells. The quiescent memory B cells and the long-lived plasma cell (LLPC). The quiescent memory B cell is reactivated quickly upon alloantigen reexposure, i.e., secondary transplantation of sensitized individuals. This accounts for generation of new DSA from their plasma cell progeny. LLPC secrete antibodies constitutively. They maintain levels of circulating DSA but are not reactivated upon alloantigen reexposure.68 The DSAs produced by B cells mediate C4d fixation and may also cause antibody-dependent cellular cytotoxicity killing by macrophages and neutrophils.77,78 While the homeostatic proliferation of B cells following immunosuppression is not as well characterized as that of T cells, it has been reported that T cell depleting antibodies can promote B cell activation, and that withdrawal from calcineurin inhibitors may result in an accelerated activation and differentiation of these cells.79,80

18.5 THE IMMUNE RESPONSE FOLLOWING IMMUNOSUPPRESSION In the setting of transplantation, an individual is deliberately immunosuppressed in order to avoid allograft rejection by the immune system. The treatment methods adopted to achieve this immunosuppression are many and varied and are discussed in detail in following chapters. However, the absolute common goal of each of these methods is tolerance. Tolerance in transplantation refers to antigen-specific unresponsiveness that is sustained in the absence of immunosuppression. Tolerance is mediated through a number of mechanisms, such as deletion, anergy, and clonal exhaustion of the alloreactive T cells and immunoregulation by other cell populations such as Tregs or other cellular therapies.81 Traditional immune-suppressants address the issue of transplant rejection by depleting the effector immune cells, inhibiting effector cell proliferation and function, and by diverting lymphocyte biodistribution. These effects can be mediated by eliminating the host’s immune response prior to transplantation, initially following alloantigen exposure, or by providing continuous tolerance to the graft. Induction therapy (IT) is administrated prior to or at the same time as transplantation in order to deplete the immune compartment to reduce the likelihood of acute rejection. The most commonly adopted agents for IT are the depleting antibodies rabbit antithymocyte globulin (ATG) and alemtuzumab (Campath). These depleting antibodies mediate their action by inducing complement-dependent cytotoxicity, antibody-dependent cellular cytotoxicity, and apoptosis.82 Campath is an anti-CD52 monoclonal antibody (mAb), which depletes CD41 and CD81 T cells, NK cells, B cells, and monocytes, while ATG is an anti-CD3 mAb specifically targeting T cells.83,84 Campath differentially depletes the different T cell subsets, with CD81 cells being the least sensitive and CD41 CD251 cells being the most sensitive to depletion.83 In contrast to Campath, ATG binds more efficiently to CD81 T cells than CD41 T cells resulting in more rapid depletion of the CD81 T cell population.85 Despite this difference, the CD41 populations that remain following both Campath and ATG depletion are rich in regulatory and memory T cells with naı¨ve T cells being preferentially depleted.85 89 The expansion of regulatory and memory T cells following rATG treatment is extensively reported.82,85,89 94 Many studies have shown that the Treg compartment is expanded following ATG-mediated lymphodepletion, and that this is dependent on T cell/monocyte interactions.90,91,95,96 Furthermore, the T cells that remain following ATG treatment have been shown to inhibit graft versus host disease in vivo.84 In 2015, Crepin et al.95 showed that ATG causes immune senescence in renal transplant recipients, resulting in prolonged CD41 T cell lymphopenia and an increase in late stage differentiated CD81 T cells, which are linked to acute rejection.97 In lymphopenic hosts, it is thought that the outcome of the immune reaction may depend on the balance of effector memory and regulatory T cells. In the lymphopenic environment following induction therapy, the effector memory T cells that remain undergo rapid homeostatic proliferation, driven by the abundant availability of

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self MHC/peptide complexes and gamma chain cytokines such as IL-7 and IL-15.97,98 Furthermore, it has been shown that the population of memory CD41 T cells skew towards a Th1 phenotype following lymphopenia, which may contribute to the Th1 acute rejection response.99 However, existing literature on the effects of homeostatic proliferation on Tregs is contradictory. Studies have shown that the homeostatic expansion of regulatory cells is slower than that of nonregulatory cells, and that higher concentrations of IL-7 are required for IL-7R signaling in Tregs than in conventional T cells.98,100 Furthermore, in the presence of IL-7 and IL-15, Tregs lose their ability to suppress proliferation of conventional T cells.99 101 In order to avoid the acute rejection that often occurs once homeostatic proliferation replenishes the immune compartment, “maintenance” immunosuppression regimens are employed. The most common of these regimens are calcineurin inhibitors, which are the mainstay of immunosuppressive therapy in renal transplantation. Calcineurin inhibitors such as cyclosporine and tacrolimus suppress T cell activation by blocking IL-2 signaling. While calcineurin inhibitors are extremely effective at suppressing inflammatory T cell activity, they similarly suppress Treg activity,102 104 which may negatively affect the homeostatic proliferation that ensues. In contrast, mammalian target of rapamycin (mTOR) inhibitors such as sirolimus and everolimus block cellular proliferation at a later stage in the proliferation pathway.105 In a comparative study of sirolimus- and cyclosporine-treated renal transplant patients following Campath induction therapy, peripheral Tregs were expanded in only the sirolimus-treated group.106 While these therapies have proven to be extremely valuable in the setting of transplantation, and significantly prolong allograft survival, there are drawbacks to their use. The effects of these drugs are systemic, causing blanket suppression of the entire immune response. This severe immune-suppression leaves the patient vulnerable to malignancies and infection. Long-term use of these calcineurin inhibitors can also be highly toxic, diverting the problem of organ rejection, to loss of function.4,107

18.6 NEW THERAPIES FOR THE INDUCTION OF TOLERANCE While traditional immune-suppressants have revolutionized transplantation and significantly improved the short-term success of the allograft, progress made in terms of long-term graft survival have not been as compelling. In order to combat the toxicity issues associated with calcineurin and mTOR inhibitors, further efforts to develop strategies to induce tolerance are ongoing. Since 2007, cellular therapies have shown great promise as facilitators of successful transplantation with extensive research being carried out to elucidate the mechanisms of immunosuppression mediated by mesenchymal stromal cells (MSCs),108 110 Tregs,81,111 and tolerogenic dendritic cells.112 MSCs have been extensively studied for a range of inflammatory disorders and have been used in a number of preclinical and clinical trials for solid organ transplantation.113 117 Adult stromal cells such as MSCs and multipotent adult progenitor cells (MAPCs) are heterogeneous populations of multipotent cells present in many tissues but most commonly derived from the bone marrow.118,119 MSCs originally garnered a lot of attention for their regenerative properties; however, it is their immunemodulatory properties that have added to their appeal in more recent years. Both MSCs and MAPCs are immune-evasive and can alter the function of DCs, T cells, and other innate immune cells, allowing them to create a more tolerogenic immune environment, without themselves being rejected.120 122 In the inflammatory environment MSCs are activated by DAMPs, inflammatory cytokines, and contact signals from immune cells to secrete antiinflammatory trophic factors such as IL-6, IDO, TSG-6, and PGE-2.123 130 MSC-derived soluble factors promote the conversion of proinflammatory M1 macrophages into an M2, IL-10 secreting regulatory phenotype,128,131 and suppress the proliferation, degranulation, and production of IFN-γ by NK cells.132 In an animal model of renal transplantation MSCs were shown to suppress DC maturation and antigen presentation.133 In vitro evidence suggests that MSCs mediate this suppression as well as directing DCs toward a more regulatory phenotype through IL-6 secretion and Notch signaling.109,125,134 136 In contrast to blanket immune-suppressants such as cyclosporine, MSCs do not completely abrogate the proliferation of all T cells, but rather modulate the population to a more regulatory phenotype.137 MSCs also alter the cytokine secretion profile of naı¨ve T cells during differentiation into Th1, Th2, and Th17 subsets, inhibiting Th1 differentiation in Th1-driven pathologies.138 141 Furthermore, MSC-DCs secrete less IL-12 than normal DCs, inhibiting Th1 differentiation.135,141,142 In animal models of transplantation MSCs inhibit lymphocyte infiltration and fibrosis in the graft, and subsequently promote long-term graft survival.110,133,143,144 In a cardiac allograft model MSCs prolonged graft survival when used in combination with mycophenolate mofetil. The positive effects of MSCs in this setting were

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attributed to the promotion of regulatory DCs and increased levels of IDO, though the source of IDO is unclear.110 Clinical studies have suggested that the success of MSC therapy is largely dependent on the timing of administration.144 145 In a humanized model of GvHD, MSCs administered on day 7 prolonged survival; however, MSCs given at the same time as PBMC were only useful when prestimulated with IFN-γ.146 IFN-γ stimulation of MSCs has been shown in countless studies to enhance their immunosuppressive properties, and it is widely accepted that MSCs require “licensing” with inflammatory signals to mediate their antiinflammatory effects.108,132,147,148 Therefore, timing of administration is vital for the efficacy of MSCs/MAPCs. Along with timing of doses, alternative routes of administration and ex vivo manipulation of MSCs prior to transplantation are being explored and must be optimized in order to enhance their efficacy in the transplantation setting.144,149 While MSCs and other cellular therapies are showing promise as tools to induce tolerance of the allograft, much remains to be elucidated regarding their safety and exact mechanisms of immunosuppression before they are introduced into the clinic.150,151

18.7 DISCUSSION The number of patients requiring kidney transplants due to end stage renal disease is ever increasing worldwide due to the aging population and the diabetes epidemic. The success of the transplant largely depends on the initial response of the immune system to the allograft.152 Initially, IRI drives the response by causing infiltration of immune cells to the graft.30,153,154 Here, recipient T cells recognize MHC molecules on donor-derived cells as foreign, while donor DCs migrate to the spleen, amplifying the allogeneic response.8,39,44 The synergistic effects of IRI and allorecognition by T cells can have disastrous effects, causing DGF, which often leads to poor allograft survival.155 While significant progress has been made in the field of transplantation immunology, there is still a lot to be desired with regards to translating this knowledge from bench to bedside. While traditional immunosuppressants have exponentially improved acute rejection rates in renal allotransplantation, the same cannot be said for longterm graft survival.4,107 Both IRI and the immediate response of immune cells to the graft have been studied for decades, with T cells being highlighted as a key mediator of acute rejection. This knowledge has resulted in improvements of graft procurement methods, surgical procedures, and immunosuppressive therapies to combat the immediate response to the allograft, and thus has resulted in significant increases in short-term survival rates.155,156 Unfortunately, although currently used immunosuppressive drugs are extremely effective at counteracting allograft rejection, patients cannot take these drugs long term due to their dangerous side effects.107 Withdrawal from these immunosuppressants and induction therapies can lead to the quick replenishment of an inflammatory T cell population, which subsequently causes allograft destruction.99 It is imperative that safe therapies are introduced to the clinic, along with the induction of immune tolerance to the allograft. Cellular therapies have garnered significant interest as safe therapies for their antiinflammatory properties, and their potential to induce tolerance in allograft recipients. Therefore, we must enhance our understanding of the immune response following induction therapy and develop a safe and effective therapy, or combination of therapies, to induce a tolerant immune state.

Acknowledgments Fiona Carty is funded by a PhD scholarship from the Irish Research Council Enterprise Partnership scheme in collaboration with Regenesys BVBA. Karen English is the recipient of a Science Foundation Ireland Starting Investigator Research Award under grant number 13/SIRG/ 2172, and a Marie Curie Career Integration Grant.

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Giaretta F, Bussolino S, Beltramo S, et al. Different regulatory and cytotoxic CD4 1 T lymphocyte profiles in renal transplants with antibody-mediated chronic rejection or long-term good graft function. Transpl Immunol 2013;28:48 56. 68. Chong AS, Sciammas R. Memory B cells in transplantation. Transplantation 2015;99:21 8. 69. Wood KJ. Is B Cell tolerance essential for transplantation tolerance? Transplantation 2005;79:S40 2. 70. Ahern AT, Artruc SB, DellaPelle P, et al. Hyperacute rejection of HLA-AB-identical renal allografts associated with B lymphocyte and endothelial reactive antibodies. Transplantation 1982;33(1):103 6. 71. Racusen LC, Haas M. Antibody-mediated rejection in renal allografts: lessons from pathology. Clin J Am Soc Nephrol 2006;1:415 20. 72. Trpkov K, Campbell P, Pazderka F, Cockfield S, Solez K, Halloran PF. Pathologic features of acute renal allograft rejection associated with donor-specific antibody: analysis using the banff grading schema1. Transplantation 1996;61:1586 92. 73. French M, Batchelor J. Immunological enhancement of rat kidney grafts. Lancet 1969;2:1103. 74. Ferguson AR, Youd ME, Corley RB. Marginal zone B cells transport and deposit IgM-containing immune complexes onto follicular dendritic cells. Int Immunol 2004;16:1411 22. 75. Ingelfinger JR. Agonistic autoantibodies and rejection of renal allografts. N Engl J Med 2005;352:617 19. 76. Lionaki S, Panagiotellis K, Iniotaki A, Boletis JN. Incidence and clinical significance of de novo donor specific antibodies after kidney transplantation. Clin Dev Immunol 2013;849835. 77. Crespo M, Pascual M, Tolkoff-Rubin N, et al. Acute humoral rejection in renal allograft recipients: I. Incidence, serology and clinical characteristics. Transplantation 2001;71:652 8. 78. Mauiyyedi S, Pelle PD, Saidman S, et al. Chronic humoral rejection: identification of antibody-mediated chronic renal allograft rejection by C4d deposits in peritubular capillaries. J Am Soc Nephrol 2001;12:574 82. 79. Kwun J, Bulut P, Kim E, et al. The role of B cells in solid organ transplantation. Semin Immunol 2012;24:96 108. 80. Kwun J, Oh BC, Gibby AC, et al. Patterns of de novo allo B cells and antibody formation in chronic cardiac allograft rejection after alemtuzumab treatment. Am J Transplant 2012;12:2641 51. 81. Wood KJ, Sakaguchi S. Antigen-induced regulatory T cells in autoimmunity. Nat Rev Immunol 2003;3:223 32. 82. Valdez-Ortiz R, Bestard O, Llaudo´ I, et al. Induction of suppressive allogeneic regulatory T cells via rabbit antithymocyte polyclonal globulin during homeostatic proliferation in rat kidney transplantation. Transpl Int 2015;28:108 19. 83. Lowenstein H, Shah A, Chant A, Khan A. Different mechanisms of Campath-1H-mediated depletion for CD4 and CD8 T cells in peripheral blood. Transpl Int 2006;19:927 36.

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84. Ruzek MC, Waire JS, Hopkins D, et al. Characterization of in vitro antimurine thymocyte globulin induced regulatory T cells that inhibit graft-versus-host disease in vivo. Blood 2008;111:1726 34. 85. Ruzek MC, Neff KS, Luong M, et al. In vivo characterization of rabbit anti-mouse thymocyte globulin: a surrogate for rabbit anti-human thymocyte globulin. Transplantation 2009;88:170 9. 86. Gallon L, Gagliardini E, Benigni A, et al. Immunophenotypic analysis of cellular infiltrate of renal allograft biopsies in patients with acute rejection after induction with alemtuzumab (Campath-1H). Clin J Am Soc Nephrol 2006;1:539 45. 87. Havari E, Turner MJ, Campos-Rivera J, et al. Impact of alemtuzumab treatment on the survival and function of human regulatory T cells in vitro. Immunology 2014;141:123 31. 88. Noris M, Casiraghi F, Todeschini M, et al. Regulatory T cells and T cell depletion: role of immunosuppressive drugs. J Am Soc Nephrol 2007;18:1007 18. 89. Xia C-Q, Chernatynskaya AV, Wasserfall CH, et al. Anti-thymocyte globulin (ATG) differentially depletes naı¨ve and memory T cells and permits memory-type regulatory T cells in nonobese diabetic mice. BMC Immunol 2012;13:70. 90. Boenisch O, Lopez M, Elyaman W, Magee CN, Ahmad U, Najafian N. Ex vivo expansion of human Tregs by rabbit ATG is dependent on intact STAT3-signaling in CD4 1 T cells and requires the presence of monocytes. Am J Transplant 2012;12:856 66. 91. Broady R, Yu J, Levings MK. ATG-induced expression of FOXP3 in human CD4 1 T cells in vitro is associated with T-cell activation and not the induction of FOXP3 1 T regulatory cells. Blood 2009;114:5003 6. 92. Feng X, Kajigaya S, Solomou EE, et al. Rabbit ATG but not horse ATG promotes expansion of functional CD4 1 rabbit ATG but not horse ATG promotes expansion of functional CD4 1 CD25 high FOXP3 1 regulatory T cells in vitro. Blood 2008;111:3675 83. 93. Meyer C, Walker J, Dewane J, et al. Impact of irradiation and immunosuppressive agents on immune system homeostasis in rhesus macaques. Clin Exp Immunol 2015. Available from: http://dx.doi.org/10.1111/cei.12646. 94. Zhang F, Wang C, Wang H, et al. Ox-LDL promotes migration and adhesion of bone marrow-derived mesenchymal stem cells via regulation of MCP-1 expression. Mediators Inflamm 2013;691023. 95. Crepin T, Carron C, Roubiou C, et al. ATG-Induced accelerated immune senescence: clinical implications in renal transplant recipients. Am J Transplant 2015;15:1028 38. 96. Gurkan S, Luan Y, Dhillon N, et al. Immune reconstitution following rabbit antithymocyte globulin. Am J Physiol Renal Physiol 2010;10:2132 41. 97. Traitanon O, Gorbachev A, Bechtel JJ, et al. IL-15 induces alloreactive CD28- memory CD8 T cell proliferation and CTLA4-Ig resistant memory CD8 T cell activation. Am J Transplant 2014;14:1277 89. 98. Neujahr DC, Chen C, Huang X, et al. Accelerated memory cell homeostasis during T Cell depletion and approaches to overcome It. J Immunol 2006;176:4632 9. 99. Moxham VF, Karegli J, Phillips RE, et al. Homeostatic proliferation of lymphocytes results in augmented memory-like function and accelerated allograft rejection. J Immunol 2008;180:3910 18. 100. Heninger A-K, Theil A, Wilhelm C, et al. IL-7 abrogates suppressive activity of human CD4 1 CD25 1 FOXP3 1 regulatory T cells and allows expansion of alloreactive and autoreactive T cells. J Immunol 2012;189:5649 58. 101. Van Belle TL, Dooms H, Boonefaes T, Wei XQ, Leclercq G, Grooten J. IL-15 augments TCR-induced CD4 1 T cell expansion in vitro by inhibiting the suppressive function of CD25 High CD4 1 T cells. PLoS One 2012;7(9):e45299. 102. Gallon L, Traitanon O, Yu Y, et al. Differential effects of calcineurin and mammalian target of rapamycin inhibitors on alloreactive Th1, Th17, and regulatory T cells. Transplantation 2015. April 22 In Press. 103. Miroux C, Morale`s O, Carpentier A, et al. Inhibitory effects of cyclosporine on human regulatory T cells in vitro. Transplant Proc 2009;41:3371 4. 104. Miroux C, Morales O, Ghazal K, et al. In vitro effects of cyclosporine A and tacrolimus on regulatory T-cell proliferation and function. Transplantation 2012;94:123 31. 105. Vilar E, Perez-Garcia J, Tabernero J. Pushing the envelope in the mTOR pathway. The second generation of inhibitors. Mol Cancer Ther 2011;10:395 403. 106. Noris M, Casiraghi F, Todeschini M, et al. Regulatory T cells and T cell depletion: role of immunosuppressive drugs. J Am Soc Nephrol 2007;18:1007 18. 107. Azzi JR, Sayegh MH, Mallat SG. Calcineurin inhibitors: 40 years later, can’t live without. J Immunol 2013;191:5785 91. 108. English K, Barry FP, Field-Corbett CP, Mahon BP. IFN-gamma and TNF-alpha differentially regulate immunomodulation by murine mesenchymal stem cells. Immunol Lett 2007;110:91 100. 109. Griffin MD, Elliman SJ, Cahill E, English K, Ceredig R, Ritter T. Concise review: adult mesenchymal stromal cell therapy for inflammatory diseases: how well are we joining the dots? Stem Cells 2013;31:2033 41. 110. Popp FC, Eggenhofer E, Renner P, et al. Mesenchymal stem cells can induce long-term acceptance of solid organ allografts in synergy with low-dose mycophenolate. Transpl Immunol 2008;20:55 60. 111. Ferrer IR, Hester J, Bushell A, Wood KJ. Induction of transplantation tolerance through regulatory cells: from mice to men. Immunol Rev 2014;258:102 16. 112. Yang J, Li R, Ren Y, Yang Y, Xie R, Fan H. Third-party tolerogenic dendritic cells reduce allo-reactivity in vitro and ameliorate the severity of acute graft-versus-host disease in allo-bone marrow transplantation. Scand J Immunol 2013;78:486 96. 113. Francesca SL, Ting AE, Sakamoto J, et al. Multipotent adult progenitor cells decrease cold ischemic injury in ex vivo perfused human lungs: an initial pilot and feasibility study. Transplant Res 2014;3:19. 114. Kovacsovics-Bankowski M, Mauch K, Raber A, et al. Pre-clinical safety testing supporting clinical use of allogeneic multipotent adult progenitor cells. Cytotherapy 2008;10:730 42. 115. Kovacsovics-Bankowski M, Streeter PR, Mauch KA, et al. Clinical scale expanded adult pluripotent stem cells prevent graft-versus-host disease. Cell Immunol 2009;255:55 60. 116. Maziarz RT, Devos T, Bachier C, et al. Prophylaxis of acute GVHD using multistems stromal cell therapy: preliminary results after administration of single or multiple doses in a Phase 1 trial. Biol Blood Marrow Transplant 2012;18:S264 5.

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117. Pileggi A, Xu X, Tan J, Ricordi C. Mesenchymal stromal (stem) cells to improve solid organ transplant outcome: lessons from the initial clinical trials. Curr Opin Organ Transplant 2013;18:672 81. 118. Caplan A. Why are MSCs therapeutic? New data: new insight. J Pathol 2009;217:318 24. 119. Sohni A, Verfaillie CM. Multipotent adult progenitor cells. Best Pract Res Clin Haematol 2011;24:3 11. 120. Jacobs SA, Pinxteren J, Roobrouck VD, et al. Human multipotent adult progenitor cells are nonimmunogenic and exert potent immunomodulatory effects on alloreactive T-cell responses. Cell Transplant 2013;22:1915 28. 121. Reading JL, Yang JHM, Sabbah S, et al. Clinical-grade multipotent adult progenitor cells durably control pathogenic T cell responses in human models of transplantation and autoimmunity. J Immunol 2013;190:4542 52. 122. Ryan JM, Barry FP, Murphy JM, Mahon BP. Mesenchymal stem cells avoid allogeneic rejection. J Inflamm 2005;2:8. 123. Burrows GG, van’t Hof W, Newell LF, et al. Dissection of the human multipotent adult progenitor cell secretome by proteomic analysis. Tissue-Specific Progenit Stem Cells 2013;2:45 757. 124. Choi H, Lee RH, Bazhanov N, Oh JY, Prockop DJ. Anti-inflammatory protein TSG-6 secreted by activated MSCs attenuates zymosaninduced mouse peritonitis by decreasing TLR2/NF-κB signaling in resident macrophages. Blood 2011;118:330 8. 125. English K, Barry FP, Mahon BP. Murine mesenchymal stem cells suppress dendritic cell migration, maturation and antigen presentation. Immunol Lett 2008;115:50 8. 126. Franc¸ois M, Romieu-Mourez R, Li M, Galipeau J. Human MSC suppression correlates with cytokine induction of indoleamine 2,3-dioxygenase and bystander M2 macrophage differentiation. Mol Ther 2012;20:187 95. 127. Iwata M, Graf L, Awaya N, Torok-Storb B. Functional interleukin-7 receptors (IL-7Rs) are expressed by marrow stromal cells: binding of IL-7 increases levels of IL-6 mRNA and secreted protein. Blood 2002;100:1318 25. 128. Maggini J, Mirkin G, Bognanni I, et al. Mouse bone marrow-derived mesenchymal stromal cells turn activated macrophages into a regulatory-like profile. PLoS One 2010;5(2):e9252. 129. Ne´meth K, Leelahavanichkul A, Yuen PST, et al. Bone marrow stromal cells attenutate sepsis via prostaglandin E2-dependent reprogramming of host macrophages to incresase their interleukin-10 production. Nat Med 2009;15:42 9. 130. Waterman RS, Tomchuck SL, Henkle SL, Betancourt AM. A new mesenchymal stem cell (MSC) paradigm: polarization into a proinflammatory MSC1 or an immunosuppressive MSC2 phenotype. PLoS One 2010;5(4):e10088. 131. Kim J, Hematti P. Mesenchymal stem cell-educated macrophages: a novel type of alternatively activated macrophages. Exp Hematol 2009;37:1445 53. 132. Noone C, Kihm A, English K, O’Dea S, Mahon BP. IFN-γ stimulated human umbilical-tissue-derived cells potently suppress NK activation and resist NK-mediated cytotoxicity in vitro. Stem Cells Dev 2013;22:3003 14. 133. Ge W, Jiang J, Arp J, Liu W, Garcia B, Wang H. Regulatory T-cell generation and kidney allograft tolerance induced by mesenchymal stem cells associated with indoleamine 2,3-dioxygenase expression. Transplantation 2010;90:1312 20. 134. Cahill EF, Tobin LM, Carty F, Mahon BP, English K. Jagged-1 is required for the expansion of CD4 1 CD25 1 FoxP3 1 regulatory T cells and tolerogenic dendritic cells by murine mesenchymal stromal cells. Stem Cell Res Ther 2015;6:1 13. 135. Chiesa S, Morbelli S, Morando S, et al. Mesenchymal stem cells impair in vivo T-cell priming by dendritic cells. Proc Natl Acad Sci USA 2011;108:17384 9. 136. Li FR, Wang XG, Deng CY, Qi H, Ren LL, Zhou HX. Immune modulation of co-transplantation mesenchymal stem cells with islet on T and dendritic cells. Clin Exp Immunol 2010;161:357 63. 137. Ghannam S, Pe`ne J, Torcy-Moquet G, Jorgensen C, Yssel H. Mesenchymal stem cells inhibit human Th17 cell differentiation and function and induce a T regulatory cell phenotype. J Immunol 2010;185:302 12. 138. Aggarwal S, Pittenger MF. Human mesenchymal stem cells modulate allogeneic immune cell responses. Blood 2005;105:1815 22. 139. Duffy MM, Pindjakova J, Hanley SA, et al. Mesenchymal stem cell inhibition of T-helper 17 cell- differentiation is triggered by cell-cell contact and mediated by prostaglandin E2 via the EP4 receptor. Eur J Immunol 2011;41:2840 51. 140. Gieseke F, Bo¨hringer J, Bussolari R, Dominici M, Handgretinger R, Mu¨ller I. Human multipotent mesenchymal stromal cells use galectin-1 to inhibit immune effector cells. Blood 2010;116:3770 9. 141. Jia Z, Jiao C, Zhao S, et al. Immunomodulatory effects of mesenchymal stem cells in a rat corneal allograft rejection model. Exp Eye Res 2012;102:44 9. 142. Zhao Z-G, Xu W, Sun L, Li W-M, Li Q-B, Zou P. The characteristics and immunoregulatory functions of regulatory dendritic cells induced by mesenchymal stem cells derived from bone marrow of patient with chronic myeloid leukaemia. Eur J Cancer 2012;48:1884 95. 143. Eggenhofer E, Hoogduijn MJ. Mesenchymal stem cell-educated macrophages. Transplant Res 2012;1:12. 144. Sivanathan KN, Gronthos S, Rojas-Canales D, Thierry B, Coates PT. Interferon-gamma modification of mesenchymal stem cells: implications of autologous and allogeneic mesenchymal stem cell therapy in allotransplantation. Stem Cell Rev 2014;10:351 75. 145. Perico N, Casiraghi F, Gotti E, et al. Mesenchymal stromal cells and kidney transplantation: pretransplant infusion protects from graft dysfunction while fostering immunoregulation. Transpl Int 2013;26:867 78. 146. Tobin LM, Healy ME, English K, Mahon BP. Human mesenchymal stem cells suppress donor CD4 1 T cell proliferation and reduce pathology in a humanized mouse model of acute graft-versus-host disease. Clin Exp Immunol 2013;172:333 48. 147. Chinnadurai R, Copland IB, Patel SR, Galipeau J. IDO-independent suppression of T cell effector function by IFN-γ-licensed human mesenchymal stromal cells. J Immunol 2014;192:1491 501. 148. Cuerquis J, Romieu-Mourez R, Franc¸ois M, et al. Human mesenchymal stromal cells transiently increase cytokine production by activated T cells before suppressing T-cell proliferation: effect of interferon-γ and tumor necrosis factor-α stimulation. Cytotherapy 2014;16:191 202. 149. Ben Nasr M, Vergani A, Avruch J, et al. Co-transplantation of autologous MSCs delays islet allograft rejection and generates a local immunoprivileged site. Acta Diabetol 2015;52(5):917 27. 150. Haarer J, Johnson CL, Soeder Y, Dahlke MH. Caveats of mesenchymal stem cell therapy in solid organ transplantation. Transpl Int 2015;28:1 9.

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C H A P T E R

19 Induction Immunosuppression in Kidney Transplantation Josep M. Grinyo´ and Oriol Bestard University of Barcelona, Barcelona, Spain

19.1 INTRODUCTION Induction therapy is the use of biological immunosuppressants, mainly in the peritransplant period, with the main goal of preventing acute rejection. As in deceased donor, and even living related renal transplantation, the degree of human leukocyte antigen (HLA) compatibility between donor and recipient may be relatively low, and the risk of acute rejection might be high only using xenobiotic small molecules immunosuppressants, as these agents have a delayed impact on the alloimmune response in the first few days after oral administration. Biological agents used in induction therapies mainly target immune-competent lymphocytes, which are the key players of the allograft reaction, and exert their pharmacodynamic effects immediately after parenteral injection. Induction agents, depletive and nondepletive, fill the gap to protect the organ from rejection in the first weeks after transplantation. The first induction agents introduced in renal transplantation were horse antilymphocyte polyclonal preparations, followed by monoclonal anti-IL-2 receptor antibodies (IL2Ra), rabbit antithymocyte globulin (rATG), OKT3, and more recently by alemtuzumab and rituximab, two agents initially approved for the treatment of lymphomas. As of 2017, the most used are Simulect, a chimeric IL2Ra, and rATG. Alemtuzumab has been explored to substitute rATG, and rituximab is mainly used to target B-lymphocytes to overcome the humoral barrier in highly sensitized kidney transplant recipients and in ABO-incompatible renal transplantation. Belatacept, a biological costimulatory blocker, recently introduced may be considered an induction agent, despite its use early after transplantation but also in maintenance immunosuppression in the long term (this agent will be reviewed in Chapter 21, Novel Drugs in Kidney Transplantation). The biologic agents currently used in kidney transplantation are summarized in Table 19.1. The efficacy of induction therapy in the prophylaxis of acute rejection in low-risk patients has derived on the sparing of concomitant immunosuppressants, such calcineurin inhibitors (CNI) or steroids, in the design of lowcomorbidity regimens. On the other hand, induction agents have been considered crucial drugs in kidney transplant recipients at high risk for acute rejection such as highly sensitized patients, those at high risk for delayed graft function (DGF) and in ethnicities with well-identified high immunological risk for graft loss. More recently, depletive agents have also been explored in tolerogenic strategies to promote graft acceptance. The systematic reviews and metaanalyses on induction therapies in renal transplantation are summarized in Table 19.2.

19.2 INDUCTION IMMUNOSUPPRESSION IN THE PREVENTION OF ACUTE REJECTION Either IL2Ra or ATG have shown effective prevention of acute rejection in low-risk kidney transplant recipients initially in association with dual therapy with CNI and steroids, and since the 1990s in quadruple regimens mainly including mycophenolate mofetil (MMF), and less frequently azathioprine. In low-risk patients the choice Kidney Transplantation, Bioengineering, and Regeneration. DOI: http://dx.doi.org/10.1016/B978-0-12-801734-0.00019-9

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TABLE 19.1

Biological Immunosuppressants in Induction Therapy in Renal Transplantation

Agent

Characteristics

Target

rATG Polyclonal antithymocyte Thymoglobulin globulins derived from ATG-Fresenius immunization with:

Doses recommended

Main MOA

Main reported uses High- and low-risk transplants CNI and steroid-sparing regimens

Surface molecules on lymphocytes and monocytes

Lympholysis T-cell apoptosis Downmodulation of surface molecules Expansion of Tregs

Basiliximab

• Thymic tissue • Jurkat T cells Chimeric mAb

1 1.5 mg/kg/ day, 4 5 doses 3 mg/kg/day, 4 doses

CD25 on T cells

20 mg on days 0 and 4

Inactivation of T cells; Low-risk transplants and down-modulation of CNI and steroid-sparing CD25 regimens

Alemtuzumab

Humanized mAb

CD52 on B and T cells 30 mg on day 0

B- and T-cell depletion

High- and low-risk transplants

Rituximab

Chimeric mAb

CD20 on B cells

B-cell depletion

Highly sensitized patients ABO-incompatible transplants

Single and double dose of 375 mg/m2

CNI, calcineurin inhibitors; MOA, mechanism of action; mAb, monoclonal antibody; rATG, rabbit antithymocyte globulin; Tregs, T-regulatory cells.

TABLE 19.2 Authors & year of publication (References)

Systematic Reviews and Metaanalyses on Induction Immunosuppression in Renal Transplantation

N patients

Source

Type of analysis

Main conclusions

1.185

4 RCT

Basiliximab versus placebo

Basiliximab RR reduction of rejection by 35%

Adu et al. (2003)2 1.871

8 RCT

IL2Ra versus placebo or no additional treatment

Webster et al. (2004)3

4.893

38 RCT

IL2Ra versus placebo Il2Ra versus other antibody therapies

Webster et al. (2010)4

10.537

71 RCT

IL2Ra versus placebo IL2Ra versus ATG

Morgan et al. (2012)5

1.223

10 RCT

Alemtuzumab versus IL2Ra Alemtuzumab versus rATG

Reduced risk of acute rejection with IL2Ra (OR 0.51) No differences on graft loss mortality, malignancies of infection No differences between basiliximab and daclizumab Significant reduction of acute rejection with IL2Ra. No differences on graft loss No differences in treatment effects Fewer side effects with IL2Ra 25% graft loss reduction with IL2Ra 28% acute rejection reduction with IL2Ra Less CMV disease ATG reduced BPAR No differences in graft loss Better tolerability of IL2Ra Lower risk of BPAR with alemtuzumab (RR 0.54) No differences on BPAR, DGF, and graft loss

Sharif et al. (2011)6

11.337

56 RCT

Depletive and nondepletive induction comparing CNI sparing versus CNI standard

Reduction of graft loss (OR 0.73), less DGF (OR 0.89), improved graft function and less NODAT in CNIsparing regimens

Pascual et al. (2012)7

1.934

9 RCT

Induction and early steroid avoidance versus conventional steroids

Less NODAT with steroid avoidance in CsA-treated patients only. No clear benefits on blood pressure and lipid abnormalities after steroid avoidance

Keown et al. (2003)1

ATG, antithymocyte globulin; BPAR, biopsy-proven acute rejection; CMV, cytomegalovirus; CNI, calcineurin inhibitors; CsA, cyclosporine; DGF, delayed graft function; IL2Ra, monoclonal anti-IL2-receptors antibodies; NODAT, new-onset diabetes mellitus after transplantation; OR, odd ratios; RCT, randomized clinical trials; RR, relative risk.

for IL2Ra or ATG varies across centers and countries, and the penetration of these two agents has changed in the last few years. According to the 2014 Annual Report of the United States Renal Data System,8 less than 20% of kidney transplant recipients received no induction, ATG was the most common agent for induction and used in more than 60% of renal transplant patients, and the use of IL2Ra had declined from 40% at the beginning of the

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2010s to less than 25%. Interestingly, the KDIGO Guidelines published in 2009 recommended IL2Ra as the firstline agents in low-risk patients, and suggest depleting antibodies, rather than IL2Ra, for high immunological risk patients,9 and the statement reported by the European Renal Best Practices (ERBP) group published in 2011 corroborated these recommendations.10

19.2.1 Induction With Anti-IL-2R Monoclonal Antibodies IL2Ra are directed against the α chain (CD25) of the IL2-R, which is expressed on the T lymphocyte surface. The binding of IL2Ra to CD25 prevents complete T cell activation. Since the 1990s, two IL2Ras have been used in prophylactic regimens in organ transplantation. Basiliximab is a chimeric high-avidity antibody with a half-life of 8 days and administered 20 mg on days 0 and 4 after transplantation.11 Daclizumab is a highly humanized antibody with a lower avidity but longer half-life administered since the time of transplantation and every 2 weeks up to 3 months after transplantation.12 The discontinuation of production of daclizumab in 2009 for commercial reasons has left basiliximab as the main IL-2Ra available in clinical practice. In the few studies comparing both IL2Ras in the prevention of acute rejection, there were no apparent differences between them.13 The clinical benefits of the use of basiliximab in cyclopsporine (CsA)-based immunosuppression in addition to double or triple therapy in comparison with placebo was evaluated in a metaanalysis with more than 1000 patients reported in 2003.1 Basiliximab reduced the relative risk (RR) and absolute risk (AR) of clinical and biopsy-proven acute graft rejection (BPAR) across all treatment regimens. The overall RR of clinical acute graft rejection was decreased by 35% in patients receiving basiliximab. AR was reduced significantly by 15.6% (pooled incidence: 28.8% vs 44.4%, P , .0001). The reduction in RR of BPAR was similar (32%) with an AR reduction of 11.7% (pooled incidence: 25.1% vs 36.8%, P , .0001). In this analysis there was a concomitant reduction in the risk of graft loss, which did not reach statistical significance (P 5 .14). The RR of graft loss was reduced by 26% with an AR reduction of 2.3% (pooled incidence: 6.4% vs 8.7%) without any impact on patient mortality. In another metaanalysis involving 1858 patients and including basiliximab and daclizumab in CsA-based therapies the use of these antibodies reduced the rates of acute rejection by 49%2 as compared to placebo or no additional treatment. In this analysis, there were no significant differences in the rate of graft loss (0.78, 0.58 1.04), mortality (0.75, 0.46 1.23), overall incidence of infections (0.97, 0.77 1.24), incidence of cytomegalovirus (CMV) infections (0.81, 0.62 1.04), or risk of malignancies at 1 year (0.82, 0.39 1.70), and no differences were observed between these antibodies on the described clinical outcomes. In another metaanalysis3 with almost 5000 patients comparing IL2Ra with placebo, graft loss was not significantly different at one (RR 0.83, 95% CI 0.66 1.04) or 3 years (RR 0.88, 95% CI 0.64 1.22). Acute rejection was significantly reduced at 6 months (RR 0.66, 95% CI 0.59 0.74) and at 1 year (RR 0.67, 95% CI 0.60 0.75). At 1 year, CMV infection (RR 0.82, 95% CI 0.65 1.03) and malignancy (RR 0.67, 95% CI 0.33 1.36) were not significantly different, and few side effects were associated with the administration of these antibodies. Because of the efficacy demonstrated and the favorable safety profile, IL2Ras were incorporated into prophylactic regimens in many centers and included in the KDIGO Guidelines recommendations.9 In 2010, the same authors conducted another metaanalysis involving more than 10,000 patients from 71 studies comparing IL2Ra with placebo or ATG.4 Where IL2Ras were compared with placebo, graft loss including death with a functioning graft was reduced by 25% at 6 months and 1 year but not in the longer follow-up. At 1 year BPAR was reduced by 28%, and there was a 19% reduction in CMV disease. There was a 64% reduction in early malignancy within 6 months. When IL2Ras were compared with ATG, there was no difference in graft loss at any time point, or for acute rejection diagnosed clinically, but there was benefit of ATG therapy over IL2Ra for BPAR. ATG patients experienced significantly more fever, cytokine release syndrome, and other adverse reactions to drug administration and more leukopenia but not thrombocytopenia, and there was no evidence that effects were different according to whether equine or rabbit ATG was used. Concerning the concomitant medication used with IL2Ra, there were no significant differences in outcomes according to CsA or tacrolimus use, azathioprine, or MMF. It has been classically considered that there are no pharmacokinetic interactions between induction antibodies and small molecules immunosuppressants. However, a recent report14 shows higher trough levels of tacrolimus in IL2Ra-treated patients than in patients receiving ATG. The explanation for such effect might be that CYP enzyme activity, which participates in tacrolimus metabolism, could be modulated by activation of IL-2 receptors expressed on hepatocytes and intestinal cells. IL-2Ra might promote preferential binding of circulating IL-2 to IL-2 receptors on these cells by blocking IL-2 receptors on activated T cells, which might downregulate CYP enzymes, leading to increased CNI levels.

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19.2.2 Induction With Antithymocyte Globulins The clinical experience and the reported analyses generally have profiled IL2Ras as induction agents with very few side effects but as less potent than polyclonal ATG, which is associated with more adverse side effects and opportunistic infections and malignancies. ATG heterologous globulin preparations are obtained by the injection of human lymphocytes, mainly thymocytes, to animals, usually rabbits. These heterologous globulins elicit transplant recipient immunization, which may reduce their efficacy and limit their reuse. However, it has been reported that a second course of rATG induction results in similar lymphocyte depletion and is as well tolerated as a first course.15 The initial empirical design of polyclonal globulin preparations aimed to merely deplete lymphocytes, which are mainly mediated by T-cell apoptosis in peripheral lymphoid tissues16 by means of caspase-3 activation.17 In the remaining lymphocytes ATG downmodulates the surface molecules CD3, CD2, CD4, and CD8, impairing the functional responses of T cells. The analysis of the composition of these polyclonal ATGs also identified functional antibodies to adhesion molecules on lymphocytes, monocytes and neutrophils, and chemokine receptors on lymphocytes and monocytes that may contribute to reduce graft cellular infiltration during rejection and in ischemia-reperfusion injury.18 ATG induces profound CD41 T-cell depletions that may persist in the long term,19 which may depend on recipient age, pretransplant CD41 T-cell counts and therapy with tacrolimus or MMF. Interestingly, the risk for severe infections after ATG induction seems to be associated with impaired T-cell proliferative capacity but not with the profound decline in CD41 cell counts that occurred after ATG induction.20 Early after the administration of ATG, cytokine-induced homeostatic proliferation is involved in T-cell repopulation of both naı¨ve and memory T cells, and at later time points, the contribution of homeostatic proliferation diminishes, which may explain the incomplete T-cell recovery.21 After T-cell depletion, both thymopoiesis and homeostatic proliferation contribute to immune reconstitution. The administration of rATG induces peripheral expansion and new thymic emigration of T cells with a T regulatory (Treg) phenotype,22 although it has also been claimed that ATG increases the proportion of Tregs, only derived from homeostatic proliferation.23 This in vivo expansion of Tregs induced by ATG is also supported by in vitro24 studies and experimental works in rodents,25 and in both instances ATG-expanded Tregs have preserved suppressor functional capacity. The ex vivo expansion of human Tregs by ATG is due to its ability to reprogram CD41 T cells in a STAT3-dependent but TGF-β-independent manner, leading to the generation of monocyte-derived dendritic cells (DCs) with a tolerogenic profile,26,27 although an analysis of the interaction of ATG with DC antigens may suggests an activation of these cells by ATGs rather than a specific induction of a tolerogenic DC phenotype.28 Functional studies indicate that ATG interferes with basic DC functions and reduces the capacity of mature DCs to stimulate allogeneic and autologous T cells.29 After the withdrawal of equine ATGs, rATGs are the only ones available for clinical use. The two accessible rATG preparations differ in the source of rabbit immunization and in the dosage recommended. rATG (Thymoglobulin) is obtained by rabbit immunization with human thymocytes,30 and is usually given at 1 1.5 mg/kg/day since the day of transplantation, usually followed by 4 5 doses. There is a gradual trend for using lower cumulative doses of ATG up to 6 7 mg/kg, or even lower.31 ATG Fresenius (ATG-F) is obtained with rabbit’s immunization with Jurkat T cells and is recommended at 3 mg/kg/day during the first 4 days after transplantation or at a single high peroperative dose of 9 mg/kg.32 Despite the differences in the source of immunization, pharmacodynamics, and dosages, there are no substantial differences in the clinical outcomes between these two ATG preparations,33 although Thymoglobulin is more widely used. Webster et al.4 in a Cochrane Database metaanalysis involving 18 studies with 1844 patients, exhaustively reviewed the risk/benefit profile of ATG induction in the prophylaxis of acute rejection. When IL2Ras were compared with ATG, there was no difference in graft loss at any time point, or for acute rejection diagnosed clinically, but there was benefit of ATG therapy over IL2Ra for BPAR at 1 year (8 studies: RR 1.30 95% CI 1.01 1.67), but at the cost of a 75% increase in malignancy (7 studies: RR 0.25 95% CI 0.07 0.87) and a 32% increase in CMV disease (13 studies: RR 0.68 95% CI 0.50 0.93). Serum creatinine was significantly lower for IL2Ra-treated patients at 6 months (4 studies: MD 211.20 μmol/L 95% CI 219.94 to 22.09). ATG patients experienced significantly more fever, cytokine release syndrome, and other adverse reactions to drug administration and more leukopenia but not thrombocytopenia. Concerning the concomitant use of CNI and antimetabolite immunosuppressants, there were no significant differences in outcomes according to cyclosporine or tacrolimus use, azathioprine, or MMF, or to the study populations’ baseline risk for acute rejection, and there was no evidence that effects were different according to whether equine or rabbit ATG was used. Despite the better tolerability and cost-effectiveness of IL2Ra compared to polyclonal induction,34 the greater effectiveness in the prevention of BPAR may account for the increasing use of ATG over IL2Ra, already described.

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19.2.3 Induction Therapy With Alemtuzumab Alemtuzumab (Campath-1H) is a humanized monoclonal antibody that reacts with the pan-lymphocyte CD52 antigen present on human lymphoid and myeloid cells that induces a profound and durable lymphopenia via an Fc-mediated mechanism, which is able to kill target cells by antibody-dependent cell-mediated cytotoxicity. CD52 is expressed at high levels on the surface of nearly all B and T lymphocytes, a majority of NK cells, monocytes, and macrophages, and a small percentage of granulocytes, but not on erythrocytes or hematopoietic stem cells.35,36 Alemtuzumab is approved for the treatment of chronic lymphocytic leukemia36 and it is also used in the treatment of some autoimmune disorders, especially multiple sclerosis.37 In renal transplantation, alemtuzumab is usually given at 30 mg the day of transplantation, and at times followed by a second dose the next day. Despite the lack of approval by the regulatory agencies for renal transplantation, alemtuzumab has been used in induction therapies, mainly in the United States. Several prospective and retrospective investigator-driven, randomized and nonrandomized clinical trials (RCTs) have been published in the last 10 years on alemtuzumab in prophylaxis regimens with variable results on the main clinical outcomes. A metaanalysis reported by the Centre for Evidence in Transplantation in 2012 only including RCT involving 1223 patients compared induction with alemtuzumab with no induction or other induction agents.5 The primary outcome evaluated in this analysis was BPAR, and secondary outcomes included graft loss, renal function, DGF, patient death, and the incidence of infection, autoimmunity, malignancy, and new-onset diabetes mellitus after transplantation. Alemtuzumab induction had a lower risk of BPAR compared with induction with the IL-2Ras, basiliximab, and daclizumab combined (RR 0.54; 95% confidence interval, 0.37 0.79; P , .01). No significant difference was observed in the risk of BPAR when alemtuzumab induction was compared with the two branded rATGs (RR 0.79; 95% CI 0.52 1.21; P 5 .28). There was no difference in graft loss, DGF, patient death, and new-onset diabetes mellitus after transplantation when alemtuzumab was compared with IL-2Ra or rATG induction. The effect of alemtuzumab induction on renal function and the incidence of infection, malignancy, and autoimmunity were limited by the data available in the RCTs. There were two trials comparing alemtuzumab with no induction, but neither trial reported a significant reduction in BPAR at 12 months. According to this metaanalysis, alemtuzumab reduces the risk of BPAR in comparison with IL2Ra, but not with rATG, and with similar incidences of DGF, graft loss, and patient death. In other reports based on retrospective studies of single-center experiences, alemtuzumab induction has been followed by poorer outcomes compared with basiliximab or rATG.38 Patients receiving alemtuzumab had reduced allograft survivals, more opportunistic infections and CMV infections, and higher cumulative antibody-mediated rejection (AMR), up to 27% versus 18% with other inductions. The observed higher rates of AMR with alemtuzumab might be due to a partial sparing of antigen-experienced and memory B cells, whereas naı¨ve B cells are profoundly depleted during the first few months after alemtuzumab infusion. B-cell repopulation is based on the emergence of pregerminal center B cells, which is followed by the expansion of naı¨ve B cells.39 These B-cell dynamics are accompanied by a higher incidence of donor-specific antibodies (DSA), which might induce AMR.40 Analysis of the US Renal Database System on the outcomes of distinct induction regimens with thymoglobulin, basiliximab, daclizumab, and alemtuzumab showed poorer outcomes in first elderly kidney transplant recipients with this last agent.41 Induction with alemtuzumab was associated with an increased risk of graft loss and death, with an adjusted hazard ratio (AHR) of 1.26 (95% CI 1.08 1.48). Risk was also present at other age cut-offs (age .60 (AHR 1.16; 95% CI 1.03 1.31; P 5 .014), age .70 (AHR 1.43; 95% CI 1.13 1.81; P 5 .003) and age .75 (AHR 1.68; 95% CI 1.07 2.63; P 5 .024)). According to recent data from the US Scientific Registry for Transplant Recipients using a propensity score analysis in retransplant recipients, alemtuzumab was also associated with a higher risk for graft loss over Thymoglobulin (AHR 1.19).42 All these data suggest that the role of alemtuzumab in renal transplantation has yet to be defined. Assuming a more potent immunosuppressive effect of alemtuzumab than IL2Ra, a multicenter British trial with a high number of patients has recently compared alemtuzumab, with low-dose tacrolimus and MMF in a steroid-free regimen, with a standard quadruple regimen consisting of basiliximab, MMF, steroids, and conventional doses of tacrolimus.43 Short-term results of this trial at 6 months after transplantation have shown a significant reduction of BPAR, mainly cellular, with alemtuzumab (7% vs 16%) and without differences on patient and graft survival. It is worth noting that the observed differences on tacrolimus trough levels between the two groups have been minimal (6.9 ng/mL in the alemtuzumab group vs 9.3 ng/mL in the basiliximab group). A second part of the study with longer follow-up with a planned conversion from CNI to mTOR inhibitors may provide the necessary information on the utility of alemtuzumab as an alternative to the classical ATG or IL2Ra. However, the removal of alemtuzumab from Europe may impair future clinical developments of this agent.

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19.3 INDUCTION IMMUNOSUPPRESSION IN LOW-COMORBIDITY REGIMENS With the low rates of acute rejection under the umbrella of induction agents, and with potent concomitant small molecules immunosuppressants, such as MMF or mTOR inhibitors, additional benefits of induction therapy may be the sparing of CNI in order to attenuate nephrotoxicity, and/or steroids to avoid the well-known cardiovascular risk factors mainly derived from the metabolic abnormalities related to these drugs, which may lead to the so-called low-comorbidity regimens.

19.3.1 Induction in Calcineurin Inhibitor Sparing Strategies After several attempts to completely avoid CNI from prophylactic regimens by using polyclonal ATG or IL2Ra,44,45 a trial addressing the feasibility of reducing or eliminating CsA with daclizumab-based induction (Caesar study) led to the conclusion that persistent minimization of CsA in association with MMF and conventional low doses of steroids efficiently prevented acute rejection and preserved renal function,46 better than complete CNI discontinuation. The results of this trial were the basis for the design of the so-called Symphony study,47 with a large number of patients exploring the feasibility of CNI (CsA or tacrolimus) or sirolimus minimization in conjunction with MMF, conventional doses of steroids under daclizumab 3-month induction. The primary end point was the estimated glomerular filtration rate (GFR) 12 months after transplantation, whereas the secondary end points included acute rejection and allograft survival. The mean calculated GFR was higher in patients receiving lowdose tacrolimus (65.4 mL/minute) than in the other therapeutic groups. The rate of BPAR was lower in patients receiving low-dose tacrolimus (12.3%) than in those receiving standard-dose CsA (25.8%), low-dose CsA (24.0%) or low-dose sirolimus (37.2%). Allograft survival differed significantly between the four groups (P 5 .02) and was highest in the low-dose tacrolimus group (94.2%), followed by the low-dose CsA group (93.1%), the standarddose CsA group (89.3%) and the low-dose sirolimus group (89.3%), and serious adverse events were more common in the low-dose sirolimus group than in the other groups. Ekberg et al.48 reported the 3-year observational results of the Symphony trial, showing that the differences between treatment groups were often no longer significant 3 years after transplantation in an intention-to-treat analysis. Renal function remained stable during followup and enabled the authors to conclude that daclizumab induction with low-dose tacrolimus and MMF attenuates the negative effects on renal function commonly reported for standard CNI regimes. However, these data should be interpreted with caution as there was probably a selection bias toward including patients with good prognosis and there were uncontrolled treatment modifications with many patients switching from CsA or sirolimus to tacrolimus. A country analysis of the Symphony trial with distinct types of transplant programs, i.e., mainly deceased donor transplants, mainly living related transplants, or a mix model, confirmed the applicability of these low-CNI regimens with induction.49 Symphony-like regimens based on basiliximab (as daclizumab production was discontinued) are very common as of 2017 and are considered a gold standard in low-risk transplant recipients in many countries. After the Symphony study, a metaanalysis on CNI-sparing regimens from the time of renal transplantation was reported in 2011,6 comprising data from more than 11,000 patients. Minimization of CNI in combination with various types of induction (depletive or nondepletive) and adjunctive agents, mainly MMF, reduced the odds of graft failure (OR 0.73; 95% CI 0.58 0.92; P 5 .009), with less DGF, improved graft function and less new-onset diabetes, without an increased rate of acute rejection. In CNI-reduction strategies, MMF with mTOR inhibitor combination has been assayed in single- and multicenter studies with variable success with monoclonal or polyclonal induction. In the referred metaanalysis,6 the use of mTOR inhibitors, in combination with MMF, increased the odds of graft failure (OR 1.43; 95% CI 1.08 1.90; P 5 .01). More recently, two main multicenter trials investigated early elimination of CNI under basiliximab or ATG. In the Zeus study,50,51 under basiliximab induction, 300 de novo kidney transplant recipients were randomized to continue receiving CsA or convert to everolimus at 4.5 months posttransplant. At 12 months, the everolimus regimen was associated with a significant improvement in GFR versus the CsA regimen (71
8 mL/minute per 1
73 m2 vs 61
9 mL/minute per 1.73 m2, respectively; mean difference 9
8 mL/minute per 1
73 m2, 95% CI 212
2 to 7
5) and this functional benefit was maintained at 5 years. Rates of BPAR, mainly mild rejections, were higher in the everolimus group than in the CsA group after randomization (15 (10%) of 154 vs 5 (3%) of 146; P 5 .036), but similar for the full study period (23 (15%) vs 22 (15%)). However in the everolimus arm there were more frequent adverse events characteristic of mTOR inhibitors, which resulted in a high rate of discontinuations. Under ATG and high doses of MMF, early switch between 10 and 24 days after transplantation from

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CsA to sirolimus was also assayed in low-risk transplant recipients.52 Similar to the previous study, the elimination of CsA resulted in higher GFR, and the incidence of BPAR in the first 12 months after transplantation was numerically higher in the sirolimus arm (15 vs 25%, P 5 .49), and mainly occurred during the first 6 months after surgery. An intent-to-treat analysis at 3 years confirmed the maintained improvement of renal function, without significant differences in patient and graft survivals.53 It is worth noting that despite the use of polyclonal induction and high doses of MMF, in this study the development of CMV infection was lower in the sirolimus arm, as it has been observed in other mTOR inhibitor clinical trials.54,55 However, and despite this advantage, more than 50% of patients in the sirolimus group discontinued therapy. This poor tolerability may account for the low penetration of induction and mTOR inhibitor-based CNI-free regimens in renal transplantation, despite the acceptable rejection rates and better renal function. The observed side effects in these studies are mainly attributed to mTOR inhibitors and MMF in combination.56 A CNI-sparing alternative currently under investigation is the combination of low-dose CNI with low-dose mTOR inhibitor and induction with IL2Ra. Recent trials,57 and others in progress using induction with basiliximab or rATG,58 may contribute to ascertain the feasibility of theses strategies.

19.3.2 Induction in Steroid-Sparing Strategies Steroid-related comorbidity is an issue in renal and other solid organ transplantation. Immunosuppressive regimens devoid of the toxicities of steroids may improve patients’ quality of life,59 and life expectancy by decreasing cardiovascular disease or infections.60 In spite of the clinical benefits of immunosuppressive regimens off steroids, steroid avoidance has entailed increased risk of rejection and even graft loss in the setting of renal transplantation. In a metaanalysis published in 2000, prednisone withdrawal significantly increased the risk of rejection by 14% and graft loss by 40%.61 Initial studies on steroid-sparing strategies identified the classical risk factors for unsuccessful steroid discontinuation, such as young age, black race, unstable renal function, sensitization, previous rejection episodes, and the timing of steroid withdrawal. Early and rapid discontinuation of steroids was associated with a high incidence of rejection episodes, which obliged the reintroduction of these agents in more than 50% of the cases.62 These results might have negatively impacted on clinical approaches attempting to avoid steroids early after transplantation. However, effective induction agents may be useful tools for implementing steroid-free immunosuppressive regimens from the beginning after transplantation allowing steroid cessation in the immediate postoperative period. This perceived more-protective initial immunosuppression has resulted in an increased trend for very early steroid discontinuation in the United States, reflected in the proportion of patients discharged off steroids, which increased from less than 4% in 2000 to more than 30% in 2006, and with no apparent increased risk of adverse clinical outcomes in the intermediate term.63 This trend for early steroid avoidance in transplant centers paralleled early steroid withdrawal in the context of prospective randomized multicenter trials. The feasibility of this approach was assessed in a trial in which 83 patients were randomized to either corticosteroid withdrawal at day 4 posttransplantation or standard steroid therapy receiving basiliximab, cyclosporine-microemulsion, and MMF.64 The incidence of BPAR at 12 months was not significantly different between the steroid withdrawal group (20%) and the standard treatment group (16%), and patient and graft survivals were very high and similar in the 2 therapeutic groups. Seventy-two percent of the steroid withdrawal group remained off steroids at 6 months posttransplant, without significant differences on allograft function and incidence of adverse events and infections between the two groups. In a step ahead, complete avoidance of steroids, versus early discontinuation on day 7 and standard use of steroids, were compared in another posterior study65 in patients treated with basiliximab, mycophenolate sodium, and CsA. Although there were no significant differences on renal function among the 3 groups, the 12-month incidence of BPAR, graft loss or death was 36.0% in the steroid-free group (P 5 .007 vs standard steroids), 29.6% with steroid withdrawal (n.s.), and 19.3% with standard steroids, and BPAR was significantly less frequent with standard steroids than either of the other two regimens. The results of this study suggested that for standard-risk renal transplant patients receiving triple therapy and basiliximab, steroid withdrawal by the end of week 1 may achieve similar 1-year renal function to a standard-steroids regimen, and may be more desirable than complete steroid avoidance, although these data raised concerns because of the relative short-term follow-up and the underrepresentation of ethnicities at high risk for steroid-sparing regimens.66 In another prospective randomized study with planned early steroid withdrawal, distinct induction agents were compared, in conjunction with MMF and tacrolimus. This trial recruited patients at high risk for rejection, such as repeated transplants, panel reactive antibodies (PRA) of 20% or more, and black race, who received induction either with rATG or alemtuzumab, and low-risk patients, who were

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treated with basiliximab or alemtuzumab.67 The rate of BPAR was significantly lower in the alemtuzumab group than in patients with basiliximab or rATG at both 6 months (3% vs 15%, P , .001) and 12 months (5% vs 17%, P , .001). At 3 years, the rate of BPAR in low-risk patients was lower with alemtuzumab than with basiliximab (10% vs 22%, P 5 .003), but among high-risk patients, no significant difference was seen between alemtuzumab and rATG (18% vs 15%, P 5 .63). The observed outcomes in this trial suggest that a very early steroid discontinuation is also feasible in high-risk patients either with alemtuzumab or rATG, and that in low-risk transplant recipients, alemtuzumab is more effective than basiliximab in preventing rejection. Other randomized trials also indicate that rATG with tacrolimus and MMF therapy may allow early elimination of corticosteroids, which is associated with metabolic benefits.68 Early elimination of steroids has also been undertaken in CNI-free regimens based on the use of sirolimus, usually under ATG or basiliximab induction with rejection rates lower than 20%.69 A systematic review and a metaanalysis of the randomized controlled studies about steroid avoidance or withdrawal within the first 2 weeks in renal transplant recipients treated with antibody induction, with a predominance of IL2Ra, and CsA or tacrolimus plus MMF comprising 1934 cases from 9 studies, showed that after steroid avoidance, acute rejection was more frequent than with conventional steroid use in CsA trials (risk ratios (RR) 1.59, 95% confidence intervals (95% CI) 1.01 2.49) and less new-onset diabetes mellitus, but not when tacrolimus was used (RR 1.06, 95% CI 0.79 1.42),7 though the metabolic benefits of steroid elimination remained unclear.7 The introduction of new immunosuppressants such as belatacept may help to refine induction therapies in order to early spare or avoid CNI and steroids. The association of belatacept with a short course of rATG was explored to avoid CNI and steroids in living or deceased donor renal transplantation. Ninety-three patients were randomized to receive belatacept-MMF, belatacept-sirolimus, or tacrolimus-MMF, and all patients received induction with 4 doses of Thymoglobulin (6 mg/kg maximum) and an associated short course of corticosteroids in the first week after surgery.70 Acute rejection occurred in 15%, 4%, and 3% of patients in the belatacept-MMF, belatacept-sirolimus, and tacrolimus-MMF groups, respectively, by month 12, and most acute rejection episodes occurred in the first 3 months. Importantly, more than two-thirds of patients in the belatacept groups remained on CNI- and steroid-free regimens at 12 months and the calculated GFR was 8 10 mL/minute higher with either belatacept regimen than with tacrolimus-MMF. This pilot study suggests that the combination of a costimulatory blocker and a depletive agent may enable the simultaneous avoidance of CNI and steroids, which might constitute a low-comorbidity immunosuppressive regimen.

19.4 INDUCTION IN HIGH-RISK PATIENTS Candidates for iterative transplants, black race, and highly sensitized patients are considered high immunological risk kidney transplant recipients for the development of cellular and antibody mediated rejection (AMR), and graft loss. Because of this relatively poor prognosis of these types of patients, the KDIGO Guidelines recommended the use of depletive agents in adult high-risk transplant recipients,9 as the benefits of lymphocytedepletive agents outweigh the harm. Sensitization is mainly defined as the presence of pretransplant anti-HLA antibodies, but interestingly transplant candidates may also have T-cell sensitization, especially elderly patients. These patients are also at high risk of early cellular rejection, which can be effectively prevented with rATG.71 ATG forms part of many induction regimens in transplant patients with known humoral sensitization despite that polyclonal ATG mainly targets T cells, but it may also have some anti-B-cell effects, as it may inhibit proliferation of activated B cells and induce apoptosis in human B-cell lines in in vitro studies.72,73 The other depletive agent, alemtuzumab, shares strong T- and B-cell effects, as previously described.35 More recently, there is an increasing interest in specifically targeting B cells with rituximab, which is an anti-CD20 monoclonal antibody, approved for the treatment of nonHodgkin lymphomas.74 Upon binding on CD20 expressed on B cells, rituximab induces direct cell death but also by means of complement-dependent and antibody-dependent cellular cytotoxicity.74 The off-label use of rituximab has been recently investigated in highly sensitized patients. According to the OPTN/UNOS registry data, even the use of nondepletive IL2Ra reduces the risk of graft loss by 48% in repeated kidney transplants.75 According to the same source, collecting data from the previous decade, the use of rATG has increased from 10% to 46% in nonsensitized patients, from 12% to 57% in PRA between 1% and 49% patients, and from 19% to 63% in PRA 5 50% 100% patients. The users of Campath, IVIg, and rituximab have been increasing and reached 16%, 20%, and 11% in highly sensitized patients.76 These trends indicate

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that the optimal induction regimen for highly sensitized patients has not been established yet, as several options are apparently feasible taking into consideration the distinct immunosuppressive properties of the biological agents available. In this regard, the more classical ATG has been compared with alemtuzumab. This comparison was undertaken in a prospective randomized study, which showed no significant differences between ATG or alemtuzumab induction on the rate of acute rejection in high-risk renal transplant recipients.67 Previous uncontrolled studies on the effect of alemtuzumab in highly sensitized patients reported rejection rates of 35% with 20% being C4d1 AMR.77 The simultaneous targeting of T and B cells by combining ATG and rituximab has also been recently explored. In a prospective pilot randomized clinical trial in 40 sensitized patients, induction with ATG was compared with ATG plus rituximab, ATG plus bortezomib, or ATG plus rituximab and bortezomib.78 The overall incidence of acute rejection at 1 year after transplantation was 25%, being 5% acute cellular rejection, 12.5% AMR, and 7.5% mixed rejections. The small numbers in this study do not allow drawing firm conclusions but suggest that the addition of rituximab or bortezomib to ATG has an acceptable safety profile. This association of rATG and rituximab has also been recently reported from a retrospective uncontrolled study with 14 patients with PRA higher than 50%. These patients received two doses of rituximab at the end of induction with ATG, and none of these patients experienced acute rejection compared to 36% of cellular rejection and 26% of AMR in a historical cohort of highly sensitized patients. Despite this positive experience these results have to be interpreted with caution, as this was not a randomized trial. The efficacy and safety of rituximab have been recently studied in a prospective randomized, double-blind, placebo-controlled trial with 280 adult patients.79 In this trial, patients were stratified according to PRA and number of transplants. Despite that rituximab did not reduce the overall incidence of BPAR, highly sensitized patients in the placebo group experienced significantly higher rates of rejection (38%), as compared with rituximab- or placebo-treated immunologically low-risk (PRA # 6% or first transplant) patients (17.9%, 16.4%, and 15.7%, P 5 .004). These data suggest that rituximab should be reserved for sensitized patients and may not add benefits in low-risk patients. Rituximab is also employed in conditioning desensitizing regimens in highly sensitized and in ABO-incompatible transplants.79

19.5 INDUCTION IN THE PREVENTION OF DELAYED GRAFT FUNCTION The mechanistic properties of polyclonal preparations containing antibodies targeting adhesion molecules involved in reperfusion injury,18 their potent immunosuppressive effect that facilitates a delayed introduction of CNI, and their demonstrated protective effect in the prophylaxis of acute rejection, provide the empirical basis for the pre- or intraoperative use of ATG before organ reperfusion in the prevention of DGF. However, a review of comparative trials showed no homogeneous benefits of ATG on this goal.80 Nevertheless, an already reported metaanalysis6 on CNI-sparing regimens under different induction agents showed reduction of DGF. Apparently there are no advantages of polyclonal induction as compared with a more selective depletion with alemtuzumab on the incidence of DGF according to data from a systematic review,5 a randomized monocentric trial,81 and data registry analyses in simultaneous kidney and pancreas transplantation.82

References 1. Keown P, Balshaw R, Khorasheh S, Chong M, Marra C, Kalo Z, et al. Meta-analysis of basiliximab for immunoprophylaxis in renal transplantation. BioDrugs 2003;17:271 9. 2. Adu D, Cockwell P, Ives NJ, Shaw J, Wheatley K. Interleukin-2 receptor monoclonal antibodies in renal transplantation: meta-analysis of randomised trials. BMJ 2003;326:789. 3. Webster AC, Playford EG, Higgins G, Chapman JR, Craig JC. Interleukin 2 receptor antagonists for renal transplant recipients: a metaanalysis of randomized trials. Transplantation 2004;77:166 76. 4. Webster AC, Ruster LP, McGee R, Matheson SL, Higgins GY, Willis NS, et al. Interleukin 2 receptor antagonists for kidney transplant recipients. Cochrane Database Syst Rev 2010;1:CD003897. 5. Morgan RD, O’Callaghan JM, Knight SR, Morris PJ. Alemtuzumab induction therapy in kidney transplantation: a systematic review and meta-analysis. Transplantation 2012;93:1179 88. 6. Sharif A, Shabir S, Chand S, Cockwell P, Ball S, Borrows R. Meta-analysis of calcineurin-inhibitor-sparing regimens in kidney transplantation. J Am Soc Nephrol 2011;22:2107 18. 7. Pascual J, Royuela A, Galeano C, Crespo M, Zamora J. Very early steroid withdrawal or complete avoidance for kidney transplant recipients: a systematic review. Nephrol Dial Transplant 2012;27:825 32. 8. United States Renal Data System. Annual Report. 2014. http://www.usrds.org/2014.

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9. Kidney Disease: Improving Global Outcomes (KDIGO) Transplant Work Group. KDIGO clinical practice guideline for the care of kidney transplant recipients. Am J Transplant 2009;9(Suppl. 3):S1 155. 10. Heemann U, Abramowicz D, Spasovski G, Vanholder R. European Renal Best Practice Work Group on Kidney Transplantation. Endorsement of the Kidney Disease Improving Global Outcomes (KDIGO) guidelines on kidney transplantation: a European Renal Best Practice (ERBP) position statement. Nephrol Dial Transplant 2011;26:2099 106. 11. Kovarik JM, Moore R, Wolf P, Abendroth D, Landsberg D, Soulillou JP, et al. Screening for basiliximab exposure-response relationships in renal allotransplantation. Clin Transplant 1999;13:32 8. 12. Mottershead M, Neuberger J. Daclizumab. Expert Opin Biol Ther 2007;7:1583 96. 13. Kandus A, Arnol M, Omahen K, Oblak M, Vidan-Jeras B, Kmetec A, et al. Basiliximab versus daclizumab combined with triple immunosuppression in deceased donor renal transplantation: a prospective, randomized study. Transplantation 2010;89:1022 7. 14. Lin S, Henning AK, Akhlaghi F, Reisfield R, Vergara-Silva A, First MR. Interleukin-2 receptor antagonist therapy leads to increased tacrolimus levels after kidney transplantation. Ther Drug Monit 2015;37:206 13. 15. Rodrı´guez-Reimundes E, Buron F, Chauvet C, Daoud S, Thaunat O, Brunet M, et al. Retreatment by antithymocyte globulin for second kidney transplantation: efficacy, tolerance and safety. Transpl Immunol 2013;28:6 8. 16. Pre´ville X, Flacher M, LeMauff B, Beauchard S, Davelu P, Tiollier J, et al. Mechanisms involved in antithymocyte globulin immunosuppressive activity in a nonhuman primate model. Transplantation 2001;71:460 8. 17. Ashokkumar C, Sun Q, Ningappa M, Higgs BW, Mazariegos G, Zeevi A, et al. Antithymocyte globulin facilitates alloreactive T-cell apoptosis by means of caspase-3: potential implications for monitoring rejection-free outcomes. Transplantation 2015;99:164 70. 18. Michallet MC, Preville X, Flacher M, Fournel S, Genestier L, Revillard JP. Functional antibodies to leukocyte adhesion molecules in antithymocyte globulins. Transplantation 2003;75:657 62. 19. Longuet H, Sautenet B, Gatault P, Thibault G, Barbet C, Marliere JF, et al. Risk factors for impaired CD4 1 T-cell reconstitution following rabbit antithymocyte globulin treatment in kidney transplantation. Transpl Int 2014;27:271 9. 20. Weimer R, Ettrich M, Renner F, Dietrich H, Su¨sal C, Deisz S, et al. ATG induction in renal transplant recipients: long-term hazard of severe infection is associated with long-term functional T cell impairment but not the ATG-induced CD4 cell decline. Hum Immunol 2014;75:561 9. 21. Bouvy AP, Kho MM, Klepper M, Litjens NH, Betjes MG, Weimar W, et al. Kinetics of homeostatic proliferation and thymopoiesis after rATG induction therapy in kidney transplant patients. Transplantation 2013;96:904 13. 22. Gurkan S, Luan Y, Dhillon N, Allam SR, Montague T, Bromberg JS, et al. Immune reconstitution following rabbit antithymocyte globulin. Am J Transplant 2010;10:2132 41. 23. Bouvy AP, Klepper M, Kho MM, Boer K, Betjes MG, Weimar W, et al. The impact of induction therapy on the homeostasis and function of regulatory T cells in kidney transplant patients. Nephrol Dial Transplant 2014;29:1587 97. 24. Lopez M, Clarkson MR, Albin M, Sayegh MH, Najafian N. A novel mechanism of action for anti-thymocyte globulin: induction of CD4 1 CD25 1 Foxp3 1 regulatory T cells. J Am Soc Nephrol 2006;17:2844 53. 25. Valdez-Ortiz R, Bestard O, Llaudo´ I, Franquesa M, Cerezo G, Torras J, et al. Induction of suppressive allogeneic regulatory T cells via rabbit antithymocyte polyclonal globulin during homeostatic proliferation in rat kidney transplantation. Transpl Int 2015;28:108 19. 26. Boenisch O, Lopez M, Elyaman W, Magee CN, Ahmad U, Najafian N. Ex vivo expansion of human Tregs by rabbit ATG is dependent on intact STAT3-signaling in CD41 T cells and requires the presence of monocytes. Am J Transplant 2012;12:856 66. 27. Gillet-Hladky S, de Carvalho CM, Bernaud J, Bendahou C, Bloy C, Rigal D. Rabbit antithymocyte globulin inhibits monocyte-derived dendritic cells maturation in vitro and polarizes monocyte-derived dendritic cells towards tolerogenic dendritic cells expressing indoleamine 2,3-dioxygenase. Transplantation 2006;82:965 74. 28. Leitner J, Grabmeier-Pfistershammer K, Majdic O, Zlabinger G, Steinberger P. Interaction of antithymocyte globulins with dendritic cell antigens. Am J Transplant 2011;11:138 45. 29. Naujokat C, Berges C, Fuchs D, Sadeghi M, Opelz G, Daniel V. Antithymocyte globulins suppress dendritic cell function by multiple mechanisms. Transplantation 2007;83:485 97. 30. Sanofi-Aventis Canada. Thymoglobulins (Anti-thymocyte Globulin [Rabbit]) Product Monograph. Version 1.0, May 6, 2013. 31. Gaber AO, Monaco AP, Russell JA, Lebranchu Y, Mohty M. Rabbit antithymocyte globulin (thymoglobulin): 25 years and new frontiers in solid organ transplantation and haematology. Drugs 2010;70:691 732. 32. Popow I, Leitner J, Majdic O, Kovarik JJ, Saemann MD, Zlabinger GJ, et al. Assessment of batch to batch variation in polyclonal antithymocyte globulin preparations. Transplantation 2012;93:32 40. 33. Gharekhani A, Entezari-Maleki T, Dashti-Khavidaki S, Khalili H. A review on comparing two commonly used rabbit anti-thymocyte globulins as induction therapy in solid organ transplantation. Expert Opin Biol Ther 2013;13:1299 313. 34. Morton RL, Howard K, Webster AC, Wong G, Craig JC. The cost-effectiveness of induction immunosuppression in kidney transplantation. Nephrol Dial Transplant 2009;24:2258 69. 35. Waldmann H. A personal history of the CAMPATH-1H antibody. Med Oncol 2002;19(Suppl):S3 9. 36. Li Y, Zhu Z. Monoclonal antibody-based therapeutics for leukemia. Expert Opin Biol Ther 2007;7:319 30. 37. Hersh CM, Cohen JA. Alemtuzumab for the treatment of relapsing-remitting multiple sclerosis. Immunotherapy 2014;6:249 59. 38. LaMattina JC, Mezrich JD, Hofmann RM, Foley DP, D’Alessandro AM, Sollinger HW, et al. Alemtuzumab as compared to alternative contemporary induction regimens. Transpl Int 2012;25:518 26. 39. Heidt S, Hester J, Shankar S, Friend PJ, Wood KJ. B cell repopulation after alemtuzumab induction-transient increase in transitional B cells and long-term dominance of naı¨ve B cells. Am J Transplant 2012;12:1784 92. 40. Todeschini M, Cortinovis M, Perico N, Poli F, Innocente A, Cavinato RA, et al. In kidney transplant patients, alemtuzumab but not basiliximab/low-dose rabbit anti-thymocyte globulin induces B cell depletion and regeneration, which associates with a high incidence of de novo donor-specific anti-HLA antibody development. J Immunol 2013;191:2818 28. 41. Hurst FP, Altieri M, Nee R, Agodoa LY, Abbott KC, Jindal RM. Poor outcomes in elderly kidney transplant recipients receiving alemtuzumab induction. Am J Nephrol 2011;34:534 41.

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42. Schold J, Poggio E, Goldfarb D, Kayler L, Flechner S. Clinical outcomes associated with induction regimens among retransplant kidney recipients in the United States. Transplantation 2015;99(6):1165 71. 43. 3C Study Collaborative Group, Haynes R, Harden P, Judge P, Blackwell L, Emberson J, Landray MJ, et al. Alemtuzumab-based induction treatment versus basiliximab-based induction treatment in kidney transplantation (the 3C Study): a randomised trial. Lancet 2014;384:1684 90. 44. Grinyo´ JM, Gil-Vernet S, Cruzado JM, Calde´s A, Riera L, Sero´n D, et al. Calcineurin inhibitor-free immunosuppression based on antithymocyte globulin and mycophenolate mofetil in cadaveric kidney transplantation: results after 5 years. Transpl Int 2003;16:820 7. 45. Vincenti F, Ramos E, Brattstrom C, Cho S, Ekberg H, Grinyo J, et al. Multicenter trial exploring calcineurin inhibitors avoidance in renal transplantation. Transplantation 2001;71:1282 7. 46. Ekberg H, Grinyo´ J, Nashan B, Vanrenterghem Y, Vincenti F, Voulgari A, et al. Cyclosporine sparing with mycophenolate mofetil, daclizumab and corticosteroids in renal allograft recipients: the CAESAR Study. Am J Transplant 2007;7:560 70. 47. Ekberg H, Tedesco-Silva H, Demirbas A, Vı´tko S, Nashan B, Gu¨rkan A, et al. Reduced exposure to calcineurin inhibitors in renal transplantation. N Engl J Med 2007;357:2562 75. 48. Ekberg H, Bernasconi C, Tedesco-Silva H, Vı´tko S, Hugo C, Demirbas A, et al. Calcineurin inhibitor minimization in the Symphony study: observational results 3 years after transplantation. Am J Transplant 2009;9:1876 85. 49. Demirbas A, Hugo C, Grinyo´ J, Frei U, Gu¨rkan A, Marce´n R, et al. Low toxicity regimens in renal transplantation: a country subset analysis of the Symphony study. Transpl Int 2009;22:1172 81. 50. Budde K, Becker T, Arns W, Sommerer C, Reinke P, Eisenberger U, et al. Everolimus-based, calcineurin-inhibitor-free regimen in recipients of de-novo kidney transplants: an open-label, randomised, controlled trial. Lancet 2011;377:837 47. 51. Budde K, Lehner F, Sommerer C, Reinke P, Arns W, Eisenberger U, et al. Five-year outcomes in kidney transplant patients converted from cyclosporine to everolimus: the randomized ZEUS study. Am J Transplant 2015;15:119 28. 52. Guba M, Pratschke J, Hugo C, Kra¨mer BK, Pascher A, Pressmar K, et al. Early conversion to a sirolimus-based, calcineurin-inhibitor-free immunosuppression in the SMART trial: observational results at 24 and 36 months after transplantation. Transpl Int 2012;25:416 23. 53. Guba M, Pratschke J, Hugo C, Kra¨mer BK, Nohr-Westphal C, Brockmann J, et al. Renal function, efficacy, and safety of sirolimus and mycophenolate mofetil after short-term calcineurin inhibitor-based quadruple therapy in de novo renal transplant patients: one-year analysis of a randomized multicenter trial. Transplantation 2010;90:175 83. 54. Nashan B, Gaston R, Emery V, Sa¨emann MD, Mueller NJ, Couzi L, et al. Review of cytomegalovirus infection findings with mammalian target of rapamycin inhibitor-based immunosuppressive therapy in de novo renal transplant recipients. Transplantation 2012;93:1075 85. 55. Andrassy J, Hoffmann VS, Rentsch M, Stangl M, Habicht A, Meiser B, et al. Is cytomegalovirus prophylaxis dispensable in patients receiving an mTOR inhibitor-based immunosuppression? a systematic review and meta-analysis. Transplantation 2012;94:1208 17. 56. Grinyo´ JM, Cruzado JM. Mycophenolate mofetil and sirolimus combination in renal transplantation. Am J Transplant 2006;6:1991 9. 57. Tedesco Silva Jr H, Cibrik D, Johnston T, Lackova E, Mange K, Panis C, et al. Everolimus plus reduced-exposure CsA versus mycophenolic acid plus standard-exposure CsA in renal-transplant recipients. Am J Transplant 2010;10:1401 13. 58. Advancing Renal TRANSplant eFficacy and Safety Outcomes With an eveRolimus-based regiMen (TRANSFORM). ClinicalTrials.gov NCT01950819. 59. Moons P, Vanrenterghem Y, van Hooff JP, Squifflet JP, Margodt D, Mullens M, et al. Steroids may compromise quality of life of renal transplant recipients on a tacrolimus-based regimen. Transplant Proc 2002;34:1691 2. 60. Opelz G, Do¨hler B. Association between steroid dosage and death with a functioning graft after kidney transplantation. Am J Transplant 2013;13:2096 105. 61. Kasiske BL, Chakkera HA, Louis TA, Ma JZ. A meta-analysis of immunosuppression withdrawal trials in renal transplantation. J Am Soc Nephrol 2000;11:1910 17. 62. Hricik DE, Whalen CC, Lautman J, Bartucci MR, Moir EJ, Mayes JT, et al. Withdrawal of steroids after renal transplantation--clinical predictors of outcome. Transplantation 1992;53:41 5. 63. Luan FL, Steffick DE, Gadegbeku C, Norman SP, Wolfe R, Ojo AO. Graft and patient survival in kidney transplant recipients selected for de novo steroid-free maintenance immunosuppression. Am J Transplant 2009;9:160 8. 64. Vincenti F, Monaco A, Grinyo J, Kinkhabwala M, Roza A. Multicenter randomized prospective trial of steroid withdrawal in renal transplant recipients receiving basiliximab, cyclosporine microemulsion and mycophenolate mofetil. Am J Transplant 2003;3:306 11. 65. Vincenti F, Schena FP, Paraskevas S, Hauser IA, Walker RG, Grinyo J, FREEDOM Study Group. A randomized, multicenter study of steroid avoidance, early steroid withdrawal or standard steroid therapy in kidney transplant recipients. Am J Transplant 2008;8:307 16. 66. Hricik DE. Comparing early withdrawal or avoidance of steroids with standard steroid therapy in kidney transplant recipients. Nat Clin Pract Nephrol 2008;4:360 1. 67. Hanaway MJ, Woodle ES, Mulgaonkar S, Peddi VR, Kaufman DB, First MR, et al. Alemtuzumab induction in renal transplantation. N Engl J Med 2011;364:1909 19. 68. Woodle ES, Peddi VR, Tomlanovich S, Mulgaonkar S, Kuo PC, TRIMS Study Investigators. A prospective, randomized, multicenter study evaluating early corticosteroid withdrawal with Thymoglobulin in living-donor kidney transplantation. Clin Transplant 2010;24:73 83. 69. Matas AJ, Granger D, Kaufman DB, Sarwal MM, Ferguson RM, Woodle ES, et al. Steroid minimization for sirolimus-treated renal transplant recipients. Clin Transplant 2011;25:457 67. 70. Ferguson R, Grinyo´ J, Vincenti F, Kaufman DB, Woodle ES, Marder BA, et al. Immunosuppression with belatacept-based, corticosteroidavoiding regimens in de novo kidney transplant recipients. Am J Transplant 2011;11:66 76. 71. Crespo E, Lucia M, Cruzado JM, Luque S, Melilli E, Manonelles A, et al. Pre-transplant donor-specific T-cell alloreactivity is strongly associated with early acute cellular rejection in kidney transplant recipients not receiving T-cell depleting induction therapy. PLoS One 2015;10:e0117618. 72. Bonnefoy-Be´rard N, Genestier L, Flacher M, Rouault JP, Lizard G, Mutin M, et al. Apoptosis induced by polyclonal antilymphocyte globulins in human B-cell lines. Blood 1994;83:1051 9. 73. Bonnefoy-Berard N, Flacher M, Revillard JP. Antiproliferative effect of antilymphocyte globulins on B cells and B-cell lines. Blood 1992;79:2164 70.

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74. Abulayha A, Bredan A, El Enshasy H, Daniels I. Rituximab: modes of action, remaining dispute and future perspective. Future Oncol 2014;10:2481 92. 75. Freitas MC. Kidney transplantation in the US: an analysis of the OPTN/UNOS registry. Clin Transpl 2011;1 16. 76. Cai J, Terasaki PI. The current trend of induction and maintenance treatment in patient of different PRA levels: a report on OPTN/UNOS Kidney Transplant Registry data. Clin Transpl 2010;45 52. 77. Vo AA, Wechsler EA, Wang J, Peng A, Toyoda M, Lukovsky M, et al. Analysis of subcutaneous (SQ) alemtuzumab induction therapy in highly sensitized patients desensitized with IVIG and rituximab. Am J Transplant 2008;8:144 9. 78. Ejaz NS, Shields AR, Alloway RR, Sadaka B, Girnita AL, Mogilishetty G, et al. Randomized controlled pilot study of B cell-targeted induction therapy in HLA sensitized kidney transplant recipients. Am J Transplant 2013;13:3142 54. 79. van den Hoogen MW, Kamburova EG, Baas MC, Steenbergen EJ, Florquin S, et al. Rituximab as induction therapy after renal transplantation: a randomized, double-blind, placebo-controlled study of efficacy and safety. Am J Transplant 2015;15:407 16. 80. Thiyagarajan UM, Ponnuswamy A, Bagul A. Thymoglobulin and its use in renal transplantation: a review. Am J Nephrol 2013;37:586 601. 81. Farney AC, Doares W, Rogers J, Singh R, Hartmann E, Hart L, et al. A randomized trial of alemtuzumab versus antithymocyte globulin induction in renal and pancreas transplantation. Transplantation 2009;88:810 19. 82. Zachariah M, Gregg A, Schold J, Magliocca J, Kayler LK. Alemtuzumab induction in simultaneous pancreas and kidney transplantation. Clin Transplant 2013;27:693 700.

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C H A P T E R

20 Maintenance Immunosuppression in Kidney Transplantation Monica Cortinovis1, Giuseppe Remuzzi1,2,3 and Norberto Perico1 1

IRCCS Mario Negri Institute for Pharmacological Research, Bergamo, Italy 2Azienda Socio Sanitaria Territoriale Papa Giovanni XXIII, Bergamo, Italy 3University of Milan, Milan, Italy

20.1 INTRODUCTION The primary challenge in organ transplantation continues to be the need to suppress the host immune system so as to ensure long-term allograft survival. Immunosuppressive agents used in kidney transplantation act by inhibiting one or more key steps in the alloimmune response that would otherwise culminate in rejection. The immune response that leads to recognition of the allograft involves a highly orchestrated action of multiple cell types and mediators.1 This process is initiated by activation of antigen presenting cells (APCs), mainly dendritic cells, through innate immune-recognition systems. Once activated, APCs of either donor or host origin move to secondary lymphoid organs, such as lymph nodes and spleen, where they present donor antigen to naı¨ve T cells. The activation of T cells requires three critical events (Fig. 20.1). In particular, the first signal is antigen-specific and it is initiated by the recognition of the donor antigen in the context of major histocompatibility complex molecules on the surface of APC by the T-cell receptor (TCR)/CD3 complex on the T cell. The second costimulatory signal (signal 2) depends on receptor ligand interactions between T cells and APCs. Although several costimulatory pathways have been identified, the best characterized is the interaction of CD28 on the T-cell surface with CD80 and CD86 expressed on activated APCs. If antigen stimulation and costimulation occur, three downstream intracellular signal transduction pathways are activated, which in turn play a key role in activating factors required for interleukin 2 (IL-2) gene transcription.1 IL-2 and other cytokines ultimately drive cell-cycle progression (signal 3) and proliferation with help from a series of kinases, including members of the mammalian target of rapamycin (mTOR) pathway. The final result is the proliferation of CD41 helper T (Th) cells and the maturation of CD81 cytotoxic T cells. Both CD41 and CD81 T cells can be divided into subsets defined by the pattern of cytokines they produce following activation. In particular, CD41 Th cells may be of the Th1 subset (producing, e.g., IL-2 and interferon-γ) or the Th2 subset (producing, e.g., IL-4 and IL-15). Activated T cells can eventually differentiate into a number of phenotypes, including memory cells, which are able to respond quickly and robustly to the original antigen, and regulatory cells, which can suppress immune response and promote tolerance.2 B cells also express antigen-specific receptors and are activated in lymphoid tissues. The majority of B cells depend on Th2 cell help for activation and production of antibodies against donor antigens.3 The contribution of B cells to the alloimmune response also involves antigen presentation to alloreactive T cells and the secretion of proinflammatory cytokines.4 Ultimately, if not recognized and managed, all these cellular and humoral factors constitute the rejection process that injures the allograft, via a T-cell-mediated and/or antibodymediated process.1 Effective immunosuppression is achieved by targeting these cells and mediators at multiple levels.

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FIGURE 20.1 The three signals required for T-cell activation. Graphic representation of the three signals required for full T-cell activation and proliferation. The first signal (signal 1) is antigen-specific and it is initiated by the recognition of the donor antigen in the context of major histocompatibility complex molecules on the surface of antigen presenting cells by the T-cell receptor/CD3 complex on the T cell. The second costimulatory signal (signal 2) depends on receptor ligand interactions between T cells and antigen presenting cells. If antigen stimulation and costimulation occurs, three downstream intracellular signaling pathways are activated, which in turn play a role in activating factors required for cytokine transcription. Interleukin 2 and other cytokines ultimately drive cell-cycle progression (signal 3) and proliferation with help from a series of kinases, including members of the mammalian target of rapamycin pathway. AP-1, activator protein-1; CDK, cyclindependent kinase; IL, interleukin; IKK, IκB kinase; MAP, mitogen-activated protein; MHC, major histocompatibility class; NFAT, nuclear factor of activated T cells; NF-κB, nuclear factor-κB; TCR, T-cell receptor.

20.2 IMMUNOSUPPRESSIVE AGENTS TO TARGET THE ALLOIMMUNE RESPONSE Although immunosuppressive protocols differ among transplantation centers, most regimens include an induction therapy and long-term maintenance immunosuppression. Induction therapy refers to using specific biologic agents (i.e., antilymphocyte antibodies) on a short-term basis during the perioperative period. Currently, two polyclonal antibody preparations (rabbit antithymocyte globulin and horse antithymocyte globulin) and two monoclonal antibodies (basiliximab and alemtuzumab) are used as induction agents. Induction therapies are covered in depth in Chapter 19, Induction Immunosuppression in Kidney Transplantation. Maintenance therapy is required for the lifetime of functioning allograft to prevent rejection of the transplanted kidney. Agents with different mechanisms of action are used in combination, so as to fully harness their synergistic effects on the immune system, while reducing the side effects associated with each medication. Since the risk of acute rejection is the greatest in the first postoperative months, more intensive immunosuppression is needed during this period, followed by progressive reduction in stable patients to minimize toxicity. The maintenance immunosuppressive agents currently used can be divided into five classes: (1) corticosteroids, (2) calcineurin inhibitors (cyclosporine and tacrolimus), (3) antiproliferative agents (azathioprine, mycophenolate mofetil, and enteric-coated mycophenolate sodium), (4) mammalian target of rapamycin inhibitors (sirolimus and everolimus), and (5) costimulatory blockers (belatacept). In renal transplant recipients, maintenance therapy generally includes a calcineurin inhibitor and an antiproliferative agent, with or without corticosteroids. Selection of the appropriate regimen should be guided by a comprehensive assessment of patient comorbidities and immunological risk, as well as the medication’s pharmacological properties, adverseevent profile, and costs.

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20.3 CORTICOSTEROIDS Corticosteroids have been used for the prevention and treatment of acute rejection since the early 1960s. Two specific medications, methylprednisolone and prednisone, are commonly employed in kidney transplantation. In addition to nonspecific antiinflammatory actions, corticosteroids have critical immunosuppressive effects by blocking APC-derived and T-cell-derived cytokine expression (Fig. 20.2). In particular, these drugs bind to cytoplasmic receptors to form a complex, which translocates into the nucleus and binds to glucocorticoid response elements in the promoter region of target genes, leading to either induction or suppression of gene transcription (e.g., of cytokines). Corticosteroids also inhibit the activity of transcription factors, such as activated protein 1 and nuclear factor-kB. The net result of these intracellular effects is inhibition of cytokine production (e.g., IL-1, IL-2, IL-6, IL-8, tumor necrosis factor-α, interferon-γ), lymphocyte proliferation (cell cycle arrest/apoptosis), and cell trafficking.5 In many transplant centers, high dose of intravenous corticosteroids (i.e., methylprednisolone) are administered in the perioperative period followed by oral prednisone, with tapering over the ensuing weeks to long-term maintenance doses of 5 10 mg/daily. Prolonged use of corticosteroids is associated with a variety of side effects, including increased susceptibility to infections, fluid retention and hypertension, posttransplant glucose intolerance/newonset diabetes mellitus, hyperlipidemia, cataracts, loss of bone mineral density, increased rate of bone fractures and avascular necrosis, weight gain, mood swings, and growth retardation in pediatric patients.6 Several protocols based on corticosteroid withdrawal or avoidance after kidney transplantation have been investigated in the attempt to circumvent these adverse events without compromising long-term graft survival. Early experiences with corticosteroid withdrawal using cyclosporine (CsA) in combination with azathioprine (AZA) or mycophenolate mofetil (MMF) were unsuccessful in preventing acute rejection or graft loss.7,8 On the other hand, it has been reported that protocol renal biopsy performed more than 1 year after kidney transplantation allowed the identification of patients without histologic lesions in whom corticosteroid withdrawal could be accomplished safely and effectively.9 The availability of potent induction agents has raised the question of whether these medications could reduce the need of or replace corticosteroid treatment. To address this issue, Woodle et al. randomized 386 kidney transplant patients receiving induction therapy with anti-IL-2 receptor antibody or thymoglobulin,

FIGURE 20.2 Maintenance immunosuppressive drugs and T cell signaling. Graphic representation of the three signals required for full T-cell activation and proliferation, and the site of action of the maintenance immunosuppressive agents currently used in the kidney transplant setting. AP-1, activator protein-1; CDK, cyclin-dependent kinase; IL, interleukin; IKK, IκB kinase; MAP, mitogen-activated protein; MHC, major histocompatibility class; MPA, mycophenolic acid; NFAT, nuclear factor of activated T cells; NF-κB, nuclear factor-κB; TCR, T-cell receptor.

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tacrolimus (TAC), and MMF to either early corticosteroid withdrawal (within 7 days posttransplant) or corticosteroid maintenance treatment. Patients who discontinued corticosteroids reported a higher rate of biopsy-proven acute rejection (BPAR) (18% vs 11%, p 5 .058) and a doubling in the incidence of chronic allograft nephropathy (10% vs 4%, p 5 .028), but 5-year patient and graft survival were comparable between the two groups.10 Moreover, in the FREDOOM trial, 336 kidney transplant patients receiving basiliximab, CsA and enteric-coated mycophenolate sodium (EC-MPS) were randomized to no corticosteroids, rapid discontinuation of corticosteroids (within 7 days posttransplant), or corticosteroid maintenance therapy.11 At 12 months the incidence of BPAR was significantly less frequent with standard-dose corticosteroids (15%) than either corticosteroid avoidance or withdrawal (32% and 26%, respectively; P , .05). Conversely, a recent meta-analysis suggested that early corticosteroid withdrawal or avoidance was well tolerated in low-immunological-risk kidney transplant patients receiving induction therapy, MMF, and TAC up to 5 years posttransplant, the longest follow-up reported.12 However, it is difficult to draw firm conclusions from these data due to heterogeneity in the studies included in the meta-analysis as for induction strategy and maintenance immunosuppressive regimen.12 The potential benefits of corticosteroid withdrawal or avoidance on the cardiovascular risk profile have been addressed in several studies. However, discontinuing or avoiding corticosteroids has not resulted in consistent effects on change in blood pressure control, serum lipid levels, or incidence of new-onset diabetes after transplantation.10 15 Due to this lack of conclusive evidence regarding early corticosteroid withdrawal or avoidance in kidney transplant recipients, recommendations given in different guidelines have been conflicting. In particular, the Kidney Disease: Improving Global Outcomes (KDIGO) clinical practice guidelines state that corticosteroids may be discontinued during the first week after transplantation in low-immunological-risk patients who received induction therapy,16 whereas the Australian Caring for Australians with Renal Impairment guidelines propose continuous treatment with low-dose corticosteroids.17

20.4 CALCINEURIN INHIBITORS 20.4.1 Cyclosporine The discovery of the first calcineurin inhibitor (CNI), cyclosporine (CsA), represents a watershed event in the history of immunosuppression, as it was the first drug shown to reversibly inhibit T-lymphocyte function, therefore allowing for one of the major breakthroughs in modern medicine, that of organ transplantation.18,19 CsA is a cyclic endecapeptide isolated from the soil fungus Tolypocladium inflatum.20 In the cytoplasm, CsA binds to cyclophylin, a protein of the immunophilin family, to form a complex that targets and inactivates the serine-threonine phosphatase calcineurin (Fig. 20.2). As a result, calcineurin fails to dephosphorylate the cytoplasmic component of the transcription factor nuclear factor of activated T cell (NFAT), thereby hampering its translocation into the nucleus. Inhibition of the NFAT passage through the nuclear membrane prevents the transcription of genes coding for IL-2 and other cytokines required for full T-cell activation.21 CsA is available in oral and intravenous formulations. The original oil-based oral CsA preparation, although representing a significant advancement in immunosuppressive therapy, was characterized by slow absorption and variable bioavailability, making dosing difficult and increasing the risk of chronic allograft rejection.22 Therefore, a modified microemulsion formulation was developed, allowing increased and more consistent CsA absorption in both de novo and stable transplant patients.23,24 In addition, randomized studies in de novo kidney transplant recipients documented lower rates of acute rejection with CsA microemulsion compared to the original preparation.23,25 Currently, CsA microemulsion is the preferred formulation for most kidney transplant centers that continue to use CsA-based maintenance regimens. The initial oral dosage of CsA after kidney transplantation is 4 5 mg/kg twice daily and monitoring of blood trough levels (12 hours after drug administration) or concentrations 2 hours after drug administration is required to adjust CsA dosage.26 Side effects of CsA include acute and chronic nephrotoxicity, hyperlipidemia, hypertension, hypertrichosis, gingival hyperplasia, thrombotic microangiopathy, electrolyte disorders (hyperkalemia, hypomagnesemia, hyperuricemia), neurotoxicity, and glucose intolerance.26

20.4.2 Tacrolimus CsA remained unchallenged until the introduction of another CNI, tacrolimus (TAC), a macrolide lactone antibiotic produced by the soil fungus Streptomyces tsukubaensis.27,28 This drug binds to the immunophilin FK506 binding protein-12 (FKBP12), to create a complex that inhibits calcineurin with greater molar potency than does

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cyclosporine (Fig. 20.2).29 TAC is available in oral and intravenous formulations. The recommended oral dosage after renal transplantation is 0.2 mg/kg daily (range, 0.1 0.3 mg/kg daily) given in two divided doses,30 and monitoring of trough levels is required to adjust TAC dose. The TAC side effect profile is somewhat similar to that of CsA, in that it can cause acute and chronic nephrotoxicity, thrombotic microangiopathy, and electrolyte disorders. However, some adverse events, including hypertension, hyperlipidemia, hirsutism, and gingival hyperplasia are more commonly encountered with CsA treatment, whereas neurotoxicity and new-onset diabetes after transplantation are more frequent with TAC.26 A once-daily extended-release tacrolimus formulation has been developed and was approved in 2013 by the Food and Drug Administration (FDA). The expectation is that a less frequent drug-dosing regimen could improve patient adherence to medication, and eventual long-term graft outcomes. A meta-analysis of six randomized controlled trials showed that prolonged-release TAC was as effective as the standard twice-daily formulation in the prevention of BPAR and graft failure up to 12 months after kidney transplantation.31 However, during the first 6 weeks posttransplant mean trough TAC levels were 40% lower in patients receiving extended-release tacrolimus compared to those given the conventional twice-daily preparation. These findings highlight the need for closely monitoring tacrolimus trough concentrations in patients receiving the prolonged-release formulation adjusting, if necessary, the dose in order to guarantee adequate systemic exposure.31

20.4.3 Studies Comparing CsA and TAC With two commercially available CNIs, CsA and TAC, the transplantation community has committed many resources in efforts to identify the superior agent. In a multicenter, randomized European trial, the rate of BPAR during the first 6 months after kidney transplant was significantly lower with tacrolimus than with the microemulsion formulation of cyclosporine (20% vs 37%, P , .0001), but there was no difference in patient or graft survival between the two groups.32 Webster et al. analyzed 30 randomized controlled trials providing data from over 4000 patients, in an attempt to differentiate the relative efficacy and safety of TAC and CsA in kidney transplant recipients.33 This meta-analysis showed that TAC treatment was associated with a significantly lower rate of graft loss compared to CsA at 6 months (relative risk (RR) 0.56, 95% confidence interval (CI) 0.36 0.86), and the benefit persisted up to 3 years posttransplantation. At 1 year, tacrolimus use also resulted in lower rate of acute rejection (RR 0.69, 95% CI 0.60 0.79) and corticosteroid-resistant acute rejection (RR 0.49, 95% CI 0.37 0.64). When considering the safety profile of the two drugs, TAC-treated patients had a higher incidence of posttransplant diabetes mellitus requiring insulin, neurologic, and gastrointestinal side effects, whereas patients receiving CsA complained of more constipation and cosmetic-related adverse events.33 By contrast, two retrospective studies of the United States Renal Data System data found that there was either no difference in allograft survival34 or improved allograft survival with CsA compared to TAC.35 Despite the lack of solid evidence favoring one agent over the other, the use of TAC has steadily increased since the 1990s, while the number of kidney transplant patients treated with CsA preparations declined during the same period.36

20.4.4 CNI-Sparing Strategies CNIs have undeniably improved short-term outcomes in renal transplantation by their ability to reduce acute rejection rates, but long-term graft survival has not increased to an appreciable extent.37 Although controversial, evidence does suggest a role of chronic CNI nephrotoxicity in the failure to improve long-term kidney graft outcome. Thus, much focus has been placed in the investigation of immunosuppressive strategies that enable reduced CNI exposure (Table 20.1). Strategies to limit CNI exposure after transplantation include CNI minimization, avoidance, and withdrawal. In the CEASAR study, 536 renal transplant patients receiving MMF and corticosteroids were randomized to one of three groups: standard-dose CsA; daclizumab induction and low-dose CsA; or daclizumab induction with low-dose CsA, which was subsequently withdrawn 6 months after transplantation.38 At 12 months mean glomerular filtration rate (GFR) was not statistically different among the groups; however, the frequency of BPAR was significantly higher in the CsA withdrawal arm (38%) compared to the group with either standard-dose or low-dose CsA (28% and 25%, respectively; P , .05).38 The same investigators also explored the use of low-dose CNI in the largest prospective study to date in de novo renal transplantation. In particular, in the ELITESYMPHONY trial 1645 renal transplant recipients were randomized to receive either standard-dose CsA, low-dose CsA, low-dose TAC, or low-dose SRL in addition to MMF and steroids, all with daclizumab induction except the standard-dose CsA group. At 1 year posttransplant, patients in the low-dose TAC arm exhibited the highest graft

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TABLE 20.1

Main Studies on CNI-Sparing Regimens in Kidney Transplant Recipients

Study

Induction therapy

Maintenance therapy in addition to corticosteroids

Patients (n )

Acute rejection (%)

Follow-up (months)

Graft survival (%)

GFR (mL/ min)

CNI Minimization Ekberg et al.38

Daclizumab in CsA withdrawal and low-dose CsA

STD-dose CsA 1 MMF

173

Low-dose CsA 1 MMF

183

CsA withdrawal 1 MMF Ekberg et al.39

Cibrik et al.40

Daclizumab in all groups except for STD-dose CsA

Basiliximab

12

27.5

94.8

48.6

25.4

96.7

50.9

93.3

50.9

a

179

38

STD-dose CsA 1 MMF

390

Low-dose CsA 1 MMF

399

27.2f

94.3

59.4e

Low-dose TAC 1 MMF

401

15.4

96.4

65.4

Low-dose SRL 1 MMF

399

STD-dose CsA 1 MMF

277

Low-dose CsA 1 EVR (3.0 mg/day) Low-dose CsA 1 EVR (1.5 mg/day)

12

f

30.1

91.9

57.1f

f

d

40.2

91.7

56.7f

19.1

96.0

50.5

279

15.1

93.9

49.4

277

19.9

94.2

52.2

246

6.5

93.9

52.1

SRL conversion 1 MMF/AZA 497

7.8

92.4

53.7

11.3

96.0

71.2

9.5

98.0

75.5

2.4

95.1

46.0

24

d

Conversion from CNI to mTOR inhibitors Schena et al.41

NA

In patients with GFR .40 mL/min CNI continuation 1 MMF/ AZA

24

Weir et al.107

Thymoglobulin or anti-IL-2 receptor antibodies or Muromonab

CNI continuation 1 MMF

151

SRL conversion 1 MMF

148

Holdaas et al.42

NA

STD-dose CNI 1 MMF/AZA

123

Low-dose CNI 1 MMF/AZA

144

5.6

94.4

46.6

EVR conversion 1 MMF/AZA 127

5.5

96.9

48.0

3

100

61.9

10b

100

71.8c

5.1

99.0

53.5

25.0c

94.8

60.1b

Budde et al.43

Basiliximab

Rostaing et al.44

Basiliximab

CNI continuation 1 EC-MPS

145

EVR conversion 1 EC-MPS

155

CsA continuation 1 STD-dose EC-MPS

98

EVR conversion 1 low-dose EC-MPS

96

24

24

12

12

P , .05 versus STD-dose CsA and CsA withdrawal. P , .05 versus CsA continuation. c P , .001 versus CsA continuation. d P , .05 versus TAC. e P 5 .001 versus TAC. f P , .001 versus TAC. AZA, azathioprine; CNI, calcineurin inhibitors; CsA, cyclosporine; EC-MPS, enteric-coated mycophenolate sodium; EVR, everolimus; GFR, glomerular filtration rate; MMF, mycophenolate mofetil; mTOR, mammalian target of rapamycin; NA, not available; SRL, sirolimus; STD, standard. a

b

function (65 6 27 mL/minute) and rate of allograft survival (94%), and the lowest incidence of BPAR (12%) compared to all other groups.39 At 3-year follow-up the low-dose TAC group continued to enjoy the highest graft function and the best graft survival rate.45 A meta-analysis of 56 randomized clinical trials providing data for 11,337 renal transplant recipients, compared three CNI-sparing strategies, i.e., avoidance, minimization, or delayed

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265

introduction, with CNI-based regimens.46 CNI avoidance, achieved by substitution of CNI with the combination of mTOR inhibitors and MMF, resulted in an increased incidence of graft failure (odd ratio (OR) 1.43, 95% CI 1.08 1.90, p 5 .01). By contrast, CNI minimization was associated with reduced graft failure rate (OR 0.73, 95% CI 0.58 0.92, p 5 .009) and improved graft function compared to standard CNI exposure.46 Recently, concern was raised about the possibility of de novo donor-specific anti-HLA antibodies (DSA) development in CNI-sparing regimens.47,48 Indeed, evidence is available of a strong association between decreased overall immunosuppression (due to either noncompliance or immunosuppressive-sparing regimens) and occurrence of de novo DSA, which are known to threaten long-term graft longevity.49,50 Along this line, two studies reported that early conversion from CNI to mTOR inhibitors was associated with increased risk of de novo DSA development and/or AMR.44,51 Overall, these studies suggest that in renal transplant recipients de novo immunosuppressive therapy based on CNI minimization, but not CNI avoidance, may have a beneficial effect on graft function while maintaining acceptable rates of acute rejection. However, it is of utmost importance to balance the risks of underimmunosuppression and eventual de novo DSA development with risks of overimmunosuppression and nephrotoxicity.

20.5 ANTIPROLIFERATIVE AGENTS 20.5.1 Azathioprine Azathioprine (AZA) was the first immunosuppressive agent to achieve widespread use in kidney transplantation. This drug inhibits T- and B-cell proliferation, and it reduces the number of circulating monocytes by arresting cell cycle of promyelocytes in the bone marrow.30 The antiproliferative effect is mediated by AZA metabolites, which include 6-mercaptopurine, 6-thiouric acid, 6-methyl-mercaptopurine, and 6-thioguanine. These compounds interfere with DNA replication and inhibit multiple enzymes involved in the de novo synthesis of purines (Fig. 20.2).52 The latter effect confers specificity of action on lymphocytes, which lack a salvage pathway for purine synthesis. Azathioprine metabolites also block CD28 costimulatory signaling, thereby inducing T-cell apoptosis.53 Azathioprine is available in oral and intravenous formulations, and its typical oral dosages as maintenance immunosuppressive agent range from 1 to 2.5 mg/kg body weight/day, adjusted for leucopenia.54 The primary adverse effects of this drug are dose-related bone marrow suppression (leucopenia, thrombocytopenia, and anemia) and gastrointestinal disturbance. Other side effects include increased susceptibility to malignancy, pancreatitis, and hepatotoxicity.30,55 Since the 1990s, AZA has largely been replaced by MMF as the antiproliferative agent of choice in most kidney transplant centers worldwide.

20.5.2 Mycophenolate Mofetil and Enteric-Coated Mycophenolate Sodium Mycophenolate mofetil (MMF) is the 2-morpholino-ethyl ester pro-drug of mycophenolic acid (MPA). MPA is a potent, selective, and reversible inhibitor of inosine-5’-monophosphate dehydrogenase (IMPDH), a key enzyme involved in the de novo synthesis of guanosine nucleotides, which are required for DNA synthesis (Fig. 20.2).56,57 T and B lymphocytes are critically dependent on the de novo synthesis of purines for their proliferation, whereas other cell types can use the salvage pathway.56,57 In addition, MPA is fivefold more potent as an inhibitor of the type II isoform of IMPDH, which is upregulated in activated lymphocytes, compared to the housekeeping type I isoform, which is expressed in most cell types.56,57 Thus, the cytostatic effects of MPA were supposed to be more selective for lymphocytes compared to other cell types. MPA also decreases the expression of glycoproteins and adhesion molecules responsible for the recruitment of monocytes and lymphocytes to sites of inflammation and graft rejection.57 MMF is available in oral and intravenous formulations, and the recommended oral dosage in renal transplant recipients is 1 2 g/day divided into two doses.58 The major side effects of MMF include gastrointestinal toxicity (nausea and vomiting, diarrhea, and colonic ulcers), bone marrow suppression (mainly leucopenia and anemia, more rarely neutropenia), and increased risk of tissue-invasive cytomegalovirus infection.59 Three landmark prospective randomized trials, published between 1995 and 1996 and conducted in a total of 1593 kidney transplant patients who received an immunosuppressive regimen including CsA and steroids, showed a significantly lower proportion of patients with a first rejection episode on MMF 2 g/day (33%) or 3 g/day (35%) than on azathioprine or placebo (50%).60 62 A pooled analysis of the three studies found that this trend in the incidence of acute rejection was still consistent at 1 year.63 Based on these findings, in subsequent years MMF progressively replaced AZA as the primary antiproliferative agent in kidney transplant immunosuppressive protocols worldwide. However, it is worth noting that in the initial trials comparing MMF versus AZA or

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placebo, the older oil-based CsA formulation was used, which was characterized by an unreliable absorption profile. In this regard, a multicenter prospective, randomized study (the MYSS study) compared the efficacy of MMF (2 g/day) and AZA (100 150 mg/day) in kidney transplant patients receiving CsA microemulsion formulation and corticosteroids. The results showed that MMF offered no advantages over AZA in preventing acute rejection during the first 6 months posttransplant (34% vs 35%, p 5 .91) or even late acute rejection in the cohort of patients who withdrew corticosteroids 9 months posttransplantation (16% vs 12%, p 5 .64).64 In the extension of MYSS, the follow-up study, graft function, incidence of late rejections, graft survival, and patient survival were comparable between the two groups at 5 years posttransplant.65 Similarly, a paired kidney analysis of 476 renal transplant recipients found no difference in graft and patient survival between MMF and AZA when used in triple immunosuppressive regimens including CNIs (TAC or CsA microemulsion) and corticosteroids, despite a higher acute rejection rate in patients given MMF (44% vs 31%, P , .01).66 Nonetheless, a subsequent meta-analysis of industry-driven and academic studies with data from 3143 kidney transplant recipients showed that MMF significantly reduced the risk of acute rejection compared with AZA (RR 0.62, 95% CI 0.55 0.87; P , .0001) and the hazard for graft loss (hazard ratio 0.76, 95% CI 0.59 0.98; P 5 .037).67 However, when the analysis was restricted to the six academic studies, the benefit of mycophenolate mofetil over azathioprine disappeared (Fig. 20.3).68 Along the same line, an analysis of data from the US Scientific Registry of Transplant Recipients has been conducted for patients who received a renal transplant between 1998 and 2006. This study showed that around 95% of patients were receiving MMF at initial discharge from hospital, but this treatment did not translate into significantly improved graft survival over AZA when the calcineurin inhibitor tacrolimus was used as a concomitant therapy.69 These findings are in line with the long-term outcomes of the Australian cohort of kidney transplant recipients originally enrolled in the Tricontinental study, one of the three landmark MMF trials. Indeed, after a median follow-up of 13.8 years, there were no significant differences in graft and patient survival, incidence of malignancy, or graft function between patients receiving MMF or AZA.70 Thus, the lack of a clear-cut benefit of long-term treatment with MMF, along with its remarkably higher cost compared to AZA, should prompt healthcare providers to reconsider the current attitude to use MMF over AZA to prevent acute rejection in kidney transplantation. This is particularly relevant for transplant programs in lowand middle-income countries where social security is often not available and cost of MMF may be unaffordable for transplant recipients. In the early 2000s, enteric-coated mycophenolate sodium (EC-MPS) was developed in an attempt to reduce the incidence of upper-gastrointestinal adverse effects associated with the use of MMF, by delaying the release of the active drug moiety until reaching the small intestine.71 Two large phase III clinical trials, one in de novo and one in stable kidney transplant recipients, showed that EC-MPS was therapeutically equivalent to MMF (i.e., comparable BPAR rates), and both drugs had a similar incidence and severity of side effects.72,73 Notably, however, these trials did not demonstrate a statistically significant difference in the incidence of gastrointestinal adverse effects when patients were given equivalent doses of MMF or EC-MPS (250 mg of MMF is equivalent to 180 mg of EC-MPS). Since then several clinical trials have attempted to compare the gastrointestinal profile of the two MPA formulations. Some studies reported beneficial effects of EC-MPS,74 76 whereas others did not document a difference in gastrointestinal-related adverse effects between MMF and EC-MPS.77 81 In some open-label studies, patients reported an improvement in their perception of gastrointestinal symptoms burden after conversion from MMF to EC-MPS.74,75,82 However, EC-MPS produces extremely variable pharmacokinetic profiles, characterized by multiple peaks of MPA concentrations and high basal drug levels.83,84 Thus, therapeutic drug monitoring is much more challenging with EC-MPS than with MMF, which is characterized by a reliable pharmacokinetic profile.

20.6 THE MAMMALIAN TARGET OF RAPAMYCIN (mTOR) INHIBITORS 20.6.1 Sirolimus Sirolimus (SRL) is a macrocyclic lactone antibiotic produced by Streptomyces hygroscopicus, an actinomycete originally isolated from a soil sample in Rapa Nui.85 Like tacrolimus, sirolimus engages the intracellular immunophilin FKBP12, but the ligand receptor complex subsequently binds to and inhibits mTOR, a serine-threonine kinase involved in the regulation of cell growth and proliferation (Fig. 20.2). The inhibition of this pivotal kinase by SRL in T cells hampers their clonal expansion in response to alloantigens.86 SRL is available as either an oilbased solution or a tablet. In de novo renal transplant recipients with low-to-moderate immunological risk, SRL is initially given at the loading dose of 6 mg, followed by a maintenance dose of 2 mg daily, adjusted for trough

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267

FIGURE 20.3 Meta-analysis of studies evaluating the effect of mycophenolate mofetil and azathioprine on the incidence of acute rejection in kidney transplant recipients. Foster plot shows the risk ratio for acute graft rejection according to whether academic and industryfunded trials were considered in the analysis.64,65 *Industry-funded trials.

concentrations.30 Early posttransplant complications of sirolimus include prolonged or increased occurrence of delayed graft function, poor wound healing and lymphocele formation.87,88 SRL use is also associated with leucopenia, thrombocytopenia, anemia, hypercholesterolemia, hypertryglyceridemia, new-onset diabetes after transplantation, and infertility. Proteinuria has been reported with SRL, especially after conversion from CNI.89 Other important, yet rare, adverse events include hemolytic uremic syndrome, thrombotic microangiopathy, and interstitial pneumonitis.89 SRL was initially proposed as an alternative antirejection therapy to calcineurin inhibitors for renal transplant recipients, in light of its immunosuppressive and antiproliferative property devoid—according to initial reports—of renal toxicity. Indeed, phase 1 study and phase 2 dose-escalation studies documented that SRL prevented allograft rejection without potentiating the nephrotoxicity of CsA.90 Then, two Phase III multicenter pivotal trials showed that SRL had a superior antirejection effect over placebo (global study91) or azathioprine (US study92) in renal transplant patients receiving corticosteroids and full-dose CsA. These findings prompted

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face-to-face comparison between SRL and CsA. When added to corticosteroid and azathioprine immunosuppression, the two drugs were associated with similar rejection rate that was close to 40%.93 However, when the two drugs were added to a corticosteroid and MMF-based immunosuppressive regimen, the rejection rate was remarkably lower in patients allocated to CsA (18%) than in those on SRL (28%).94 Differences in the antirejection potency between SRL and CNI became subsequently clearer when induction therapy with anti-CD25 antibody was added to the immunosuppressive armamentarium. A large randomized, prospective study, the ELITESYMPHONY trial, showed that among renal transplant patients receiving induction therapy with daclizumab and maintenance therapy with MMF and corticosteroids, those randomized to SRL had the highest rate of BPAR at 12 months, with the largest difference reported between the SRL and the TAC arms (37% vs 12%, P , .001). Notably, 5.3% of patients on low-dose SRL therapy developed proteinuria compared with 2.0% of those on lowdose CsA.39 The increased rate of acute rejection associated with de novo use of SRL was also evident in the ORION study. In this trial, 443 renal transplant recipients were randomized to one of three groups: SRL plus TAC, with TAC discontinuation at postoperative week 13 (Group 1), SRL plus MMF (Group 2), or TAC plus MMF (Group 3). At 12 and 24 months there was no difference among groups in graft function or survival; however, Group 2 was sponsor terminated due to a high rate of BPAR within the first 6 months posttransplant (26% in Group 2 vs 7% in Group 3, P , .001). Moreover, at 12 months proteinuria was significantly higher in SRLtreated patients.95 Of note, in both the ELITE-SYMPHONY and the ORION trials, withdrawal of the study drug due to adverse effects occurred more frequently in patients receiving SRL.39,95 Altogether, these data indicate that in renal transplant patients, de novo SRL therapy resulted in an increased risk of acute rejection and graft loss along with poorer tolerability, compared to CNI-based immunosuppressive regimens (Table 20.2).39,94,95,98 101 These findings are in line with experimental and clinical data showing that SRL, besides expanding regulatory T cells,102 also fosters the generation of memory CD81 T cells.103 106 This, along with the evidence of a direct nephrotoxic effect, documented by proteinuria development, made SRL a nonsuitable alternative to CsA or TAC. Recent studies have also investigated regimens that entailed CNI withdrawal and conversion to SRL to limit CNI-associated chronic nephropathy. This approach was first addressed in the CONVERT study, in which kidney transplant patients who were on immunosuppressive therapy with a CNI in the last 6 120 months were randomized to continue their current treatment or exchange CNI for SRL. Among patients with baseline estimated GFR .40 mL/minute, conversion to SRL was associated with increased proteinuria and no GFR improvement compared to maintenance on CNI therapy. Furthermore, enrollment of patients with estimated GFR of 20 40 mL/minute was stopped when the primary safety outcome of BPAR, graft loss, or death was reached by 8 of 48 patients in the SRL arm and none of the 25 patients in CNI group.41 The CONVERT investigators hypothesized that the conversion from CNI to SRL may have occurred after too much allograft dysfunction had accrued for any benefit to be appreciated. This led subsequent trials to investigate earlier conversion from CNI to SRL. In particular, in the Spare the Nephron study, kidney transplant recipients who had started a CNI-based regimen within the preceding 30 180 days were randomized to exchange CNI for SRL or continue CNI treatment. After 12 months, the mean percentage increase in measured GFR was greater in the SRL than in the CNI arm (24% vs 5%, p 5 .012), but this benefit was no longer evident at 24 months. Although at 12 and 24 months the rates of BPAR were comparable between groups, urinary protein excretion was significantly higher in patients converted to SRL compared to those on CNI.107 These data suggest that in patients without markedly compromised renal function, conversion from a CNI to SRL may initially preserve GFR to a small degree, a benefit not sustained on longer follow-up period. Nevertheless, this effect needs to be balanced by an increased risk to develop proteinuria.

20.6.2 Everolimus Everolimus (EVR) was developed as a semisynthetic analog of SRL with similar antiproliferative and immunosuppressive properties, but with a shorter half-life and higher bioavailability.108 Like SRL, this agent acts by forming a complex with FKBP12, which binds to and inhibits the kinase mTOR (Fig. 20.2). EVR, initially dosed at 0.75 mg orally twice daily followed by monitoring of blood trough concentrations, has an antirejection and safety profile similar to that of SRL.89 In two early trials, one in Europe and one in the United States, EVR (1.5 or 3.0 mg/day) was compared to MMF (2 g/day) in de novo renal transplant patients also receiving standard-dose CsA and corticosteroids.109,110 In both studies, all the groups showed similar graft survival and rates of BPAR, but patients assigned to EVR had significantly lower creatinine clearance. Subsequent studies explored EVRbased regimens with CNI minimization in the attempt to reduce CsA-related nephrotoxicity.40,111,112 In particular, in a phase III trial, de novo renal transplant recipients receiving basiliximab were randomized to EVR (1.5 or

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TABLE 20.2

Study

Main Randomized Clinical Trials of SRL Versus CsA or TAC in De Novo Renal Transplant Patients

Immunosuppressants in addition to corticosteroids

Flechner et al.96

Basiliximab, MMF

Flechner et al.95

Basiliximab

Glotz et al.97

Thymoglobulin, MMF

Durrbach et al.98

Thymoglobulin, MMF

Ekberg et al.39

Daclizumab, MMF

Acute rejection (%)

Graft survival (%)

Estimated GFR (mL/ min)

Study drug withdrawal (%)

Patients Study drugs (n )

Follow-up (months)

DGF (%)

SRL

310

6

20.4

21.4b

96.2

67.0

17.4c

CsA

161

22.4

6.1

96.6

67.2

6.8

SRL 1 TACElim

152

14.8

17.4

88.5

58.3

34.2

SRL 1 MMF

152

17.1

32.8g

89.9

63.4

33.6

TAC 1 MMF 139

15.1

12.3

95.4

62.2

22.3h

NA

16.9

85.9d

56.1

28.2e

12.9

95.7

58.4

4.3

45.4

12.1

87.5

44.7

48.5a

30.6

8.3

41.9

16.7

SRL

71

TAC

70

SRL

33

CsA

36

24

12

6

97.0

21.1

e

40.2

d

91.7

e

56.7

6.8

Low-dose SRL

399

Low-dose CsA

399

32.4

27.2e

94.3

59.4f

5.1

Low-dose TAC

401

35.7

15.4

96.4

65.4

4.5

71

12

18.6

14.3

93

59.9

28.2

12.3

8.6

96

56.9

14.9

6

23.3

40.0

100

82.7

17

13.3

13.3

100

77.8

0

NA

12.9

96.4a

66.7a

NA

23.3

79.7

50.7

25

27.5

92.5

NA

24

18.4

89.5

Buchler et al.99

Thymoglobulin, MMF

SRL CsA

74

Pescovitz et al.100

Daclizumab, MMF

SRL

30

CsA

15

Flechner et al.101

Basiliximab, MMF

SRL

31

CsA

30

Kreis et al.94

MMF

SRL

40

CsA

38

12

f

60

12

43 26

P , .05 versus CsA. P , .001 versus CsA. c P 5 .001 versus CsA. d P , .05 versus TAC. e P , .001 versus TAC. f P 5 .001 versus TAC. g P , .001 versus TAC 1 MMF. h P , .05 versus SRL-based regimens. AZA, azathioprine; CsA, cyclosporine; DGF, delayed graft function; Elim, elimination; GFR, glomerular filtration rate; MMF, mycophenolate mofetil; NA, not available; SRL, sirolimus. a

b

3.0 mg/day) with reduced-exposure CsA, or MPA plus standard-dose CsA. At 24 months the rates of the composite efficacy endpoint (treated BPAR, graft loss, death, or loss to follow-up) were similar in the two EVR groups (33% and 27%) and in the MMF arm (27%). The mean estimated GFR was also comparable among the three groups. However, adverse events leading to drug discontinuation were more frequently reported in patients receiving EVR than in those on MPA.40 In summary, available data indicate that de novo use of EVR in combination with induction therapy and low-dose CNI may prevent acute graft rejection at a rate comparable to that with conventional immunosuppressive regimens, although adverse effects were common. Other studies investigated the efficacy and the safety of regimens based on conversion from CNI to EVR at different time points posttransplantation. In the ASCERTAIN study, 398 kidney transplant patients were randomized (mean 5.6 years after transplant) to continue CNIs (CsA or TAC), to minimize CNI therapy with the addition of EVR, or to exchange CNI for EVR. After 24 months, there were no significant between-group

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differences for the composite efficacy endpoint (biopsy-proven acute rejection, graft loss, death, or loss to followup). The mean measured GFR at 24 months did not differ at significant extent among the three groups, while proteinuria was significantly higher in the CNI withdrawal group compared to the standard-dose CNI arm at 12 months.42 Regarding trials focused on earlier conversion, the ZEUS study assessed the efficacy of exchanging CsA for EVR at 4 5 months after transplantation.43 At 12 months posttransplant there was a statistically significant difference in the mean estimated GFR in favor of EVR (mean difference 9.8 mL/minute/1.73 m2). However, after randomization the rate of BPAR was higher in the EVR group than in the CsA arm (10% vs 3%, P 5 .036).43 Notably, a ZEUS substudy documented that the incidence of de novo DSA development and AMR beyond 3 years after transplantation were significantly higher in patients converted to EVR compared to those remaining on CsA.51 In a 2015 study, 3 months after kidney transplantation patients receiving CsA, EC-MPS, and corticosteroids were stratified according to graft fibrosis and randomized to start EVR with half-dose EC-MPS and CsA withdrawal, or continue CsA with standard-dose EC-MPS. At 12 months, the rate of fibrosis progression was similar between groups. At the same time point, estimated GFR was in favor of the EVR group (60 6 20 mL/ minute/1.73 m2 vs 54 6 17 mL/minute/1.73 m2, P 5 .037), but at the expense of significantly higher rates of BPAR (25% vs 5%, P , .001) and de novo DSA development versus CsA continuation.44 These data provide evidence that in kidney transplant recipients late conversion from CNI to EVR (more than 6 months posttransplant) is associated with an increase in proteinuria, similar to what has been reported with SRL. Furthermore, the modest short-term improvement in graft function following early exchange of CNI for EVR has been shadowed by the higher rates of BPAR, de novo DSA development, and AMR.

20.6.3 Antineoplastic Properties of mTOR Inhibitors Besides being involved in T-cell activation, the mTOR pathway is often dysregulated in many types of malignancies.113 As such, there has been interest in the use of mTOR inhibitors to reduce the risk of cancer in kidney transplant recipients.114 In a multicenter study, renal transplant patients on a CNI-based regimen with a prior history of posttransplant squamous cell carcinoma were randomized to either continue CNI treatment or convert to SRL. Over 2 years of follow-up, new squamous cell carcinomas developed in 22% of patients in the SRL group and in 39% in the CNI arm (median time until onset 15 vs 7 months, P 5 .02).115 A multivariate analysis suggested that the use of mTOR inhibitors also associated with a decreased rate of noncutaneous malignancy.116 However, these findings have not been consistently observed in all studies, with a large retrospective cohort trial demonstrating a significantly increased risk of posttransplant lymphoproliferative disorders (PTLD) associated with de novo use of SRL (adjusted hazard ratio 1.22, 95% CI 1.03 1.45).117 Notably, a recent meta-analysis showed that sirolimus reduced the overall risk of malignancy and nonmelanoma skin cancer compared with non-SRL immunosuppressive regimens, but was also associated with a 43% increased risk of death (adjusted hazard ratio 1.43, 95% CI 1.21 1.71; P , .001).118 These data raise caution in adopting SRL as part of the immunosuppressive regimen in all kidney transplant recipients. Instead, this drug could be considered for patients who develop nonmelanoma skin cancer in the attempt to help lower or discontinue CNIs, which promote tumor development, while limiting the risk of kidney allograft rejection and simultaneously exerting its antineoplastic effect.

20.7 COSTIMULATION BLOCKERS Costimulatory blockade has been proposed as part of the therapeutic armamentarium for maintenance immunosuppression in kidney transplantation. Belatacept is a human fusion protein combining a modified extracellular portion of cytotoxic T-lymphocyte-associated antigen 4 with the constant-region fragment of human IgG1.119 This agent works by binding to CD80 and CD86 receptors on APCs, eventually blocking the CD28-mediated costimulation of T cells, a critical step for T-cell activation (Fig. 20.2). Belatacept is administered as intravenous infusion. The recommended dosing is 10 mg/kg administered prior to transplant, on day 5, at the end of weeks 2, 4, 8, and 12 posttransplant, followed by 5 mg/kg every 4 weeks.120 Two pivotal randomized, Phase III trials with a similar study design, the BENEFIT121 and BENEFIT-EXT122 studies, have compared more intensive or less intensive regimens of belatacept to CsA in de novo kidney transplant patients receiving basiliximab induction, MMF, and corticosteroids. The BENEFIT study involved kidney transplants from either living or standard criteria deceased donors, whereas the BENEFIT-EXT study included extended criteria donor kidneys. In both trials, at 12 months posttransplant the belatacept groups showed similar patient and graft survival rates compared with

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the CsA arm, and enjoyed better renal function until 3 years after transplantation. In the BENEFIT trial, however, at 12 months the incidence of acute rejection was higher in the belatacept arms (22% in more intensive and 17% in less intensive group) than in the CsA group (7%).121 At the same time point in the BENEFIT-EXT study the incidence of acute rejection was not significantly different among the three groups (18% in both belatacept arms vs 14% in the CsA group), but numerically more type IIb acute rejections were reported with belatacept than with CsA.122 An analysis of the pool data from the BENEFIT and BENEFIT-EXT trials at month 12 posttransplant showed that belatacept-based regimens were associated with a better cardiovascular and metabolic risk profile compared to the CsA group, reflecting lower blood pressure, less increase in atherogenic serum lipids, and less occurrence of new-onset diabetes after transplantation.123 Notably, in both trials belatacept treatment was associated with higher incidence of PTLD, especially in patients’ seronegative for Epstein Barr virus. Five-year outcomes of patients who entered the two BENEFIT long-term extension studies at 3 years showed that, compared to the CsA group, the belatacept arms maintained a higher GFR with a difference of 21 23 mL/minute in BENEFIT and 11 14 mL/minute in BENEFIT-EXT studies, respectively.124,125 Conversion from CNI to belatacept-based immunosuppressive regimen in renal transplant recipients was also investigated in a Phase II study. In particular, patients with stable allograft function between 3 and 36 months after transplantation were randomized to either continue CNI treatment or switch to belatacept. At 12 months, the mean change in estimated GFR from baseline was about 5 mL/minute higher in the belatacept group, but at the expense of higher acute rejection rate compared to CNI continuation (7% vs 0%).126 Overall, the available data suggest that belatacept use could be associated with better preservation of graft function and a more favorable cardiovascular risk profile compared to CNI in kidney transplant recipients. It remains to be established whether these effects will eventually translate into improved long-term graft and patient survival. The areas of concern, however, include the higher incidence of acute graft rejection (mostly of higher Banff grades) and of PTLD during the first 12 months posttransplant in patients receiving belatacept compared to CNI.

20.8 GENERIC IMMUNOSUPPRESSIVE MEDICATIONS The introduction of generic immunosuppressive medications may present an opportunity for cost savings in kidney transplantation. CsA, TAC, prednisone, MMF, EC-MPS, SRL, and AZA are now available as generic products. In order to meet the standard for FDA and European Medicines Agency (EMA) approval, generic medications must demonstrate therapeutic equivalence and bioequivalence to their brand name counterpart. As for therapeutic equivalence, the generic formulation must be identical in the active ingredient(s), strength and dosage form, intended route of administration, and therapeutic indication. To be deemed bioequivalent, the generic product must show that rate and extent to which the active ingredient becomes available at the site of drug action in the body is comparable with the branded formulation under similar conditions.127 Under current regulations, there is no requirement for generic products to demonstrate bioequivalence in the target population (e.g., in transplant patients) or to confirm that bioequivalence is maintained in the setting of multiple comedications or with chronic use. Moreover, although generic formulations must demonstrate bioequivalence with the branded product, they are not required to show bioequivalence with each other. Thus, transplant organizations offered guidelines on the use of generic immunosuppressive agents. The European Society for Organ Transplantation (ESOT) published its advisory committee recommendations to ensure that substitution of the brand name for the generic product is performed with the appropriate therapeutic monitoring to guarantee that adequate drug exposure is maintained and outcomes are unaffected.128 The KDIGO clinical practice states that the use of generics offers the opportunity to reduce direct costs to the patients and potentially improve adherence to immunosuppressive therapy.16 There is conflicting evidence as to whether change from brand name to generic immunosuppressive agents results in similar blood concentrations and clinical outcomes. In fact, although most of the studies evaluating conversion from the branded microemulsion formulation of CsA to the generic product in stable kidney transplant recipients showed the tested products to be similar,129 131 two prospective, randomized, controlled studies reported significant differences in CsA blood levels between generic and innovator compounds.132,133 Moreover, two studies in de novo renal transplant recipients showed higher rates of acute rejection with generic CsA compared to the innovator product.134,135 To date, few clinical trials have compared branded and generic tacrolimus products. One study reported that conversion from the reference to a generic TAC formulation resulted in a small but significantly drop in trough concentrations in liver and kidney transplant recipients, even though no cases of acute rejection occurred following substitution.136 Two subsequent studies have shown

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similar trough levels before and after conversion from branded to generic tacrolimus.137,138 However, McDevittPotter et al. documented an increase in the proportion of patients requiring dose adjustment after switching from the innovator to the generic formulation compared with the 6-month period prior to conversion (21% vs 7%, P 5 .028).137 These data suggest that in kidney transplant recipients conversion from brand name to generic immunosuppressive medications should be accompanied by close monitoring of drug concentrations and biochemical indices of graft function to guarantee adequate immunosuppression while avoiding toxicity.

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100. Pescovitz MD, Vincenti F, Hart M, et al. Pharmacokinetics, safety, and efficacy of mycophenolate mofetil in combination with sirolimus or ciclosporin in renal transplant patients. Br J Clin Pharmacol 2007;64:758 71. 101. Flechner SM, Goldfarb D, Solez K, et al. Kidney transplantation with sirolimus and mycophenolate mofetil-based immunosuppression: 5-year results of a randomized prospective trial compared to calcineurin inhibitor drugs. Transplantation 2007;83:883 92. 102. Macedo C, Turquist H, Metes D, Thomson AW. Immunoregulatory properties of rapamycin-conditioned monocyte-derived dendritic cells and their role in transplantation. Transplant Res 2012;1:16. 103. Araki K, Turner AP, Shaffer VO, et al. mTOR regulates memory CD8 T-cell differentiation. Nature 2009;460:108 12. 104. Brouard S, Puig-Pey I, Lozano JJ, et al. Comparative transcriptional and phenotypic peripheral blood analysis of kidney recipients under cyclosporin A or sirolimus monotherapy. Am J Transplant 2010;10:2604 14. 105. Saemann MD, Remuzzi G. Transplantation: time to rethink immunosuppression by mTOR inhibitors? Nat Rev Nephrol 2009;5:611 12. 106. Cravedi P, Ruggenenti P, Remuzzi G. Sirolimus for calcineurin inhibitors in organ transplantation: contra. Kidney Int 2010;78:1068 74. 107. Weir MR, Mulgaonkar S, Chan L, et al. Mycophenolate mofetil-based immunosuppression with sirolimus in renal transplantation: a randomized, controlled Spare-the-Nephron trial. Kidney Int 2011;79:897 907. 108. Schuler W, Sedrani R, Cottens S, et al. SDZ RAD, a new rapamycin derivative: pharmacological properties in vitro and in vivo. Transplantation 1997;64:36 42. 109. Vitko S, Margreiter R, Weimar W, et al. Three-year efficacy and safety results from a study of everolimus versus mycophenolate mofetil in de novo renal transplant patients. Am J Transplant 2005;5:2521 30. 110. Lorber MI, Mulgaonkar S, Butt KM, et al. Everolimus versus mycophenolate mofetil in the prevention of rejection in de novo renal transplant recipients: a 3-year randomized, multicenter, phase III study. Transplantation 2005;80:244 52. 111. Tedesco Silva Jr. H, Cibrik D, Johnston T, et al. Everolimus plus reduced-exposure CsA versus mycophenolic acid plus standardexposure CsA in renal-transplant recipients. Am J Transplant 2010;10:1401 13. 112. Salvadori M, Scolari MP, Bertoni E, et al. Everolimus with very low-exposure cyclosporine a in de novo kidney transplantation: a multicenter, randomized, controlled trial. Transplantation 2009;88:1194 202. 113. Khokhar NZ, Altman JK, Platanias LC. Emerging roles for mammalian target of rapamycin inhibitors in the treatment of solid tumors and hematological malignancies. Curr Opin Oncol 2011;23:578 86. 114. Geissler EK, Schlitt HJ, Thomas G. mTOR, cancer and transplantation. Am J Transplant 2008;8:2212 18. 115. Euvrard S, Morelon E, Rostaing L, et al. Sirolimus and secondary skin-cancer prevention in kidney transplantation. N Engl J Med 2012;367:329 39. 116. Kauffman HM, Cherikh WS, Cheng Y, Hanto DW, Kahan BD. Maintenance immunosuppression with target-of-rapamycin inhibitors is associated with a reduced incidence of de novo malignancies. Transplantation 2005;80:883 9. 117. Nee R, Hurst FP, Dharnidharka VR, Jindal RM, Agodoa LY, Abbott KC. Racial variation in the development of posttransplant lymphoproliferative disorders after renal transplantation. Transplantation 2011;92:190 5. 118. Knoll GA, Kokolo MB, Mallick R, et al. Effect of sirolimus on malignancy and survival after kidney transplantation: systematic review and meta-analysis of individual patient data. BMJ 2014;349:g6679. 119. Larsen CP, Pearson TC, Adams AB, et al. Rational development of LEA29Y (belatacept), a high-affinity variant of CTLA4-Ig with potent immunosuppressive properties. Am J Transplant 2005;5:443 53. 120. Martin ST, Tichy EM, Gabardi S. Belatacept: a novel biologic for maintenance immunosuppression after renal transplantation. Pharmacotherapy 2011;31:394 407. 121. Vincenti F, Charpentier B, Vanrenterghem Y, et al. A phase III study of belatacept-based immunosuppression regimens versus cyclosporine in renal transplant recipients (BENEFIT study). Am J Transplant 2010;10:535 46. 122. Durrbach A, Pestana JM, Pearson T, et al. A phase III study of belatacept versus cyclosporine in kidney transplants from extended criteria donors (BENEFIT-EXT study). Am J Transplant 2010;10:547 57. 123. Vanrenterghem Y, Bresnahan B, Campistol J, et al. Belatacept-based regimens are associated with improved cardiovascular and metabolic risk factors compared with cyclosporine in kidney transplant recipients (BENEFIT and BENEFIT-EXT studies). Transplantation 2011;91:976 83. 124. Rostaing L, Vincenti F, Grinyo J, et al. Long-term belatacept exposure maintains efficacy and safety at 5 years: results from the long-term extension of the BENEFIT study. Am J Transplant 2013;13:2875 83. 125. Charpentier B, Medina Pestana JO, Del CRM, et al. Long-term exposure to belatacept in recipients of extended criteria donor kidneys. Am J Transplant 2013;13:2884 91. 126. Rostaing L, Massari P, Garcia VD, et al. Switching from calcineurin inhibitor-based regimens to a belatacept-based regimen in renal transplant recipients: a randomized phase II study. Clin J Am Soc Nephrol 2011;6:430 9. 127. Garcia-Arieta A, Gordon J. Bioequivalence requirements in the European Union: critical discussion. AAPS J 2012;14:738 48. 128. van Gelder T. European Society for Organ Transplantation Advisory Committee recommendations on generic substitution of immunosuppressive drugs. Transpl Int 2011;24:1135 41. 129. Diarra DA, Riegersperger M, Saemann MD, Sunder-Plassmann G. Maintenance immunosuppressive therapy and generic cyclosporine A use in adult renal transplantation: a single center analysis. Kidney Int Suppl 2010;S8 11. 130. Vitko S, Ferkl M. Interchangeability of ciclosporin formulations in stable adult renal transplant recipients: comparison of Equoral and Neoral capsules in an international, multicenter, randomized, open-label trial. Kidney Int Suppl 2010;S12 16. 131. Pamugas GE, Danguilan RA, Lamban AB, Mangati VB, Ona ET. Safety and efficacy of generic cyclosporine arpimune in Filipino lowrisk primary kidney transplant recipients. Transplant Proc 2012;44:101 8. 132. Qazi YA, Forrest A, Tornatore K, Venuto RC. The clinical impact of 1:1 conversion from Neoral to a generic cyclosporine (Gengraf) in renal transplant recipients with stable graft function. Clin Transplant 2006;20:313 17. 133. Hibberd AD, Trevillian PR, Roger SD, et al. Assessment of the bioequivalence of a generic cyclosporine A by a randomized controlled trial in stable renal recipients. Transplantation 2006;81:711 17.

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134. Taber DJ, Baillie GM, Ashcraft EE, et al. Does bioequivalence between modified cyclosporine formulations translate into equal outcomes? Transplantation 2005;80:1633 5. 135. Spasovski G, Masin-Spasovska J, Ivanovski N. Do we have the same clinical results with Neoral and Equoral treatment in kidney transplant recipients? A pilot study. Transpl Int 2008;21:392 4. 136. Momper JD, Ridenour TA, Schonder KS, Shapiro R, Humar A, Venkataramanan R. The impact of conversion from prograf to generic tacrolimus in liver and kidney transplant recipients with stable graft function. Am J Transplant 2011;11:1861 7. 137. McDevitt-Potter LM, Sadaka B, Tichy EM, Rogers CC, Gabardi S. A multicenter experience with generic tacrolimus conversion. Transplantation 2011;92:653 7. 138. Spence MM, Nguyen LM, Hui RL, Chan J. Evaluation of clinical and safety outcomes associated with conversion from brand-name to generic tacrolimus in transplant recipients enrolled in an integrated health care system. Pharmacotherapy 2012;32:981 7.

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21 Novel Drugs in Kidney Transplantation Sindhu Chandran and Flavio Vincenti University of California, San Francisco, CA, United States

21.1 INTRODUCTION The field of transplantation has made significant progress in developing immunosuppressive drugs and applying them to the prevention and control of acute rejection and secondarily the prolongation of graft life. Despite these achievements however, there exist several unmet needs, including the prevention and treatment of ischemia reperfusion injury in the perioperative period, the control of the humoral arm of the immune system, and the mitigation of toxicities associated with currently used agents. It has become clear that there is a pressing clinical need to develop novel agents that can replace calcineurin inhibitors that, although highly efficacious, have been associated with significant nonimmune toxicities and consequent shortening of late graft and patient survival. An area which has received somewhat less attention as a potential target of drug therapies is that of intragraft inflammation prior to and at the time of transplantation which is induced by ischemia and reperfusion injury. A growing body of evidence indicates a key role of this inflammatory cascade in potentially instigating and fomenting the immune response to the graft. The presence of inflammation in grafts following transplantation has also been shown to be detrimental to long-term graft outcome. We will provide here an overview of targets and strategies to down-regulate graft inflammation prior to and after transplantation. One of the most intensively researched areas in immunology in the past quarter century has been costimulation, which has led to the development of biologic agents designed to block or manipulate the interactions between T cells and antigen presenting cells. The first approved agent in this class is belatacept, a rationally designed CTLA4-Ig fusion protein whose success has been built on the absence of metabolic and nephrotoxic side effects in the short-term. However, full potential of improved specificity and efficacy offered by these agents is yet to be realized and in this chapter we will discuss several promising new drugs in this category. Finally, therapeutic innovation in the prevention and control of antibody-mediated injury to the graft, which has lagged behind T cell directed therapies, is now coming to the forefront.

21.2 THERAPIES TARGETING DELAYED GRAFT FUNCTION Ischemia-reperfusion injury (IRI) is a complex pathophysiological process that occurs inevitably in transplanted kidneys and affects graft function and survival. The process starts with the death of the donor, leading to direct effects of hypoxia and metabolic stress such as mitochondrial damage and ATP depletion and continues through the organ procurement and preservation procedures. Upon reperfusion, the organ is then further damaged by a reactive inflammatory process which had been primed during the earlier injuries. The cascade of proinflammatory cytokines and chemokines and the up-regulation of adhesion molecules lead to increased trafficking of host immune cells to the graft and a shift towards an adaptive immune response.1 Clinically, the damage from microvascular dysfunction and cytotoxic agents generated by the innate immune

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278 TABLE 21.1

21. NOVEL DRUGS IN KIDNEY TRANSPLANTATION

Therapies Targeting Delayed Graft Function in Kidney Transplantation Current status in clinical trials

Drug

Molecular target

Mechanism of action

Carbon monoxide as gas or from CO-releasing molecules (CORM)

Inhibition of oxidative injury,2 inflammation,6 and apoptosis7,8

Phase II

Infliximab (Remicade, Janssen Biotech Inc.)

Cytochrome C oxidase2 Cytochrome P4503 HMGB-14 P38 MAPK pathway5 TNFα

Inhibition of inflammation

Phase II

OPN-305 (Opsona Therapeutics Ltd.)

TLR-2

Inhibition of innate immunity

Phase II

Eculizumab (Soliris, Alexion Pharmaceuticals Inc.)

C5

Inhibition of complement activation

Phase II

C1 esterase inhibitor

C1 esterase

Inhibition of complement activation

(Berinert, CSL Behring)

Phase I/II

(Cinryze, Shire plc)

Phase I

15NP or QPI-1002 (Quark Pharmaceuticals)

P53

Inhibition of apoptosis

Phase I/II

response manifests as delayed graft function and sometimes primary nonfunction. Several drugs are currently in clinical trials (see Table 21.1).

21.2.1 Strategies to Prevent Ischemia-Induced Injury and Mitigate Inflammation Two approaches that are currently being tested to decrease the early inflammatory response induced by ischemia are the use of carbon monoxide (CO) and the inhibition of TNFα. CO can be synthesized during the conversion of heme to biliverdin by heme oxygenase (HO) and is involved with the regulation of endothelial cell survival and proliferation, protection against IRI, vasorelaxation, and inhibition of proinflammatory responses.2,4,5,9 HO-1, normally expressed in cortical tubules and the renal vasculature, is up-regulated in several stressful events such as brain death and organ procurement and is protective against IRI,7,10 suggesting beneficial effects of CO. Exposure to low concentrations of CO (20 250 ppm) has cytoprotective effects equal to that attained by HO-1 induction in transplant-induced IRI.6,7 A limitation of the clinical use of CO is its narrow therapeutic window. High concentrations ( . 100 ppm) are associated with toxicity due to interference with oxygen delivery. However, low doses of CO as donor treatment revealed beneficial effects on graft immunogenicity and chronic graft function in a rat kidney transplant model.11 Another study of CO (20 ppm) with or without biliverdin in rat heart and kidney transplant models showed that combination therapy improved graft function and survival versus CO alone.3 A phase I trial of inhaled CO utilized an investigational device (Covox DS, Ikaria Inc.) that delivered CO at 3 mg/kg/hour for 1 hour either once or daily for 10 days. COHb elevated reliably to 12% and no adverse effects were reported.12 A phase II trial of inhaled CO delivered intraoperatively in kidney transplant recipients (NCT00531856) is currently underway. Slow CO-releasing molecules have been created as well with reduced risk of toxicity due to high COHb levels and impaired oxygen delivery to organs and tissues. In murine renal transplant models, treatment with CORM-2 (in the donor) and CORM-3 (in the perfusate) had protective effects against IRI and improved early graft function and survival.6,8 These agents have not yet been tested in clinical trials. TNFα, a potent proinflammatory cytokine, is an important mediator of IRI in various organ types, including the kidneys.13 Recombinant TNFR:Fc was first shown to prolong graft survival in a NHP renal transplant model.14 Etanercept (Enbrel, Amgen) is a soluble fusion protein that inhibits TNFα by binding TNFα and lymphotoxin and is currently approved for multiple autoimmune diseases including rheumatoid arthritis. Treatment with etanercept attenuated IRI in a rat kidney transplant model15 and down-regulated cytokines resulting in improved engraftment in a syngeneic rat hepatocyte transplant model.16 The efficacy of islet transplant protocols including etanercept infusions has been reported extensively17 20 and etanercept is part of the CIT07 protocol developed by the NIH and sponsored by the Clinical Islet Transplantation Consortium.21 There are currently no ongoing clinical trials of this agent in kidney transplantation. Experiments using anti-TNF monoclonal antibody at the time of allograft reperfusion in murine cardiac transplant models found that it reduced graft infiltration

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with neutrophils, macrophages, and donor reactive memory CD8 T cells, priming of alloreactive CD4 and CD8 T cells,22 decreased production of antidonor MHC IgG antibodies, and prolonged graft survival.23 Published reports on clinical administration of anti-TNF antibodies in transplantation are lacking thus far. The CTOT-19 trial is a multicenter phase II randomized controlled trial of a single dose of infliximab (Remicade, Janssen Biotech Inc.), a chimeric monoclonal antibody to TNF-alpha, administered at the time of kidney transplantation. The trial, currently underway, is designed to answer multiple questions including the impact of this therapy on DGF as well as 2-year graft function.

21.2.2 Strategies to Down-Regulate Innate Immunity Toll-like receptors (TLRs) are an evolutionarily conserved group of transmembrane proteins which are rapidly up-regulated24,25 and interact with endogenous, cell-derived ligands displayed on the cell surface following IRI.26 These interactions lead to downstream effects such as cytokine and chemokine release and augmentation of costimulatory molecule expression.27 TLR2 deficiency or blockade with antisense oligonucleotides is protective against IRI in mice.28 Blockade of TLR2 with a monoclonal antibody (OPN-301 and OPN-305) was subsequently found to mitigate IRI in murine renal and cardiac transplant models.29,30 A phase II dose escalation trial (NCT01794663) of OPN-305 (Opsona Therapeutics Ltd.) is currently underway in deceased donor kidney transplant recipients at high risk of DGF. Complement activation plays a significant role in IRI through membrane attack complex (MAC) formation, endothelial cell activation, cytokine production, and augmentation of alloimmunity through activation of complement receptor-bearing adaptive immune cells.30 32 Expression of complement genes was found to be high in all transplanted kidneys postperfusion and higher in deceased donor kidneys compared to living donor kidneys prior to perfusion,33 speaking to the roles of both brain death and ischemic injury in complement activation.34 Eculizumab (Soliris, Alexion Pharmaceuticals Inc.) is a humanized monoclonal antibody directed against the complement component C5, which prevents its cleavage and thus the formation of MAC. It has received FDA approval to treat paroxysmal nocturnal hemoglobinuria and is currently being tested for efficacy in preventing postkidney transplant DGF in two separate phase II trials (NCT01403389; NCT01919346). Plasma-derived concentrates of human C1 esterase inhibitor currently approved for the treatment of acute attacks of hereditary angioedema. A single center placebo-controlled trial of C1-INH (Berinert, CSL Behring) for prevention of antibody-mediated rejection in 20 highly sensitized patients found that delayed graft function developed in only 1 C1-INH subject versus 4 in the placebo arm.35 A phase I/II trial of C1-INH in deceased donor kidney transplant recipients is currently underway (NCT02134314) with the primary endpoint being delayed graft function. Anticomplement therapies in the donor may be the most effective prophylactic therapy to prevent IRI, given the role of brain death in complement activation. A phase I trial for the prevention of DGF in kidney transplant recipients (NCT02435732) will test the administration of a different C1 esterase inhibitor (Cinryze, C1-INH, Shire plc.) to deceased donors with KDPI .85% prior to organ procurement.

21.2.3 Antiapoptotic Strategies Apoptosis is widely believed to play a major role in IRI. The proapoptotic gene, p53, is activated in the setting of hypoxia and oxidative stress, resulting in cell cycle arrest, cell senescence, and apoptosis.36 QPI-1002 or I5NP (Quark Pharmaceuticals) is a siRNA that acts via RNA interference to temporarily inhibit the expression of protein p53. I5NP is trophic to the kidneys and following intravenous administration, undergoes rapid glomerular filtration, with subsequent binding to endothelial cells and endocytosis by peritubular capillary cells.37 Experiments in animal models showed that p53 inhibition provided significant protective effects in proximal tubule cells.38 QPI-1002 has been granted orphan drug designation in the USA and Europe for the prevention of DGF in kidney transplant recipients. The results of a Phase I/II clinical trial in kidney transplant recipients (n 5 331) were presented at the 2014 World Transplant Conference. The primary study endpoint was to achieve at least 30% relative risk reduction of DGF in QPI-1002-treated patients compared to placebo. While the primary endpoint for the study was not met in the total study population (15.1% relative risk reduction of DGF), there was a 30.5% relative risk reduction in patients included in the largest of the prospectively defined study strata—ECD kidneys entirely cold stored (ECD/CS, n 5 177). In this cohort, QPI-1002 treatment significantly increased the time to first dialysis in the first posttransplant month, reduced the mean duration of dialysis dependency, and reduced the number of dialysis

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sessions required in the first 30 days posttransplant. Similar results were obtained in all recipients of kidney grafts from donors older than 45 and in patients receiving kidneys of donors older than 35 years of age. The overall QPI-1002 safety profile was consistent with the expected profile for transplant recipients, and similar in both treated and placebo groups. In summary, QPI-1002 was effective only in ECD kidneys that were susceptible to ischemia-induced apoptosis. A phase III trial is yet to be planned.

21.3 THERAPIES TARGETING CELLULAR IMMUNITY 21.3.1 Targeting the T Cell Receptor T cells are key effectors of the adaptive immune response and T cell recognition of antigenic peptide/MHC on antigen presenting cells plays a pivotal role in the initiation of the effector response. T10B9 (the predecessor antibody to TOL101) is a murine IgM monoclonal antibody targeting the αβ TCR. Unlike other T cell targets, the αβ TCR has no known intracellular signaling domains and may provide a nonmitogenic target for T cell inactivation. A single center phase II trial comparing T10B9 to OKT3 for the treatment of acute rejection in renal allograft recipients (n 5 76) showed that T10B9 was as effective as OKT3 in reversing acute rejection,39 while being associated with fewer adverse effects, less cytokine release, and fewer serious infections. Unfortunately, the development of human antimouse antibodies with T10B9 occurred at approximately the same high frequency as with OKT3 (30% with titer 1:100) although these were not cross-reactive with OKT3. TOL101 (Tolera Therapeutics) is a next-generation antibody that has two nucleotide mutations and a different glycosylation pattern to T10B9. In addition, the manufacturing process was optimized, incorporating serum and animal-free components. A multicenter phase II clinical trial with an initial dose escalation component40 in de novo renal transplant recipients (n 5 36) consisted of TOL101 administered daily for 5 10 doses (target CD3 T cell counts ,25 mm23) in addition to MMF, tacrolimus, and tapering steroids. In this study, doses of TOL101 above 28 mg resulted in prolonged CD3 modulation, with rapid recovery observed 7 days after therapy cessation. There were no cases of patient or graft loss and few significant adverse events were reported. There were five biopsy-confirmed acute cellular rejections (13.9%); however, no donor-specific antibodies were detected. Overall TOL101 was well tolerated, supporting continued clinical development, although funding challenges to advance TOL101 to a phase III trial have not yet been resolved.

21.3.2 Costimulation Blockade 21.3.2.1 Blockade of CD28/CTLA-4: CD80/86 Pathway The observation by Linsley of Bristol Myers Squibb in 1991 that CTLA-4 binds and is a powerful antagonist to CD28 ushered in an era of intense clinical development of costimulation blockade, first with recombinant CTLA-4 Ig (abatacept) and then the higher affinity variant LEA29Y or belatacept. On the strength of the initial human studies with CTLA-4 Ig and NHP studies with belatacept, a larger phase II trial was launched using belatacept in a CNI-free regimen for kidney transplantation. The promising results from this study led to the conduct of two phase III registration trials (BENEFIT and BENEFIT-EXT) to evaluate the efficacy and safety of belatacept in a CNI-free regimen consisting of basiliximab induction and maintenance therapy with belatacept, mycophenolate mofetil, and steroids (Fig. 21.1). The 1-, 3- and 5-year results of both studies have been published41,42 and the final 7-year outcomes were presented at the American Transplant Congress in Philadelphia in May 2015. This is the first time since the introduction of cyclosporine that an improvement in long-term outcome (reaching statistical significance in the BENEFIT trial) with belatacept has been demonstrated in a prospective randomized trial. While belatacept in the lower intensity regimen has been approved for use by the FDA since 2011 as part of de novo immunosuppressive regimens in patients who are EBV seropositive, there remain several challenges to the widespread adoption of belatacept in renal transplantation (Table 21.2). Modifications to the regimens used in the phase II and III trials may be able to address some of these challenges and potentially contribute to greater acceptance and utilization of belatacept in the transplant community. As noted in Table 21.2, these modifications include the judicious use of depleting induction (with Thymoglobulin or alemtuzumab), conversion from mycophenolic acid to mTOR inhibitor as concomitant therapy for better synergism, and possibly reduced frequency of belatacept dosing. As we continue to gain greater understanding of the costimulation tract from in vitro and experimental studies, it has become clear that better costimulatory inhibition

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CLINICAL ENDPOINTS (months) Primary

Transplantation

Day 1

6 months

12

24

36

84

10 mg/kg Belatacept MI

5 mg/kg every 4 weeks DAY 1 514 28 42 56 70 80

Belatacept LI Approved Regimen

168

10 mg/kg 5 mg/kg every 4 weeks DAY 1 514 28

Cyclosporine (7 ± 3 mg/kg daily)

112 140

150–300 ng/mL DAY 1

56

84

100–250 ng/mL 28

Placebo infusions All patients received basiliximab induction, mycophenolate mofetil, and corticosteroid taper LI/MI: Less intensive or more intensive belatacept dosing regimen

FIGURE 21.1

TABLE 21.2

BENEFIT Study: regimens and dosing.

Why Belatacept Has Not Fulfilled Its Promise as a Transformational Immunosuppressive Agent

Challenge

Potential solution

Higher rates of early acute rejection

Use of depleting antibody induction

Histologically more severe acute rejection Posttransplant lymphoproliferative disorder

Restriction to EBV seropositive population

Intravenous administration requiring IV access and infusion appointment

Reducing frequency to every 8 weeks may be an option in some patients. IV administration can be an advantage with respect to ensuring compliance

Higher cost

May be offset by lower cost of monitoring and superior graft function (if confirmed). Every 8 weeks administration regimen may render the therapy practical and cost effective

can be achieved through the use of more selective blockade of CD28 (through direct nonagonistic blockade of CD28) or combining belatacept with blockade of other targets that play a role in costimulation. Direct antagonism of CD28 allows the CD80/CD86 ligands to interact with CTLA-4 Ig in order to augment inhibitory signals in effector T cells and the suppressor function in Tregs (see Fig. 21.2). Selective CD28 blockade was indeed attempted in the phase I trial of the agonistic anti-CD28 monoclonal antibody, TGN1412.43 Unfortunately, in this trial, the use of TGN1412 resulted in massive T cell activation and “cytokine storm” in all six patients, highlighting the complexities of developing CD28-specific blocking reagents.44 Subsequent advances in the production of domain antibodies, in which the Fc portion is completely removed, have permitted the development of novel blocking, nonactivating reagents that can safely and specifically block CD28 costimulatory signals, while leaving CTLA-4 coinhibitory signals intact. Along these lines, selective blockade of CD28 for 2 weeks post-engraftment with α28scFv, a nonactivating single-chain Fv-based reagent, was found to promote allograft survival in a murine cardiac transplant model and attenuate chronic rejection in a CTLA-4 dependent manner.45 Sc28AT, a chimeric human/primate monovalent CD28-specific fusion antibody was found to significantly prolong cardiac and renal allograft survival in a NHP model both alone and when used in combination with a CNI.46 Treatment with Sc28AT was also associated with increased intragraft and peripheral blood Tregs. The same group used FR104 (by Effimune), an antagonist PEGylated anti-CD28 Fab’ antibody fragment, in combination with low doses of tacrolimus or steroid free with rapamycin in a NHP renal transplant model.47

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4 LA CT L 1 D P

L OS IC

Selective CD28 Blocker

CD28 Teff

CD80 TCR CD86

MHC

R

TC

APC MHC

Treg CD80

CTLA-4 PDL1 CD28

CD86

FIGURE 21.2 Selective CD28 blockade allows inhibitory signals via CTLA4 and PD1 to remain intact while blocking T cell activation via CD28.

MOA of CD40-CD54 IFN-γ Anbibody production

B-cell CD40

APC

CD40L

CD80/86

CD40

T-cell

Up-regulation

Macrophage

CD28 CD40 IL-12

T-helper and CTL APC: Antigen Presenting Cell

Cytokines: TNF-α

Tissue destruction

FIGURE 21.3 Multiple sites of action of anti-CD40 therapies.

Treatment with FR104 prevented acute rejection and alloantibody development and prolonged allograft survival in this model and was associated with an increase in Tregs in circulation and in the graft. Another study of FR104 alone or in combination with CD154 antibody in a NHP cardiac transplant model found a decrease in acute rejection rates and increase in the frequency of circulating Tregs when compared to anti-CD154 alone, but no significant difference in graft survival.48 Finally, lulizumab pegol or BMS-931699 (Bristol Myers Squib) is a pegylated humanized antibody to CD28 which is currently undergoing phase II clinical trials for the treatment of SLE. 21.3.2.2 Concomitant Blockade of Other Costimulatory Pathways Targeting the interaction between CD154 (CD40 ligand) on activated antigen-specific CD41 T cells and CD40 on dendritic cells (Fig. 21.3) has been long recognized as a means to attenuate alloreactive immune responses.49 51 The emergence of data supporting a role of CD154/CD40 pathway blockade in enhancing Treg

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mediated immune suppression52 has made it an even more attractive strategy. Unfortunately, development of novel agents in this area was somewhat slowed by the experience in early clinical trials of Fc-intact CD154 antagonists hu5C8 and IDEC-131 which resulted in unanticipated thromboembolic complications associated with the expression of CD154 on platelets.53 Subsequent research has uncovered new aspects of CD154 and CD40 biology, including a role for CD154 in CD4 T cell independent priming of CD8 T cells54 and the contribution of CD40 based T cell interactions to CD8 T cell memory generation,55 the rescue of exhausted CD8 T cells56 and the optimal expansion and differentiation of donor-reactive CD8 T cell responses. These improvements in our understanding, coupled with advances in technology, have allowed the development of several new antagonists to CD154 and CD40. Based on evidence suggesting that intact CD154 antibodies stabilize aggregates of activated platelets and that this effect requires biological activity of the Fc region,57,58 a humanized IgG1 Fc-disabled, aglycosylated anti-CD154 heavy chain variant was created which was less able to bind Fc receptors and activate complement. This antibody was able to prolong graft survival with a similar efficacy to the wild-type glycosylated form in a murine transplant model.59 Domain antibodies are the smallest known Ag-binding fragments of antibodies, ranging from 11 to 15 kDa. An Fc-silent antimouse CD154 domain antibody was found to be equivalent to Fc-intact anti-CD154 monoclonal antibodies in its ability to inhibit alloreactive T cell expansion, attenuate cytokine production of antigen-specific T cells and promote the conversion of Tregs.60 This domain antibody is currently being developed for transplantation by Bristol Myers Squibb. Early studies of CD40 blockade using chimeric anti-CD40 monoclonal antibodies were shown to prolong kidney allograft survival in NHPs and functioned at the level of depleting APCs in vivo.61,62 However, they were less effective than anti-CD154 monoclonal antibodies for prolonging graft survival. CD40 blockade was subsequently found to synergize with CTLA4-Ig to promote long-term allograft survival in mouse models of skin and bone marrow transplantation63 independent of the depletion of APCs. Nondepleting CD40 blockade agents have now been developed that show efficacy in NHP models of renal and islet transplantation.64 66 One of these agents is ASKP1240 or 4D11 (Astellas Pharma US, Inc.), a fully human anti-CD40 monoclonal IgG4 antibody, which interrupts the CD40:CD154 axis by masking and does not cause antibody-dependent cell-mediated cytotoxicity or complement-dependent cytotoxicity.66 Use of ASKP1240 in both the induction (2-week) and maintenance (6-month) therapy arms markedly prolonged renal allograft survival in cynomolgus monkeys without causing apparent side effects.64,67 A phase Ib trial in renal transplant recipients who received a single dose of ASKP1240 with conventional immunosuppressive therapy showed that the 200 and 300 mg doses achieved prolonged occupancy of the CD40 receptor without any significant side effects. The results of a large phase II study of ASKP1240 performed to assess both safety and efficacy (Fig. 21.4) were reported at the American Transplant Congress in Philadelphia in May 2015. While ASKP1240 provided evidence of efficacy with reduced dose tacrolimus and steroids, it was associated with an unacceptably high rate of acute rejection in the CNI-free arm (Table 21.3). No subjects experienced any thromboembolic events. Additional studies with ASKP1240 in Arm 1: Standard of Care

De novo kidney transplant recipients Randomized, open label, multicenter trial ASKP1240 Phase IIb

Basiliximab induction Tacrolimus trough 4–11 ng/mL MMF 1000 mg bid Steroids

Arm 2: CNI Avoidance Basiliximab induction ASKP1240 MMF 1000 mg bid Steroids Arm 3: CNI Minimization with MMF Avoidance ASKP1240 Tacrolimus trough 4–11 ng/mL (Day 0–30) Tacrolimus trough 2–5 ng/mL (Day 31 onward) Steroids

FIGURE 21.4

Design of phase IIb study ISN7163-CL-0108 to assess the efficacy and safety of ASKP1240 in de novo kidney transplant

recipients.

I. KIDNEY TRANSPLANTATION

284 TABLE 21.3

21. NOVEL DRUGS IN KIDNEY TRANSPLANTATION

Key Outcomes at 180 Days in a Phase IIb Trial of ASKP1240 in Kidney Transplant Recipients (n 5 138)

Parameter

Arm 1 (n 5 48) Standard of Care Tac, MMF, steroids

Arm 2 (n 5 46) ASKP1240 MMF, steroids

Arm 3 (n 5 44) ASKP1240 Tac minimization, steroids

BPAR

3 (6.3%)

17 (37%)

4 (9.1%)

BKV infection

6 (12.2%)

7 (15.2%)

12 (27.3%)

2 (4.1%)

4 (8.7%)

3 (6.8%)

Mean MDRD eGFR in mL/min/1.73 m

63.5

63.9

62.6

Patient survival

48 (100%)

45 (97.8%)

43 (97.7%)

Death-censored graft survival

47 (97.9%)

46 (100%)

43 (97.7%)

CMV infection 2

There were 3 malignancies in ASKP1240 groups: one renal cell carcinoma and 2 squamous cell carcinomas. No cases of PTLD were reported. There were no cases of graft loss from BKV nephropathy. Anti-ASKP1240 antbodies were infrequent (3.3%).

renal transplantation are currently on hold. Another anti-CD40 antibody in clinical development is OM11-62MF (Novartis International AG), a fully human Fc silent antibody. Phase I trials (n 5 48) were recently completed and there were no serious adverse events reported. OX40 (CD134) is transiently induced on both CD468 and CD869 T cells after activation and plays a key role in the survival and homeostasis of effector and memory T cells.70 72 Interestingly, OX40 also appears to be a key negative regulator of Tregs.73 Consistent with this biology, blockade of OX40:OX40L has been shown to inhibit the generation of an optimal effector T cell pool by promoting activation induced cell death,74 while concomitantly aiding the induction of Tregs.75,76 Blocking the OX40-OX40L pathway using an OX40-Ig fusion protein in a murine cardiac transplant model was found to result in prolonged cardiac allograft survival when donor and recipient were mismatched at a minor histocompatibility antigen locus but not across a full MHC mismatch.77 In contrast, in a fully-MHC mismatched model where TCR transgenic CD81 T cells were adoptively transferred into syngeneic T cell deficient recipients, anti-OX40 was able to significantly prolong skin graft survival, although tolerance was not achieved.74 Oxelumab (huMAbOX40L or R4930 or RO4989991, Roche Pharmaceuticals), a once promising humanized monoclonal antibody to OX40L, is not currently in clinical development. Whether anti-OX40 antibodies will next be evaluated for clinical development is unclear.

21.3.3 B Targeting Cytokines Targeting cytokines to module the immune response has been a promising approach experimentally but has delivered weak results clinically. Anti-IL-2 receptor antibodies have had limited efficacy overall but are useful for induction in low immunologic risk patients.78,79 IL-15 and IL-17 has yet to be exploited as clinically safe and effective targets. IL-6 is a pleiotropic cytokine produced early by a wide variety of immune and nonimmune cells in response to acute injury and elicits its cellular actions by binding to the IL6R. IL-6 was initially identified as B cell stimulatory factor 2, which is important for the development of antibody-producing plasma cells.80 Excessive activity of IL-6 causes polyclonal B cell activation, plasmacytosis, and B cell neoplasia. Subsequently, IL-6 was found to have major effects on the cellular compartment as well. IL-6 has been found to promote specific differentiation of naı¨ve CD41 T cells into Th17 cells,81 while at the same time inhibiting TGF-β-induced Treg differentiation.82 Thus IL-6 acts as link between the innate to acquired immune response, promotes the production of allograft antibody, and tips the balance of the T cell response from a tolerant to an effector phenotype. Inhibition of IL-6, directly or through blocking its receptor, has been shown to be effective at controlling inflammation both experimentally, in models of autoimmunity and transplantation, as well as clinically, in patients with rheumatoid arthritis. Data from murine cardiac transplant model83 showed that production of IL-6 is up-regulated during allograft rejection and that IL-6 production affects Th1/Th2 balance. Allograft acceptance appeared to result from the combined effect of costimulatory molecule blockade and IL-6-deficiency, which limited the differentiation of effector cells and promoted the migration of regulatory T cells into the grafts. Therefore, the blockade of IL-6, or its signaling pathway, when combined with strategies that inhibit Th1 responses, may have a synergistic effect on the promotion of allograft acceptance.

I. KIDNEY TRANSPLANTATION

285

21.4 THERAPIES TARGETING HUMORAL IMMUNITY

Tocilizumab (Actemra, Roche Pharmaceuticals) is a humanized monoclonal antibody against the interleukin-6 receptor (IL-6R), which is currently approved by the FDA for the treatment of rheumatoid arthritis. A study of the B-cell compartment in patients with rheumatoid arthritis found that tocilizumab induced a significant reduction in the frequency of peripheral preswitch and postswitch memory B cells, a decline in the number of IgG and IgA B cells, and reduced serum immunoglobulin levels.84 Other studies have found an increase in the number of peripheral Tregs in patients receiving tocilizumab which is associated with the degree of clinical response in patients with rheumatoid arthritis.85 A phase I/II pilot study (n 5 10) of a desensitization strategy using tocilizumab and IVIg, followed by 6 months of posttransplant treatment, resulted in the transplantation of 5 out of 10 highly sensitized patients and was associated with a reduction in the strength and number of donor-specific antibodies.86 A randomized clinical trial (NCT02108600) of 6 months of tocilizumab therapy in kidney transplant recipients with early allograft inflammation is currently underway.

21.4 THERAPIES TARGETING HUMORAL IMMUNITY Insidious humoral immune responses have become increasingly recognized as a major contributor to long-term graft loss in kidney transplant recipients. Achieving control of alloantibody response has therefore gained in importance as an unmet need in transplantation. Fig. 21.5 shows targets to control humoral alloimmunity.

21.4.1 Targeting B Cells 21.4.1.1 CD20 Rituximab, a monoclonal antibody directed against the CD20 molecule found on pre-B cells and mature B cells (but not on plasma cells), was introduced in the late 1990s for the treatment of non-Hodgkin’s lymphoma Belimunab BAFF (BLyS)

Atacicept

APRIL Atacicept

BAFF-R BCMA CD22

TACl

BCR Bortezomib/Carfilzomib CD19

CD4

26S Proteasome

0

AS

KP

124

CD

CD1

20

CD

0

54

T-Cell

80

CTLA4

Obinutuzumab

B-Cell

CD

86

1

CD28

FIGURE 21.5

Novel targets to control humoral alloimmunity.

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286

21. NOVEL DRUGS IN KIDNEY TRANSPLANTATION

and later gained favor as a component of induction and desensitization regimens to reduce or prevent the development of alloantibody as well as one of the treatments for antibody mediated rejection. However, the incorporation of B-cell depleting agents at induction had unexpected results with higher than expected rates of acute rejection.87 Surprisingly, these rejection episodes frequently encompassed humoral as well as cellular responses. Rituximab has also been used for the treatment of acute antibody mediated rejection, and as part of desensitization strategies, with limited success.88 Obinutuzumab (Gazyva, Genentech), also known as afutuzumab until 2009, is a fully humanized monoclonal antibody to CD20 on mature B cells, currently approved by the FDA for the treatment of chronic lymphocytic leukemia in combination with chlorambucil. An early phase trial is currently planned to test preliminary efficacy as a desensitization strategy in kidney transplant recipients. 21.4.1.2 Targeting BAFF/BLYS B cell activation factor (BAFF; also known as BLYS and TNFSF13B) and its homologue APRIL (a proliferationinducing ligand) promote B cell development, class switching, and survival.89 91 BAFF, but not APRIL, also binds to BAFF receptor (BAFFR) to promote the development and survival of B2 cells and marginal zone B cells.92 Of note, B1 B cells, memory B cells and a small population of mature splenic B cells do not require BAFF or APRIL for survival. BAFF levels are controlled by production but also by B cell absorption.93 The increase in BAFF levels after widespread B cell depletion94 may provide a promiscuous environment that promotes the survival of developing alloreactive B cells that would otherwise die upon encountering graft antigen in the periphery. This provides a possible explanation for the apparent late augmentation of the humoral alloimmune response following rituximab induction. In 2011, belimumab (Benlysta, GlaxoSmithKline plc.), a human IgG1 monoclonal antibody targeting BAFF, was approved by the FDA for the treatment of SLE based on data from two large phase III trials BLISS-52 and BLISS-76. Belimumab sequesters soluble and membrane-bound BAFF and suppresses B cell proliferation and antibody production. Belimumab-treated patients experienced significant decreases in the numbers of naive and activated B cells, as well as plasma cells, whereas memory B cells and T cell populations did not decrease.95 The use of belimumab alone as an induction agent in transplantation is likely to be impractical because it takes several weeks to complete B cell depletion after blockade of BAFF signaling. However, combination therapy consisting of a standard B cell depleting agent (such as rituximab) at induction with the administration belimumab after transplantation may, by increasing the stringency of selection, ensure that the recovering alloreactive B cell population is robustly deleted and prevent the generation of late alloantibody responses. In a murine diabetic model, indefinite allograft survival of a fully MHC-mismatched islet transplants was achieved after a transient period of mature peripheral B cell depletion was induced by means murine analog of belimumab.96 The tolerant state was associated with abrogation of the donor-specific antibody response, transient preponderance of immature/transitional B cells in all lymphoid organs, impaired CD4 T cell activation during the period of B cell depletion, and presence of a “regulatory” cytokine milieu. Whether blocking BAFF signaling improves kidney graft survival is as yet untested, and there are several reasons why it may be ineffective, such as a potential negative impact on regulatory B97 and T cells98 and germinal center expansion of alloreactive B cells that escape negative selection pressures induced by the kidney graft. Hopefully, some of these questions will be answered by a phase II clinical trial (NCT01536379) of belimumab currently underway in kidney transplant recipients for the prevention of allograft rejection. 21.4.1.3 TACI Receptor The relationship between the long-lived plasma cells (source of alloantibody) and the memory B-cell populations is complex but it is relatively clear that the two populations survive independently of each other.99,100 Survival of long-lived plasma cells in their bone marrow niche has been recently shown to be dependent on eosinophils that secrete interleukin-6 and APRIL.101 BAFF and APRIL share two receptors: transmembrane activator and cyclophilin ligand interactor (TACI) and B cell maturation antigen (BCMA). APRIL binds strongly to BCMA and moderately to TACI, whereas BAFF binds weakly to BCMA and strongly to TACI.102 Additionally, BAFF binds strongly to BAFF receptor (BAFF-R). These three main receptors have distinct expression patterns based on B cell development stages, related to their separate functions. TACI appears to be the dominant receptor regulating plasma cell.103,104 Atacicept (Merck Serono) is a chimeric recombinant fusion protein comprising the extracellular domain of the TACI receptor linked to the human IgG1 Fc domain. Atacicept inhibits B lymphocyte stimulation by neutralizing BAFF, APRIL, and heterotrimer activity and induces significant depletion of plasma cells.105 However, TACI-Ig

I. KIDNEY TRANSPLANTATION

REFERENCES

287

treatment does not reduce the numbers of memory B cells, which are active in long-term humoral immunity, as their survival is independent of BAFF or APRIL.89 Although the data from two Phase Ib studies appeared promising,106,107 the results of phase II/III trials in lupus were disappointing, with limited efficacy and an increased rate of infections.108,109 Within the context of transplantation, BAFF blockade using atacicept in a NHP renal transplant model in combination with an AMR inducing regimen showed reduced levels of DSA and lower peripheral B cell numbers at 6 weeks posttransplant, albeit with only a marginal increase in graft survival.110 One potential area where atacicept may find application is that of desensitization. Clearly there exists redundancy for BAFF and APRIL in maintaining long-lived plasma cell survival and it may be that targeting both APRIL and BAFF (e.g., with atacicept) rather than simply blocking BAFF signaling alone (e.g., with belimumab) is the best strategy to achieve depletion of these cells.

21.4.2 Targeting Plasma Cells Bortezomib (Velcade, Millennium Pharmaceuticals) was approved for the treatment of multiple myeloma in 2010. It binds the catalytic site of the 26S proteasome with high affinity and specificity and inhibits the degradation of cell-cycle regulatory proteins, resulting in cell-cycle arrest and apoptosis. Bortezomib has been anecdotally used for the treatment of acute antibody mediated rejection111,112 and as part of desensitization strategies113 with mixed success. Its use has been limited by the associated toxicities including cytopenias, peripheral neuropathy, and heart failure. Carfilzomib (Krypolis, Onyx Pharmaceuticals) is a second-generation proteasome inhibitor that irreversibly binds to and inhibits the chymotrypsin-like activity of the 20S proteasome. Interim analysis of data from a phase III clinical trial in patients with relapsed multiple myeloma showed that treatment with carfilzomib was associated with doubling of the progression free survival. An advantage of carfilzomib over bortezomib is the reduced risk of neuropathy. A phase I clinical trial of carfilzomib (NCT02442648) as part of a desensitization strategy in kidney transplant candidates is currently underway.

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The role of CD40 in CD40L- and antibody-mediated platelet activation. Thromb Haemost 2005;93(6):1137 46. 58. Mirabet M, Barrabes JA, Quiroga A, Garcia-Dorado D. Platelet pro-aggregatory effects of CD40L monoclonal antibody. Mol Immunol 2008;45(4):937 44. 59. Daley SR, Cobbold SP, Waldmann H. Fc-disabled anti-mouse CD40L antibodies retain efficacy in promoting transplantation tolerance. AmJ Transplant 2008;8(11):2265 71. 60. Pinelli DF, Wagener ME, Liu D, et al. An anti-CD154 domain antibody prolongs graft survival and induces Foxp3(1) iTreg in the absence and presence of CTLA-4 Ig. Am J Transplant 2013;13(11):3021 30. 61. Haanstra KG, Ringers J, Sick EA, et al. Prevention of kidney allograft rejection using anti-CD40 and anti-CD86 in primates. Transplantation 2003;75(5):637 43. 62. Pearson TC, Trambley J, Odom K, et al. Anti-CD40 therapy extends renal allograft survival in rhesus macaques. Transplantation 2002;74(7):933 40. 63. Gilson CR, Milas Z, Gangappa S, et al. Anti-CD40 monoclonal antibody synergizes with CTLA4-Ig in promoting long-term graft survival in murine models of transplantation. J Immunol 2009;183(3):1625 35. 64. Aoyagi T, Yamashita K, Suzuki T, et al. A human anti-CD40 monoclonal antibody, 4D11, for kidney transplantation in cynomolgus monkeys: induction and maintenance therapy. Am J Transplant 2009;9(8):1732 41. 65. Badell IR, Thompson PW, Turner AP, et al. Nondepleting anti-CD40-based therapy prolongs allograft survival in nonhuman primates. Am J Transplant 2012;12(1):126 35. 66. Imai A, Suzuki T, Sugitani A, et al. A novel fully human anti-CD40 monoclonal antibody, 4D11, for kidney transplantation in cynomolgus monkeys. Transplantation 2007;84(8):1020 8. 67. Song L, Ma A, Dun H, et al. Effects of ASKP1240 combined with tacrolimus or mycophenolate mofetil on renal allograft survival in Cynomolgus monkeys. Transplantation 2014;98(3):267 76. 68. Calderhead DM, Buhlmann JE, van den Eertwegh AJ, Claassen E, Noelle RJ, Fell HP. Cloning of mouse Ox40: a T cell activation marker that may mediate T-B cell interactions. J Immunol 1993;151(10):5261 71. 69. al-Shamkhani A, Birkeland ML, Puklavec M, Brown MH, James W, Barclay AN. OX40 is differentially expressed on activated rat and mouse T cells and is the sole receptor for the OX40 ligand. Europ J Immunol 1996;26(8):1695 9. 70. Gramaglia I, Weinberg AD, Lemon M, Croft M. Ox-40 ligand: a potent costimulatory molecule for sustaining primary CD4 T cell responses. J Immunol 1998;161(12):6510 17. 71. Rogers PR, Song J, Gramaglia I, Killeen N, Croft M. OX40 promotes Bcl-xL and Bcl-2 expression and is essential for long-term survival of CD4 T cells. Immunity 2001;15(3):445 55. 72. Vu MD, Clarkson MR, Yagita H, Turka LA, Sayegh MH, Li XC. Critical, but conditional, role of OX40 in memory T cell-mediated rejection. J Immunol 2006;176(3):1394 401. 73. Vu MD, Xiao X, Gao W, Degauque N, Chen M, Kroemer A, et al. OX40 costimulation turns off Foxp3 1 Tregs. Blood 2007;110(7):2501 10. 74. Kinnear G, Wood KJ, Marshall D, Jones ND. Anti-OX40 prevents effector T-cell accumulation and CD8 1 T-cell mediated skin allograft rejection. Transplantation 2010;90(12):1265 71. 75. Chen M, Xiao X, Demirci G, Li XC. OX40 controls islet allograft tolerance in CD154 deficient mice by regulating FOXP3 1 Tregs. Transplantation 2008;85(11):1659 62. 76. Valzasina B, Guiducci C, Dislich H, Killeen N, Weinberg AD, Colombo MP. Triggering of OX40 (CD134) on CD4(1)CD25 1 T cells blocks their inhibitory activity: a novel regulatory role for OX40 and its comparison with GITR. Blood 2005;105(7):2845 51. 77. Curry AJ, Chikwe J, Smith XG, et al. OX40 (CD134) blockade inhibits the co-stimulatory cascade and promotes heart allograft survival. Transplantation 2004;78(6):807 14. 78. Kahan BD, Rajagopalan PR, Hall M. Reduction of the occurrence of acute cellular rejection among renal allograft recipients treated with basiliximab, a chimeric anti-interleukin-2-receptor monoclonal antibody. United States Simulect Renal Study Group. Transplantation 1999;67(2):276 84. 79. Webster AC, Playford EG, Higgins G, Chapman JR, Craig JC. Interleukin 2 receptor antagonists for renal transplant recipients: a meta-analysis of randomized trials. Transplantation 2004;77(2):166 76. 80. Muraguchi A, Hirano T, Tang B, et al. The essential role of B cell stimulatory factor 2 (BSF-2/IL-6) for the terminal differentiation of B cells. J Exper Med 1988;167(2):332 44. 81. Korn T, Bettelli E, Oukka M, Kuchroo VK. IL-17 and Th17 cells. Ann Rev Immunol 2009;27:485 517. 82. Bettelli E, Carrier Y, Gao W, et al. Reciprocal developmental pathways for the generation of pathogenic effector TH17 and regulatory T cells. Nature 2006;441(7090):235 8. 83. Zhao X, Boenisch O, Yeung M, et al. Critical role of proinflammatory cytokine IL-6 in allograft rejection and tolerance. Am J Transplant 2012;12(1):90 101. 84. Roll P, Muhammad K, Schumann M, et al. In vivo effects of the anti-interleukin-6 receptor inhibitor tocilizumab on the B cell compartment. Arthritis Rheum 2011;63(5):1255 64.

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85. Kikuchi J, Hashizume M, Kaneko Y, Yoshimoto K, Nishina N, Takeuchi T. Peripheral blood CD4(1)CD25(1)CD127(low) regulatory T cells are significantly increased by tocilizumab treatment in patients with rheumatoid arthritis: increase in regulatory T cells correlates with clinical response. Arthritis Res Ther 2015;17(1):10. 86. Vo AA, Choi J, Kim I, et al. A Phase I/II trial of the interleukin-6 receptor specific humanized monoclonal (tocilizumab) 1 intravenous immunoglobulin in difficult to desensitize patients. Transplantation 2015. 87. Clatworthy MR, Watson CJ, Plotnek G, et al. B-cell-depleting induction therapy and acute cellular rejection. New Engl J Med 2009;360 (25):2683 5. 88. Macklin PS, Morris PJ, Knight SR. A systematic review of the use of rituximab for desensitization in renal transplantation. Transplantation 2014;98(8):794 805. 89. Benson MJ, Dillon SR, Castigli E, et al. Cutting edge: the dependence of plasma cells and independence of memory B cells on BAFF and APRIL. J Immunol 2008;180(6):3655 9. 90. Mackay F, Silveira PA, Brink R. B cells and the BAFF/APRIL axis: fast-forward on autoimmunity and signaling. Curr Opin Immunol 2007;19(3):327 36. 91. Schneider P, MacKay F, Steiner V, et al. BAFF, a novel ligand of the tumor necrosis factor family, stimulates B cell growth. J Exper Med 1999;189(11):1747 56. 92. Mackay F, Schneider P. Cracking the BAFF code. Nat Rev Immunol 2009;9(7):491 502. 93. Kreuzaler M, Rauch M, Salzer U, et al. Soluble BAFF levels inversely correlate with peripheral B cell numbers and the expression of BAFF receptors. J Immunol 2012;188(1):497 503. 94. Bloom D, Chang Z, Pauly K, et al. BAFF is increased in renal transplant patients following treatment with alemtuzumab. Am J Transplant 2009;9(8):1835 45. 95. Stohl W, Hiepe F, Latinis KM, et al. Belimumab reduces autoantibodies, normalizes low complement levels, and reduces select B cell populations in patients with systemic lupus erythematosus. Arthritis Rheum 2012;64(7):2328 37. 96. Parsons RF, Yu M, Vivek K, et al. Murine islet allograft tolerance upon blockade of the B-lymphocyte stimulator, BLyS/BAFF. Transplantation 2012;93(7):676 85. 97. Yang M, Sun L, Wang S, et al. Novel function of B cell-activating factor in the induction of IL-10-producing regulatory B cells. J Immunol 2010;184(7):3321 5. 98. Walters S, Webster KE, Sutherland A, et al. Increased CD4 1 Foxp3 1 T cells in BAFF-transgenic mice suppress T cell effector responses. J Immunol 2009;182(2):793 801. 99. Ahuja A, Anderson SM, Khalil A, Shlomchik MJ. Maintenance of the plasma cell pool is independent of memory B cells. Proc Nat Acad Sci USA 2008;105(12):4802 7. 100. DiLillo DJ, Hamaguchi Y, Ueda Y, et al. Maintenance of long-lived plasma cells and serological memory despite mature and memory B cell depletion during CD20 immunotherapy in mice. J Immunol 2008;180(1):361 71. 101. Chu VT, Fro¨hlich A, Steinhauser G, Scheel T, Roch T, Fillatreau S, et al. Eosinophils are required for the maintenance of plasma cells in the bone marrow. Nat Immunol 2011;12(2):151 9. 102. Day ES, Cachero TG, Qian F, et al. Selectivity of BAFF/BLyS and APRIL for binding to the TNF family receptors BAFFR/BR3 and BCMA. Biochemistry 2005;44(6):1919 31. 103. Ou X, Xu S, Lam KP. Deficiency in TNFRSF13B (TACI) expands T-follicular helper and germinal center B cells via increased ICOS-ligand expression but impairs plasma cell survival. Proc Nat Acad Sci USA 2012;109(38):15401 6. 104. Tsuji S, Cortesao C, Bram RJ, Platt JL, Cascalho M. TACI deficiency impairs sustained Blimp-1 expression in B cells decreasing longlived plasma cells in the bone marrow. Blood 2011;118(22):5832 9. 105. Munafo A, Priestley A, Nestorov I, Visich J, Rogge M. Safety, pharmacokinetics and pharmacodynamics of atacicept in healthy volunteers. Europ J Clin Pharmacol 2007;63(7):647 56. 106. Dall’Era M, Chakravarty E, Wallace D, et al. Reduced B lymphocyte and immunoglobulin levels after atacicept treatment in patients with systemic lupus erythematosus: results of a multicenter, phase Ib, double-blind, placebo-controlled, dose-escalating trial. Arthritis Rheum 2007;56(12):4142 50. 107. Pena-Rossi C, Nasonov E, Stanislav M, et al. An exploratory dose-escalating study investigating the safety, tolerability, pharmacokinetics and pharmacodynamics of intravenous atacicept in patients with systemic lupus erythematosus. Lupus 2009;18(6):547 55. 108. Ginzler EM, Wax S, Rajeswaran A, et al. Atacicept in combination with MMF and corticosteroids in lupus nephritis: results of a prematurely terminated trial. Arthritis Res Ther 2012;14(1):R33. 109. Isenberg DA, Rahman A. Systemic lupus erythematosus in 2013. Taking a closer look at biologic therapy for SLE. Nat Rev Rheumatol 2014;10(2):71 2. 110. Kwun J, Page E, Hong JJ, et al. Neutralizing BAFF/APRIL with atacicept prevents early DSA formation and AMR development in T cell depletion induced nonhuman primate AMR model. Am J Transplant 2015;15(3):815 22. 111. Flechner SM, Fatica R, Askar M, et al. The role of proteasome inhibition with bortezomib in the treatment of antibody-mediated rejection after kidney-only or kidney-combined organ transplantation. Transplantation 2010;90(12):1486 92. 112. Waiser J, Budde K, Schutz M, et al. Comparison between bortezomib and rituximab in the treatment of antibody-mediated renal allograft rejection. Nephrol Dial Transplant 2012;27(3):1246 51. 113. Everly MJ. An update on antibody reduction and rejection reversal following bortezomib use: a report of 52 cases across 10 centers. Clin Transpl 2010;353 62.

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22 Hematopoietic Stem Cell Transplantation for Induction of Allograft Tolerance Kiyohiko Hotta, Tetsu Oura, A. Benedict Cosimi and Tatsuo Kawai Massachusetts General Hospital, Boston, MA, United States

22.1 INTRODUCTION The short-term results of organ transplantation have significantly improved in recent years with the advent and availability of potent immunosuppressive medications. However, long-term survival following organ transplantation has been less than satisfactory as a result of chronic rejection and complications associated with the multiple medications necessary to suppress allograft rejection. Patients with suppressed immune function are vulnerable to numerous pathologies, including cardiovascular disease, infection, malignancies, and metabolic derangements, all of which contribute to the high rate of patient mortality, 4.8% during the first year and 7.7% during years 2 5, despite the presence of a well-functioning graft.1,2 Thus, a critical goal of organ transplantation is to obviate the need for chronic immunosuppression through the induction of specific allograft tolerance. This chapter reviews the representative basic studies in rodents and preclinical studies in nonhuman primates (NHPs) that presaged the development of strategies for clinical tolerance induction (Fig. 22.1), followed by a brief summary of the three major ongoing clinical trials in kidney transplantation.

22.2 BASIC STUDIES IN RODENTS Early studies by Ray D. Owen published in 1945 led to the discovery that intrauterine cross-circulation in dizygotic bovine twins, so called the freemartin cattle, results in hematopoietic chimerism (Fig. 22.2). Subsequent demonstration that freemartin cattle are able to accept genetically disparate skin allografts from their twin3 5 led Billingham and Medawar to initiate a series of seminal studies on neonatal tolerance. These demonstrated that skin allograft tolerance also could be induced in the early postnatal period by injecting donor cells intravenously into the eventual graft recipient.6 In developing his original hypothesis about acquired immunological tolerance, Medawar had relied on Owen’s earlier findings that the chimerism and immunological tolerance demonstrated in the freemartin cattle was stable and long lasting. Unfortunately, most subsequent studies revealed that neonatal tolerance reported by Billingham et al. was not a direct translation of the freemartin cattle and determined that chimerism induced in neonatal mice was not, in fact, stable, and the number of circulating donor hematopoietic cells eventually decreased to the point where they could no longer be detected.7,8 As time went on, it became clear that postnatal induction of allograft tolerance was successful only if full donor chimerism was established in the recipient, as demonstrated by Main and Prehn who achieved skin allograft tolerance in animals undergoing donor bone marrow transplantation (BMT) after a lethal-dose of total body irradiation (TBI).9 In contrast to freemartin cattle, the chimerism induced in these radiation chimeras was full donor chimerism. The recipient hematopoietic cells were completely replaced with donor hematopoietic cells.

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© 2017 Elsevier Inc. All rights reserved.

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Transient chimerism

MGH

Neonatal tolerance

Radiation transient chimerism

Medawar et al. (1953)

Freemartin

Kidney tolerance by transient chimerism In NHP

Balner (1964)

Persistent mixed chimerism

Owen (1945) permanent mixed chimerism

Kidney tolerance (HLA mismatched) by transient chimerism Kawai et al. (2008)

Kawai et al. (1995)

Stanford Mixed chimerism by TLI

Mixed Mixed chimerism chimerism by TBI+mAb by TBI+TI+mAb

Slavin et al. (1977)

Cobbold et al. (1986)

Kidney tolerance (HLA matched) by persistent mixed chimerism

Sharabi et al. (1989)

Sacndling et al. (2008)

Full donor chimerism

Northwestern

Full donor chimerism by lethal TBI

Kidney tolerance (HLA mismatch) by full chimerism

Main (1955)

Leventhal et al. (2012)

1950

1960

1970

1980

1990 rodents

2000 NHPs

2010 clinical

FIGURE 22.1 Evolution of allograft tolerance via induction of chimerism. Three strategies have evolved to induce allograft tolerance since Owen first discovered persistent mixed chimerism in dizygotic bovine twins, so-called freemartin cattle, in 1945: (1) persistent mixed chimerism, (2) transient mixed chimerism, and (3) full donor chimerism. As a direct translation of Owen’s earlier work, Billingham et al. achieved neonatal tolerance in mice and chicks in 1953. Most studies following this report, however, observed that the chimerism in these mice was only transient, which suggested that the mechanism of tolerance in these mice might not be identical to freemartin cattle.7,8 In 1964, Balner10 also suggested the possibility of inducing allograft tolerance through transient chimerism. Allograft tolerance via persistent mixed chimerism was first achieved by Slavin et al.,13 who used a conditioning regimen with TLI. This work was followed by numerous studies in murine models with various nonmyeloablative conditioning regimens. However, induction of persistent mixed chimerism has been extremely difficult to induce in primates. The MGH has induced renal allograft tolerance with only transient mixed chimerism. Stanford University continue to pursue allograft tolerance by achieving persistent mixed chimerism. On the other hand, as safer conditioning regimens have been developed, tolerance induction via full donor chimerism has also shown some success. Using this approach, the Northwestern group has recently achieved renal allograft tolerance via induction of persistent full donor chimerism in HLA-mismatched kidney transplant recipients without an unacceptable incidence of GVHD. TLI, total lymphoid irradiation; TBI, total body irradiation; TI, thymic irradiation; mAb, monoclonal antibody.

Not surprisingly, however, a significant number of these animals developed graft-versus-host disease (GVHD), rendering this approach unacceptable for most clinical protocols. In 1964, Balner reported a series of studies in adult rat radiation chimeras pretreated with high-dose TBI and donor BMT. Interestingly, he found that several of the animals which had developed transient chimerism only were still able to permanently accept skin allografts.10 Recognizing the implications of this finding, Balner emphasized the potential advantages of avoiding durable chimerism in terms of minimizing the risk of GVHD. His observations showed the clear possibility that specific tolerance (i.e., tolerance without the necessity of immunosuppression) could be induced independently of establishing a permanent chimeric state. Surprisingly, tolerance induced by transient mixed chimerism was not extensively pursued, and the majority of researchers focused on establishing durable mixed chimerism in adult animals (Fig. 22.1), equivalent to the state Owen described in the freemartin cattle (Fig. 22.2). It was Slavin and Strober who first reported successful induction of persistent chimerism in adult rodents. Using total lymphoid irradiation (TLI) conditioning, persistent mixed chimerism and skin or heart allograft

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B. Stanford Persistent mixed chimerism

A. Freemartin C. MGH Transient mixed chimerism

Persistent Mixed chimerism Host cells Donor cells

D. Northwestern Persistent full chimerism

FIGURE 22.2 Three clinical strategies. Three top strategies for clinical tolerance induction have emerged since the possibility of acquired neonatal tolerance through persistent mixed chimerism was first suggested in freemartin cattle. These strategies are being pursued independently by three major groups, with variable results. The Stanford group has been studying persistent mixed chimerism to induce HLA-matched renal allograft tolerance. MGH has successfully induced HLA mismatched renal allograft tolerance using the transient mixed chimerism strategy. Most recently, the Northwestern group was able to achieve HLA mismatched allograft tolerance with persistent full donor chimerism (solid circle: host cells, open circle: donor cells).

tolerance were successfully induced following MHC-mismatched BMT.11 13 In these studies, the investigators found that (1) a cumulative TLI dose of 3400 rad, (2) higher doses (30 3 108 rather than 10 3 108) of bone marrow (BM) cells, and (3) thymic irradiation were all necessary for consistent engraftment of BM cells. Although GVHD was not observed in most TLI recipients, the mechanisms by which TLI prevents GVHD were not clarified. In 1984, Ildstad and Sachs reported the induction of stable mixed chimerism without GVHD in MHC-mismatched donor-recipient combinations of BMTs.14 In this study, a mixture of T cell depleted syngeneic and allogeneic BM cells was infused into mice treated with lethal-dose TBI. All recipients that were reconstituted with a mixture of these BM cells survived, and close to 90% of the recipients accepted skin allografts from the BM donor indefinitely. This study indicated that stable mixed chimerism could be induced without risk of GVHD, if the T cells were removed from both recipient and donor BM grafts. However, since lethal-dose TBI was included in the conditioning regimen, this approach was not directly applicable to clinical transplantation. To achieve BM engraftment with reduced-dose TBI, Cobbold et al. reported a new strategy using T cell depleting monoclonal antibody (mAb). After a conditioning regimen that consisted of sublethal-dose TBI (6 Gy) and T cell depleting mAb, induction of persistent mixed chimerism and donor-specific skin allograft tolerance was successfully achieved in mice.15 With lower dose (3 Gy) TBI, only limited levels of chimerism with moderately prolonged skin allograft survival (,30 days) could be induced. Sharabi and Sachs subsequently reported successful induction of persistent mixed chimerism by administering a combination of 7 Gy local thymic irradiation, 3 Gy TBI, and T cell depleting mAbs. They reported that, without thymic irradiation, consistent induction of chimerism and allograft tolerance could not be achieved. The authors speculated that a significant number of residual T cells survived without thymic irradiation and prevented consistent engraftment of BM cells.16 In their subsequent mechanistic studies, specific deletion of antidonor T cell clones in the thymus was found to be responsible for skin allograft tolerance induced by persistent chimerism.17,18 To develop a mixed chimerism approach that might be more clinically applicable, Sykes et al. sought a conditioning regimen without TBI. They found that high-dose (174 3 106) BM cell infusions combined with anti-CD4 and anti-CD8 mAbs and thymic irradiation induced stable mixed chimerism and skin allograft tolerance without the need for TBI.19,20 Weckerle et al. subsequently reported successful induction of stable mixed chimerism without TBI or T cell depletion by adding double costimulatory blockade with anti-CD40L mAb and CTLA4Ig.21 23 This group more recently induced persistent mixed chimerism and skin allograft tolerance without cytoreductive treatment by infusing regulatory T cells (Tregs).24 In this study, polyclonal recipient Tregs were infused along

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with fully mismatched allogeneic donor BM cells into recipients conditioned solely with a short-course of costimulatory blockade and rapamycin. This treatment led to long-term multilineage chimerism and donor-specific skin graft tolerance. Cippa et al. have reported another novel strategy to induce mixed chimerism and tolerance without myelosuppressive therapy by directly modulating the intrinsic apoptosis pathway of peripheral lymphocytes. Their conditioning protocol, which includes a Bcl-2 inhibitor, CD154 blockade, low-dose cyclosporine (CyA), and BMT-induced stable chimerism and skin allograft tolerance without myelosuppressive therapy.25 Extensive studies over last 60 years of hematopoietic chimerism in mice have provided essential information regarding the mechanisms by which hematopoietic stem cell transplantation can achieve tolerance of allogeneic transplants. However, translating these observations to large animals and humans has been difficult. It has become apparent that inbred laboratory mice are essentially devoid of bona fide allospecific memory T and B cells, which represents a major barrier to chimerism and induction of tolerance in large animals.26 29 Nevertheless, some of the protocols designed to achieve transplantation tolerance via hematopoietic chimerism in laboratory mice have finally begun to be validated in a “nonlaboratory” milieu, i.e., NHPs, and even clinical protocols.

22.3 PRECLINICAL STUDIES IN NONHUMAN PRIMATES As summarized above, many murine studies have shown that stable mixed chimerism and skin allograft tolerance can be readily achieved across full MHC barriers after treatment with nonmyeloablative conditioning regimens. However, in contrast to murine studies, it has been extremely difficult to induce persistent mixed chimerism in primates. Investigators at Emory University have attempted to induce stable mixed chimerism in NHPs treated with a nonmyeloablative regimen consisting of busulfan, costimulatory blockade (CTLA4Ig plus anti-CD154 mAb), and rapamycin. They achieved high-level myeloid, but not lymphoid, chimerism. The duration of chimerism was correlated with the degree of MHC matching between donors and recipients. However, donor BM cell engraftment was consistently lost after recovery of T cell-mediated alloreactivity, even in an MHCmatched cohort.30,31 More recently, the Emory group reported the synergistic effect of CTLA4Ig and anti-CD40 mAb on chimerism induction, but persistent chimerism still was not achieved.32 Similar to the Emory experience, studies by our group at the Massachusetts General Hospital (MGH) also revealed the difficulty of inducing persistent chimerism with a nonmyeloablative conditioning regimen. Nevertheless, we have found that renal allograft tolerance can be induced with only transient chimerism. After a conditioning regimen based on the earlier murine model,16 which includes TBI (300 rads), thymic irradiation (700 rads), horse antithymocyte globulin (hATG), as well as splenectomy and a 1-month course of CyA, the recipients of combined kidney and bone marrow transplantation (CKBMT) achieved renal allograft tolerance after induction of only transient chimerism (Fig. 22.3).33,34 Mixed chimerism was subsequently improved by adding costimulatory blockade with anti-CD154 mAb in place of splenectomy.35 However, since anti-CD154 mAb is not clinically available, the conditioning regimen has been further modified to include CTLA4Ig (belatacept) in place of anti-CD154 mAb.36 A major limitation of our initial regimen was that it could not be used for deceased donor transplantation, since conditioning treatments were initiated 1 week before CKBMT. We therefore recently developed a “delayed

FIGURE 22.3 Histopathological findings in a renal allograft specimen from a tolerant monkey. Renal allograft biopsy taken 10 years after combined kidney and bone marrow transplantation (CKBMT) showed no diagnostic abnormality.

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22.4 CLINICAL STUDIES

Anti donor response Tolerance 8.18%

105

Acute rejector 106

Q2 1.56 Comp-FL4-A :: FoxP 3–A

Foxp3

Comp-FL4-A :: FoxP 3–A

106

104

103 0

-103

105

3.04%

Q2 1.41

104

103 0

-103

Q4 24.4

-104

102

CD4 gated

Q3 65.8 103 104 105 106 Comp-FL1-A :: CFSE–A

-104 107

Q4 14.4 102

Q3 81.2 103 104 105 106 Comp-FL1-A :: CFSE–A

107

CFSE

FIGURE 22.4 Regulatory T cells in allograft tolerance induced by transient chimerism. CD31 cells isolated from the monkeys that achieved renal allograft tolerance, or developed acute rejection, were labeled with carboxyfluorescein succinimidyl ester (CFSE) and cultured with irradiated donor cells for 5 days. Cultured cells were then stained with CD4 and FoxP3. After 5 days of culture, significant Treg expansion was observed in the tolerant monkey, but not in the acute rejection monkey.

tolerance” approach, in which the kidney transplant is performed first with conventional immunosuppression, followed by conditioning and donor BMT at a later time. With this approach, we found that additional CD81 T cell depletion by anti-CD8 mAb was necessary to induce mixed chimerism, probably because allo-reactive T cells were activated by the kidney allograft.37,38 The timing of donor BMT was also found to be important as recipients who underwent donor BMT at 1 month failed to achieve tolerance despite consistent induction of chimerism. Higher inflammatory responses detected at 1 month after kidney transplantation might have prevented tolerance induction.38 This delayed tolerance approach has recently been extended to NHP lung transplantation. By adding anti-IL6R mAb to the conditioning regimen, three of four recipients of MHC-mismatched lung allografts achieved long-term lung allograft tolerance. Interestingly, chimerism was more durable in the lung transplant recipients with persistent lymphoid chimerism observed in two recipients.39 A better understanding of the mechanisms involved in allograft tolerance induction via transient chimerism is critically important to improving the efficacy of this approach. Recent in vitro peripheral blood studies of recipients who achieved tolerance have demonstrated donor-specific expansion of Tregs in the CFSE-MLR assay40 (Fig. 22.4). Enrichment of Tregs in the renal allograft also has been consistently observed in NHPs and humans who achieve stable renal allograft tolerance.41 These observations suggest that tolerance induced by transient chimerism may be Treg-dependent, but further studies to elucidate the significance of these observations are clearly necessary.

22.4 CLINICAL STUDIES “Operational tolerance” is generally defined as stable graft function without a destructive immune response in the absence of any maintenance immunosuppressive drugs.42,43 Although the number is small, there have been reports of transplant recipients who spontaneously achieved such “operational tolerance.” Starzl et al. proposed the concept of microchimerism as an explanation for operational tolerance in such individuals. In their studies, very small levels of chimerism (microchimerism), detectable only with sensitive assays (e.g., PCR or FISH), was often associated with excellent liver or kidney allograft function despite minimal or no immunosuppression.44 They hypothesized that immune responses between coexisting donor and recipient cells led to bidirectional clonal exhaustion, followed by peripheral clonal deletion of donor reactive T cells.45 In an effort to enhance such “microchimerism” after organ transplantation, the Pittsburgh and Miami groups have evaluated DBM infusion with conventional immunosuppression in various organ transplants.46 49 Although a lower incidence of acute rejection and lower maintenance immunosuppressive drug dosages have been reported, clear evidence of tolerance induction via this approach has not been demonstrated.46 50

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22. HEMATOPOIETIC STEM CELL TRANSPLANTATION FOR INDUCTION OF ALLOGRAFT TOLERANCE

Conditioning Regimens for CKBMT Stanford

HLA match (n)

Mismatched (6)

Matched (22)

Massachusetts General Hospital Haplo-match (10)

Northwestern

Haplo-match (10)

Haplo-match (2)

Mismatched (19)

Chemotherapy None

Cyclophosphamide 60 mg/kg, d 25, 24

None

Antibody therapy

anti-CD2 mAb 0.1 mg/kg (d 2 2) 0.6 mg/kg, d 1 to 1 Rituximab, 375 mg/m2 BSAa Thymic irradiation

anti-CD2 mAb 0.1 mg/kg (d 2 2) 0.6 mg/kg, d 1 to 1 Rituximab, 375 mg/m2 BSAb Low-dose TBI

Fludarabine 30 mg/kg, d4, 23, 22 Cyclophosphamide 50 mg/kg on d 23, 13 None

700 cGy

150 cGy 3 2 (d 25, 26)

200 cGy

Thymic irradiation

(d 2 1)

CONDITIONING

Rabbit antithymocyte globulin (1.5 mg/kg/day, d 0 4)

Radiation type TLI 80 or 100 cGy/day 3 10 (d 1 10)

TLI

TLI

120 cGy/day 3 10 (d 1 10)

120 cGy/day 3 10 (d 1 2 10)

TBI

700 cGy 1

1

1

HSCT type Infusion day

CD34 CD31 T cells (d10)

CD34 CD31 T cells (d10)

CD34 CD31 T cells (d10)

Donor bone marrow (d0)

Donor bone marrow (d0)

HSC plus “Facilitating cell” (d1)

Initial IS

CyA/steroids

CyA/MMF/ steroids

Tac/MMF

CNI/steroids

CNIc/steroids

Tac/MMF

No rituximab was administered in Case 1 3, two doses on d 7 and 2 in Case 4, 5 and four doses on d 7, 2, 15 and 112 in Cases 5 10. Four doses on d 7, 2, 15 and 112. c Tac was converted to Rapamycin after 1 month due to toxicity to CNI. TLI, total lymphoid irradiation; TBI, total body irradiation; HSCT, hematopoietic stem cell transplantation; IS, immunosuppression; CyA, cyclosporin; Tac, tacrolimus; CNI, calcineurin inhibitor; MMF, mycophenolate mofetil. a

b

Intentional induction of “operational tolerance” has thus far been achieved only by the induction of macrochimerism, also termed high-level chimerism, which can only be reliably detected (typically .1%) by assays, such as flow cytometry. Although the approach is performed quite differently at various centers, Stanford University, MGH, and Northwestern University are currently actively pursuing renal allograft tolerance via induction of hematopoietic chimerism (Table 22.1).

22.5 STANFORD UNIVERSITY The basic protocol underlying the Stanford approach to tolerance induction was initially developed in 1977 by Slavin and Strober, who demonstrated successful induction of skin allograft tolerance in mice with persistent chimerism established by TLI. However, the efficacy of chimerism induction was less clearly demonstrated when attempts were made to induce allograft tolerance in large animal models and humans.51 53 In the initial clinical trial using TLI and rabbit antithymocyte globulin (rATG), but without donor BMT, successful induction of renal allograft tolerance was initially reported in three patients. Two of the participants, however, eventually lost graft function as a result of ureteral stenosis and chronic rejection.54,55 Beginning in 2000, the Stanford team revised their TLI-based conditioning regimen by adding donor CD341 and CD31 cell infusions in an effort to induce stable mixed chimerism and renal allograft tolerance. In clinical trials performed between 2000 and 2003, six HLAmismatched kidney transplant recipients were treated with TLI (80 or 120 cGy 3 10), rATG, and 3.1 11.1 3 106 CD341 cells/kg with less than 0.1 3 106 CD31 cells/kg, followed by posttransplant CyA and prednisone.

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Only two of the six recipients developed transient chimerism lasting up to 3 months. Withdrawal of immunosuppression was attempted in these two patients but both developed rejection at 3.5 and 5.5 months after the drugs were discontinued.56,57 They subsequently revised the protocol by (1) increasing the dose of TLI (120 cGy 3 10), (2) increasing the dose of CD31 cells (1 10 3 106 cells/kg), and (3) adding mycophenolate mofetil (MMF) after the infusion of donor cells. This revised protocol was evaluated in 22 HLA-matched kidney transplant recipients between 2005 and 2013. Sixteen of these subjects developed chimerism that persisted longer than 6 months and their immunosuppression was successfully withdrawn. Persistent chimerism after discontinuation of immunosuppression was observed in only seven of the 16 subjects. Thus, while renal allograft tolerance was achieved with this approach, even with transient chimerism in the HLA-matched cohort, the Stanford studies continued to demonstrate a requirement for persistent chimerism in the HLA-mismatched allograft tolerance cohort (Fig. 22.2).56 Subsequently, they again modified the protocol by increasing the dose of CD31 cells to 3 2 50 3 106 kg 1, and chimerism was successfully induced in 5/10 recipients with the highest chimerism levels being observed in the last recipients who received the greatest number (50 3 106 cells/kg) of CD31 cells. The Stanford group is just now in the process of withdrawing immunosuppression from the recipients who developed chimerism.

22.6 MASSACHUSETTS GENERAL HOSPITAL With support from the Immune Tolerance Network (ITN), a collaborative clinical network for clinical research funded by the National Institute of Allergy and Infectious Diseases, a total of 10 patients with end-stage renal disease have been enrolled in a clinical trial. The initial conditioning regimen included cyclopshosphamide, thymic irradiation, anti-CD2 mAb with or without rituximab, and a posttransplant calcineurin inhibitor (CNI). Of the 10 subjects enrolled into these studies, all recipients developed transient mixed chimerism for up to 3 weeks, without GVHD. Seven of them successfully discontinued their immunosuppression by 14 months. Although all 7 recipients remained immunosuppression-free for 5 or more years (the longest currently being over 12 years), 3 resumed immunosuppression at 6 8 years after kidney transplantation because of chronic rejection or recurrence of membranoproliferative glomerulonephritis.58 60 These results have been compared with 21 living donor kidney recipients of comparable age and disease, who received their transplant during the same time period and were treated with conventional immunosuppression. The patients treated with conventional immunosuppression had a higher incidence of posttransplant complications including hypertension, hyperlipidemia, new-onset diabetes, infection, and skin cancer.60 A major limitation observed in this initial clinical trial was acute kidney injury (AKI), which occurred in 9 subjects between 10 and 20 days posttransplant. AKI was associated with recipient hematopoietic cell recovery and rapid loss of chimerism. Since AKI had not been observed in NHP studies that utilized TBI rather than cyclophosphamide in the conditioning regimen, a revised regimen, in which low-dose TBI replaces cyclophosphamide, has recently been evaluated in two human recipients. Both recipients have done well, without evidence of the AKI syndrome. Immunosuppression in the first patient was discontinued 1 year posttransplant, and the patient remains off immunosuppression after a follow-up period of 2 years. Since MEDI-507 mAb, a component of the conditioning regimen in the initial trials, is no longer available, further clinical trials are planned using a new regimen with belatacept, which was developed in NHP studies.36 Since allograft tolerance was achieved by transient chimerism, and the presence of chimerism cannot itself be used as a surrogate maker of tolerance, there is a pressing need for reliable assays to detect tolerance in such patients so that they may be safely tapered from immunosuppression.

22.7 NORTHWESTERN UNIVERSITY Until recently, full donor chimerism was not regarded as a viable strategy for tolerance induction in patients without malignancies because of the unacceptable risk of GVHD. However, Johns Hopkins Hospital recently developed a conditioning regimen, which can both induce durable chimerism and significantly reduce the risk of GVHD, even in recipients of HLA-mismatched BMT. The BMT conditioning regimen includes rabbit antithymoglobulin, fludarabine, cyclophosphamide, and 200 Gy TBI. After BMT, immunosuppression is delayed for 3 days to elicit maximal proliferation of allo-reactive T cells. Cyclophosphamide is then administered on days 3 and 4 to delete these activated allo-reactive T cells. This treatment is followed by maintenance immunosuppression with MMF and tacrolimus. In 17 BMT recipients (14 HLA-mismatched and

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Host thymus

Foreign antigens (e.g., viral, bacterial)

Precursor T cell Full chimerism

Mixed chimerism

donor T cell

donor T cell

donor APC

donor T cell

donor T cell

donor APC

host T cell

host T cell

host APC

FIGURE 22.5 Differences in T cell development in full and mixed chimerism. In full donor chimerism, donor precursor T cells that develop in the recipient thymus are restricted to recipient type MHC as a result of positive selection. Thus, donor type T cells may not recognize antigens effectively when presented by donor-type APCs, which are the only APCs available in full donor chimerism. In mixed chimerism, since recipient type APCs are also available, both host and donor T cells restricted to recipient MHC can properly recognize the antigens expressed on recipient-type APC. APC, antigen presenting cell.

3 HLA-matched) treated with this conditioning regimen, only one recipient developed self-limiting skin-only acute GVHD.61 Using a similar conditioning regimen but with an additional infusion of tolerogenic CD8-positive and T cell receptor-negative (CD81/TCR-) facilitating cells (FC), Northwestern University has successfully achieved HLAmismatched renal allograft tolerance by inducing durable full donor chimerism (Fig. 22.2). After conditioning with TBI (2 Gy), fludarabine (30 mg/kg 3 3), and cyclophosphamide (50 mg/kg), the recipients received kidney transplantation on day 0 and donor hematopoietic stem cell (HST) and FC infusion on day 1. In contrast to the Johns Hopkins protocol, maintenance immunosuppression with MMF and tacrolimus was started on Day 0, followed by posttransplant cyclophosphamide on day 3.62 64 The facilitating cell population65,66 was found to have various immunomodulatory effects that appeared to enhance engraftment of allogeneic hematopoietic stem cells and to limit the risk of GVHD.65,67 71 Thirty-one recipients have been enrolled in this trial, and to date, 25 recipients have received kidney transplants with their protocol. The first 19 recipients have attained 18 months or more of follow-up. Two of the 19 subjects lost their renal allografts at 3 and 9 months owing to infectious complications. Twelve subjects of the remaining 17 developed persistent chimerism and have been successfully weaned off all immunosuppression. Four patients developed transient chimerism only, and one failed to develop any chimerism. These five patients were unable to taper off their immunosuppression.64 Although these achievements are encouraging, there are several concerns related to the use of full donor chimerism for achieving allograft tolerance. First, the risk of GVHD is not completely eliminated and two GVHD cases, including one fatal case, have been reported in their study. Any risk of GVHD would seem to be difficult to justify in patients without malignancy for the purpose of inducing allograft tolerance. Another concern is the risk of infection. In this trial, various viral, bacterial, and fungal infections have been reported, with two recipients losing their allografts.64 Finally, patients with full donor chimerism might be expected to suffer from varying degrees of immunoincompetence, since donor T cells that develop in the recipient thymus would be restricted to recipient type MHC as a result of positive selection. Thus, donor type T cells may not recognize antigens effectively when presented by the donor type APCs72,73 (Fig. 22.5). Longer-term follow-up is imperative to ensure the safety of durable full donor chimerism.

22.8 CONCLUSION Despite the short-term success of organ transplantation, the true benefit of clinical transplantation can only be gained through immunosuppression-free, specific operational tolerance, which has yet to be fully realized. The

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stepwise evolution of strategies to induce allograft tolerance using hematopoietic stem cells in animal models of increasing immunological complexity has been arduous and long. At a minimum, we can say with certainty that renal allograft tolerance can be intentionally induced, even in humans, and three plausible clinical protocols have been developed to meet this goal: persistent mixed chimeras (Stanford University); transient mixed chimeras (MGH); and full donor chimeras (Northwestern University). Note the latter strategy uses a conditioning regimen developed at Johns Hopkins, but with an additional infusion of tolerogenic facilitating cells. More effort is needed to improve the results of these clinical protocols, including methodological refinements that can achieve the goal of allograft specific tolerance with less clinical morbidity.

Acknowledgement We wish to thank Ann S. Adams for editorial assistance.

References 1. Opelz G, Dohler B. Association of HLA mismatch with death with a functioning graft after kidney transplantation: a collaborative transplant study report. Am J Transplant 2012;12:3031. 2. Opelz G, Dohler B. Effect of human leukocyte antigen compatibility on kidney graft survival: comparative analysis of two decades. Transplantation 2007;84:137. 3. Owen RD. Immunogenetic consequences of vascular anastomoses between bovine twins. Science 1945;102:400. 4. Billingham RE, Lampkin GH, Medawar PB, Williams HL. Tolerance to homografts, twin diagnosis, and the freemartin condition in cattle. Heredity 1952;6:201. 5. Anderson D, Billingham RE, Lampkin GH, Medawar PB. The use of skin grafting to distinguish between monozygotic and dizygotic twins in cattle. Heredity 1951;5:379. 6. Billingham RE, Brent L, Medawar PB. Actively acquired tolerance of foreign cells. Nature 1953;172:603. 7. Doria G. Identification of the immune system responsible for the specificity of actively acquired tolerance in mice. Proc Natl Acad Sci U S A 1963;49:281 6. 8. Michie D, Woodruff MF, Zeiss IM. An investigation of immunological tolerance based on chimaera analysis. Immunology 1961;4:413 24. 9. Main JM, Prehn RT. Successful skin homografts after the administration of high dosage X radiation and homologous bone marrow. J Natl Cancer Inst 1955;15:1023. 10. Balner H. Persistence of tolerance towards donor-type antigens after temporary chimerism in rats. Transplantation 1964;2:464 74. 11. Slavin S, Fuks Z, Kaplan HS, Strober S. Transplantation of allogeneic bone marrow without graft-versus-host disease using total lymphoid irradiation. J Exp Med 1978;147:963. 12. Slavin S, Reitz B, Bieber CP, Kaplan HS, Strober S. Transplantation tolerance in adult rats using total lymphoid irradiation: permanent survival of skin, heart, and marrow allografts. J Exp Med 1978;147:700. 13. Slavin S, Strober S, Fuks Z, Kaplan HS. Induction of specific tissue transplantation tolerance using fractionated total lymphoid irradiation in adult mice: long-term survival of allogeneic bone marrow and skin grafts. J Exp Med 1977;146:34 48. 14. Ildstad ST, Sachs DH. Reconstitution with syngeneic plus allogeneic or xenogeneic bone marrow leads to specific acceptance of allografts or xenografts. Nature 1984;307:168. 15. Cobbold SP, Martin G, Qin S, Waldmann H. Monoclonal antibodies to promote marrow engraftment and tissue graft tolerance. Nature 1986;323:164. 16. Sharabi Y, Sachs DH. Mixed chimerism and permanent specific transplantation tolerance induced by a nonlethal preparative regimen. J Exp Med 1989;169:493. 17. Tomita Y, Khan A, Sykes M. Role of intrathymic clonal deletion and peripheral anergy in transplantation tolerance induced by bone marrow transplantation in mice conditioned with a nonmyeloablative regimen. J Immunol 1994;153:1087. 18. Khan A, Tomita Y, Sykes M. Thymic dependence of loss of tolerance in mixed allogeneic bone marrow chimeras after depletion of donor antigen. Peripheral mechanisms do not contribute to maintenance of tolerance. Transplantation 1996;62:380. 19. Sykes M, Szot GL, Swenson KA, Pearson DA. Induction of high levels of allogeneic hematopoietic reconstitution and donor-specific tolerance without myelosuppressive conditioning. Nat Med 1997;3:783. 20. Wekerle T, Nikolic B, Pearson DA, Swenson KG, Sykes M. Minimal conditioning required in a murine model of T cell depletion, thymic irradiation and high-dose bone marrow transplantation for the induction of mixed chimerism and tolerance. Transpl Int 2002;15:248. 21. Wekerle T, Kurtz J, Ito H, et al. Allogeneic bone marrow transplantation with co-stimulatory blockade induces macrochimerism and tolerance without cytoreductive host treatment. Nat Med 2000;6:464. 22. Durham MM, Bingaman AW, Adams AB, et al. Cutting edge: administration of anti-CD40 ligand and donor bone marrow leads to hemopoietic chimerism and donor-specific tolerance without cytoreductive conditioning. J Immunol 2000;165:1. 23. Wekerle T, Sayegh MH, Hill J, et al. Extrathymic T cell deletion and allogeneic stem cell engraftment induced with costimulatory blockade is followed by central T cell tolerance. J Exp Med 1998;187:2037. 24. Pilat N, Baranyi U, Klaus C, et al. Treg-therapy allows mixed chimerism and transplantation tolerance without cytoreductive conditioning. Am J Transplant 2010;10:751. 25. Cippa PE, Gabriel SS, Chen J, et al. Targeting apoptosis to induce stable mixed hematopoietic chimerism and long-term allograft survival without myelosuppressive conditioning in mice. Blood 2013;122:1669. 26. Valujskikh A. The challenge of inhibiting alloreactive T-cell memory. Am J Transplant 2006;6:647. 27. Adams AB, Pearson TC, Larsen CP. Heterologous immunity: an overlooked barrier to tolerance. Immunol Rev 2003;196:147.

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28. Lakkis FG, Sayegh MH. Memory T cells: a hurdle to immunologic tolerance. J Am Soc Nephrol 2003;14:2402. 29. Nadazdin O, Boskovic S, Murakami T, et al. Host alloreactive memory T cells influence tolerance to kidney allografts in nonhuman primates. Sci Transl Med 2011;3:86ra51. 30. Larsen CP, Page A, Linzie KH, et al. An MHC-defined primate model reveals significant rejection of bone marrow after mixed chimerism induction despite full MHC matching. Am J Transplant 2010;10:2396. 31. Kean LS, Adams AB, Strobert E, et al. Induction of chimerism in rhesus macaques through stem cell transplant and costimulation blockade-based immunosuppression. Am J Transplant 2007;7:320. 32. Page A, Srinivasan S, Singh K, et al. CD40 blockade combines with CTLA4Ig and sirolimus to produce mixed chimerism in an MHCdefined rhesus macaque transplant model. Am J Transplant 2012;12:115. 33. Kawai T, Cosimi AB, Colvin RB, et al. Mixed allogeneic chimerism and renal allograft tolerance in cynomolgus monkeys. Transplantation 1995;59:256. 34. Kawai T, Poncelet A, Sachs DH, et al. Long-term outcome and alloantibody production in a non-myeloablative regimen for induction of renal allograft tolerance. Transplantation 1999;68:1767. 35. Kawai T, Sogawa H, Boskovic S, et al. CD154 blockade for induction of mixed chimerism and prolonged renal allograft survival in nonhuman primates. Am J Transplant 2004;4:1391. 36. Yamada Y, Ochiai T, Boskovic S, et al. Use of CTLA4Ig for induction of mixed chimerism and renal allograft tolerance in nonhuman primates. Am J Transplant 2014;14:2704. 37. Koyama I, Nadazdin O, Boskovic S, et al. Depletion of CD8 memory T cells for induction of tolerance of a previously transplanted kidney allograft. Am J Transplant 2007;7:1055. 38. Yamada Y, Boskovic S, Aoyama A, et al. Overcoming memory T-cell responses for induction of delayed tolerance in nonhuman primates. Am J Transplant 2012;12:330. 39. Tonsho M, Lee S, Aoyama A, et al. Tolerance of lung allografts achieved in nonhuman primates via mixed hematopoietic chimerism. Am J Transplant 2015;15:2231. 40. Hotta K, Aoyama A, Oura T, et al. Donor specific expansion of regulatory T-cells was associated with allograft tolerance induced by transient mixed chimerism in nonhuman primates. Am J Transplant 2015;15(Suppl. 3). Available from: http://www.atcmeetingabstracts.com/. 41. Tonsho M, Benichou G, Nadazdin O, et al. Successful tolerance induction of cardiac allografts in nonhuman primates through donor kidney co-transplantation. Am J Transplant 2013;13(Suppl. 5):181. 42. Roussey-Kesler G, Giral M, Moreau A, et al. Clinical operational tolerance after kidney transplantation. Am J Transplant 2006;6:736. 43. Brouard S, Pallier A, Renaudin K, et al. The natural history of clinical operational tolerance after kidney transplantation through twentyseven cases. Am J Transplant 2012;12:3296. 44. Starzl TE, Murase N, Abu-Elmagd K, et al. Tolerogenic immunosuppression for organ transplantation. Lancet 2003;361:1502. 45. Starzl TE, Demetris AJ. Transplantation tolerance, microchimerism, and the two-way paradigm. Theor Med Bioeth 1998;19:441. 46. Barber WH, Mankin JA, Laskow DA, et al. Long-term results of a controlled prospective study with transfusion of donor-specific bone marrow in 57 cadaveric renal allograft recipients. Transplantation 1991;51:70. 47. Ciancio G, Burke GW, Garcia-Morales R, et al. Effect of living-related donor bone marrow infusion on chimerism and in vitro immunoregulatory activity in kidney transplant recipients. Transplantation 2002;74:488. 48. Tryphonopoulos P, Tzakis AG, Weppler D, et al. The role of donor bone marrow infusions in withdrawal of immunosuppression in adult liver allotransplantation. Am J Transplant 2005;5:608. 49. Light J, Salomon DR, Diethelm AG, et al. Bone marrow transfusions in cadaver renal allografts: pilot trials with concurrent controls. Clin Transplant 2002;16:317. 50. Delis S, Burke 3rd GW, Ciancio G. Bone marrow-induced tolerance in the era of pancreas and islets transplantation. Pancreas 2006;32:1. 51. Slavin S, Gottlieb M, Strober S, et al. Transplantation of bone marrow in outbred dogs without graft-versus-host disease using total lymphoid irradiation. Transplantation 1979;27:139. 52. Myburgh JA, Smit JA, Stark JH, Browde S. Total lymphoid irradiation in kidney and liver transplantation in the baboon: prolonged graft survival and alterations in T cell subsets with low cumulative dose regimens. J Immunol 1984;132:1019. 53. Strober S, Dhillon M, Schubert M, et al. Acquired immune tolerance to cadaveric renal allografts. A study of three patients treated with total lymphoid irradiation. N Engl J Med 1989;321:28. 54. Strober S, Benike C, Krishnaswamy S, Engleman EG, Grumet FC. Clinical transplantation tolerance twelve years after prospective withdrawal of immunosuppressive drugs: studies of chimerism and anti-donor reactivity. Transplantation 2000;69:1549. 55. Saper V, Chow D, Engleman ED, et al. Clinical and immunological studies of cadaveric renal transplant recipients given total-lymphoid irradiation and maintained on low-dose prednisone. Transplantation 1988;45:540. 56. Millan MT, Shizuru JA, Hoffmann P, et al. Mixed chimerism and immunosuppressive drug withdrawal after HLA-mismatched kidney and hematopoietic progenitor transplantation. Transplantation 2002;73:1386. 57. Scandling JD, Busque S, Shizuru JA, et al. Chimerism, graft survival, and withdrawal of immunosuppressive drugs in HLA matched and mismatched patients after living donor kidney and hematopoietic cell transplantation. Am J Transplant 2015;15:695. 58. Kawai T, Cosimi AB, Spitzer TR, et al. HLA-mismatched renal transplantation without maintenance immunosuppression. N Engl J Med 2008;358:353. 59. Kawai T, Sachs DH, Sykes M, Cosimi AB. HLA-mismatched renal transplantation without maintenance immunosuppression. N Engl J Med 2013;368:1850. 60. Kawai T, Sachs DH, Sprangers B, et al. Long-term results in recipients of combined HLA-mismatched kidney and bone marrow transplantation without maintenance immunosuppression. Am J Transplant 2014;14:1599. 61. Bolanos-Meade J, Fuchs EJ, Luznik L, et al. HLA-haploidentical bone marrow transplantation with posttransplant cyclophosphamide expands the donor pool for patients with sickle cell disease. Blood 2012;120:4285. 62. Leventhal J, Abecassis M, Miller J, et al. Chimerism and tolerance without GVHD or engraftment syndrome in HLA-mismatched combined kidney and hematopoietic stem cell transplantation. Sci Transl Med 2012;4:124ra28.

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23 Regulatory T Cell Therapy in Transplantation Scott McEwen1 and Qizhi Tang2 1

Case Western Reserve University School of Medicine, Cleveland, OH, United States 2 University of California, San Francisco, San Francisco, CA, United States

23.1 INTRODUCTION For kidney transplantation, most centers now report excellent short-term graft and patient survival that is mainly attributable to improvements in surgical management and immunosuppressive therapy.1,2 It is becoming clear, however, that these improved short-term outcomes from nonspecific immunosuppression have increased the long-term disease burden to patients, leading to increased rates of diabetes, cardiovascular disease, and malignancy.1 Furthermore, the average lifespan of a standard risk kidney transplant has not exceeded 15 years, with this lack of improvement attributable to late graft failure from chronic rejection and calcineurin inhibitor (CNI) toxicity.1,3 While the exact cause of chronic rejection remains unclear, an immunological process is responsible for a large part.3 Preventing chronic rejection and minimizing nonspecific immunosuppression are primary treatment goals for improving late graft outcomes. Immune tolerance has frequently been called the “holy grail” of transplantation since its success would simultaneously achieve both goals. Transplant tolerance is defined by the absence of an immune response toward the graft while maintaining overall immune competence without the need for exogenous immunosuppression.4 Operationally, tolerance typically means stable graft function in the absence of immunosuppression.5,6 Since the initial experiments in the 1950s to induce tolerance to skin grafts,5,7 two theories of the mechanism of tolerance have prevailed: Deletion of antigen-specific T cells, and active control of immune responses by immunosuppressive cells.8 It is now recognized that both mechanisms are necessary for durable transplant tolerance, and neither alone is sufficient.8 12 As early as the 1970s, research indicated the existence of a T cell population with suppressive properties that were responsible for transplant tolerance.13 16 By the mid-1990s, this cell population was better defined and named regulatory T cells (Tregs).17 19 Tregs are a subset of CD41 T cells that suppress peripheral immune responses and are essential for prevention of autoimmunity. Congenital defects in the FoxP3 transcription factor, which is crucial for Treg development and function, result in fatal systemic autoimmune diseases in mice, and a similar condition called IPEX (immune dysregulation, polyendocrinopathy, enteropathy, X-linked) syndrome in humans.20,21 Extensive research in Treg biology in the past 20 years has led to a deep understanding of their antigen recognition, activation, and cell signaling, and the mechanism of their suppressive function. In addition to their natural role in ensuring self-tolerance, it is now established that Tregs can also suppress alloimmune responses and are important for transplant tolerance. Therapeutic use of Tregs have been shown to be effective in animal models for the treatment of autoimmune diseases, inflammation, graft-versus-host disease (GvHD), and to induce transplant tolerance.22 25 Early phase clinical trials are currently ongoing to assess the safety and efficacy of Treg cell therapy in humans. This chapter discusses the use of Tregs as a cell-based therapy in kidney transplant recipients, reviewing necessary Treg biology before discussing the practical considerations in therapeutic development and the current efforts in clinical application of these cells.

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23.2 Treg BIOLOGY AND THERAPEUTIC IMPLICATIONS 23.2.1 Treg Development Tregs can develop from immature thymocytes in the thymus and from mature T cells in the periphery after the cells have exited the thymus.26 Thymic Treg (tTreg) development is a multistep process controlled by the T cell receptor (TCR) and IL-2 signaling. Immature thymocytes with higher affinity to antigens expressed in the thymus increase CD25 expression as a result of antigen stimulation through their TCR. CD25 is a component of the high affinity IL-2 receptor, and its expression enables developing Tregs to respond to a low level of IL-2 to induce expression of the transcription factor FoxP3. Further stimulation through the TCR then stabilizes FOXP3 expression through epigenetic modification, committing the cells to Treg lineage.27,28 The process of peripheral Treg (pTreg) differentiation is distinct from that of tTregs. pTregs are generated under low antigenic stimulation either by low dose or low affinity antigens, reduced costimulation, or through the action of natural immune suppressive cytokines such as TGF-β or immunosuppressive drugs (effect of immunosuppression, below).26,29,30 This conversion to a regulatory phenotype occurs in secondary lymphoid organs and peripheral tissues. Phenotypically, pTregs and tTregs are indistinguishable and suppress immune responses with similar strength by the same mechanisms.31 33 One distinction of pTregs is that they require continual stimulation with antigens and regulatory cytokines to maintain their phenotype, otherwise they lose FoxP3 expression and suppressive function and revert back to a conventional CD41 T cell (Tconv). Thus, pTregs are a more dynamic population providing additional Treg antigen specificities not present during thymic development. For example, pregnancy is associated with a dramatic rise of Tregs in response to alloantigens from the developing fetus and it has been suggested that pTreg development originated in placental mammals due to the need to establish maternal-fetal tolerance during pregnancy.34 36 In addition, the gastrointestinal tract is an active site of pTreg development in response to food antigens and changes in intestinal microbiota. In the context of transplantation, tTregs contain alloantigen specificities and continued exposure to an allograft in a tolerogenic environment will allow acquisition of new alloantigen specificities through pTreg development (see infectious tolerance below).37 Both tTregs and pTregs are vital to transplant tolerance.

23.2.2 Mechanisms of Action The potency of Tregs in suppressing unwanted immune response lies in their functional versatility.38,39 Tregs can act directly on other immune cells and indirectly by modulating the tissue microenvironment. By virtue of their constitutive expression of high level of CD25, Tregs are the first responders to IL-2 in the tissue, depriving other T cells and NK cells of this important activating cytokine and growth factor.40,41 Tregs also highly express CTLA-4 on their cell surface in steady state and use CTLA-4 to remove B7 costimulatory molecules on antigen presenting cells (APCs) through transendocytosis (engulfing the part of neighboring cells), rendering these APCs tolerogenic by reducing their costimulatory ability.42 After exposure to Tregs, APCs in lymph nodes do not effectively interact with Tconv cells.43,44 Tregs further affect the interaction between APC and effector T cells by altering APC metabolism. For example, Tregs induce APC to express enzymes that deplete essential amino acids required by effector T cells and increase tryptophan metabolites that make T cells unresponsive.45 Tregs also highly express surface ectoenzymes that metabolize proinflammatory ATP to immunosuppressive adenosine.46,47 Activated Tregs secrete the immunosuppressive cytokines IL-10, TGF-β, IL-35, and IL-948 50 and produce exosomes that release microRNA to silence Th1-specific genes in effector cells.51 Lastly, activated Tregs express granzyme and perforin and can directly kill effector T cells and APCs.52 54 Of these many suppressive mechanisms, some are constitutively active nonspecific to a particular antigen, such as IL-2 competition and CTLA-4-mediated transendocytosis, and important for preserving immune homeostasis at rest; whereas others require prior activation of Tregs through their TCR and thus are more important for suppressing an active immune response in an antigen-specific manner such as allograft rejection. This multitude of suppressive mechanisms used by Tregs allows them to tune their suppressive activity depending on the inflammatory context. This therapeutic characteristic is highly desirable in the context of transplantation when just the right degree of suppression is needed to prevent rejection without compromising protective immunity.

23.2.3 Alloantigen Recognition Treg activity is regulated through their TCR. Tregs are CD41 T cells, therefore, they recognize alloantigens presented by MHC class II molecules. CD41 T cells can recognize alloantigen in two different ways; direct and

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indirect. Direct alloantigen recognition refers to host CD41 T cells recognizing intact donor MHC molecules on donor cells; whereas indirect recognition refers to host CD41 T cells recognizing antigenic peptides derived from donor presented by host MHC class II on host APCs. It is worth mentioning that all CD41 T cells have been selected in the thymus to see antigens presented by self MHC class II. Therefore, the indirect pathway is the common way T cells sense antigens, whereas the direct pathway is an aberration unique to transplantation. TCRs capable of directly binding to allogeneic MHC class II have additional antigen specificities presented by self MHC class II. Direct alloantigen recognition is due to structural similarities between an allogeneic MHC and a self MHC with a foreign peptide and can be viewed as an alternative specificity of the T cell. Strictly speaking, direct T cells are not alloantigen specific and it is more accurate to refer to them as alloantigen reactive. The frequencies of T cells capable of direct allorecognition can be as high as 10%,55 whereas frequency of indirect alloreactive T cells is orders of magnitude lower and estimated to be 0.01% 0.1% for a specific allogeneic peptide.56 MHC class II are expressed on donor APCs and at lower levels on endothelial cells and other cell types. Since APCs express both MHC and costimulatory molecules, they are the main stimulators of the direct alloreactive T cells. Donor MHC may also be transferred to host APCs producing direct alloreactive T cells via a “semidirect” pathway.57,58 In general, as donor APCs die away over time and less intact donor MHC class II are available, the direct pathway becomes less important.56 On the other hand, the indirect pathway can present peptides from MHC class I, class II, and minor histocompatibility antigens from the grafts. The indirect alloantigen stimulation persists, thus T cells with indirect alloreactivity become increasingly prevalent over time.56 The above paradigm of alloantigen recognition by CD41 T cells also applies to Tregs. The frequencies of Tregs recognizing direct and indirect alloantigen are similar to that for Tconvs before transplant. Due to the strength of antigraft response driven by the high frequency of directly alloreactive T cells early after transplant, grafts are often irreversibly damaged before alloreactive Tregs reach the grafts. Thus, Treg therapy alone is unlikely to completely counteract this potent immediate antidonor response, and attenuation of alloimmune responses is needed to control graft damage and create the therapeutic window for Tregs.59,60 Due to their higher frequency and the higher abundance of donor MHC class II antigens early after transplant, directly alloreactive Tregs are more likely to contribute to graft protection than indirectly alloreactive Tregs. Over time, with the waning of donor MHC class II antigens and expansion of indirect alloreactive Tregs, indirect Tregs become more important, and experimental evidence suggests that long-term immune tolerance is maintained by indirect Tregs.61 64

23.2.4 Dominant Regulation, Linked Suppression, and Infectious Tolerance Tregs have several characteristics that make them effective instigators of tolerance. First, tolerance conferred by Tregs is dominant, in contrast to passive mechanisms that delete T cells or render T cells unresponsive (anergic).65 Passive mechanisms can be broken by the arrival of new T cells capable of rejecting the graft, whereas tolerance by Tregs actively suppresses other cells and therefore persists despite new production of T cells from the thymus. Moreover, Tregs are known to exert linked or bystander suppression. This means that Tregs specific for one antigen on the graft not only suppress other T cells that recognize the same antigen, but also T cells that see distinct antigens on the same graft.66,67 Therefore, therapeutic Tregs of a single specificity can suppress T cells with a wide variety of other specificities. However, for linked suppression to work, the antigens have to be expressed by the same graft or in the same tissue and present on the same APC (Fig. 23.1A). Thus Treg-mediated suppression is localized to where the Treg activating antigens are, making it unlikely for Tregs to induce global immunosuppression. Not only can Tregs dominantly suppress T cells of other specificities, but by creating a tolerogenic milieu Tregs can help these other T cells to become Tregs—a phenomenon referred to as infectious tolerance (Fig. 23.1B).37,67,68 Similar to linked suppression, infectious tolerance is also limited to antigens present within the same tissue.37,68 This infectious spread of tolerance has been robustly demonstrated in inbred animal models. Animals made tolerant to a graft from strain A can reject a graft from the unrelated strain B. Because of linked suppression, these animals accept grafts from F1 offspring of strains A and B because the B antigen is linked to the A antigen in the same F1 graft. After the F1 AB graft has been accepted for a month or more, a separate graft from strain B is accepted due to the generation of strain-B-reactive Tregs in the AB grafts.37 Infectious tolerance allows tolerance to persist even when the original Tregs or the antigens responsible for inducing tolerance are no longer present. For example, direct alloantigen-reactive Tregs activated by donor APCs may create a tolerogenic microenvironment for indirect alloantigen-specific Tregs to emerge. Over time, with decreasing donor APCs, direct Tregs become less important and indirect Tregs will be mostly responsible for maintaining tolerance to the graft as observed in many animal models.61,62,69

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FIGURE 23.1 Two cardinal features of immune regulation by Tregs. (A) Linked suppression. APC display more than one antigen on surface MHC molecules. Tregs recognize one antigen can suppress effector T cells that recognize a distinct antigen presented by the same APC. (B) Infectious tolerance. Tregs induce tolerance to antigens they recognize and also secrete cytokines that promote peripheral conversion of T cells to become pTregs. Through this process Tconv that recognize a distinct antigen on the same APC can become Tregs, therefore leading to an infectious spread of tolerance to other antigens. Infectious tolerance expands the antigen-specificity of Tregs and allows tolerance to persist beyond the survival of the original Tregs. The pTregs produced through infectious tolerance can then propagate the allograft-specific antigen tolerance through additional cycles of linked suppression and infectious tolerance, as long as the antigens are coexpressed locally within the same tissue.

23.3 Tregs AS PHARMACEUTICALS FOR TRANSPLANTATION In 2015 when this textbook is written, cell therapy is still a nascent field aside from hematopoietic cell transplantation. Since cells are alive, many pharmaceutical development issues such dosing, potency, pharmacokinetics, pharmacodynamics, stability, and cost are not only unknown, but also demand a very different mindset when considering these important issues. In this section of the chapter, we will summarize our current thinking on these issues based on preclinical experiences with Treg cell therapy in transplantation.

23.3.1 Potency As described above, Treg can suppress in an antigen nonspecific manner by sopping up IL-2 and removing costimulation on APCs, but a wider range of suppressive mechanisms are unleashed by TCR activation of Tregs, increasing their potency to control an active immune response. In preclinical models of transplantation, alloantigenreactive Tregs are more effective; however, polyclonal Tregs have a clearly demonstrable therapeutic effect in preventing transplant rejection. The activity of polyclonal Tregs is likely due to both nonantigen specific suppressive mechanisms and the antigen-stimulated activation of the alloreactive Tregs among the polyclonal Tregs. As many as 10% 20% of polyclonal Tregs are alloreactive and are activated in the presence of an allograft, allowing polyclonal Tregs to increase suppressive activity in an antigen-specific manner. By enriching for alloantigen-reactive Tregs, potency of therapeutic Tregs increases 5- to 10-fold over polyclonal Tregs proportional to the frequency of alloreactive Tregs, suggesting that alloantigen reactivity is a dominant determinant of Treg potency.

23.3.2 Dosing An important concept in considering Treg therapy is that Tregs function by providing a counterbalance to effector cells, therefore an effective dose is not simply determined by their absolute number or number per kilogram of body weight, but by the ratio between Tregs and effector cells. In in vitro assays of Treg suppression, inhibition of Tconv cell proliferation can consistently be observed at a ratio of 1 Tregs per 2 Tconv cells (33%), but mostly lost at 1 Treg per 4 Tconv cells (20%). Quantitative experiments in animal models have shown that it requires at least 1 Treg per 2 Tconv cells, i.e., 33% of all CD41 cells must be Tregs, to prevent transplant rejection.65,70,71 Moving to human therapy, it is expected that a similar ratio of cells will be necessary.72 A 70 kg adult human has approximately 460 3 109 lymphocytes, 160 3 109 CD41 cells, and 13 3 109 Tregs.72 Thus, to achieve the 30% Tregs needed for tolerance induction, delivering 50 3 109 Tregs would be required. Since enough Tregs need to be present to control effector T cell activation this dose needs to be given at one time. In fact, in most animal models of Treg therapy in transplantation a single large dose is sufficient in preventing rejection. I. KIDNEY TRANSPLANTATION

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There are two limitations to the use of such a large dose of Tregs. First, total lymphocyte number and Treg frequency in the body are tightly controlled by the availability of MHC molecules and cytokines needed for lymphocyte survival. A large bolus dose of 50 3 109 Tregs is unlikely to engraft efficiently, and will thus fail to reach the therapeutic threshold. In addition, producing such large number of Tregs would require repeated stimulation,73 75 and the stability and longevity of Tregs after massive expansion is uncertain.76 Lymphodepletion is a currently available solution to overcome these limitations. Common lymphocyte depleting agents such as antithymocyte globulin or alemtuzumab decrease lymphocyte counts by 95% 99% at standard doses, leaving 4.6 23 3 109 CD41 Tconv cells. Thus, delivering 2 11 3 109 Tregs would reach the tolerance induction threshold, which falls within current manufacturing possibilities.72,74,75,77 The required dose of Tregs can be further reduced by 80% 90%, to 0.2 2.2 3 109, if alloantigen-reactive Tregs are used.72,77 All these are estimates based on preclinical models and theoretical considerations. Results from clinical trials designed with these considerations will be needed to determine the actual effective doses.

23.3.3 Timing Tregs are an integral part of a T cell immune response. Their activation is typically slower than that of CD81 T cells and Tconv cells. This delayed activation of Tregs allows a protective immune response to proceed where Tregs provide negative feedback control to limit excessive immune activation and collateral tissue damage. This has been beautifully illustrated in mouse models using intravital imaging of transplant rejection.78 Consistently, Tregs are often found to be a signature associated with acute rejections in transplant patients.79,80 However, Treg activation often is too late to stop irreversible graft damage, especially for grafts that have limited ability to regenerate. Therefore, for Treg therapy to be effective, the cells should be infused early before effector T cells are activated. Effective Treg therapy protocols in mouse models of transplantation nearly invariably infuse cells before or at the time of transplantation. However, there are several limitations to this strategy when considering translating to humans. Grafts are likely to be highly inflamed at the time of transplant due to an ischemiareperfusion reaction and surgical trauma. Acute inflammatory mediators interfere with Treg suppression.81 This may explain why a Treg to Tconv ratio of more than 1 often is needed to prevent rejection in these models. This dose of Tregs is difficult to achieve for human patients and raises concern of prolonged global immunosuppression. Additionally, most experimental models of Treg therapy do not use immunosuppression. This will not be possible in early trials in humans when the efficacy of Treg therapy remains unproven. At the time of transplant, patients usually receive a much higher dose of immunosuppression and many of these drugs interfere with Treg survival and function (see below). A better design for Treg cell therapy in humans would be to initiate immunosuppression at the time of transplant to keep effector response in control and introduce Tregs after the acute inflammation has subsided and immunosuppression has been reduced to a low maintenance level.

23.3.4 Pharmacokinetics and Pharmacodynamics With traditional small molecule and biologic drugs, both the drug level measured by pharmacokinetics and the impact of the drug measured by the effect of the drug on the target molecule are important determinants of drug efficacy. Since Tregs preferentially accumulate in inflamed tissue to exert their effects, and their therapeutic impact can last beyond their own persistence through infectious tolerance, measuring Treg pharmacokinetics and pharmacodynamics in humans requires new thinking and technological development. Experiments in preclinical animal models found that Tregs migrate to the lung and liver soon after infusion before redistribution into lymphoid organs.82 84 Infused Tregs also traffic efficiently to allografts, likely due to the high amount of inflammatory chemoattractants expressed by the ischemic tissue.85 Tregs protect the grafts from acute injuries by suppressing effector cell infiltration and activation in the grafts.60 They are also shown to migrate to draining lymph nodes where they function to maintain tolerance.86 These results from small animal models are consistent with findings in nonhuman primate models that show two-phased clearance of infused Tregs from circulation—first through redistribution into primary and secondary lymphoid organs then followed by slower elimination correlating with cell death.87,88 A unique pharmacokinetic issue of cell-based therapy is the functional stability of the cells. Tregs can lose their regulatory ability and even acquire effector functions in highly inflamed tissue with low levels of IL-2 and an abundance of proinflammatory cytokines such as IL-6.89 Sustained high expression of FoxP3 is central for a durable regulatory phenotype, and one critical element to stable FoxP3 expression is the DNA demethylation of an

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enhancer in the FoxP3 gene locus called the Treg-specific demethylation region (TSDR).90,91 This region is fully methylated in Tconv cells and fully demethylated in tTregs.27 pTregs are partially demethylated at TSDR and rapidly lose FoxP3 expression if they are activated without a continued supply of exogenous TGF-β.27 While pTregs serve important role in maintaining immunological tolerance in vivo, therapeutic utility of in vitro generated pTregs is limited due to their instability.92 Research in preclinical models on the immunological impact of Tregs provides guidance for assessing pharmacodynamics of therapeutic Tregs. The immediate targets of Tregs are mostly other T cells and APCs, and alterations in these cells can be used to assess pharmacodynamics of therapeutic Tregs. Tregs are known to reduce the costimulatory molecules CD80, CD86, and CD40 on APCs. Depending on the activation status of the targeted T cells, Tregs can prevent their proliferation and clonal expansion, effector cytokine expression, expression of chemokine receptors for trafficking and retention at the site of inflammation, and their cytotoxicity.93 Alterations of these molecular targets can happen within days of Treg infusion and ultimately lead to reduction of inflammatory cell load in the tissue, and restoration of normal tissue function. Tregs preferentially traffic to inflamed tissue and their draining lymph nodes to exert a suppressive effect.94,95 Thus, monitoring the above parameters in peripheral blood will not provide complete information on the Treg pharmacodynamics, and analysis of the target tissue will be necessary.

23.3.5 Potential Toxicity of Treg Therapy 23.3.5.1 Infusion Reaction An infusion reaction is an immediate immunological response to the infused drug that can occur during the minutes, hours, and, in rare situations, days after infusion. The cause of an infusion reaction can be an allergic response to infused cells or excipients, inflammatory intermediates released by dead cells,96,97 histamine release induced by cryoprotectant dimethyl sulfoxide if frozen cell products are used,98 or alloimmune responses if nonautologous cells are infused or allogeneic sera is present in the infusion solution. For these reasons, cells of high viability and nonimmunogenic excipient should be preferentially used and cryoprotectant should be minimized if not washed out. Prophylactic antipyretic and histamine antagonist medications are often given to patients prior to cell infusion in order to prevent possible infusion reaction. 23.3.5.2 Opportunistic Infections As immunosuppressive cells, Tregs have the capacity to impair antimicrobial immune responses. However, the impact of Tregs on infection is rather complex. During acute infections, microbial products often activate pathogen pattern recognition receptors on innate immune cells to induce the production of inflammatory mediators such as IL-1, IL-6, and TNF-α.99,100 These factors counteract the suppressive function of the Tregs by inhibiting Tregs and/or making effector cells resistant to inhibition, so that a protective immune response can be mounted. When the invading pathogens are brought under control, tissue inflammation subsides, and Tregs are able to function to limit bystander damage to surrounding tissue. In chronic infections Tregs are often a component of the infection site along with protective immune infiltrates. The presence of Tregs limit the ability of effector cells to eliminate the pathogen, contributing to the chronicity of the infection.101,102 Interestingly, deletion of Tregs in a mouse model of chronic cutaneous leishmaniasis led to sterilization of the infection site but also loss of immunological memory and made the host susceptible to secondary infections. A few mouse studies have addressed the role of Tregs on infections relevant to transplantation. Depletion of Tregs does not affect NK responses, but augments T cell responses to acute mouse CMV infections.103,104 Global Treg depletion leads to systemic nonspecific immune activation, but it cannot be inferred that Treg infusion leads to global immunosuppression. One study specifically addressed Treg therapy on influenza infection in a mouse model of heart transplant and found no impairment of viral clearance by Treg infusion.105 Overall, preclinical results show that Tregs are generally ineffective in suppressing a highly inflammatory acute infection, but can contribute to the persistence of chronic infections. The impact of Treg therapy in humans should be closely monitored in clinical trials to more definitely address this concern. 23.3.5.3 Malignancy Immune surveillance is one of our most important defenses against malignant cells. As Tregs inhibit the CD81 lymphocytes responsible for this surveillance, higher numbers of tumor-infiltrating Tregs often correlate with worse outcomes.106,107 The number of tumor-infiltrating Tregs correlates with circulating Tregs in many cancers,

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allowing for assessment of circulating Tregs with cancer outcomes.108,109 Skin cancer and posttransplant lymphoproliferative disease (PTLD) are the most common posttransplant malignancies, and the associations between circulating Tregs and clinical outcomes also apply to this transplant population. A clear association between higher circulating Tregs after kidney transplantation and the development of cutaneous squamous cell carcinoma exists.110 Furthermore, increased posttransplant Tregs correlate with a worse prognosis in squamous cell carcinoma.111 An association with increased Treg number in EBV-associated PTLD has been reported, as well as a worse prognosis with an increased frequency of EBV antigen-specific Tregs in this malignancy.112 However, with PTLD there is also conflicting evidence that tumor-infiltrating Tregs are rare and do not correlate with patient survival.113 Currently, there are no data on the effect of adoptive Treg therapy on the subsequent development of malignancy, but possible increased risk of skin cancer and PTLD following Treg therapy remains a concern.

23.3.6 The Effect of Immunosuppression on Infused Tregs As all immunosuppressive medications currently used to prevent allograft rejection target lymphocytes, these medications will also affect Tregs. Importantly, however, different immunosuppressive drugs have differential effects on Treg and Tconv cell function and survival, and therefore impact the balance between these cells. 23.3.6.1 Induction Immunosuppression Currently there are two categories of induction immunosuppression, lymphocyte depleting and nondepleting. Alemtuzumab and antithymocyte globulin are the primary lymphodepleting agents currently in use, and both appear to have similar effects on Treg proliferation and function with small differences observed in some trials.114 Tregs are depleted in proportion to other circulating lymphocytes by both agents, but there is some evidence that Tregs and memory cells recover more quickly than other cell subsets with increased circulating Treg/Teff ratio for the first year posttransplant.115,116 There is also contradictory evidence that alemtuzumab leads to long-term decrease in Treg/Teff ratio through homeostatic proliferation of the effector memory cells.117 Since the increased Tregs in lymphocyte depleted patients are not derived from homeostatic proliferation of CD251 cells or increased tTreg production, the increased Tregs are speculated to arise from peripheral conversion of CD41CD252 cells (pTregs).116 Differences in the conditions allowing for peripheral Treg conversion may explain these contradictory findings and may actually reflect the effects of maintenance immunosuppression more than the induction agent. The most common nonlymphodepleting induction agents are IL-2 receptor antagonists (anti-CD25) with basiliximab the only agent currently in use, although daclizumab has been used and studied in the past. As Tregs highly express CD25 it is not surprising that these antagonists have a significant effect on circulating Tregs. Most evidence demonstrates a transient reduction in the number of Tregs with anti-CD25 induction. The loss of CD41CD251FoxP31 cells is more pronounced than the loss of CD41FoxP31 due to down-modulation of CD25 from the surface of Tregs.116,118 120 While there appears to be a reduction in the absolute and relative quantity of CD41CD251FoxP31 Tregs following anti-CD25 treatment, these cells retain suppressive function in all studies.114,116 It is not clear why an agent this effective at reducing Treg numbers is not harmful to transplants. One possible explanation is that anti-CD25 mAbs delete CD251 effector T cells activated by the allograft shortly after transplant.118 In addition, daclizumab greatly prevents disease relapse in multiple sclerosis patients, where the mechanism involves deletion of CD251 Tregs but also an increase of CD561 NK cells that kill activated effector cells.121 123 This switch to NK-mediated regulation has not been reported in transplant patients yet. Together, all current induction agents directly target Tregs and should not be used at the same time as Treg infusion. Depleting agents may be used first to reduce the total number of Tconv and CD81 T cells to create the favorable setting for therapeutic Tregs to engraft and control the residual T cells. This would require a delay in the infusion of Tregs until the depletion agents have been cleared in the body. Ideal approaches should selectively delete donor-reactive T cells while leaving other T cells intact to preserve protective immunity. For example, chemical fixed donor cells can effectively inactivate donor-reactive T cells and induce long-term graft acceptance without immunosuppression.124 However, these approaches are still at a preclinical experimental stage. One induction agent that has been used in clinical trials, alefacept, is found to selectively delete effector memory T cells while preserving Tregs. Short course of alefacept in patients newly diagnosed with type 1 diabetes preserved beta cell function and showed long-term benefit.125 Such induction agents should be superior to global T cell deletion in transplantation.

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23.3.6.2 Maintenance Immunosuppression Most current immunosuppressive regimens include CNI as their central component. CNI have a detrimental effect on Treg survival and proliferation,126 although animal evidence suggests that at low concentrations tacrolimus can be used with Tregs to prolong graft survival.127 CNI adversely affect Tregs in two ways. First, Tregs are critically dependent on the calcineurin pathway for maintaining FoxP3 expression and for cellular activation following antigen encounter. Second, CNI inhibit effector T cell production of IL-2, thereby depriving Tregs of this essential survival factor. These findings suggest that Treg therapy will likely require a CNI-reduction or CNI-free immunosuppression regimen to be successful. In contrast, mTOR inhibitors exert stronger inhibitory effects on effector T cells than Treg and favor the conversion of Tconv to Tregs.128,129 When switched from a CNI-based immunosuppression to mTOR inhibitor-based immunosuppression, patients consistently demonstrate an increase in circulating Tregs.128 130 Therefore, mTOR inhibitors are currently the favored immunosuppressant over CNI to be used together with Treg cell therapy. It should be noted that mTOR inhibitors do inhibit Treg proliferation and function and should not be simply regarded as a Treg inducer and promoter. Their “Treg friendliness” lies in their relatively stronger inhibitory effects on Tconv and CD81 T cells than on Tregs. Taken together, preclinical data and experiences in patients strongly suggest that successful Treg therapy would benefit from mTOR inhibitors in place of a CNI.

23.4 CLINICAL EXPERIENCE USING Tregs As of mid-2016 when this chapter is finalized, clinical trials of Treg therapy in kidney transplantation have just begun, with 5 trials registered in the US clinicaltrials.org website and likely more outside the US not reflected in the registry. Currently, most published clinical work using Tregs therapeutically has been to prevent GvHD and to treat autoimmune diabetes. Thus, it is worth considering their use, safety, and efficacy in these contexts for a full understanding of their current uses and applications in transplantation.

23.4.1 Manufacturing Considerations All currently published, ongoing, and planned Treg therapy trials use Tregs specifically made for the recipient. This highly personalized therapy poses challenges to manufacture cells with consistent yield, identity, purity, and potency. We will briefly review the process of Treg manufacturing and relate how manufacturing choices may impact their clinical use. 23.4.1.1 Treg Purification None of the markers expressed on Tregs are unique to Tregs; rather, it is the combination of markers that uniquely identify Tregs (Table 23.1). When selecting markers to purify Tregs for clinical use, the practical issue of having access to good manufacturing practice (GMP)-grade reagents poses a limit. Currently, antibodies against CD4, CD25, CD45RA, and CD127 are available for GMP Treg manufacturing. What combination of markers can be used depends on the instrument of choice for the purification process, with magnetic activated cell sorters (MACS) or fluorescence activated cell sorting (FACS) being the two currently available instruments. MACS is available as a closed GMP-compliant instrument but FACS remains to be a research instrument. MACS has the advantage of better throughput by processing billions of cells in one batch as opposed to FACS selecting cells one at a time, although at very high speed. However, MACS is not as precise as FACS so the resulting cell population is not as pure. This is a severe limitation to MACS when the isolated Tregs are to be expanded. Contaminating Tconv cells and CD81 T cells proliferate much faster than Tregs, so the purity of cell preparation progressively declines in culture. FACS, by virtue of its precision and the ability to easily select cells based on multiple markers, can isolate highly pure Tregs. For example, using the phenotype of CD41CD127lo/-CD251, 1 3 106 highly pure Tregs can be isolated from 100 mL of blood on average.131 These cells remain highly pure after expansion and show superior suppressive function in a humanized mouse model of transplantation.132 Speed and the lack of a closed system are the major limitations to FACS. It currently takes 8 12 hours for a high speed FACS to process one unit of blood. Ideally, the best Treg purification process should incorporate the high throughput of MACS and the precision of FACS in one GMP-compliant instrument.

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TABLE 23.1

Cellular Markers Relevant to Treg Immunotherapy

Cell marker

Alternative name

Function

Relevance to Treg therapy

CD3

TCR coreceptor complex

TCR signal transduction

Stimulation required for cell activation and proliferation

Interacts with MHC class II molecules to strengthen TCR binding

Identifies CD41 lymphocyte subset

CD4

FoxP3

Forkhead box protein 3

Transcription factor, critical regulator of Treg function

Identifies Tregs in CD41 cells

CD25

IL-2 receptor a-chain

IL-2 receptor

Highly expressed in CD41 Tregs

CD127

IL-7 receptor α-chain

IL-7 receptor

Negative Treg marker

Costimulation molecule necessary for T cell activation

Stimulation required for Treg proliferation

CD28 CTLA-4

Cytotoxic T lymphocyte antigen-4, CD152

Inhibitory signal that prevents T cell activation

Highly expressed in Tregs, important for suppressive function of Tregs

PD-1

Programmed death 1

Inhibitory signal that prevents T cell activation

Highly expressed in Tregs, marker of cell exhaustion

GITR

Tumor necrosis factor receptor (TNFR) superfamily member 18, glucocorticoid-induced TNFR-related protein

Cell signaling

Important mechanism of Treg suppression

GARP

Glycoprotein-A repetitions predominant

Cell signaling

Marker of activated Tregs, identifies thymic-derived Tregs

ICOS

Inducible T cell costimulator, CD278

Costimultory molecule on T cells

Stimulation leads to Treg proliferation and activation

LAG-3

Lymphocyte activation gene 3, CD223

CD4 homolog with MHC class II binding properties

Expressed on Tregs

LAP

Latency-associated peptide

Component of TGF-β latent complex

Identifies Treg subset with TGF-β-mediated function

CD45RA

Leukocyte common antigen (RA isoform)

Protein tyrosine phosphatase, receptor type, C

Identifies naı¨ve cells

CD45RO

Leukocyte common antigen (RO isoform)

Protein tyrosine phosphatase, receptor type, C

Identifies experienced and memory cells

CD69

Transmembrane C-type lectin

Cell signaling

Marker of activated Tregs

CD62L

L-selectin

Lymphocyte cell adhesion molecule

Marker of activated Tregs

CD49b

Integrin VLA-4 α4β1α-chain

Cell adhesion and signaling

Expressed on Tregs

DNA binding protein

Marker of thymic-derived Tregs

Cell adhesion and signaling

Identifies a Treg subset with increased suppressive function

Helios CD103

Integrin αE

23.4.1.2 Treg Expansion Approximately 200 3 106 Tregs are circulating in the blood. Using leukapheresis, about 100 3 106 of the circulating Tregs can be collected. This number may be sufficient for prevention of GvHD when the number of Tconv cells to be infused can be controlled, but in organ transplant without systemic lymphoablative preconditioning, short-term ex vivo expansion of Tregs will be needed to effectively change the balance between Tregs and Tconv. Tregs can be expanded polyclonally using nonspecific stimulants such as antibodies to CD3 and CD28. This process is relatively simple by using off-the-shelf reagents, but the cells have less specific activity in controlling graft

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rejection and are more likely to induce generalized immunosuppression. Using donor-derived APCs as stimulators, donor alloantigen-reactive Tregs can be selectively expanded. For example, by using activated donor B cells billions of alloantigen-reactive Tregs can be produced after two rounds of expansion, reaching the estimated therapeutic dose.77 The improved specificity and potency of alloantigen-reactive Tregs makes these cells a desirable first choice. However, prospective collection of donor blood or spleen under GMP conditions is required to enable this approach. 23.4.1.3 Quality Control Properly defining the identity, purity, and potency of Tregs is an integral part of the pharmaceutical development of Tregs. While Tregs should express CD4, CD25, and FoxP3, these markers are shared with activated T cells, which can lead to overestimation of the purity of the Tregs, especially when ex vivo expansion is used. Currently, the unequivocal marker for bona fide human Tregs is the demethylated TSDR and a quantitative assay is available for its measurement.133 This marker not only distinguishes Tregs from activated Tconv cells, it also provides additional assurance of the stability of the cell product. Measuring the potency of Treg products is even more challenging than determining their identity and purity. Typical in vitro suppression assays take 3 7 days to complete, which means the Treg cell product has to be cryopreserved while waiting for the result. Cryopreservation leads to selective loss of Tregs and reduction of FoxP3 expression.134 A fast suppression assay has been developed based on suppression of Tconv expression of activation markers CD69 and CD154 after overnight stimulation in the presence of Tregs. However, since cell-based assays are intrinsically variable, defining a potency cutoff is difficult. Currently, most clinical trials of Treg therapy do not have a potency criterion for product release and more development in this area is clearly needed. Taken together, remarkable advances have been made in the academic setting to manufacture Tregs for therapeutic evaluation. The processes developed by adopting various existing technologies are operational, but cumbersome. Technological investment will be needed should this therapy be used beyond academic research.

23.4.2 Clinical Experiences 23.4.2.1 Graft-Versus-Host Disease The earliest clinical use of Tregs was in the treatment and prevention of GvHD. As of mid-2016, the results of one case study and four clinical trials evaluating the use of Tregs for the treatment and prevention of GvHD following allogeneic bone marrow transplantation have been reported in the literature.135 139 All studies for GvHD thus far have used polyclonal Tregs. The first trial was a Phase I, dose escalation trial evaluating the safety of CD41CD251 Tregs isolated from umbilical cord blood and given to 23 patients who underwent a nonmyeloablative double umbilical cord blood transplant for leukemia or lymphoma.136 In this study, Tregs from a third umbilical cord blood unit were activated and expanded with anti-CD3 and anti-CD28 beads along with IL-2 and introduced via one or two infusions of up to 3 3 106 Treg/kg. After infusion, an increase in the proportion of CD41FoxP31CD1272 cells in circulation was observed, and donor Tregs were detected in peripheral blood of patients for up to 14 days. The median time to development of GvHD in these patients was longer compared to the normal disease course in historical controls, although the delay was not statistically significant. Importantly, the infusion of ex vivo expanded Tregs was reported to be safe at all doses tested without an increase in opportunistic infections. This group of researchers expanded their investigation with an additional 11 patients and increased Treg dose up to 100 3 106 kg21.137 This new study broke many new grounds by demonstrating that it is feasible to produce more than 10 3 109 of cGMP-grade Tregs and infusion of 100 3 106 kg21 expanded polyclonal Tregs is well tolerated. Moreover, with a higher dose of Tregs, the research observed more definitive efficacy signal in preventing acute GvHD and chronic GvHD with no increase in cancer relapse or similar infections. A second clinical study evaluated the use of freshly isolated CD41CD251 Tregs from the stem cell donor for the prevention of GvHD.138 In this study, 28 patients with high-risk hematologic malignancies undergoing HLA-haploidentical hematopoietic stem-cell transplantation received an infusion of donor Tregs at doses of up to 4 3 106 kg21 followed by conventional donor T cell infusion at the time of stem-cell transplantation. No posttransplantation immunosuppression was given. Full donor-type engraftment was seen in 26 of the 28 patients. Two of these 26 patients developed grade 2 acute GvHD; no patients had developed chronic GvHD at a median follow-up of 11.2 months. The investigators concluded that the donor Tregs, combined with conventional T cell therapy, prevented GvHD, improved immune reconstitution, and did not weaken the graft-versus-leukemia

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effect. The same group has since conducted a phase I study of 43 with similar trial design and concluded that prophylactic use of Tregs allowed infusion of higher dose of Tconv cells, resulting in better engraftment of donor cell and lower disease relapse.139 23.4.2.2 Type 1 Diabetes Beyond GvHD, two studies have thus far evaluated the safety and efficacy of ex vivo expanded CD31CD41CD25hiCD1272 Tregs in patients with type 1 diabetes.140,141 In the first trial, 12 children received 10-30 3 106 Tregs/kg in one or two infusions. Compared to untreated children, the Treg-treated patients had significantly elevated C-peptide levels 6 months after disease onset. One year after inclusion into the study, 66% patients still met the criteria for clinical remission with two patients remaining insulin independent. When data of the treated and untreated patients were analyzed together after 4-month and 1-year follow-ups, the investigators observed that C-peptide levels correlated with percentage of Tregs in peripheral blood. No serious infections, acute hyperglycemia, or hypoglycemia were associated with the treatment. The investigators concluded that Treg therapy in children was well tolerated and potentially efficacious. In the second published trial, 14 adult patients received 5 3 106 2.6 3 109 polyclonally expanded Tregs in a single infusion.142 The therapy was again well tolerated with no safety concerns and most patients were metabolically stable for up to 5 years after Treg therapy. This trial involved two clinical sites in the United States, one at the University of California, San Francisco (UCSF) that also served as the Treg manufacturing site and the other site was at Yale University. For patients at Yale, blood was shipped to UCSF for manufacturing and Treg products were shipped back for infusion. This demonstrates feasibility of multicenter study using a centralized manufacturing facility. Moreover, some patients received Treg products that were manufactured in a medium containing deuterated glucose, which resulted in incorporation of deuterium in the DNA of the Treg product. This allowed monitoring of the persistence and stability of the infused Tregs in patients. Tregs peaked in the circulation during the first 2 weeks after infusion, declined to 25% of the peak level by 3 months after infusion and then stabilized. In all four patients who received 2.6 3 109 Tregs, deuterium can be detected at 1 year after infusion, the last time point of the follow-up. Importantly, all the deuterium signals were contained in the Treg population and never detected among conventional T cells, demonstrating the stability of the Treg product in patients. 23.4.2.3 Organ Transplantation The first human study to give Tregs to transplant recipients to induce tolerance was conducted in 10 livingdonor liver recipients.143 In this study, patients received induction immunosuppression with cyclophosphamide and conventional maintenance immunosuppression with CNI, mycophenolate, and steroids beginning at the time of transplant. Autologous pTregs expanded with irradiated donor cells in the presence of antibodies to CD80 and CD86 were given 13 days after transplant. An average of 2.3 3 108 (max 4.6 3 108) Tregs were administered. The maintenance immunosuppression was quickly tapered to CNI monotherapy by 3 months posttransplant. At 6 months posttransplant a step-wise reduction of CNI was initiated with the goal of complete cessation of immunosuppression by 1 year posttransplant. Remarkably, 6 of 10 patients successfully completed immunosuppression withdrawal. In comparison, the rate of spontaneous liver transplant tolerance is 15% at 2 years posttransplant. This encouraging result suggests that Treg infusion combined with a carefully selected immunosuppressive regimen can induce transplant tolerance in humans. Currently, no results have been reported using of Tregs in kidney transplant patients, but a number of trials are ongoing in the United States, European Union, and China. The ONE Study is currently the largest consortium in clinical trials to evaluate Tregs and other regulatory cells in kidney transplantation (onestudy.org). These trials began in 2014 to test the safety of regulatory cell therapy in kidney transplant recipients receiving standard immunosuppression. Preliminary results from the ongoing trials are expected in 2016.

23.5 CONCLUSIONS AND FUTURE DIRECTIONS Treg therapy is a promising method to induce transplant tolerance and improve the treatment of ESRD with kidney transplantation, but its clinical use is still in its infancy with much refinement yet to come. However, the potential benefits of this personalized cell therapy to decrease the medication side effect burden and prolong graft survival make this lengthy process worthwhile. This approach is feasible, and results from ongoing development efforts in GvHD, autoimmune diseases, and transplantation suggest that it can be efficacious. Wider

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clinical evaluation would require more robust data from phase I and phase II trials. Major challenges in designing clinical trials in transplantation are the required concurrent immunosuppression and the long follow-up period needed to observe potential improvement over the current standard of care. Therefore, sophisticated clinical trial designs by integrating knowledge of clinical transplantation and Treg biology are needed to expedite development in this field. Additionally, technological advancements in Treg manufacturing and quality control are also needed. For example, chimeric CAR T cell therapy has achieved the goal of antigen-specific targeting of cancer cells by growing patient’s autologous cells using off-the-shelf reagents,144 and CAR-engineered Tregs have been shown to be effective in animal models of autoimmunity and GvHD.145 147 It is conceivable that anti-HLA antibodies can be used as part of a CAR construct to direct Tregs to allografts. A limited number of such antiHLA-CAR constructs can cover a large number of donor-recipient combinations and can be made in advance for off-the-shelf use. By focusing on selectively promoting Tregs and refining the protocols involving their use, it is possible that transplant tolerance—the “holy grail” of transplant medicine—may finally be achieved.

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134. Golab K, Leveson-Gower D, Wang X-J, et al. Challenges in cryopreservation of regulatory T cells (Tregs) for clinical therapeutic applications. Int Immunopharmacol 2013;16(3):371 5. ´ 135. Trzonkowski P, Bieniaszewska M, Ju´scinska J, et al. First-in-man clinical results of the treatment of patients with graft versus host disease with human ex vivo expanded CD41 CD251 CD127- T regulatory cells. Clin Immunol 2009;133(1):22 6. 136. Brunstein CG, Miller JS, Cao Q, et al. Infusion of ex vivo expanded T regulatory cells in adults transplanted with umbilical cord blood: safety profile and detection kinetics. Blood 2011;117(3):1061 70. 137. Brunstein CG, Miller JS, McKenna DH, et al. Umbilical cord blood-derived T regulatory cells to prevent GVHD: kinetics, toxicity profile, and clinical effect. Blood 2016;127:1044 51. 138. Di Ianni M, Falzetti F, Carotti A, et al. Tregs prevent GVHD and promote immune reconstitution in HLA-haploidentical transplantation. Blood 2011;117(14):3921 8. 139. Martelli MF, Di Ianni M, Ruggeri L, et al. HLA-haploidentical transplantation with regulatory and conventional T-cell adoptive immunotherapy prevents acute leukemia relapse. Blood 2014;124(4):638 44. 140. Marek-Trzonkowska N, Mysliwiec M, Dobyszuk A, et al. Administration of CD41 CD25highCD127- regulatory T cells preserves β-cell function in type 1 diabetes in children. Diabetes Care 2012;35(9):1817 20. 141. Marek-Trzonkowska N, My´sliwiec M, Dobyszuk A, et al. Therapy of type 1 diabetes with CD4(1)CD25(high)CD127-regulatory T cells prolongs survival of pancreatic islets - results of one year follow-up. Clin Immunol 2014;153(1):23 30. 142. Bluestone JA, Buckner JH, Fitch M, et al. Type 1 diabetes immunotherapy using polyclonal regulatory T cells. Sci Transl Med 2015;7:315ra189. 143. Todo S, Yamashita K, Goto R, et al. A Pilot Study of Operational Tolerance with a Regulatory T Cell-Based Cell Therapy in Living Donor Liver Transplantation. Hepatology 2016. published online ahead of print. 144. Barrett DM, Singh N, Porter DL, Grupp SA, June CH. Chimeric antigen receptor therapy for cancer. Annu Rev Med 2014;65:333 47. 145. Elinav E, Adam N, Waks T, Eshhar Z. Amelioration of colitis by genetically engineered murine regulatory T cells redirected by antigenspecific chimeric receptor. Gastroenterology 2009;136(5):1721 31. 146. Fransson M, Piras E, Burman J, et al. CAR/FoxP3-engineered T regulatory cells target the CNS and suppress EAE upon intranasal delivery. J Neuroinflam 2012;9:112. 147. MacDonald KG, Hoeppli RE, Huang Q, et al. Alloantigen-specific regulatory T cells generated with a chimeric antigen receptor. J Clin Invest 2016;126(4):1413 24.

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24 Mesenchymal Stromal Cells to Improve Solid Organ Transplant Outcome: Lessons From the Initial Clinical Trials Marlies E.J. Reinders, Johannes W. De Fijter, Maarten L. Zandvliet and Ton J. Rabelink Leiden University Medical Center, Leiden, The Netherlands

24.1 INTRODUCTION Overall kidney graft survival has markedly improved over the past decades, mainly as a result of improvement of first-year graft survival. Long-term graft survival, however, has remained in essence unaltered over the past two decades. This has been attributed to graft loss from interstitial fibrosis and tubular atrophy (IF/TA).1 The mechanism for the development of IF/TA includes both allo immunity/rejection and nonimmunologic causes such as drug toxicity (e.g., calcineurin inhibitors), recurrent or de novo renal diseases, and (viral) infections.2 There is therefore un unmet need to develop new ways to modulate the immune system and fibrosis process and to govern the balance between over- and under-immune suppression. Administration of mesenchymal stromal cells (MSCs) has been put forward as a possible strategy. In contrast to most current pharmacological agents that target only a single pathophysiological pathway, MSCs potentially affect immunologic, inflammatory, vascular, and regenerative pathways (Fig. 24.1). In vitro studies demonstrate that MSCs may play a role in modulation of immune responses and beneficial immune modulatory effects of MSCs have been shown in experimental models of inflammatory disorders, including allograft rejection. In clinical kidney transplantation, initial trials have focused mainly on safety and feasibility of MSC treatment. MSCs possibly could serve as induction therapy to stimulate graft tolerance, in the treatment and/or prevention of IF/TA, and in calcineurin minimization protocols. In the current chapter we summarize the current state-of-the-art of MSCs in kidney transplantation and we discuss lessons learned from the initial clinical trials for design of future studies.

24.2 MESENCHYMAL STROMAL CELLS MSCs are nonhematopoietic progenitor cells that can differentiate into several mesenchymal tissues, including osteoblasts, adipocytes, and chondrocyte progenitors. MSCs can be isolated from various tissues including bone marrow (BM), adipose tissue (AT) and umbilical cord. In the BM, MSCs control hematopoietic stem cell (HSC) homeostasis in the endosteal and the perivascular niche. In the endosteal niche, MSCs mainly mediate HSC retention, maintenance, and quiescence, via various factors including CXCL12, stem cell factor, angiopoeietin-1, and vascular cell adhesion molecule. The perivascular niche includes MSCs that secrete factors that promote self-renewal

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FIGURE 24.1 MSC isolation, expansion, infusion, and follow-up in a clinical study: A complex process. MSCs isolated from the bone marrow undergo the following process: (1) Transport and quality control of bone marrow starting material; (2) Good Manufacturing Practice compliant isolation of mononuclear cells; (3) Good Manufacturing Practice compliant expansion in controlled incubators; (4) Culture processing and in process quality controls: e.g., morphology, sterility; (5) Final product quality controls: Spindle shaped morphology; (6) Final product quality controls: e.g., sterility, viability, surface markers; (7) Final product: Mesenchymal Stromal Cells cryopreserved in liquid nitrogen; (8) Patient administration: Intravenous infusion of thawed viable Mesenchymal Stromal Cells.

of active HSC.3 Besides the BM, MSCs have also been detected in perivascular tissues of several organs, including the kidney, and in the fat,4 and it is hypothesized that these cells may contribute to local tissue repair. While several attempts have been made to select a homogeneous MSC population by using surface markers such as, e.g., STRO-1, no unique phenotype has been identified that allows the reproducible isolation of MSC precursors with predictable differentiation potential. Functional characterization still relies primarily on their ability to adhere to plastic and their differentiation potential. The International Society of Cellular Therapy stated that MSCs should bear at least the stromal markers CD73, CD90, and CD105 and be negative for hematopoietic markers CD14, CD34, and CD45.5 Important for their possible clinical application is that MSCs are easily isolated as they adhere to plastic and are capable of substantial proliferation and expansion in culture.6

24.3 THE ROLE OF MSCs IN IMMUNE MODULATION Several studies suggest that MSCs play a role in modulation of immune responses.7,8 These immunomodulatory properties make MSCs especially attractive for potential use in treating immune mediated diseases, including allograft rejection.7 12 MSCs are poorly equipped for induction of alloreactivity. Human MSCs express a low-intermediate level of HLA class I and LFA-3, and do not express costimulatory molecules, such as B7-1, B7-2, CD40, and CD40L.7,11,13 MSCs affect several of the functional properties of T cells. They efficiently suppress the proliferation of CD41 and CD81 T cells.8,10,14,15 This reduction of T-cell proliferation is dependent on a cell-cycle arrest in G0/G1 phase.16 They can prevent T cells from activation-induced cell death by down-regulation of Fas receptor and Fas ligand on T cells and inhibition of endogenous proteases involved in cell death.17 When present in an inflammatory microenvironment, MSCs could inhibit interferon (IFN)-γ secretion from Th1, interleukin (IL)17 release by CD41 Th17, and IL-4 secretion from Th2 cells, thereby promoting a Th1 to Th2 shift. The cytolytic potential of cytotoxic lymphocytes (CTLs) was also efficiently impaired by MSCs.18 Several cell membraneassociated and soluble molecules have been identified to contribute to MSC-mediated inhibition of T-cell proliferation and function, including prostaglandin E2 (PGE2), transforming growth factor-β (TGF-β), indoleamine 2,3-dioxygenase (IDO), soluble HLA-G, nitric oxide (NO), hepatic growth factor (HGF), IL-10, programmed death-ligand 1 (PD-L1), Jagged-1, and B7-H1.9,11,19 Besides their direct effects on T cells, MSCs have profound effects on the antigen-presenting milieu. They inhibit the IL-2 and IL-15 driven NK cell proliferation and IFN-γ production,7,20 22 as well as the dendritic cell (DC) generation from peripheral blood monocytes in vitro.23 25 Interestingly, intravenous injection of MSCs

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significantly affected the ability of DCs to prime T cells in vivo because of their inability to home to draining lymph nodes.26 In addition, MSCs have been shown to inhibit the differentiation of B cells. AT derived MSCs exert an indirect effect on B cell proliferation through immunomodulation of T cells and a direct effect on B cells by inhibiting plasmablast differentiation and induction of IL-10-producing regulatory B cells.27 Cell-cell contact and soluble factors synthesized by MSCs are thought to suppress B cell function. PD-1/PD-L1 ligations have been shown to enact B cell suppression by MSCs, with soluble factors largely remaining unidentified.27,28 Recently, several studies investigated the impact of MSCs on regulatory cells, which play an important role in the induction of peripheral tolerance and the inhibition of proinflammatory immune responses.11,19 MSCs have the ability to induce regulatory FoxP31Tregs, T regulatory 1 cells (Tr1) IL101 producing cells and Th3 TGF B1 regulatory T cells.29 MSCs mediated a switch to alternatively activated M2 macrophages.30 These macrophages, induced by Th2 cytokines such as IL-4 and IL-13, possess antiinflammatory activities and are considered the regulatory type macrophage subsets.31 Of interest, MSCs were shown to attenuate sepsis via PGE2 dependent reprogramming of host macrophages, resulting in improved organ function after sepsis induction.32 Like many cells of the immune system, it is becoming clear that MSCs do not always display an antiinflammatory response. Dependent upon the timing and the local milieu where they home to, they may lose their immune modulatory properties. As an example, in the presence of low IFN-γ levels, MSCs express MHC-class II molecules and acquire phagocytic function and antigen presentation activity to CD41 T cells.33,34 In turn, high IFN-γ levels downregulate the expression of MHC class II molecules by MSCs, thus leading to a shift in MSC function to immunosuppression.33,34 These findings can be translated to improve design of clinical MSC studies, in which either profylactic administration, or therapeutic administration in the inflammatory setting may result in immune suppression. However, MSCs may lose their immune modulatory properties when they reach low grade inflammation, which may correspond to a more preemptive clinical setting.

24.4 THE ROLE OF MSCs IN TISSUE REPAIR Because of their broad tissue distribution, multipotent differentiation capacity and well-established effects in experimental studies, MSCs are believed to have critical roles in repairing damaged tissues. In line with this, MSCs were originally evaluated for their capacity to repair skeletal defects in experimental models and subsequently in patients with osteogenesis imperfecta.35,36 Different studies have suggested that the ability of MSCs to produce paracrine factors, rather than their transdifferentiation, plays a prominent role in effecting tissue repair.9,37 In animal models, MSC transplantation decreased fibrosis in the heart38 and other organs such as the lung, liver, and kidney.39 41 Different cytokines have been shown to mediate the antifibrotic properties, including BMP-742 and HGF.43 After kidney transplantation, a therapeutic effect of MSCs attenuating the progression of IF/TA was seen when this process is already in progress.41 Besides a reduction in IF/TA, MSC treated animals demonstrated also less macrophages infiltrating the parenchyma and lowered expression of inflammatory cytokines while increasing the expression of antiinflammatory factors.41 Of note, MSCs may not only be antifibrotic but, probably beyond the point of regeneration, may also contribute to fibrosis. Indeed, Humphreys et al. showed nicely with lineage tracing studies of FoxD11 pericytes that perivascular stromal cells can transform into myofibroblasts upon severe kidney injury, and contribute to renal fibrosis as a last resort repair mechanism.44 MSCs are closely related to endothelial cells (ECs) not only in the BM where they control the vascular niche, but also in the peripheral tissue where they stabilize the microvascular architecture. MSCs interact directly with ECs via the production of paracrine factors, including vascular endothelial growth factor and angiopoeitins.45 47 Not only paracrine mechanisms, but also cell-cell contacts, are of importance for vessel stabilization. As an example, knock down of the α6β1 integrin receptor in BM MSCs leads to decreased capillary sprouting and failure of vessels to associate with nascent vessels.48 In the allograft inhibition of EC injury and stabilization of the microvasculature, leads to physiological remodeling and restoration of the allograft tissue.49,50 Therefore, protecting the vasculature and/or enabling physiologic homeostatic repair of the microvasculature will prevent tissue fibrosis. In different models it was shown that MSC-derived exosomes have functions similar to those of MSCs, such as repairing tissue damage and modulating the immune system. In a cisplatin induced acute kidney injury (AKI) model it was shown that the tissue regenerative effects of MSCs were caused by the combination of IGF-1 release and transfer of mRNA of the corresponding IGF-1 receptor via exosomes.51 In addition, microvesicles isolated

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from MSCs protected against AKI induced by ischemia reperfusion injury and subsequent chronic renal damage.52 These suggest that microvescicles from MSCs could be exploited as a potential new therapy.

24.5 EXPERIMENTAL STUDIES WITH MSCs IN RENAL TRANSPLANTATION A large number of experimental models have been used to evaluate the in vivo MSC immune regulatory properties related to alloreactive responses in solid organ transplantation.11,12,53 Most studies focused their endpoints on efficacy, including prolonged graft survival and inhibition of the rejection process.12,54 57 In addition, an important aim of these studies was to obtain mechanistic insight. In a study on MSCs from nonhuman primates, baboon MSCs suppressed the proliferative activity of allogeneic peripheral blood lymphocytes in vitro.54 A prolongation of skin graft survival was found with a single dose of MSCs administered intravenously. Of interest, in a rat cardiac transplant model, heart grafts tolerized through third-party rat MSCs could be retransplanted to secondary hosts with no immunosuppression.58 Moreover, in another rat cardiac transplant model donor rat MSCs suppressed allogeneic T-cell responses both in vitro and in vivo and intravenous administration of MSCs prolonged the survival of transplanted hearts, possibly by induction of allograft tolerance through changing the Th1/Th2 balance.55 In this model MSCs vigorously migrated to the site of inflammation during the chronic rejection process, suggesting that the MSCs may be attracted to participating in the process of active tissue repair. After rat kidney transplantation, MSCs could downregulate the immune response and control acute cellular rejection.59 Rats treated with MSCs had an improved renal function and histologically a diminished tubular damage and vasculitis. In addition, MSCs reduced the number of ED11 and CD81 cells, showing that MSCs indeed could downregulate the immune response and attenuated histological damage. Different experimental studies have demonstrated that the majority of MSCs get entrapped in the lung after intravenous infusion and do not migrate to the kidney.60 Therefore it is assumed that they cause a systemic immune modulatory response in the kidney. In a study by Casiraghi et al., pretransplant infusion of MSCs prolonged the survival of semiallogeneic (B6C3 in B6) murine heart transplants through the generation of T regs. In this model, pretransplant infusion of MSCs of recipient origin was as effective in inducing long-term acceptance of cardiac allografts as donor-derived MSCs.57 A single recipient-derived MSC infusion given peri-transplant was marginally effective, and a single MSC dose given 1 day after transplantation was not effective at all. Interestingly, the same group investigated the optimal timing for MSC infusion to promote immune tolerance in a murine kidney transplant model.56 Posttransplant MSC infusion caused premature graft dysfunction and failed to prolong graft survival. In contrast, pretransplant MSC infusion induced a significant prolongation of kidney graft survival by inducing regulatory T cells.56 These studies show that the therapeutic use of the immunomodulatory properties of MSCs depend on the timing of their infusion.61 The need for the appropriate timing is probably related to the necessity for the appropriate microenvironment to allow MSCs to acquire their immunosuppressive properties. Of interest to these observations, studies demonstrate that MSCs indeed need to be “licensed” in an appropriate cytokine environment before they exert their actions. Only supernatants obtained from cultures in which MSCs were incubated with activated T cells displayed an immunosuppressive effect.62 There was no effect detectable using supernatants from cultures of MSC alone.62 IFN-γ induced the immunosuppressive activity markedly,63 probably via the upregulation of IDO.64 In vivo studies also demonstrate that IFN-γ activation of MSCs increases their therapeutic efficacy.65,66 Like IFN-γ, TNF-α has been observed to induce immunosuppressive activity by MSCs through the production of PGE2 and COX-2.67 Of importance for the transplant setting, IFN-γ stimulated BM MSC and AT MSCs markedly inhibited the proliferation of activated peripheral blood mononuclear cells (PBMC) in a dose dependent manner.65 In a humanized mouse allograft rejection model, alloreactivity was marked by pronounced CD451 T-cell infiltrates consisting of CD41 and CD81 T cells and increased IFN-γ expression in the skin grafts which were all significantly inhibited by both BM MSC and AT MSC.65

24.5.1 MSCs and Concurrent Immune Suppressive Drugs Current immunosuppressive drugs cannot be withheld from renal transplant recipients receiving MSCs. Therefore, it is of importance that an optimal concurrent immunosuppressive regimen is chosen in which drugs have no negative impact on MSC function and vice versa.68 This interaction has mainly been assessed by in vitro studies,68 and only a few studies have elucidated the interaction of MSCs with concurrent immune suppression

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in vivo.69 72 Zhang et al. studied in a kidney allograft rat model the contribution of rat MSCs to the development of acute rejection with or without Cyclosporin A. They demonstrated that MSCs could downregulate the immune response and reduce the production of inflammatory mediators, IL-1β, TNF-α, and TGF-1β. MSCs preserved graft function in the initial stage after transplantation and prolonged animal survival, however Cyclosporin A was more effective. When MSCs were combined with low dose Cyclosporin A, animal survival was comparable with high dose Cyclosporin A monotherapy, suggesting an additional effect of MSC and Cyclosporin A.72 Eggenhofer et al. administered MSCs in a fully allogeneic heart transplantation rat model in combination with Mycophenolic acid (MMF). They found that the combination of MSCs with MMF leads to a significant prolonged survival of allografts compared to MSCs alone and they suggested that it was most likely through differential secretion of IFN-γ.70 In a heart transplantation model Ge et al. found that MSC monotherapy inhibited acute graft rejection and in combination with Rapamycin induced donor-specific allograft tolerance. In tolerant recipients, MSCs migrated to the transplanted heart and various lymphoid organs, and furthermore, a high frequency of Tol-DCs and CD41CD251Foxp31 T cells was found.71 These results support the clinical applicability of MSCs in combination with a mammalian target of rapamycin inhibitor or MMF in clinical transplantation. The above-described experimental studies provide us with important mechanistic information on MSCs in the transplant setting. Translation of the findings of experimental studies into the human setting is however challenging. The underlying disease of the renal recipient, concomitant use of immune suppressives, occurrence of infections, the difference in inflammatory responses between animals and humans, and numerous other factors which are of relevance for the human situation setting are not taken into account in the animal studies.

24.6 CLINICAL TRIALS WITH MSCs AFTER RENAL TRANSPLANTATION 24.6.1 MSC Product Isolation and Procedures for Clinical Use As MSC products contain viable human cells that have been cultured ex vivo and may have undergone functional and phenotypic changes during manufacturing, defined as “substantially manipulated,” these products are classified as cell therapy medicinal products. The regulatory framework of cell therapy medicinal products only permits application in clinical trials or under a marketing authorization. Since MSCs are medicinal products, they should be manufactured in full compliance with Good Manufacturing Practice guidelines, to ensure quality and reproducibility. Both the processes of manufacturing and quality control are tightly controlled and fully documented, to allow final release of a MSC product batch for clinical application with maximal quality, safety, and efficacy (Fig. 24.1). MSC for clinical interventions can be sourced from either human BM or AT samples. As these human tissues contain only a very low percentage (0.001% 0.01%) of primary MSCs, the expansion of MSCs ex vivo is necessary. Among the cells in the BM or AT starting material, MSCs have the specific property to adhere to plastic culture flasks. After a standard density separation to isolate mononuclear cells, the starting material cell suspension can be directly plated in culture flasks. Cells are plated at densities ranging from 1 3 104 cells/cm2 to 0.4 3 106 cells/cm2,73 and generally cultured in Dulbecco’s modified Eagle’s medium. To culture and differentiate MSCs an optimal combination of several factors, including cytokines and serum supplements, is essential. Clinical trials so far have mainly used fetal calf serum-expanded cells. However, the use of a culture raw material from bovine origin results in an increased risk in xenogeneic transmission, which is minimized by full material traceability and an extensive package of viral and quality control. For these reasons, studies are investigating animal serum-free culture conditions.74 These animal-free additives include platelet lysate (PL)/platelet rich plasma (PRP), cytokines and growth factors and human serum. Currently, more efficient methods for manufacturing of MSC for clinical application are being developed. Several companies have introduced bioreactor systems that are specifically suited for MSC expansion. For third party allogeneic MSC products, these advanced manufacturing systems will significantly increase cell growth efficiency, and result in much larger product batch sizes to supply large clinical trials and possibly the market. Final MSC products contain viable cells, and cannot be sterilized by heat, filtration or irradiation. Therefore, all starting and raw materials, disposables, and equipment need to be sterile, and manufacturing is performed in laminar airflow units in a clean room background. During the process, environmental monitoring of viable and nonviable particles is performed to minimize the risk for a potential product batch contamination. During the manufacturing process and after packaging of the MSC product, in-process and final product quality control testing is performed to demonstrate batch quality and consistency. This includes the rate of proliferation,

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morphology, viability, flow cytometric analyses of presence of MSC surface markers and absence of non-MSC surface markers, sterility, endotoxin, mycoplasma, and genetic stability.

24.6.2 Route, Dosage, and Frequency of Administration of MSC Infusion Intravenous infusion has been the route of administration of choice for clinical studies with MSC for immune modulation after solid organ transplantation so far, and has been shown to be safe. Theoretically, MSCs can also be directly injected into the kidney or underneath the kidney capsula. The rationale here is that MSCs home directly to the site of injury and do not accumulate in microvasculature, such as in the lungs. However, possible side effects might include the development of embolisms. Doses of MSC are chosen above what is considered the minimal effective dose, and below a potential toxic dose. The studies in renal transplantation have administered 0.5 3 106 to 5.0 3 106 MSCs/kg; however in graftversus-host-disease (GVHD) patients higher amounts (0.4 9.0 3 106) of MSCs were administered.75 Doses from 0.8 3 106 were effective in GVHD, but no clear dose-dependent effect was obtained. In patients that underwent myeloablation, infusion of 1 3 106 and 2.2 3 106 MSC/kg body weight showed no toxic effects.76 The frequency of two infusions might be preferable compared to one infusion. A long-lasting response was hardly observed in patients with steroid resistant graft versus host disease who received one infusion, while most responders had two or more infusions.77 In most studies low passage numbers (2 3) were used. It is of importance to note that in most studies the dosage and frequency of MSCs is chosen empirically, since no large dose finding studies have been performed. Unfortunately there is no potency assay that can guide us in the best dosage and frequency of administration.

24.6.3 Safety Aspects in Clinical Trials With MSCs In the vulnerable population of kidney transplant recipients, patient safety and prevention of adverse (immune) reactions is essential. Since transplant recipients have already an increased risk of (opportunistic) infections and malignancies due to the concomitant immunotherapy, it is very difficult to determine the additional risk of MSC infusions. Clinical studies should be performed under ethically approved protocols and serious adverse events (SAEs) and suspected unexpected serious adverse reactions (SUSARs) should be carefully recorded and reported to the proper authorities. Important potential risks in renal transplant recipients include direct toxicity of the MSC infusion, malignancies, and risks for over immune suppression and immunogenicity, as also extensively reviewed elsewhere.12,78

24.6.4 Toxicity Related to the Infusion To date, no direct toxicity related to the infusion itself or immediate adverse effects have been described9 in the numerous clinical trials with MSCs for clinical indications. We are however still awaiting long-term effects. In terms of assessing for toxicity, adverse events are graded using scales that base the adverse events of the study treatment on the known side effects of the therapy and the patient’s medical history. Long-term assessment for late toxicity is of importance here. Of interest, a scoring system, the so-called MiSOT-1 score, was designed to evaluate safety of intravenous and intraportal infusion stem cell products after liver transplantations.79 This score was developed to identify very SAEs. It is however also of importance to identify the less severe adverse events. There is no uniform score yet, which can be used to assess safety in cell-based trials in renal transplantation.

24.6.5 Risk for Malignant Transformation With MSCs, there has been initial concern with respect to the risk of malignant transformation during the expansion period, as extensively reviewed.78 In humans quite a large cohort of patients is exposed to MSC therapy, and none of them have developed new malignancies so far. However, most clinical trials have a short follow-up period and the inclusion of ill patients with poor prognosis could have biased the outcomes as well. It is clear that long-term follow up should be performed in MSC treated patients and one should discuss the extent of screening protocols which should be applied before MSC infusions. It will be a difficult task to link the development of carcinomas to the use of MSC therapy in transplant recipients, since these patients are exposed to such an increased risk for malignancies.

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24.6.6 Risks for Over Immune Suppression As MSCs are particularly known for their immune suppressive functions, a possible side effect of MSC treatment is over-immune suppression. In the study by Tan et al., a significant decrease in opportunistic infections was seen with MSC induction. However, 151 of the 154 patients in this study had a cytomegalovirus (CMV) negative serological status, probably explaining the low incidence of CMV infections in their population.80 In a phase I trial in renal transplant recipients, 3 out of 6 patients developed opportunistic virus infections after MSC infusions that are typically associated with too much immune suppression, including BK nephropathy and CMV infections.81 Other clinical trials in the context of GVHD or hematopoietic stem cell transplantation also showed a trend to more infections after MSC therapy. In a study by Von Bahr et al. of 31 patients with GVHD and MSCs there was a high rate of infections. Most patients in this study had steroid-refractory GVHD and, since there was no matching control group, infections could not be fairly compared.82 MSC coinfusion after hematopoietic stemcell transplantation caused a higher 1-year incidence of infections, particularly fungal infections.83 Clearly, frequent and accurate monitoring of infectious complications remains essential in cell-based therapy.

24.6.7 Source of MSCs Most clinical studies so far in renal transplant recipients have used autologous MSCs. A few studies have specifically investigated whether MSCs from patients with end stage renal disease are suitable for autologous therapy. It was shown that both human BM and AT MSCs are not affected by renal disease.84,85 Indeed, MSCs from both groups showed similar morphology, phenotype, and differentiation capacity compared to MSCs from healthy donors. In addition, functional capacities, including the ability of inhibiting allo-antigen- and anti-CD3/ CD28-activated PBMC proliferation, were similar in MSCs from patients with renal disease compared to their healthy counterpart.85 Due to the expansion period, quality controls, and logistics, it takes several weeks to months to manufacture autologous cells, which is undesirable for patients in urgent need of treatment, e.g., in patients with rejection. In these indications autologous MSC therapy is only possible when the cells are harvested in advance, but this is financially and logistically impossible. Allogeneic MSCs offer the advantage of immediate availability for clinical use. Another benefit of using allogeneic MSCs is that the age of the donor is controlled, and cells can be selectively derived from young donors. This is important because MSC number and functionality has been shown to decrease with age.86 There are potential disadvantages to the clinical use of allogeneic MSCs especially in the transplant setting. A potential danger could be the provocation of an antidonor immune response, which may increase the incidence of rejection/graft loss and impact the allograft survival on the long term.87 The importance of these de novo HLA donor specific antibodies (DSA) as a major cause of allograft failure in the long term has recently been confirmed.88,89 Of importance, it was shown that DSA with the ability to activate complement, as determined by this novel C1q assay, are associated with greater risk of acute rejection and allograft loss.90 Indeed, assessment of the complement-binding capacity of donor-specific anti-HLA antibodies appears to be useful in identifying patients at high risk for kidney allograft loss. These safety issues should be studied accurately in patients receiving allogeneic MSCs. Of interest, risks of immune sensitization could be reduced by selecting the allogeneic MSCs. One could think of criteria that include no HLA sharing with the kidney donor and the recipient should have no antibodies directed to the MSCs. The objective of such a strategy is to decrease the risk for development of anti-MSC antibodies, which might have a negative impact on the donor kidney. Of interest for the development of future studies is the great therapeutic potential of allogeneic stem cells harvested from Wharton’s Jelly of the human umbilical cord, which can be very easily isolated and cultured. A “bank” with allogeneic MSCs would be available with the possibility to select the MSCs for the patient in need for treatment.

24.6.8 Measuring Efficacy in Trials With MSC Since patient and kidney survival have markedly improved, and acute rejection rates have declined, these endpoints are nowadays difficult to use in the clinical setting. Indeed, the production of MSCs is labor-intensive and costly, and such a design would be a great, if not an almost impossible, challenge. Therefore, short-term surrogate endpoint markers are used that can predict biopsy proven acute rejection (BPAR) and graft survival. These endpoints include in the first place measurement of renal function and histological analysis before and after MSC treatment, with a focus on fibrosis quantification and inflammatory processes. Secondly, measurement of biomarkers and immune monitoring strategies are used in the different clinical trials to evaluate the efficacy of MSC

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treatment and to elucidate underlying mechanisms. A biomarker is defined as an objectively measurable parameter, which can be used to quantify a normal biological or pathological process. Intensive research has been done studying several biomarkers in kidney disease, including neutrophil gelatinase-associated lipocalin, kidney injury molecule-1, Interleukin-18, and liver-type fatty acid-binding protein. In addition, genomic and proteomic platforms have provided various promising new biomarkers in recent years. A strong focus on the development of biomarkers that can monitor safety, immune modulation, and regeneration should be the aim in MSC based trials. It is of importance that there is no routine application of any of the biomarkers markers in clinical transplantation yet. In addition, the validation is still insufficient, probably due to the heterogeneity of the patients with kidney injury, the patients’ comorbidities, and the underlying etiologies and treatment strategies. In addition, kidney injury is not a single entity but a multifactorial process. Therefore a single biomarker that reflects physiological and pathophysiological processes in the injured kidney is a difficult quest. Immune monitoring by flow cytometry is crucial in the evaluation of new therapies in renal transplantation. The One study consortium has recently developed a robust immune monitoring strategy, including procedures for whole blood leukocyte subset profiling by flow cytometry.91 In this strategy, 6 panels are used to analyze the immune response: panel 01 includes the general immune status; panel 02 the T cell subsets and the αβ1 T cells and γδ1 T cells; panel 03 the T cell activation; panel 04 the T cell memory and regulatory T cells; panel 05 the B cell subsets; panel 06 the DC subsets. This panel may eventually be adopted as a standardized method for monitoring patients in clinical trials enrolling transplant patients, particularly trials of therapies which include the MSC based studies, to facilitate fair and meaningful comparisons between trials. Of interest, MSCs have also been used for several cardiovascular indications.12 In renal transplantation the administration of MSCs might also benefit cardiac function, and it is of interest to monitor the cardiac function in MSC treated patients. In addition, improvement of renal function by the administration of MSCs might also have an indirect effect on cardiac function.

24.6.9 What Have We Learned so Far From the Clinical Trials The in vitro immunosuppressive and reparative properties of MSCs and the positive results in experimental studies have led to the start of a number of clinical trials in kidney transplantation. Most clinical studies in renal recipients have focused so far on safety and feasibility endpoints.81,92 95 Safety in the different trials was defined as MSC infusion toxicity and/or adverse events related to MSCs with a follow-up period of 12 months posttransplantation. Although the primary focus was on safety, the different studies have also assessed endpoints that provided insights into the mechanisms of actions of MSCs (Fig. 24.2). Different studies have focused on the role of autologous MSCs in the early induction phase after transplantation. MSC infusion was safe and clinically feasible,80,81,92 95 although timing of the infusion seemed of major importance. Indeed, patients given autologous BM MSCs posttransplant developed renal insufficiency, which was not observed when MSCs were administered before transplantation.93 MSC infusion allowed enlarging of T Regs in the peripheral blood and control of memory CD81 T cell function.92 In a larger study in 159 patients undergoing renal transplantation, the use of autologous MSCs compared with anti-IL-2 receptor antibody induction therapy resulted in lower incidence of acute rejection, decreased risk of opportunistic infections, and better estimated renal function at 1 year.80 In another safety and feasibility study autologous MSCs were infused at a later time point. Patients with subclinical rejection or an increase in IF/TA in their renal biopsy at 24 weeks after renal transplantation (compared to the renal biopsy at 4 weeks) received MSCs, with the aim to inhibit inflammation (rejection) and fibrosis and to protect the renal structure. In total 6 of the 15 patients received MSC treatment, since not all patients met the inclusion criteria.81 The MSC infusion was well tolerated and there were no adverse events related to the treatment itself. In addition, the initial results suggested immune suppression after MSC therapy. All patients that received MSCs demonstrated a profound reduction in proliferation of patient PBMC 12 weeks after MSC infusion upon stimulation with donor specific PBMCs, while the response to third party PBMCs was more variable. Three patients developed opportunistic viral infections (2 CMV, 1 BK nephropathy), which might be related to the MSC treatment. In two patients with allograft rejection, there was a clinical indication to do a third biopsy. In both patients the infiltrate had disappeared and there were no signs of fibrosis after the MSC infusion. In a follow up study autologous BM MSCs will be used in combination with Everolimus with the aim to facilitate early Tacrolimus withdrawal and to preserve renal structure.96 The primary endpoint is to compare fibrosis by

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24.6 CLINICAL TRIALS WITH MSCs AFTER RENAL TRANSPLANTATION

Clinical

Preclinical

Small animals

Humanized animals

Large animals

Mechanisms of action

Proof of concept in humanized situation

Safety and feasibility Dose finding

Phase I

Phase II

Phase III

Safety and feasibility

Safety and efficacy Directing continuing research

Comparison to existing therpies in larger cohort

• MSC therapy is safe • MSC therapy is feasible • Timing is of paramount importance Important lessons learned so far

• Step by step approach is needed • Longer follow up is needed • Standardization of safety, feasibility, and efficacy endpoints is needed

Safety endpoints • Adverse events • Patient and graft survival • BPAR • Renal function

Efficacy endpoints • Histology • BPAR • Renal function • Cardiovascular disease Mechanisms of action • Characteristics MSCs • Immune monitoring • Functional immune assays • Histology • Biomarkers

FIGURE 24.2

Lessons learned from initial clinical trials. Preclinical studies with small animals have been performed to investigate mechanisms of actions of MSCs in renal transplantation. A few humanized and larger animals have performed in the setting of MSC therapy in renal transplantation to proof the concept. Human phase I studies have addressed safety and feasibility in a low number of patients. In addition, the aim was to provide insights in mechanisms of actions and to determine the direction of further research. Lessons learned so far indicate that MSC therapy is safe and feasible; that timing of MSC therapy is of paramount importance, and that a step-by-step approach is needed. Of importance for the future a longer follow up is needed and standardization of safety, feasibility, and efficacy endpoints should be implemented between the different centers to make meaningful comparisons. Phase II studies have started which focus on both safety and efficacy parameters.

quantitative Sirius Red scoring of MSC treated and untreated groups at 6 months compared to 4 weeks posttransplantation. Secondary endpoints focus on adverse events (including infections), BPAR and graft loss, renal function measured by iohexol, and progression of subclinical cardiovascular disease. In addition, immune monitoring will be performed according to the methods as standardized and validated in the One study.91 Few studies are focusing on allogeneic MSCs. Allogeneic MSC infusion was safe and prevented acute rejection after renal transplantation in a small cohort of patients,94 however no accurate monitoring of sensitization was performed. In a newly-developed protocol, renal transplant recipients will receive two doses of allogeneic third party MSCs 6-month posttransplantation. The allogeneic MSCs will meet specific criteria to minimize the risk of sensitization (NCT02387151), including: (1) no HLA sharing with the kidney donor; and (2) no antibodies to the MSCs. The primary objective is to evaluate whether allogeneic MSCs are safe by assessing acute rejection and graft loss after MSC treatment. In addition, the development of de novo DSA will be monitored. These safety issues should be studied before further studies are planned with allogeneic MSCs in the transplant setting.

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24.6.10 Large Randomized Controlled Trials or Step-By-Step Approach Uncertainties regarding the safety issues raise the question of what study design is most suitable in this first phase of clinical trials with MSC therapy. Randomized controlled trials (RCTs) are considered the most powerful trial set up. However, RCT are not designed to pick up signals that infringe patient safety, and they require large amounts of patients, making them less suitable for addressing safety considerations and optimizing treatments in the first stages of clinical trials. Another disadvantage of RCTs is the high risk of failure in relation to the costs, particularly in the setting of cell therapy. Therefore study designs that harbor adaptive features, i.e., changes in design or analyses guided by examination of the accumulated data at interim points are preferred, so that studies can be more efficient, prevent unnecessary exposure to risk, and be more informative. A Data Safety Monitoring Board with well described roles and responsibilities to assess the safety and efficacy of the interventions during the trial, and to monitor the overall conduct of the clinical trial, is needed to maximize safety for the patient.

24.7 CONCLUSION AND FUTURE DIRECTIONS MSCs could potentially play an important role after renal transplantation in the prevention and treatment of rejection episodes, in the induction of tolerance and in the prevention of fibrosis. Several animal models have investigated MSCs for those different indications and demonstrated prolongation of allograft survival and an inhibition of the rejection process. In humans, the first clinical trials have been performed mainly with autologous BM MSCs, showing safety and feasibility. An important lesson of these trials is that timing of the infusion is of paramount importance, as early posttransplant infusion may induce deterioration of renal function. Although in current clinical trials no major side-effects have been reported, longer follow-up of the MSC-treated patients is necessary in order to identify the possible long-term effects. In addition, initial results indicate efficacy in preventing acute rejection, inducing stable graft function and reducing tubulitis and IF/TA in small groups of patients. Currently the first phase II trials with autologous BM MSCs have started, with an important focus on the minimization of immune suppressive drugs to reduce fibrosis and to prolong allograft survival.87,96 In addition, safety studies with (matched) allogeneic MSCs are planned, which offer the advantage of availability for clinical use without the delay required for expansion. In these trials accurate analysis of sensitization should be performed. To compare the effectiveness of MSCs in the different trials, well-defined end-points and appropriate controls are needed. In this perspective, the described standardized and validated methods for immune monitoring are a nice example. In general, sharing of procedures and protocols for MSC culture and follow-up after treatment will allow for more reliable comparisons between the different clinical trials.

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The impact of mesenchymal stem cell therapy in transplant rejection and tolerance. Curr Opin Organ Transplant 2012;17(4):355 61. 20. Spaggiari GM, Capobianco A, Abdelrazik H, Becchetti F, Mingari MC, Moretta L. Mesenchymal stem cells inhibit natural killer-cell proliferation, cytotoxicity, and cytokine production: role of indoleamine 2,3-dioxygenase and prostaglandin E2. Blood 2008;111(3):1327 33. 21. Spaggiari GM, Capobianco A, Becchetti S, Mingari MC, Moretta L. Mesenchymal stem cell-natural killer cell interactions: evidence that activated NK cells are capable of killing MSCs, whereas MSCs can inhibit IL-2-induced NK-cell proliferation. Blood 2006;107(4):1484 90. 22. Sotiropoulou PA, Perez SA, Gritzapis AD, Baxevanis CN, Papamichail M. Interactions between human mesenchymal stem cells and natural killer cells. Stem Cells 2006;24(1):74 85. 23. Jiang XX, Zhang Y, Liu B, et al. Human mesenchymal stem cells inhibit differentiation and function of monocyte-derived dendritic cells. Blood 2005;105(10):4120 6. 24. Nauta AJ, Kruisselbrink AB, Lurvink E, Willemze R, Fibbe WE. Mesenchymal stem cells inhibit generation and function of both CD341derived and monocyte-derived dendritic cells. J Immunol 2006;177(4):2080 7. 25. Zhang W, Ge W, Li C, et al. Effects of mesenchymal stem cells on differentiation, maturation, and function of human monocyte-derived dendritic cells. Stem Cells Dev 2004;13(3):263 71. 26. Chiesa S, Morbelli S, Morando S, et al. Mesenchymal stem cells impair in vivo T-cell priming by dendritic cells. Proc Natl Acad Sci USA 2011;108(42):17384 9. 27. Franquesa M, Mensah FK, Huizinga R, et al. Human adipose tissue-derived mesenchymal stem cells abrogate plasmablast formation and induce regulatory B cells independently of T helper cells. Stem Cells 2014;33(3):880 91. 28. Franquesa M, Hoogduijn MJ, Bestard O, Grinyo JM. Immunomodulatory effect of mesenchymal stem cells on B Cells. Front Immunol 2012;3:212. 29. Di Ianni M, Del Papa B, De Ioanni M, et al. Mesenchymal cells recruit and regulate T regulatory cells. Exp Hematol 2008;36(3):309 18. 30. Kim J, Hematti P. Mesenchymal stem cell-educated macrophages: a novel type of alternatively activated macrophages. Exp Hematol 2009;37(12):1445 53. 31. Mannon RB. Macrophages: contributors to allograft dysfunction, repair, or innocent bystanders? Curr Opin Organ Transplant 2012;17(1):20 5. 32. Nemeth K, Leelahavanichkul A, Yuen PS, et al. Bone marrow stromal cells attenuate sepsis via prostaglandin E(2)-dependent reprogramming of host macrophages to increase their interleukin-10 production. Nat Med 2009;15(1):42 9. 33. Chan JL, Tang KC, Patel AP, et al. Antigen-presenting property of mesenchymal stem cells occurs during a narrow window at low levels of interferon-gamma. Blood 2006;107(12):4817 24. 34. Stagg J. Immune regulation by mesenchymal stem cells: two sides to the coin. Tissue Antigens 2007;69(1):1 9. 35. Horwitz EM, Gordon PL, Koo WK, et al. Isolated allogeneic bone marrow-derived mesenchymal cells engraft and stimulate growth in children with osteogenesis imperfecta: implications for cell therapy of bone. Proc Natl Acad Sci USA 2002;99(13):8932 7. 36. Horwitz EM, Prockop DJ, Fitzpatrick LA, et al. Transplantability and therapeutic effects of bone marrow-derived mesenchymal cells in children with osteogenesis imperfecta. Nat Med 1999;5(3):309 13. 37. Prodromidi EI, Poulsom R, Jeffery R, et al. Bone marrow-derived cells contribute to podocyte regeneration and amelioration of renal disease in a mouse model of Alport syndrome. Stem Cells 2006;24(11):2448 55. 38. Ohnishi S, Sumiyoshi H, Kitamura S, Nagaya N. Mesenchymal stem cells attenuate cardiac fibroblast proliferation and collagen synthesis through paracrine actions. FEBS Lett 2007;581(21):3961 6. 39. Reinders ME, de Fijter JW, Rabelink TJ. Mesenchymal stromal cells to prevent fibrosis in kidney transplantation. Curr Opin Organ Transplant 2014;19(1):54 9. 40. Souidi N, Stolk M, Seifert M. Ischemia-reperfusion injury: beneficial effects of mesenchymal stromal cells. Curr Opin Organ Transplant 2013;18(1):34 43. 41. Franquesa M, Herrero E, Torras J, et al. Mesenchymal stem cell therapy prevents interstitial fibrosis and tubular atrophy in a rat kidney allograft model. Stem Cells Dev 2012;21(17):3125 35. 42. Ninichuk V, Gross O, Segerer S, et al. Multipotent mesenchymal stem cells reduce interstitial fibrosis but do not delay progression of chronic kidney disease in collagen4A3-deficient mice. Kidney Int 2006;70(1):121 9. 43. Li L, Zhang Y, Li Y, et al. Mesenchymal stem cell transplantation attenuates cardiac fibrosis associated with isoproterenol-induced global heart failure. Transpl Int 2008;21(12):1181 9. 44. Humphreys BD, Lin SL, Kobayashi A, et al. Fate tracing reveals the pericyte and not epithelial origin of myofibroblasts in kidney fibrosis. Am J Pathol 2010;176(1):85 97. 45. Khairoun M, van der Pol P, de Vries DK, et al. Renal ischemia/reperfusion induces a dysbalance of angiopoietins, accompanied by proliferation of pericytes and fibrosis. Am J Physiol Renal Physiol 2013;305(6):F901 10. 46. Chen L, Tredget EE, Wu PY, Wu Y. Paracrine factors of mesenchymal stem cells recruit macrophages and endothelial lineage cells and enhance wound healing. PLoS One 2008;3(4):e1886. 47. Zacharek A, Chen J, Cui X, et al. Angiopoietin1/Tie2 and VEGF/Flk1 induced by MSC treatment amplifies angiogenesis and vascular stabilization after stroke. J Cereb Blood Flow Metab 2007;27(10):1684 91.

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48. Carrion B, Kong YP, Kaigler D, Putnam AJ. Bone marrow-derived mesenchymal stem cells enhance angiogenesis via their α6β1 integrin receptor. Exp Cell Res 2013;319(19):2964 76. 49. Babu AN, Murakawa T, Thurman JM, et al. Microvascular destruction identifies murine allografts that cannot be rescued from airway fibrosis. J Clin Invest 2007;117(12):3774 85. 50. Contreras AG, Briscoe DM. Every allograft needs a silver lining. J Clin Invest 2007;117(12):3645 8. 51. Tomasoni S, Longaretti L, Rota C, et al. Transfer of growth factor receptor mRNA via exosomes unravels the regenerative effect of mesenchymal stem cells. Stem Cells Dev 2013;22(5):772 80. 52. Gatti S, Bruno S, Deregibus MC, et al. Microvesicles derived from human adult mesenchymal stem cells protect against ischaemiareperfusion-induced acute and chronic kidney injury. Nephrol Dial Transplant 2011;26(5):1474 83. 53. Reinders ME, Fibbe WE, Rabelink TJ. Multipotent mesenchymal stromal cell therapy in renal disease and kidney transplantation. Nephrol Dial Transplant 2010;25(1):17 24. 54. Bartholomew A, Sturgeon C, Siatskas M, et al. Mesenchymal stem cells suppress lymphocyte proliferation in vitro and prolong skin graft survival in vivo. Exp Hematol 2002;30(1):42 8. 55. Zhou HP, Yi DH, Yu SQ, et al. Administration of donor-derived mesenchymal stem cells can prolong the survival of rat cardiac allograft. Transplant Proc 2006;38(9):3046 51. 56. Casiraghi F, Azzollini N, Todeschini M, et al. Localization of mesenchymal stromal cells dictates their immune or proinflammatory effects in kidney transplantation. Am J Transplant 2012;12:2373 83. 57. Casiraghi F, Azzollini N, Cassis P, et al. Pretransplant infusion of mesenchymal stem cells prolongs the survival of a semiallogeneic heart transplant through the generation of regulatory T cells. J Immunol 2008;181(6):3933 46. 58. Eggenhofer E, Popp FC, Mendicino M, et al. Heart grafts tolerized through third-party multipotent adult progenitor cells can be retransplanted to secondary hosts with no immunosuppression. Stem Cells Transl Med 2013;2(8):595 606. 59. De Martino M, Zonta S, Rampino T, et al. Mesenchymal stem cells infusion prevents acute cellular rejection in rat kidney transplantation. Transplant Proc 2010;42(4):1331 5. 60. Schrepfer S, Deuse T, Reichenspurner H, Fischbein MP, Robbins RC, Pelletier MP. Stem cell transplantation: the lung barrier. Transplant Proc 2007;39(2):573 6. 61. Bernardo ME, Fibbe WE. Mesenchymal stromal cells: sensors and switchers of inflammation. Cell Stem Cell 2013;13(4):392 402. 62. Jones S, Horwood N, Cope A, Dazzi F. The antiproliferative effect of mesenchymal stem cells is a fundamental property shared by all stromal cells. J Immunol 2007;179(5):2824 31. 63. Krampera M, Cosmi L, Angeli R, et al. Role for interferon-gamma in the immunomodulatory activity of human bone marrow mesenchymal stem cells. Stem Cells 2006;24(2):386 98. 64. Meisel R, Zibert A, Laryea M, Gobel U, Daubener W, Dilloo D. Human bone marrow stromal cells inhibit allogeneic T-cell responses by indoleamine 2,3-dioxygenase-mediated tryptophan degradation. Blood 2004;103(12):4619 21. 65. Roemeling-van Rhijn M, Khairoun M, Korevaar SS, et al. Human bone marrow- and adipose tissue-derived mesenchymal stromal cells are immunosuppressive in a humanized allograft rejection model. J Stem Cell Res Ther 2014;6(Suppl. 1):20780. 66. Roemeling-van Rhijn M, Reinders ME, Franquesa M, et al. Human allogeneic bone marrow and adipose tissue derived mesenchymal stromal cells induce CD81 cytotoxic T cell reactivity. J Stem Cell Res Ther 2014;3(Suppl. 6):004. 67. English K, Barry FP, Field-Corbett CP, Mahon BP. IFN-gamma and TNF-alpha differentially regulate immunomodulation by murine mesenchymal stem cells. Immunol Lett 2007;110(2):91 100. 68. Hoogduijn MJ, Crop MJ, Korevaar SS, et al. Susceptibility of human mesenchymal stem cells to tacrolimus, mycophenolic acid, and rapamycin. Transplantation 2008;86(9):1283 91. 69. Eggenhofer E, Renner P, Soeder Y, et al. Features of synergism between mesenchymal stem cells and immunosuppressive drugs in a murine heart transplantation model. Transpl Immunol 2011;25(2-3):141 7. 70. Eggenhofer E, Steinmann JF, Renner P, et al. Mesenchymal stem cells together with mycophenolate mofetil inhibit antigen presenting cell and T cell infiltration into allogeneic heart grafts. Transpl Immunol 2011;24(3):157 63. 71. Ge W, Jiang J, Baroja ML, et al. Infusion of mesenchymal stem cells and rapamycin synergize to attenuate alloimmune responses and promote cardiac allograft tolerance. Am J Transplant 2009;9(8):1760 72. 72. Zhang W, Qin C, Zhou ZM. Mesenchymal stem cells modulate immune responses combined with cyclosporine in a rat renal transplantation model. Transplant Proc 2007;39(10):3404 8. 73. Phinney DG, Prockop DJ. Concise review: mesenchymal stem/multipotent stromal cells: the state of transdifferentiation and modes of tissue repair current views. Stem Cells 2007;25(11):2896 902. 74. Schallmoser K, Bartmann C, Rohde E, et al. Human platelet lysate can replace fetal bovine serum for clinical-scale expansion of functional mesenchymal stromal cells. Transfusion 2007;47(8):1436 46. 75. Le Blanc K, Frassoni F, Ball L, et al. Mesenchymal stem cells for treatment of steroid-resistant, severe, acute graft-versus-host disease: a phase II study. Lancet 2008;371(9624):1579 86. 76. Koc ON, Gerson SL, Cooper BW, et al. Rapid hematopoietic recovery after coinfusion of autologous-blood stem cells and cultureexpanded marrow mesenchymal stem cells in advanced breast cancer patients receiving high-dose chemotherapy. J Clin Oncol 2000;18(2):307 16. 77. Ball LM, Bernardo ME, Roelofs H, et al. 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25 Renal Transplantation Across HLA and ABO Barriers Shaifali Sandal1 and Robert A. Montgomery2 1

McGill University Health Centre, Montreal, QC, Canada 2NYU Langone Transplant Institute, New York, NY, United States

25.1 INTRODUCTION Kidney transplantation (KT) offers clear benefits in terms of quality of life, survival, and cost to the healthcare system when compared with dialysis.1,2 Living donor kidney transplant (LDKT) recipients when compared with deceased-donor kidney transplant (DDKT) recipients have superior immediate and long-term patient and graft survival.3 Despite this, LDKT rates in the United States have declined since 2002.3 Human leukocyte antigen (HLA) sensitization and ABO incompatibility (ABOi) remain the most significant barriers to further expansion of live donation. In 2012, amongst the waitlisted patients, 15.8% had a PRA (panel reactive antibody) of .80% and 51.2% had blood group O.3 Currently, patients with an incompatible live donor have three options: wait on the list for a suitable deceased donor; undergo desensitization; kidney-paired donation (KPD). In this chapter we will discuss these strategies in detail and how they can be optimally utilized to transplant across HLA and ABO barriers.

25.2 TYPES OF ANTIBODIES Pathological antibodies can be generated by allogeneic or autologous molecules and through cross reactivity with naturally occurring antigens. Antibodies can be directed against the HLA antigens by previous exposure to allogeneic tissue through transfusion, pregnancy, or a previous transplant. They can also occur as a result of tissue injury, environmental exposure, and immune dysregulation. Cytotoxic antibodies most often have targets in the microcirculation and cause injury through activation of complement. The antibodies are typically produced by long-lived plasma cells residing in the bone marrow after a vaccine type of immune response. These can be high titer antibody that cause direct tissue injury or can be a marker for B-cell memory that can result in an anamnestic response.

25.2.1 ABO Antibodies A and B are blood group antigens expressed on erythrocytes, vascular endothelium, and other tissues. A-transferase adds N-acetylgalactosamine to the H oligosaccharide while the B-transferase adds galactose.4 This subtle molecular difference leads to production of natural antibodies typically during the first 4 6 months of life.5,6 Anti-A or -B antibodies can result in normal engraftment, hyperacute rejection (HAR), or antibody mediated rejection (AMR) depending upon the titer of the isohemagglutinins. Kidney Transplantation, Bioengineering, and Regeneration. DOI: http://dx.doi.org/10.1016/B978-0-12-801734-0.00025-4

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25.2.1.1 HLA Antibodies There are 20 class I genes in the HLA region, 3 of which, HLA-A, B, and C, are present on all somatic cells.7 Class II MHC molecule nomenclature is designated by class (D), family (M, O, P, Q, R), and chain (A or B) and is present on B cells, macrophages, and dendritic cells.7 The function of the HLA molecule is the presentation of short, pathogen-derived peptides to T cells and then to activate the immune system causing cytokine production, B-cell activation and formation of a germinal center.4,7,8 In germinal centers, the activated B-cells rapidly divide and undergo somatic hypermutation, affinity maturation, and isotype switching.4 Some of the high affinity clones also differentiate into long-lived memory B-cells and antibody-secreting plasma cells that produce anti-HLA antibodies for long periods of time.4 25.2.1.2 Other Non-HLA Antibodies Non-HLA antibodies directed against antigens that are expressed on endothelial cells but not lymphocytes are increasingly being recognized as important contributors to all AMR phenotypes.9 17 These antibodies are not routinely detected by complement-dependent lymphocytotoxic assays. Many of these immunostimulatory antigens appear to be polymorphic and can be detected by performing a crossmatch in which donor endothelial precursor cells are tested against recipient serum.13 Most relevant endothelial antigens have probably not yet been identified15 but some examples of known molecules that can cause AMR include agrin, vimentin, MHC class I related chain A, and angiotensin II type 1 receptor.18

25.3 ADVANTAGES OF DESENSITIZATION Presence of antibodies is a major risk for development of HAR and AMR with subsequent graft loss. Desensitization is a process in which various immunomodulating therapies and in some cases antibody depleting modalities are administered to transplant recipients to eliminate or reduce unacceptable antibody levels in order to enable transplantation. It has the following advantages (Table 25.1).

25.3.1 Enables Transplantation Amongst Incompatible Donor and Recipient Pairs Several protocols that entail removal of preformed ABO antibodies and inhibition of ongoing antibody production have allowed successful ABOi KT and will be discussed in detail below. Outcomes of three registry analysis are summarized in Table 25.2.19 21 The largest experience with the longest follow up comes from Japan.19 The registry of the Japanese ABO-Incompatible Kidney Transplantation Committee has reported 2434 ABOi LDKTs across 120 Japanese centers with excellent graft and patient survival. The heightened risk of graft loss from preformed anti-HLA antibodies was recognized as early as the 1960s.22 The cytotoxic crossmatch (CXM) identified individuals at risk allowing these transplants to be averted. Unfortunately, highly sensitized patients were relegated to the wait list where they were likely to remain for TABLE 25.1

Advantages and Disadvantages of Desensitization

Advantages of desensitization • • • •

Enables transplantation amongst incompatible donor and recipient pairs Prevention of antibody mediated rejection (hyperacute and acute) Survival advantage Costeffective

Disadvantages and issues with desensitization • • • • • • • • •

Lack of randomized controlled trials and evidence for long-term outcomes Risk of antibody mediated rejection still exists Risk of transplant glomerulopathy and graft loss Lack of efficacy in very high risk patients Lack of standard testing guideline and treatment protocols Risk of being cited by regulatory agencies Infection risk Malignancy risk Interference with crossmatch

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TABLE 25.2

ABO Incompatible Transplants: Registry Analyses (Ref.19

21

)

Europe/Australia/New Zealand

Japan

United States

Time period

2005 12

1989 2012

1995 2010

# of centers

101

120

120

ABOi

ABOc

ABOi

ABOc

ABOi

ABOc

n

1420

1420 matched controls (MC) and 27845 center controls (CC)

2434

N/A

738

3679 (matched 5:1)

Rejection

16% (1 year)

18% (1 year) in MC

No HAR since 2001

N/A

N/A

N/A

Graft outcomes

Graft survival: 90% (3 year), DCGS 93% (3 years)

Graft survival: MC 1 CC 90% (3 years), DCGS: MC 93% (3 years), CC 92% (3 years)

Graft survival 86% (5 years), 71% (10 years), 52% (20 years)

N/A

a

Graft loss: 10% (3 years), 17% (5 years), 27% (10 years)

a Graft loss: 6% (3 years), 11% (5 years), 24% (10 years)

Patient survival

96% (3 years)

96% (3 years) for MC 1 CC

93% (5 years), 90% (10 years), 73% (20 years)

N/A

94% (3 years), 88% (5 years), 74% (10 years)

95% (3 years), 91% (5 years), 75% (10 years)

a

incidence of graft loss (with death treated as a competing risk). ABOc: ABO compatible, ABOi: ABO incompatible, DCGS: death censored graft survival, HAR: hyperacute rejection, N/A: not available.

many years with high levels of mortality. The development of several different desensitization protocols has allowed for increased rates of transplantation with good graft outcomes. At Cedars-Sinai Medical Center, the mean wait time on dialysis for sensitized patients prior to desensitization was 114 6 56 months and after desensitization was 4.4 6 4.9 months.23

25.3.2 Prevention of Antibody Mediated Rejection Several different phenotypes of AMR are reported in the transplant literature. Desensitization and sophisticated techniques for detecting a wide array of clinically significant donor specific antibodies (DSA) has nearly abolished HAR and has decreased the rates of AMR in incompatible transplants. HAR occurs in the first 24 hours after transplantation due to preformed antibodies.24,25 It is now very rare. Accelerated AMR occurs in the first week after KT. A humoral anamnestic response causes a sudden sharp rise in HLA DSA or isohemagglutinins and results in microcirculation inflammation and injury, which can be severe. This is responsible for early graft loss following desensitization and in our experience occurs at a rate of 9%.26,27 Focused therapy at the first sign of oliguric accelerated AMR has been shown to be successful in rescuing most of these organs and allograft loss due to severe AMR is now a rarity.27 30 A less aggressive early (,30 days posttransplant) AMR phenotype has also been recognized in about 16% of patients, responds well to standard of care desensitization therapies, and rarely leads to immediate graft loss. Lastly, the emergence of de novo DSA in a previously nonsensitized patient can lead to AMR and its impact will be discussed later in the chapter entitled “Impact of de novo donor-specific alloantibody in primary renal allografts.” Pretransplant third party HLA antibody is a risk factor as it may be a marker of an alloreactive patient at risk for a recall response or non-DSA could be related to future appearance of DSA through epitope spreading.31 Desensitization of patients with a high burden of antibodies but no DSA may lead to lower incidence of de novo DSA and chronic AMR (CAMR) posttransplant.32 34

25.3.3 Survival Advantage Results of desensitization for ABOi are similar to ABO compatible transplants.35 39 Beyond the first month a phenomenon called accommodation occurs in which an organ transplant functions normally, despite the presence of ABO antibodies, albeit generally at relatively low levels (titer ,32).40,41 Graft survival rates following desensitization in HLA incompatible (HLAi) individuals have generally been inferior when compared with compatible LDKT.42,43 However, outcomes of desensitization protocols when compared with options that are actually available to this subset of patients have shown a profound survival benefit.44,45 When compared to matched control patients who remained on dialysis waiting for a compatible kidney, desensitized patients doubled their survival after 8 years of follow-up, as illustrated in Fig. 25.1.44

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100 90 80 Patient survival

70 60 50 40 30 20

Overall

FXM + only

CXM +

8-year

5-year

3-year

1-year

8-year

5-year

3-year

1-year

8-year

5-year

3-year

0

1-year

10

Desensitized group Dialysis only Dialysis or Transplantation

FIGURE 25.1 Survival benefit of desensitization: This graph was drawn from data collected and analyzed by our center. These results were eventually published in NEJM in 2011.44 We compared rates of death between 3 groups: one group undergoing desensitization treatment and two matched control groups of patients on a waiting list for kidney transplantation who continued to undergo dialysis (dialysis-only group) or who underwent either dialysis or transplantation (dialysis-or-transplantation group). FXM, flow-cytometric cross-match; CXM, complement dependent cytotoxicity cross-match

25.3.4 Costeffective Several studies have reported that desensitization is cost effective when compared to staying on dialysis.35,45 47 Cost savings of more than $300,000 per patient over a 5-year observation period have been proposed.47 However, this will also depend on the actual modalities employed; for instance, a study from India reported that desensitization is only affordable with cheaper modalities.48 Similarly, the use of complement inhibitor eculizumab might be cost prohibitive as it costs $5000 per 300 mg vial, dosing may be frequent and therapy may be life-long.49

25.4 DESENSITIZATION MODALITIES The aim of desensitization is to decrease DSA to acceptable levels and prevent AMR. The definition of an “acceptable level” varies from one institution to another based on the variability of DSA testing techniques and the risk tolerance of the physicians. Most centers would not proceed to transplantation unless the CXM is negative; i.e., mean fluorescent intensity (MFI),10,000 on Luminex testing for class I DSA. Some centers are comfortable with a positive flow crossmatch (FXM), i.e., MFIs between 5000 and 10,000, while others require a negative FXM (MFI,5000). In ABOi transplants most centers will deplete until the IgG titer is #16 by antihuman globulin for isohemagglutination. Potential targets and desensitization therapies are identified in Fig. 25.2 and summarized in Table 25.3. The Johns Hopkins’ desensitization protocol for crossmatch positive living and deceased kidney donor recipients is summarized in Fig. 25.3.

25.4.1 Removal of Antibodies Two main strategies to remove antibodies include plasmapheresis or plasma exchange and immunoadsorption (IA) (Fig. 25.2). Neither strategy has been compared in randomized trials, hence use is center-dependent based on local expertise. 25.4.1.1 Plasmapheresis and Plasma Exchange Plasma exchange or double-filtration plasmapheresis nonselectively removes immune complexes, protein bound toxins, circulating antibodies, complement components, and coagulation factors.50 52 Complications are I. KIDNEY TRANSPLANTATION

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25.4 DESENSITIZATION MODALITIES

Splenectomy

Proteosomal inhibitors

Complement inhibitors

Plasmablasts plasma cells

Complement activation

Anti-CD20 Clonal expansion

IVIg, immunoadsorption & plasmapheresis Induction agents

Alloreactive antibody

T-Cell Memory B cells Endothelial destruction

FIGURE 25.2 Targets and therapeutic modalities for desensitization: Desensitization protocols involve an alloantibody depleting modality, IVIg, and a B cell depleting therapy. Complement inhibition and splenectomy are some of the other methodologies available. TABLE 25.3

Therapies Used in Desensitization Protocols

Desensitization modalities • Removal of antibodies • Plasmapheresis and plasma exchange • Immunoadsorption • IVIga • B cell depleting therapies • Anti-CD 20 monoclonal antibodies (Rituximab) • Proteosome inhibitors (Bortezomib) • Splenectomy • Emerging therapies • Inhibiting complement • Anti-C5 monoclonal antibodies (Eculizumab) • C1 esterase inhibitor • Antiinterleukin antibodies (Tocilizumab) • Auxiliary liver transplant • Cryofiltration • BAFF inhibitors (Belimumab) a

Multiple mechanisms of action; see text.

not common and are usually related to complications of vascular access, allergic and infectious reactions to transfusion products, nausea, hypotension, increased risk of bleeding, and symptoms related to hypocalcemia.44,50,53,54 Arrhythmias and pulmonary edema are some of the major reported adverse effects55 and major events occurred in 1.4% of patients in a series of 221 patients44 (Plasma exchange and plasmapheresis will be used interchangeably and abbreviated PP.). PP is a reliable method of reducing both HLA and ABO antibody strength and often forms the backbone of antibody removal in most desensitization protocols.56,57 The number of treatments required is estimated based on the initial antibody strength.54,58 However, antibody rebound is common if PP is discontinued without transplantation or if IVIg is not given after a PP treatment. Plasmapheresis is most often coupled with low dose IVIg (100 mg/kg). It is thought that repletion of IgG at near physiologic levels (low dose IVIg) prevents endogenous antibody production and rebound. 25.4.1.1.1 Efficacy in HLAi Transplants The most popular desensitization protocol used is PP followed by IVIg (100 mg/kg). This protocol was more effective in abrogating a positive crossmatch with lower AMR rates than a single high-dose of IVIg.59 We have reported good 5-year graft survival in 211 HLA-sensitized patients who underwent desensitization using this protocol.44 Other centers have also reported successful KT using PP based regimens in highly sensitized patients with a positive crossmatch.42,43,57,59 74 I. KIDNEY TRANSPLANTATION

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Steroids ATG Anti-CD20*

(A)

Continue FK506 Continue MMF (2 gm/day) Prednisone Taper

FK506 (0.1 mg/kg/day) MMF (2 gm/day)

–10 –9 –8 –7 –6 –5 –4 –3 –2 –1

0

1

2

3

4

5

6

7

8

9

10

Post-operative PP/IVIg Goal: negative flow crossmatch

Pre-operative PP/IVIg Goal: negative cytotoxiccrossmatch

Day of transplant * Used if combined ABO and HLA incompatible, high starting HLA DSA titer, multiple repeat mismatches, and increased level of donor HLA-specific B cells.

(B)

Steroids ATG Anti-CD20 FK506 (0.1 mg/kg/day) MMF (2 gm/d)

0

1

2

3

Continue FK506 Continue MMF (2 gm/day) Prednisone Taper

4

5

6

7

8

9

10

Postoperative PP/IVIg Goal: negative flow crossmatch

Day of transplant FIGURE 25.3 (A) Desensitization protocol for crossmatch positive living kidney donor recipients. (B) Desensitization Protocol for crossmatch positive deceased kidney donor recipients: These are the protocols employed at the Johns Hopkins’ Hospital. ATG, antithymocyte globulin; MMF, mycophenolate mofetil; PP, plasmapheresis.

25.4.1.1.2 Efficacy in ABOi Transplants The role of PP in ABOi transplantation has been recognized since the 1970s.54 We have previously reported 53 successful ABOi KT using PP preconditioning with excellent graft performance.54 Other centers have reported similar short-term outcomes when using PP based desensitization regimens.75 77 Perhaps the most impactful data comes from registry analyses,20,21 in particular the afore-mentioned Japanese registry19 (Table 25.2). 25.4.1.2 Immunoadsorption IA is an extracorporeal hemofiltration technique in which immunoglobulins are selectively removed via binding to a Staphylococcal protein A column for HLA specific antibodies or an ABO antigen-specific column for isohemagglutinins.4,78,79 Potent immunosuppression is then needed for the sustained reduction of the pathogenic antibodies.79 IA offers the advantage of no significant loss of clotting factors or albumin, requires no substitution of plasma, antibody specificities against previously encountered antigens are preserved, and columns can in some cases be reused.79 82 Issues related to IA include higher cost, citrate reaction, hypotonia, reduced protective antibodies, and anaphylaxis.49,79,81,83 Lastly, IA columns are not FDA approved in the United States.57

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339

25.4.1.2.1 Efficacy in HLAi Transplants Several studies have reported the successful use of IA based protocols in sensitized patients.84 89 Desensitization using 10 16 IA sessions effectively reduced or eliminated DSA in 71% recipients of KT.89 Ten patients were desensitized and then successfully transplanted using IA before and after transplantation in addition to one dose of anti-CD20 therapy prior to transplantation.88 25.4.1.2.2 Efficacy in ABOi Transplants As mentioned above, the Japanese groups have demonstrated excellent outcomes when using IA or PP to remove antibodies.19 Clinical outcomes of ABOi patients desensitized with IA and anti-CD20 therapy do not differ from that of matched ABO-compatible patients.38 In addition, a multicenter study of 60 ABOi kidney transplants using preconditioning methods with IA, IVIg, anti-CD20, and conventional immunosuppression demonstrated an excellent graft survival.90 Similar results were reported in other studies.35 An analysis of the 703 ABOi recipients in the Collaborative Transplant Study showed no difference in 3-year death censored graft survival in patients who received PP or IA.21 25.4.1.3 IVIg Intravenous immunoglobulin (pooled polyclonal IgG from healthy volunteers) has emerged as an important component of virtually all desensitization protocols and is now recognized for its antiinflammatory and immunomodulatory actions.91,92 Its role has been implicated in regulation of both innate and cellular immunity such as inhibition of B-cell and T-cell activation, Fc receptor-mediated interactions, complement cascade blockade, antiidiotypic blockade of alloantibodies and modulation of proinflammatory cytokines.47,57,59,91,93 Also, low dose IVIg after PP may prevent endogenous antibody rebound, and reconstitute depleted normal antimicrobial immunoglobulin.4,57 Multiple different preparations of IVIg are available and features that affect tolerability include volume load, infusion rates, osmolality, pH, and sodium or sugar content.94 Possible adverse effects of IVIg include volume overload and infusion-related complications such as aseptic meningitis, thrombotic events and bronchospasms.92 IVIg preparations containing sucrose can cause reversible acute renal failure57,94 and certain preparation can cause hemolytic anemia.92 The most common adverse event in one controlled trial was headache.47 25.4.1.3.1 Efficacy in HLAi Transplants IVIg alone or in combination with other agents has been used successfully to desensitize patients undergoing both LDKT and DDKT.45,47,56,57,65,71,73,74,95 104 A multicenter prospective placebo-controlled trial of high dose IVIg in sensitized waitlisted patients demonstrated a reduction in PRA and a higher transplant rate among the patients who received IVIg.47 Two protocols have been widely implemented for desensitization: IVIg and PP/IA with low-dose IVIg. An analysis of all studies involving IVIg and desensitization reported that for kidney transplant recipients with a positive CXM, both were considered equivalent.105,106 However, IVIg alone is usually not sufficient to render enduring desensitization over the long term.34,59,100 Loupy et al.100 compared the outcomes using two strategies: combined posttransplant quadritherapy/IVIg and the second that added anti-CD20/PP to the above protocol. They noted that the latter group had a lower rate of transplant glomerulopathy, more decline in DSA-MFI and a higher eGFR at 1 year. Another trial had to be halted as more episodes of AMR were noted in the group desensitized with IVIg alone compared with IVIg and anti-CD20 therapy.34 25.4.1.3.2 Efficacy in ABOi Transplants Desensitization for ABOi transplants has not been reported using IVIg alone. Reports of IVIg in patients undergoing ABOi KT concurrently with other immune modulating agents report good graft survival.90,106,107 However, the Japan group has stated that the use of IVIg for ABOi transplants is not necessary.19

25.4.2 B Cell Depleting Therapies B cells are central to the production of antibody, can act as immune effectors and negatively regulate or modulate immune responses.108 In the field of transplantation, strategies to target B cells include the following: B cell depletion, reducing B cell activation, enhancing the generation of regulatory B cells and depleting plasma cells108 (Fig. 25.2). Three of the most commonly used strategies are anti-CD20 monoclonal antibody, proteosome inhibiting therapy and splenectomy.

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25. RENAL TRANSPLANTATION ACROSS HLA AND ABO BARRIERS

25.4.2.1 Anti-CD20 Monoclonal Antibodies Rituximab is a chimeric monoclonal anti-CD20 antibody directed against B cells (immature and mature B cells but not on plasma cells) causing them to undergo apoptosis and lysis.109 Rituximab has minimal to no effect on circulating antibody but is an important adjunct for desensitization.34 Some centers have even reported that B-cell depletion prior to transplant prevents posttransplant B cell activation, de novo DSA production, and CAMR.32 34 Reported adverse effects include lung toxicity, greater need for IVIg supplementation, hematologic toxicity, infectious and infusion complications, and, rarely, JC polyomavirus viremia.32,57,109 111 25.4.2.1.1 Efficacy in HLAi Transplants Rituximab in conjunction with IVIg, PP and IA has been successfully used in transplanting HLAi living and deceased-donor kidney recipients.34,65,71,73,74,88,97 100,112 In a study of 15 highly sensitized patients, randomized to IVIg and placebo versus IVIg and rituximab, no patients in the latter arm experienced AMR and protocol biopsies at 1 year posttransplant showed no evidence of TG.34 However, rituximab has no effect on plasma cells that are the primary source of antibody production and hence therapy failure has been noted especially in those with high antibody burden.113 115 Rituximab’s main utility may be in preventing anamnestic responses by depleting the pool of memory B-cells in sensitized patients at risk for AMR who have high alloreactive B-cell precursor frequencies but are not actively making DSA before transplantation.116 Also, we recently reported that compared to controls, HLAi recipients with or without rituximab had similar DSA persistence, and no significant difference in renal function, graft survival, or AMR rates.115 The efficacy of rituximab might hinge on timing and accessibility to HLA-specific B cells via increased mobilization of B cells from protective niches of the bone marrow.117,118 25.4.2.1.2 Efficacy in ABOi Transplants Rituximab has largely replaced splenectomy in ABOi transplantation.32,35,38,39,76,119,120 Studies comparing rituximab to historical controls that underwent splenectomy in ABOi transplants, report similar 3-year graft survival.119 and lower rates of CAMR and DSA appearance rates in the rituximab group.32 In 22 ABOi KT recipients, rituximab induction and PP resulted in sustained suppression of B cell count and immunoglobulins.120 We, however, have questioned whether rituximab is even needed at all in ABOi KT recipients and have reported excellent outcomes with PP/IVIg alone, without splenectomy or rituximab.121 25.4.2.2 Proteosome Inhibitors Bortezomib is a proteasome inhibitor that can cause mature plasma cell apoptosis.122 The mechanism of action of the drug requires that the target cell is metabolically active. It may have limited efficacy for killing long-lived plasma cells in bone marrow niches, although there is some evidence that these cells are susceptible.123 PP may increase long-lived plasma cell antibody production and make these cells better targets for killing. Bortezomib has been primarily used as a rescue treatment for AMR refractory to standard therapies and desensitization protocols.122,124,125 Also, combining rituximab and bortezomib may have a synergistic effect because of the combined deletion of plasma cells and their precursors.124 Bortezomib-related side effects include gastrointestinal toxicity, thrombocytopenia, paresthesias and painful neuropathy.122,126 128 25.4.2.2.1 Efficacy in HLAi Transplants Use of bortezomib in desensitization is still developing. Efficacy evidence is as follows. A prospective iterative trial of proteasome inhibitor based therapy for reducing HLA antibody levels was conducted in five phases differing in bortezomib dosing density and PP timing.129 Immunodominant antibody (highest HLA antibody level) reductions were observed in 86% of patients, allowing KT in over 40%. Others have shown that bortezomib depleted DSA-producing plasma cells, reduced crossmatch intensities and allowed successful transplants in highly sensitized patients.126,128,130 However, in isolation, bortezomib has modest effects on circulating antibodies and these affects are not sustained.131,132 This is not that surprising because the mechanism of action of bortezomib would predict that activated plasma cells and plasmablasts with high protein turnover would be the most responsive to the drug.132 Lastly, bortezomib was shown to decrease HLA class I antibodies but not HLA class II antibodies.133

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341

25.4.2.2.2 Efficacy in ABOi Transplants There are minimal data to support the use of bortezomib for ABOi transplants. Two cases have been reported where bortezomib in addition to other therapies was used to desensitize ABOi transplants.127,134 25.4.2.3 Splenectomy The spleen is the largest lymphoid organ in the body. Splenectomy is thought to have the effect of debulking plasmablasts, plasma cells, and activated B cells that traffic from regional lymphoid tissue during an AMR episode and hence dramatically diminish antibody production.29,125,135 Analyses of spleens explanted from patients with AMR have demonstrated donor-specific plasmablasts and plasma cells.125,136,137 Since the advent of rituximab, splenectomy is not used preemptively and is now reserved as a rescue therapy. The major concern with splenectomy is sepsis; infection related deaths were double in splenectomized patients when compared with nonsplenectomized patients,138 however recent developments in immunosuppression and antibiotics are believed to have decreased this.29 25.4.2.3.1 Efficacy in HLAi Transplants In patients with severe oliguric AMR following desensitization, several centers have demonstrated the utility of splenectomy as a rescue treatment.29,30,136 Rituximab does not completely deplete the spleen of B cells and has no effect on plasma cells.137 During severe AMR episodes due to anamnestic responses in the first week or two after a HLAi transplant, PP/IVIg and rituximab have been shown to be ineffective at rescuing the allograft.29 It is thought that antibody production during this type of AMR outpaces the ability of PP to remove the antibody burden. Splenectomy provides source control so that PP/IVIg is able to clear the remaining DSA, preventing irreversible cortical necrosis. Splenectomy has been reported to be about 80% effective in rescue, however, many of the patients will go on to develop tg at 1 year and have a truncated graft half-life.27 25.4.2.3.2 Efficacy in ABOi Transplants Splenectomy has historically been a part of ABOi transplants. However, now its use is relegated to rescue in the rare event of a severe AMR. In 4 patients in whom conventional therapies could not decrease the antidonor blood-type antibody titer below 1:16, a regimen consisting of splenectomy enabled successful transplantation.137

25.4.3 Emerging Therapies 25.4.3.1 Inhibiting Complement 25.4.3.1.1 Anti-C5 Monoclonal Antibodies The complement system is a key mediator of graft injury in incompatible transplants. Eculizumab is a humanized anti-C5 monoclonal antibody that causes terminal complement inhibition (Fig. 25.2). Its use as a rescue therapy in accelerated AMR refractory to conventional therapies has been reported.27,139 One of the biggest disadvantages of this medication is high costs.49 Also, efficacy data in desensitization is limited. In eculizumab treated crossmatch positive patients, incidence of AMR was lower; however, eculizumab did not have an impact on DSA levels and the development of CAMR and tg.140,141 In ABOi transplants, a trial involving eculizumab was terminated due to poor enrollment (NCT01095887). Two other trials are ongoing evaluating the safety & efficacy of eculizumab in LDKT recipients requiring desensitization (NCT01399593), and another evaluating the dosing regimen in CXM positive recipients (NCT00670774). Our center has shown that DSA removal with PP/IVIg is essential to the prevention of tg after severe AMR and splenectomy. Using a combination of splenectomy, rituximab, PP/IVIg, and eculizumab, tg was prevented.27 25.4.3.1.2 C1 Esterase Inhibitor C1 esterase inhibitor is a multifunctional member of the serpin family of protease inhibitors that regulates both complement and contact (kallikren-kinin) system activation.142,143 A recent randomized, placebo-controlled study of 20 highly sensitized kidney transplant recipients who were randomized to receive C1-inhibitor preemptively reported that at 1 month no patient in the C1-inhibitor group had AMR.143 We have also performed a multicenter, randomized, placebo-controlled study of C1 esterase inhibitor for the treatment of AMR and found a good safety profile and low rate of tg (unpublished data).

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25.4.3.2 Antiinterleukin Antibodies Interleukin-6 (IL-6) is a pleiotropic cytokine that stimulates B cell growth and differentiation and Th-17 cell activation.144,145 Tocilizumab is a humanized monoclonal antibody against IL-6 receptor.144,145 A phase I/II pilot study using Tocilizumab and IVIg in 10 patients unresponsive to IVIg 1 Rituximab was recently published.145 Five of 10 patients were transplanted, DSA strength and number were reduced, 6-month protocol biopsies showed no AMR, and renal function at 12 months was good. 25.4.3.3 Auxiliary Liver Transplant Liver allograft appears to be protective for renal graft when simultaneously transplanted in highly sensitized patients.146,147 In 5/7 crossmatch positive patients with broadly reactive HLA antibodies, crossmatches turned negative after combined partial-auxiliary liver and kidney transplant, with excellent kidney function and no rejections during a follow-up of 24 60 months.147 In 2011, this group reported 23 patients with positive crossmatch who received KT using this procedure.148 Acceptance of a kidney graft seems to be associated with a proinflammatory reaction within the liver graft after ischemia-reperfusion.148 25.4.3.4 Cryofiltration Cryofiltration is an alternative therapy to IA and PP. It is carried out by separating plasma from blood and then cooling the plasma to create a “cryogel” consisting of heparin, fibronectin, fibrinogen, immunoglobulins, and other proteins.149 This cryogel is retained by filtration before the plasma is rewarmed and returned to the patient.149 Use of cryofiltration has been described in older studies involving ABOi KT150,151 and recently in HLAi transplants.149 25.4.3.5 BAFF Inhibitors B cell activating factor (BAFF) is a cytokine belonging to the tumor necrosis factor ligand family.144 It is immunostimulatory and potentiates the growth, proliferation and differentiation of B cells. The role of Belimumab, a monoclonal antibody against BAFF, as a desensitizing agent was being studied (NCT01025193). However, the study was terminated as it did not demonstrate its primary goal. 25.4.3.6 Drawbacks and Issues With Desensitization Several issues exist with the use of desensitization and they are summarized from here on (Table 25.1).

25.4.4 Lack of RCT and Evidence for Long-Term Outcomes While exact protocols vary across centers, they often entail an alloantibody depleting modality, IVIg, and a B cell depleting therapy.23,58,144,152 There are very few well-designed studies looking at the efficacy of desensitization or comparing different protocols.57,153 The protocols are complicated and the individual components have not undergone rigorous testing. Often many different interventions are made simultaneous and establishing best practices has been a challenge (Table 25.4). Given the heterogeneity in study design, variable immunologic risk of the recipients, and variations in DSA quantification, meta-analyses have not been feasible.57 What can be said about HLAi transplants is that the outcomes are not as good as those reported for compatible transplants, but considerably better than remaining on dialysis or waiting for a compatible transplant.44 However, multiple studies have shown comparable outcomes between ABOi and ABO compatible transplants.35 39

25.4.5 Risk of AMR While desensitization can decrease the risk of AMR it does not abrogate it (Table 25.4). Some have even reported rates as high 61%.65 AMR is a risk factor for graft loss154,155 and early tg.156 In ABOi KT, there is an early risk of AMR, ranging from 10% to 30% of recipients and 0% 10% will have irreversible rejection.49

25.4.6 Risk of tg and Graft Loss One-year biopsies from desensitized recipients showed that tg was present in 25% of the patients and was associated with worse graft survival.156 In another analysis of patients transplanted with a positive crossmatch, tg developed in 47% of patients and was preceded by glomerulitis in more than 90% of cases.157 A multicenter

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TABLE 25.4 Summary of Studies Published on Desensitization by Center

N

Protocol (excluding induction and maintenance immunosuppression) Acute rejection

Graft survival

Patient survival

Center

Year Author

Follow up

Desensitization Control

Desensitization Control

Johns Hopkins, MD

2000 Montgomery 4

PP/IVIg

100%

Up to 2.2 years

100%

100%

Johns Hopkins, MD

2002 Sonnenday

18

PP/IVIg

28%

17 months

94%

Johns Hopkins, MD

2004 Montgomery 80

PP/IVIg

3-year

80%

Johns Hopkins, MD

2011 Montgomery 211

PP/IVIg

Mayo Clinic, MN

2003 Gloor

14

PP/IVIg/RTX/SPX

43%

37 months

79%

86%

Mayo Clinic, MN

2006 Stegall

61

PP/IVIg/RTX

29 80%

1-year

82%

93%

Mayo Clinic, MN

2010 Gloor

119

RTX/SPX

41%

Mayo Clinic, MN

2011 Stegall

26

ECU/PP

8%

3-month

100%

96%

Mayo Clinic, MN

2015 Cornell

30

ECU/PP

7%

3-year

86%

96%

UK

1984 Taube

5

CTX/PP

80%

3 16 months 80%

80%

UK

1989 Palmer

7

CTX/IA/PP

1-year

71%

100%

UK

1996 Higgins

13

IA

69%

26 months

54%

92%

UK

2007 Higgins

24

PP

42%

3-month

88%

92%

UK

2011 Higgins

84

PP/IVIg

22 53%

40 months

3-year 94% 5-year 80%

Cedar Sinai, CA

2008 Vo

16

IVIg/RTX

AMR: 31%, acute rejection 50%

5 month

94%

100%

Cedar Sinai, CA

2010 Vo

76

IVIg/RTX

37%

2-year

84%

95%

Cedar Sinai, CA

2013 Vo

146

IVIg/RTX

ACR: 7%, AMR: 22%

4-year

88%

95%

Brigham and Women’s Hospital, 2008 Magee MA

28

PP/IVIg

39%

22 months

89%

93%

Brigham and Women’s Hospital, 2014 Riella MA

39

PP/IVIg/RTX

61%

5.2-year

3-year 79% 5-year 72%

3-year 88% 5-year 84%

France

2002 Glotz

13

IVIg

1-year

85%

France

2014 Aubert

11

BOR/PP

0

18 months

France

2015 Rostaing

6

IA/IVIg/RTX

67%

1-year

5-, 8-year 81% (see Fig. 25.1)

80%

83%

100% (Continued)

TABLE 25.4 (Continued)

Center

Year Author

N

Protocol (excluding induction and maintenance immunosuppression) Acute rejection

Graft survival

Patient survival

Follow up

Desensitization Control

Desensitization Control

100%

STUDIES FROM OTHER CENTERS University of Maryland, MD

2000 Schweitzer

11

PP/IVIg

36%

13 months

100%

University of Maryland, MD

2009 Haririan

41

PP/IVIg

ACR: 12%, AMR: 12%

9-years

5-year 69%

Mount Sinai, NY

2003 Akalin

8

IVIg

13%

1-year

88%

100%

Multicenter, CA

2003 Jordan

42

IVIg

31%

2-year

89%

98%

Multicenter, US

2004 Jordan

16 (total 101)

IVIg (high dose)

25%

2-year

80%

Austria

2005 Lorenz

40

IA

ACR: 11 20%, AMR: 32 33%

3-year

78%

89-94%

Paris

2007 Anglicheau

38

IVIg

10 25%

1-year

95%

97%

5-year 81%

NS

NS

75%

University of Illinois, IL

2009 Thielke

51

PP/IVIg

ACR: 10%, AMR: 23%

23 months

95%

Mayo Clinic, FL

2009 Mai

20

IVIg

30%

3-year

89%

79 80%

93%

China

2010 Yuan

35

PP/IVIg

total 32%, AMR 20%

52 months

NS

NS

France

2010 Loupy

54

PP/IVIg/RTX

19%

upto 35 months

89%

Austria

2010 Bartel

68

IA

24 30%

5-year

76%

Switzerland

2010 Bachler

37

IVIg

11 38%

1-year

97%

Australia

2011 Rogers

10

PP/IVIg

ACR: 30%, AMR: 0

upto 4.2 years

80%

90%

India

2011 Kute

34

BOR/PP/IVIg

ACR: 14%, AMR: 17%

1-year

88%

100%

Korea

2012 Huh

86

PP/IVIg/RTX

21%

3-year

94%

99%

Korea

2012 Jin

7

PP/IVIg/RTX

0%

33 months

100%

100%

Germany

2012 Morath

10

IA/RTX

30%

2-year

100%

100%

Germany

2013 Klein

23

IAorPP/RTX

22%

2-year

100%

Portugal

2014 Santos

8

PP/IVIg/RTX

38%

30 months

88%

University of Cincinnati

2015 Woodle

19

BOR/RTX/PP

AMR: 12%, acute rejection 19%

36 months

95%

94%

91 94%

87% 92%

88%

100%

100% 100%

ACR, acute cellular rejection; AMR, antibody mediated rejection; BOR, bortezomib; CTX, cyclophosphamide; DCGS, death censored graft survival; ECU, eculizumab; IA, immunoadsorption; NS, not significant; PP, plasmapheresis; RTX, rituximab.

100%

88%

25.5 KIDNEY-PAIRED DONATION

345

study of incompatible LDKT reported a 1.64- and 5.01-fold increase in the risk of graft loss in positive FXM but negative CXM and positive CXM patients, respectively.158

25.4.7 Lack of Efficacy in Very High Risk Patients Several studies have noted desensitization failure in high risk patients with higher DSA levels.26,113,114,159,160 In a 22-center US study of incompatible LDKTs, increased anti-HLA DSA strength was associated with worse graft outcomes and higher mortality.158 Risk of AMR generally increases with increasing baseline DSA levels.161 However, even in the highest risk patients, survival was far superior to remaining on dialysis.44

25.4.8 Lack of Standard Testing Guideline and Treatment Protocols A survey of directors at centers that perform LDKTs revealed that a large number of centers perform incompatible transplants.162 Hence, there is a need for a national incompatible registry and capturing data so as to create appropriate risk adjustment models and novel approaches to quality assurance.162,163 Also, there is an absence of Food and Drug Administration approved drugs for desensitization.153

25.4.9 Risk of Being Cited by Regulatory Agencies Regulatory agencies in the US use data from the Scientific Registry of Transplant Recipients to create multivariate regression models for risk adjustment, and expected survival is calculated by mathematical formula based on donor and recipient variables.162 But these are likely not an accurate estimate for the high-risk desensitized population.162 Centers that perform more than 20% of incompatible LDKTs are at increased odds of being flagged by Centers for Medicare and Medicaid Services.158

25.4.10 Infection Risk Infection from regular and opportunistic infections is common after transplantation and increased immunosuppression is a risk factor, as discussed in the chapter on Infections after Transplantation. Common infections include pneumonia, bacteremia, urinary tract infections, BK viremia/nephropathy, and cytomegalovirus viremia/disease.39,65,164 166 Sustained and higher immunosuppression could potentially increase the infection rates in desensitized individuals but data is conflicting.38,39,164,165

25.4.11 Malignancy Risk There is paucity of data regarding the long-term malignancy risks. We identified 318 ABOi recipients in the Cancer Transplant Match Study and compared them with 37,643 ABO compatible recipients.167 We found no demonstrable association between ABOi kidney transplant and cancer. 25.4.11.1 Interference With Crossmatch Assays Desensitization modalities can interfere with crossmatch assays; hence caution should be exercised when interpreting a positive or negative crossmatch test.99,168 Recently it was reported that rituximab can turn the CXM positive on B cells.168 Pronase or DTT treatment of donor cells can reduce or eliminate this interference.99,168 IVIg also interferes with crossmatch testing but the effect disappears within a few days.

25.5 KIDNEY-PAIRED DONATION KPD is a rapidly growing segment of live donation.3 The concept of KPD was first proposed by Rapaport in 1986.169 Several forms of KPD exist.

I. KIDNEY TRANSPLANTATION

346

25. RENAL TRANSPLANTATION ACROSS HLA AND ABO BARRIERS

25.5.1 Types of KPD A 2-way KPD is the simplest form, in which two recipient and donor pairs with reciprocal incompatibilities participate in a simultaneous exchange (Fig. 25.4A). A 3-way KPD would involve 3 incompatible donor and recipient pairs allowing increased proportion of incompatible pairs finding suitable matches (Fig. 25.4B).170 List donation is a variation on KPD in which a live donor kidney from an incompatible pair is allocated to the deceased donor list and in return, the intended recipient of the live donor receives priority for a deceased-donor kidney (Fig. 25.4C).171 For a small population, more patients are served by list donation than KPD, however for a large population and national level matching programs, list donation will yield fewer matches.172 To maximize the benefits of KPD several other more complicated strategies utilizing sophisticated algorithms have been created. The goal is to create a better set of matches such that more recipients undergo KT or one in which the same number of recipients were matched, but with more highly sensitized recipients.173 In domino paired donation (DPD), the exchange is initiated by a nondirected donor that could be an altruistic donor and multiple donor and recipients pairs are transplanted (Fig. 25.4D and E).174 The last kidney donor could donate to a patient on the deceased donor wait-list, a closed-chain DPD, or become a bridge donor at a later time via a nonsimultaneous extended altruistic donation (NEAD) chain.175 The advantage of the NEAD is that it can unfold over a longer period of time, enables multicenter participation and reduces logistic challenges associated with simultaneous transplants.4 However, a comparison of the potential impact of DPD versus NEAD chains showed that each would be predicted to result in a similar number of transplants.176 Poor outcome in the recipient pair or long wait time increases the risk that the donor will reconsider their donations or become ineligible for health reasons.175,176

25.5.2 Advantages Through proper planning, communication, and logistical arrangements, KPD has been successfully conducted across 30 centers throughout the United States and Canada.177 In reasonably sized registries, match rates for incompatible pairs can be as high as 49%.178 Using a simulated national optimized matching algorithm we have shown that KPD would result in more transplants, better HLA concordance, more grafts surviving at 5 years, (B)

(A) D1

D2

R1

(C) D1

R1

D2

R2

R1

D1

R2

R2 D3

R3

Priority on deceased donor list Deceased donor waitlisted recipient

Bridge donor

Non directed donor

D1

D1 D2

R2

D2

R2

D3

R3

D3

R3

D4

R4

D4

R4

D5

R5

D5

R5

Wait list

D6

R6

(D)

Bridge donor to another NEAD chain

(E)

FIGURE 25.4 Kidney-paired donation: (A) 2-way, (B) 3-way, (C) List Donation, (D) Domino Paired Donation (closed chain) (E) Domino Paired Donation. D, donor; R, recipient; NEAD, nonsimultaneous extended altruistic donation.

I. KIDNEY TRANSPLANTATION

25.6 THE FUTURE

347

a sixfold benefit to highly sensitized patients, and significant cost benefit.179 National KPD program is also predicted to offer similar waiting times between races for most PRA ,80% blood type subgroups.180 When comparing KPD and standard transplants, patient and graft survival rates were similar.181,182

25.5.3 Issues KPD has only realized a fraction of its promise. Ethical issues related to KPD were recognized since 1997.183 These include benefits and risks for donors and recipients, coercion and informed consent, right to withdraw consent, privacy and confidentiality, public acceptance, and legality and exploitation.183 These issues have been vetted and are no longer a limitation to implementation in most countries. Other issues include donor reneging, simultaneous donor nephrectomy requirements, geographic barriers related to donor travel or shipping kidneys, legal and ethical barriers, living donor safety and fragmented small registries.173,184,185 Also, KPD is realistically not feasible for most highly sensitized recipients.172 Two sets of patients have low match rates (,15%); highly sensitized patients (PRA .80%) and O recipients with A donors.172,179 In traditional KPD pools of incompatible pairs, match rates for type O recipients with non type O donors are B15%, whereas rates for other pairs with donors of other blood group types are B50%.180 Broadly sensitized patients frequently have antibodies that are specific for epitopes shared by multiple antigens; hence they have low match rates as well.179,186,187

25.5.4 Expansion Some ways to increase match rates have been suggested. Participation by compatible pairs would have a significant effect on the match rate for incompatible pairs. For a small pool of 25 incompatible pairs, even 10% participation by compatible pairs would result in a 26% increase in the number of incompatible pairs matched.188 If half of compatible pairs registered for a national KPD program, about 70% increase in the incompatible pair match rate could be achieved.188 Potential benefits of compatible pairs participating in a KPD include finding a younger donor, finding a better size match and a better immunologic match.173 Pairs could be motivated by altruism to help incompatible pairs who otherwise could not find a match.173 Additionally, individuals who desire to donate a kidney but do not have a designated recipient (nondirected donors) should be included in KPD pools to maximize transplants.152 Match rates can be increased by larger KPD pools, greater genotype variation (extending geographic reach), increasing the number of new registrants, relaxing reciprocity and simultaneity requirements, and using improved matching algorithms.173,189

25.6 THE FUTURE 25.6.1 Individualized Medicine Good long-term patient and graft survival can be achieved if strategies to transplant across HLA and ABO barriers are individualized to each donor/recipient pair.158 For instance at our center, for an ABOi recipient we base our transplant strategy on blood type, isohemagglutinin titer and PRA (Fig. 25.5). We have reported a case of a patient with cPRA of 100% and impending total loss of dialysis access, who received a FXM-positive, negative CXM kidney at the end of an eight-way multiinstitution domino chain after undergoing desensitization.190 A center in Heidelberg, Germany has developed an integrative algorithm to identify high-risk patients, good HLA matches and appropriate desensitization and posttransplant monitoring.191

25.6.2 Combination Therapy Very broadly sensitized patients with high HLA reactivity are both difficult-to-match and difficult-todesensitize.152 For such patients, optimal transplant opportunities can be realized by finding the best donor in a KPD pool, for whom the recipient has the lowest strength DSA, and then desensitize them against that KPD donor27,82,115,152,189 (Fig. 25.6). Patients who are broadly sensitized but with low-titer DSA will benefit from desensitization alone, while KPD alone is well suited for narrowly sensitized patients who have high-titer DSA to their donor.152,189 Using data from our program, we have proposed a generic Bayes’ calculation to estimate the likelihood of successful transplantation through KPD or combined KPD-desensitization, and propose a nearly 95% probability of transplantation through one of these options.192

I. KIDNEY TRANSPLANTATION

348

25. RENAL TRANSPLANTATION ACROSS HLA AND ABO BARRIERS

ABO-incompatible live donor

Recipient O

Recipient non-O

Titer ≤ 128 or PRA > 80 Kidney paired donation

Desensitization

Titer >128 No match

Kidney paired donation

Transplant

FIGURE 25.5 Individualizing Medicine for an ABO Incompatible Donor-Recipient Pair: This algorithm summarizes the approach to an ABO incompatible recipient at the Johns Hopkins Hospital. We base our strategy on blood type, isohemagglutinin titer and PRA. Incompatible live donor positive crossmatch

Easy to match and/or difficult to desensitize

Kidney paired donation

Difficult to match and difficult to desensitize

Difficult to match and/or easy to desensitize

Kidney-paired donation followed by desensitization

Desensitization

Transplant

FIGURE 25.6 Combining therapies and individualizing medicine to high-risk recipients: For easy to match patients (O donor, narrow sensitization, low PRA) and/or difficult to desensitize (high titer DSA, high immunologic risk such as repeat mismatches) we recommend kidney-paired donation alone. For difficult to match (AB donor, broad sensitization, high PRA) and/or easy to desensitize (low DSA strength, flow crossmatch positive but cytotoxic crossmatch negative, low immunologic risk such as no repeat mismatch) we recommend desensitization alone. For difficult to match (AB donor, broad sensitization, high PRA) and difficult to desensitize (high titer DSA, high immunologic risk, repeat mismatches) we recommend finding a suitable donor via kidney-paired donation and then desensitization.

25.6.3 Immunogenetics Results of assays to detect DSA vary between and within laboratories because of the differences in reagents, instruments, and the specific procedures followed. Standardizing the results between different laboratories is challenging.144 Also, more needs to be learned about the immunogenicity of HLA mismatches and the breadth of immune memory generated following HLA sensitizing events.115,192 Lastly, identifying risk factors for DSA rebound may help with targeted intensive therapy; such as previous sensitization, high MFI before desensitization, anticlass II antibodies, and history of previous transplants.114,115,155 Eighty-seven percent of the DSA detected posttransplant was directed at immunogenic epitopes on HLA antigens the patient had been previously exposed to. Repeated HLA antigen mismatches in high-risk patients should be avoided or receive personalized therapy.115 Others have proposed a DSA scoring system using MFI intensities142 and reported on when exactly to transplant patients after desensitization (“window of opportunity”).98

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Cost analysis of kidney transplantation in highly sensitized recipients compared to intermittent maintenance hemodialysis. Ann Transplant 2012;17(4):82 91. 47. Jordan SC, Tyan D, Stablein D, et al. Evaluation of intravenous immunoglobulin as an agent to lower allosensitization and improve transplantation in highly sensitized adult patients with end-stage renal disease: report of the NIH IG02 trial. J Am Soc Nephrol 2004;15(12):3256 62. 48. Varma PP, Hooda AK, Kumar A, Singh L. Highly successful and low-cost desensitization regime for sensitized living donor renal transplant recipients. Ren Fail 2009;31(7):533 7. 49. Crew RJ, Ratner LE. ABO-incompatible kidney transplantation: current practice and the decade ahead. Curr Opin Organ Transplant 2010;15(4):526 30. 50. Rahman T, Harper L. Plasmapheresis in nephrology: an update. Curr Opin Nephrol Hypertens 2006;15(6):603 9. 51. Pusey CD, Levy JB. Plasmapheresis in immunologic renal disease. Blood Purif 2012;33(1-3):190 8. 52. Higgins R, Lowe D, Hathaway M, et al. Double filtration plasmapheresis in antibody-incompatible kidney transplantation. Ther Apher Dial 2010;14(4):392 9. 53. Allen NH, Dyer P, Geoghegan T, Harris K, Lee HA, Slapak M. Plasma exchange in acute renal allograft rejection. A controlled trial. Transplantation 1983;35(5):425 8. 54. Tobian AA, Shirey RS, Montgomery RA, Ness PM, King KE. The critical role of plasmapheresis in ABO-incompatible renal transplantation. Transfusion 2008;48(11):2453 60. 55. Gungor O, Sen S, Kircelli F, et al. Plasmapheresis therapy in renal transplant patients: five-year experience. Transplant Proc 2011;43 (3):853 7. 56. Zachary AA, Montgomery RA, Ratner LE, et al. Specific and durable elimination of antibody to donor HLA antigens in renal-transplant patients. Transplantation 2003;76(10):1519 25. 57. Abu Jawdeh BG, Cuffy MC, Alloway RR, Shields AR, Woodle ES. Desensitization in kidney transplantation: review and future perspectives. 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Am J Transplant 2003; 3(8):1017 23. 63. Magee CC, Felgueiras J, Tinckam K, Malek S, Mah H, Tullius S. Renal transplantation in patients with positive lymphocytotoxicity crossmatches: one center’s experience. Transplantation 2008;86(1):96 103. 64. Thielke JJ, West-Thielke PM, Herren HL, et al. Living donor kidney transplantation across positive crossmatch: the University of Illinois at Chicago experience. Transplantation 2009;87(2):268 73. 65. Riella LV, Safa K, Yagan J, et al. Long-term outcomes of kidney transplantation across a positive complement-dependent cytotoxicity crossmatch. Transplantation 2014;97(12):1247 52. 66. Rogers NM, Eng HS, Yu R, et al. Desensitization for renal transplantation: depletion of donor-specific anti-HLA antibodies, preservation of memory antibodies, and clinical risks. Transpl Int 2011;24(1):21 9. 67. Schweitzer EJ, Wilson JS, Fernandez-Vina M, et al. A high panel-reactive antibody rescue protocol for cross-match-positive live donor kidney transplants. Transplantation 2000;70(10):1531 6. 68. Taube DH, Williams DG, Cameron JS, et al. Renal transplantation after removal and prevention of resynthesis of HLA antibodies. Lancet 1984;1(8381):824 8. 69. Montgomery RA, Zachary AA, Racusen LC, et al. Plasmapheresis and intravenous immune globulin provides effective rescue therapy for refractory humoral rejection and allows kidneys to be successfully transplanted into cross-match-positive recipients. Transplantation 2000;70(6):887 95. 70. Sonnenday CJ, Ratner LE, Zachary AA, et al. Preemptive therapy with plasmapheresis/intravenous immunoglobulin allows successful live donor renal transplantation in patients with a positive cross-match. Transplant Proc 2002;34(5):1614 16. 71. Huh KH, Kim BS, Yang J, et al. Kidney transplantation after desensitization in sensitized patients: a Korean National Audit. Int Urol Nephrol 2012;44(5):1549 57. 72. Higgins R, Hathaway M, Lowe D, et al. 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107. Gloor JM, Lager DJ, Moore SB, et al. ABO-incompatible kidney transplantation using both A2 and non-A2 living donors. Transplantation 2003;75(7):971 7. 108. Clatworthy MR. B-cell regulation and its application to transplantation. Transpl Int 2014;27(2):117 28. 109. Waiser J, Budde K, Schutz M, et al. Comparison between bortezomib and rituximab in the treatment of antibody-mediated renal allograft rejection. Nephrol Dial Transplant 2012;27(3):1246 51. 110. Fehr T, Rusi B, Fischer A, Hopfer H, Wuthrich RP, Gaspert A. Rituximab and intravenous immunoglobulin treatment of chronic antibody-mediated kidney allograft rejection. Transplantation 2009;87(12):1837 41. 111. Toyoda M, Thomas D, Ahn G, et al. JC polyomavirus viremia and progressive multifocal leukoencephalopathy in human leukocyte antigen-sensitized kidney transplant recipients desensitized with intravenous immunoglobulin and rituximab. Transpl Infect Dis 2015;17 (6):838 47. 112. Klein K, Susal C, Schafer SM, et al. Living donor kidney transplantation in patients with donor-specific HLA antibodies enabled by antiCD20 therapy and peritransplant apheresis. Atheroscler Suppl 2013;14(1):199 202. 113. Marfo K, Ling M, Bao Y, et al. Lack of effect in desensitization with intravenous immunoglobulin and rituximab in highly sensitized patients. Transplantation 2012;94(4):345 51. 114. Lobashevsky AL, Higgins NG, Rosner KM, Mujtaba MA, Goggins WC, Taber TE. Analysis of anti-HLA antibodies in sensitized kidney transplant candidates subjected to desensitization with intravenous immunoglobulin and rituximab. Transplantation 2013;96 (2):182 90. 115. Jackson AM, Kraus ES, Orandi BJ, Segev DL, Montgomery RA, Zachary AA. A closer look at rituximab induction on HLA antibody rebound following HLA-incompatible kidney transplantation. Kidney Int 2015;87(2):409 16. 116. Zachary AA, Lucas DP, Montgomery RA, Leffell MS. Rituximab prevents an anamnestic response in patients with cryptic sensitization to HLA. Transplantation 2013;95(5):701 4. 117. Odendahl M, Mei H, Hoyer BF, et al. Generation of migratory antigen-specific plasma blasts and mobilization of resident plasma cells in a secondary immune response. Blood 2005;105(4):1614 21. 118. Gonzalez-Garcia I, Ocana E, Jimenez-Gomez G, Campos-Caro A, Brieva JA. Immunization-induced perturbation of human blood plasma cell pool: progressive maturation, IL-6 responsiveness, and high PRDI-BF1/BLIMP1 expression are critical distinctions between antigenspecific and nonspecific plasma cells. J Immunol 2006;176(7):4042 50. 119. Tanabe K, Ishida H, Shimizu T, Omoto K, Shirakawa H, Tokumoto T. Evaluation of two different preconditioning regimens for ABOincompatible living kidney donor transplantation. A comparison of splenectomy vs. rituximab-treated non-splenectomy preconditioning regimens. Contrib Nephrol 2009;162:61 74. 120. Tsai MK, Wu MS, Yang CY, et al. B cells and immunoglobulin in ABO-incompatible renal transplant patients receiving rituximab and double filtration plasmapheresis. J Formos Med Assoc 2015;114(4):353 8. 121. Segev DL, Simpkins CE, Warren DS, et al. ABO incompatible high-titer renal transplantation without splenectomy or anti-CD20 treatment. Am J Transplant 2005;5(10):2570 5. 122. Everly MJ, Terasaki PI. The state of therapy for removal of alloantibody producing plasma cells in transplantation. Semin Immunol 2012;24(2):143 7. 123. Perry DK, Burns JM, Pollinger HS, et al. Proteasome inhibition causes apoptosis of normal human plasma cells preventing alloantibody production. Am J Transplant 2009;9(1):201 9. 124. Everly MJ, Everly JJ, Susskind B, et al. Bortezomib provides effective therapy for antibody- and cell-mediated acute rejection. Transplantation 2008;86(12):1754 61. 125. Tzvetanov I, Spaggiari M, Oberholzer J, et al. Cell population in spleens during antibody-mediated rejection: pathologic and clinical findings. Transplantation 2012;94(3):255 62. 126. Kute VB, Vanikar AV, Trivedi HL, et al. Desensitization protocol for highly sensitized renal transplant patients: a single-center experience. Saudi J Kidney Dis Transpl 2011;22(4):662 9. 127. Yang KS, Jeon H, Park Y, et al. Use of bortezomib as anti-humoral therapy in kidney transplantation. J Korean Med Sci 2014;29(5):648 51. 128. Aubert O, Suberbielle C, Gauthe R, Francois H, Obada EN, Durrbach A. Effect of a proteasome inhibitor plus steroids on HLA antibodies in sensitized patients awaiting a renal transplant. Transplantation 2014;97(9):946 52. 129. Woodle ES, Shields AR, Ejaz NS, et al. Prospective iterative trial of proteasome inhibitor-based desensitization. Am J Transplant 2015;15 (1):101 18. 130. Diwan TS, Raghavaiah S, Burns JM, Kremers WK, Gloor JM, Stegall MD. The impact of proteasome inhibition on alloantibodyproducing plasma cells in vivo. Transplantation 2011;91(5):536 41. 131. Wahrmann M, Haidinger M, Kormoczi GF, et al. Effect of the proteasome inhibitor bortezomib on humoral immunity in two presensitized renal transplant candidates. Transplantation 2010;89(11):1385 90. 132. Guthoff M, Schmid-Horch B, Weisel KC, Haring HU, Konigsrainer A, Heyne N. Proteasome inhibition by bortezomib: effect on HLA-antibody levels and specificity in sensitized patients awaiting renal allograft transplantation. Transpl Immunol 2012;26(4):171 5. 133. Philogene MC, Sikorski P, Montgomery RA, Leffell MS, Zachary AA. Differential effect of bortezomib on HLA class I and class II antibody. Transplantation 2014;98(6):660 5. 134. Wong NL, O’Connell P, Chapman JR, et al. Bortezomib in ABO-incompatible kidney transplant desensitization: a case report. Nephrology (Carlton) 2015;20(Suppl. 1):22 4. 135. Tzvetanov I, Spaggiari M, Jeon H, et al. The role of splenectomy in the setting of refractory humoral rejection after kidney transplantation. Transplant Proc 2012;44(5):1254 8. 136. Kaplan B, Jie T, Diana R, et al. Histopathology and immunophenotype of the spleen during acute antibody-mediated rejection. Am J Transplant 2010;10(5):1316 20. 137. Sawada T, Fuchinoue S, Kawase T, Kubota K, Teraoka S. Preconditioning regimen consisting of anti-CD20 monoclonal antibody infusions, splenectomy and DFPP-enabled non-responders to undergo ABO-incompatible kidney transplantation. Clin Transplant 2004;18 (3):254 60.

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138. Alexander JW, First MR, Majeski JA, et al. The late adverse effect of splenectomy on patient survival following cadaveric renal transplantation. Transplantation 1984;37(5):467 70. 139. Stewart ZA, Collins TE, Schlueter AJ, et al. Case report: eculizumab rescue of severe accelerated antibody-mediated rejection after ABOincompatible kidney transplant. Transplant Proc 2012;44(10):3033 6. 140. Stegall MD, Diwan T, Raghavaiah S, et al. Terminal complement inhibition decreases antibody-mediated rejection in sensitized renal transplant recipients. Am J Transplant 2011;11(11):2405 13. 141. Cornell LD, Schinstock CA, Gandhi MJ, Kremers WK, Stegall MD. Positive crossmatch kidney transplant recipients treated with eculizumab: outcomes beyond 1 year. Am J Transplant 2015;15(5):1293 302. 142. Jordan SC, Choi J, Vo A. Kidney transplantation in highly sensitized patients. Br Med Bull 2015;114(1):113 25. 143. Vo AA, Zeevi A, Choi J, et al. A phase I/II placebo-controlled trial of C1-inhibitor for prevention of antibody-mediated rejection in HLA sensitized patients. Transplantation 2015;99(2):299 308. 144. Iyer HS, Jackson AM, Zachary AA, Montgomery RA. Transplanting the highly sensitized patient: trials and tribulations. Curr Opin Nephrol Hypertens 2013;22(6):681 8. 145. Vo AA, Choi J, Kim I, et al. A Phase I/II Trial of the Interleukin-6 Receptor Specific Humanized Monoclonal (Tocilizumab) 1 Intravenous Immunoglobulin in Difficult to Desensitize Patients. Transplantation 2015. 146. Alqurashi S, Alsayyari AA, Abdullah K, Alwan A, Hajeer AH. Combined liver and kidney transplantation in a highly sensitized and positively cross-matched patient. Saudi J Kidney Dis Transpl 2011;22(4):757 60. 147. Olausson M, Mjornstedt L, Norden G, et al. Successful combined partial auxiliary liver and kidney transplantation in highly sensitized cross-match positive recipients. Am J Transplant 2007;7(1):130 6. 148. Ingelsten M, Karlsson-Parra A, Granqvist AB, et al. Postischemic inflammatory response in an auxiliary liver graft predicts renal graft outcome in sensitized patients. Transplantation 2011;91(8):888 94. 149. Sinha D, Lambie M, Krishnan N, et al. Cryofiltration in the treatment of cryoglobulinemia and HLA antibody-incompatible transplantation. Ther Apher Dial 2012;16(1):91 6. 150. Kawamura A, Osanai M, Yonekawa M. Immunomodulation in transplant patients by cryofiltration. Ther Apher 1998;2(3):205 9. 151. Tamaki T, Tanaka M, Katori M, et al. Cryofiltration apheresis for major ABO-incompatible kidney transplantation. Ther Apher 1998;2 (4):308 10. 152. Montgomery RA. Renal transplantation across HLA and ABO antibody barriers: integrating paired donation into desensitization protocols. Am J Transplant 2010;10(3):449 57. 153. Archdeacon P, Chan M, Neuland C, et al. Summary of FDA antibody-mediated rejection workshop. Am J Transplant 2011;11(5):896 906. 154. Gloor JM, Sethi S, Stegall MD, et al. Transplant glomerulopathy: subclinical incidence and association with alloantibody. Am J Transplant 2007;7(9):2124 32. 155. Vo AA, Sinha A, Haas M, et al. Factors predicting risk for antibody-mediated rejection and graft loss in highly human leukocyte antigen sensitized patients transplanted after desensitization. Transplantation 2015;99(7):1423 30. 156. Sharif A, Kraus ES, Zachary AA, et al. Histologic phenotype on 1-year posttransplantation biopsy and allograft survival in HLAincompatible kidney transplants. Transplantation 2014;97(5):541 7. 157. Bagnasco SM, Zachary AA, Racusen LC, et al. Time course of pathologic changes in kidney allografts of positive crossmatch HLAincompatible transplant recipients. Transplantation 2014;97(4):440 5. 158. Orandi BJ, Garonzik-Wang JM, Massie AB, et al. Quantifying the risk of incompatible kidney transplantation: a multicenter study. Am J Transplant 2014;14(7):1573 80. 159. Kozlowski T, Andreoni K. Limitations of rituximab/IVIg desensitization protocol in kidney transplantation; is this better than a tincture of time? Ann Transplant 2011;16(2):19 25. 160. Alachkar N, Lonze BE, Zachary AA, et al. Infusion of high-dose intravenous immunoglobulin fails to lower the strength of human leukocyte antigen antibodies in highly sensitized patients. Transplantation 2012;94(2):165 71. 161. Burns JM, Cornell LD, Perry DK, et al. Alloantibody levels and acute humoral rejection early after positive crossmatch kidney transplantation. Am J Transplant 2008;8(12):2684 94. 162. Garonzik Wang JM, Montgomery RA, Kucirka LM, Berger JC, Warren DS, Segev DL. Incompatible live-donor kidney transplantation in the United States: results of a national survey. Clin J Am Soc Nephrol 2011;6(8):2041 6. 163. Neumayer HH, Budde K, Liefeldt L. Human leukocyte antigen-incompatible kidney transplantation after “desensitization”--hope and reality. Transplantation 2014;98(8):819 20. 164. Kahwaji J, Sinha A, Toyoda M, et al. Infectious complications in kidney-transplant recipients desensitized with rituximab and intravenous immunoglobulin. Clin J Am Soc Nephrol 2011;6(12):2894 900. 165. Sharif A, Alachkar N, Bagnasco S, et al. Incidence and outcomes of BK virus allograft nephropathy among ABO- and HLA-incompatible kidney transplant recipients. Clin J Am Soc Nephrol 2012;7(8):1320 7. 166. Egawa H, Teramukai S, Haga H, et al. Impact of rituximab desensitization on blood-type-incompatible adult living donor liver transplantation: a Japanese multicenter study. Am J Transplant 2014;14(1):102 14. 167. Hall EC, Engels EA, Montgomery RA, Segev DL. Cancer risk after ABO-incompatible living-donor kidney transplantation. Transplantation 2013;96(5):476 9. 168. Milongo D, Vieu G, Blavy S, et al. Interference of therapeutic antibodies used in desensitization protocols on lymphocytotoxicity crossmatch results. Transpl Immunol 2015;32(3):151 5. 169. Rapaport FT. The case for a living emotionally related international kidney donor exchange registry. Transplant Proc 1986;18(3, Suppl. 2):5 9. 170. Saidman SL, Roth AE, Sonmez T, Unver MU, Delmonico FL. Increasing the opportunity of live kidney donation by matching for twoand three-way exchanges. Transplantation 2006;81(5):773 82. 171. Delmonico FL, Morrissey PE, Lipkowitz GS, et al. Donor kidney exchanges. Am J Transplant 2004;4(10):1628 34. 172. Gentry SE, Segev DL, Montgomery RA. A comparison of populations served by kidney paired donation and list paired donation. Am J Transplant 2005;5(8):1914 21.

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173. Gentry SE, Montgomery RA, Segev DL. Kidney paired donation: fundamentals, limitations, and expansions. Am J Kidney Dis 2011; 57(1):144 51. 174. Montgomery RA, Gentry SE, Marks WH, et al. Domino paired kidney donation: a strategy to make best use of live non-directed donation. Lancet 2006;368(9533):419 21. 175. Rees MA, Kopke JE, Pelletier RP, et al. A nonsimultaneous, extended, altruistic-donor chain. N Engl J Med 2009;360(11):1096 101. 176. Gentry SE, Montgomery RA, Swihart BJ, Segev DL. The roles of dominos and nonsimultaneous chains in kidney paired donation. Am J Transplant 2009;9(6):1330 6. 177. Segev DL, Veale JL, Berger JC, et al. Transporting live donor kidneys for kidney paired donation: initial national results. Am J Transplant 2011;11(2):356 60. 178. de Klerk M, Witvliet MD, Haase-Kromwijk BJ, Claas FH, Weimar W. A highly efficient living donor kidney exchange program for both blood type and crossmatch incompatible donor-recipient combinations. Transplantation 2006;82(12):1616 20. 179. Segev DL, Gentry SE, Warren DS, Reeb B, Montgomery RA. Kidney paired donation and optimizing the use of live donor organs. Jama 2005;293(15):1883 90. 180. Segev DL, Gentry SE, Melancon JK, Montgomery RA. Characterization of waiting times in a simulation of kidney paired donation. Am J Transplant 2005;5(10):2448 55. 181. Segev DL, Kucirka LM, Gentry SE, Montgomery RA. Utilization and outcomes of kidney paired donation in the United States. Transplantation 2008;86(4):502 10. 182. Montgomery RA, Zachary AA, Ratner LE, et al. Clinical results from transplanting incompatible live kidney donor/recipient pairs using kidney paired donation. Jama 2005;294(13):1655 63. 183. Ross LF, Rubin DT, Siegler M, Josephson MA, Thistlethwaite Jr. JR, Woodle ES. Ethics of a paired-kidney-exchange program. N Engl J Med 1997;336(24):1752 5. 184. de Klerk M, Witvliet MD, Haase-Kromwijk BJ, Claas FH, Weimar W. Hurdles, barriers, and successes of a national living donor kidney exchange program. Transplantation 2008;86(12):1749 53. 185. Chkhotua A. Paired kidney donation: outcomes, limitations, and future perspectives. Transplant Proc 2012;44(6):1790 2. 186. Zachary AA, Montgomery RA, Leffell MS. Factors associated with and predictive of persistence of donor-specific antibody after treatment with plasmapheresis and intravenous immunoglobulin. Hum Immunol 2005;66(4):364 70. 187. Mao Q, Terasaki PI, Cai J, El-Awar N, Rebellato L. Analysis of HLA class I specific antibodies in patients with failed allografts. Transplantation 2007;83(1):54 61. 188. Gentry SE, Segev DL, Simmerling M, Montgomery RA. Expanding kidney paired donation through participation by compatible pairs. Am J Transplant 2007;7(10):2361 70. 189. Montgomery RA. Living donor exchange programs: theory and practice. Br Med Bull 2011;98:21 30. 190. Lonze BE, Dagher NN, Simpkins CE, et al. Eculizumab, bortezomib and kidney paired donation facilitate transplantation of a highly sensitized patient without vascular access. Am J Transplant 2010;10(9):2154 60. 191. Morath C, Beimler J, Opelz G, et al. An integrative approach for the transplantation of high-risk sensitized patients. Transplantation 2010;90(6):645 53. 192. Jackson AM, Leffell MS, Montgomery RA, Zachary AA A. GPS for finding the route to transplantation for the sensitized patient. Curr Opin Organ Transplant 2012;17(4):433 9.

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C H A P T E R

26 Pathology of the Renal Allograft Loredana Melchiorri1, Christian C. Morrill2, Gino Coletti1 and Paul Persad3 1

San Salvatore Hospital of L’Aquila, L’Aquila, Italy 2Utah State University, Logan, UT, United States 3 Wake Forest School of Medicine, Winston Salem, NC, United States

26.1 INTRODUCTION Renal allograft pathology is perhaps one of the most complex and challenging areas of surgical pathology. While a thorough understanding of the various types and intricacies of rejection forms the cornerstone of evaluating the transplanted kidney biopsy, still additional factors such as the patient’s primary medical disease, de novo diseases, drug toxicities, infectious agents, and all other afflictions of the native kidney must all be kept in mind.1 After all, a transplanted kidney is still a kidney. Other aspects to be mindful of include donor source, initial function of the organ, and time since transplant as all these factors can impact the pathologist’s interpretation of biopsy findings.2

26.2 BIOPSY AND SPECIMENS 26.2.1 Specimen Adequacy By Banff criteria, an adequate biopsy for the evaluation of rejection must consist of two cores of renal parenchyma containing 10 glomeruli and 2 arteries. The sensitivity for detecting acute rejection from one core is about 90%. Two cores approach 99% sensitivity.3 Put another way, diagnostic lesions can be found even in the smallest of biopsies. Alternatively, with respect to acute rejection in small biopsies, the absence of evidence is not evidence of absence.

26.2.2 Routine Tissue Processing Standard evaluation of renal allograft biopsies includes dividing the tissue for light microscopic and immunofluorescent studies with electron microscopic evaluation performed as dictated by the IF studies as well as clinical indications such as proteinuria.4 The specimen allocated for light microscopy is fixed in formalin, embedded in paraffin, multiple levels cut and routinely stained with hematoxylin and eosin (H&E), Periodic Acid Schiff (PAS), a silver stain such as Jones silver aka Periodic Acid Methanamine Silver (PAMS), and Trichrome stain. Each of these has unique strengths in highlighting various pathologies. For example, the silver stain delineates the glomerular basement membranes (GBM), mesangium, Bowman’s capsule and tubular basement membranes (TBM), whereas Trichrome stains can make various immune complex deposits more apparent. Immunofluorescence microscopy entails freezing a portion of tissue and incubating with antibodies labeled with a fluorescent tag. This slide is then viewed under a fluorescent microscope revealing the location and intensity of the specific molecule of interest. IF studies for C4d, a breakdown product of complement that is covalently bound to various proteins, provide evidence of antibody-mediated rejection (AMR) (see below). Electron microscopy is not routinely used for transplant evaluation unless there is some specific indication of glomerular pathology, such as transplant glomerulopathy Kidney Transplantation, Bioengineering, and Regeneration. DOI: http://dx.doi.org/10.1016/B978-0-12-801734-0.00026-6

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(TGP), recurrent or de novo disease as may be indicated by new or worsening proteinuria.5 Occasional renal pathologists advocate routine EM on all transplant specimens but most opt for a case by case evaluation.

26.2.3 Biopsy Evaluation A systematic approach is paramount. The classic teaching is to evaluate renal parenchyma as four separate yet interrelated compartments. Tubules and interstitium can be considered as a unit, as pathologies of one compartment often affect the other. Similarly, vessels and glomeruli may be evaluated together since endothelial damage is frequently a common denominator. It is customary to report on the number of cores, glomeruli, arteries, and to quantitate in a systematic fashion various aspects of the above described compartments such tubulitis, interstitial fibrosis/inflammation, glomerulitis, glomerulosclerosis, arteritis, and fibrinoid necrosis. In an effort to unify the reporting of biopsy specimens between pathologists, minimize confusion/frustration amongst clinicians, and facilitate meaningful research, various scoring systems have been devised.

26.3 BANFF The first Banff Classification was published in 1993 by Solez and Racusen.6 Since then this schema has undergone several revisions.

26.3.1 Banff Timeline 1991 First Conference in Banff, Canada 1993 First Kidney International (KI) publication7 1997 Integration with Collaborative Clinical Trials in Transplantation (CCTT) 2001 Classification of antibody-mediated rejection 2005 Gene chip analysis 2007 “Banff Working Group” meeting in Spain8 2009 “Banff Working Group” meeting in Banff, Canada 2011 “Banff Working Group” meeting in Paris, France 2013 “Banff Working Group” meeting in Comandatuba, Brazil9 2017 “Banff Working Group” meeting in Spain 2019 “Banff Working Group” meeting in Pennsylvania, USA

26.3.2 Summary of Banff Classification 1. 2. 3. 4. 5. 6.

Normal Antibody-mediated rejection (AMR) Borderline changes “suspicious” for acute T-cell mediated rejection T-cell mediated rejection Interstitial fibrosis and tubular atrophy (IFTA) Other

Trying to combine and understand the pathogenesis, histologic and serologic findings can be somewhat overwhelming at first. Our understanding of various aspects of rejection has undergone much growth over the years. Rejection may be divided into acute and chronic phases with an intermediary or transitional phase in between. The effector mechanisms of the immune system may theoretically be separated into T-cell mediated and antibody-mediated mechanisms, although they likely operate together.1 The figure below will help one put together the various terminologies currently in use with the corresponding histologic findings. Tubules damaged by mononuclear inflammatory cells insinuating themselves between tubular epithelial cells is called “tubulitis,”1 accompanied by mononuclear cells in the interstitium and acute tubular injury. This process, primarily driven by T-cells, is called Acute Cellular Rejection (Figs. 26.1 and 26.2). Further, such injury is graded as follows:

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FIGURE 26.1

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Diffuse lymphocytic interstitial infiltrate with frequent tubulitis and acute tubular injury (H&E).

FIGURE 26.2 Acute cellular rejection, Banff Ib: Tubulitis characterized by lymphocyte infiltrating between tubular epithelial cells with resulting tubular injury and tissue edema (PAS).

IA. Cases with significant interstitial inflammation ( .25% of parenchyma affected) and tubulitis graded as follows: T0: No mononuclear inflammatory cells in tubules T1: Foci with 1 4 cells per tubular cross section IB. Cases with significant interstitial inflammation ( .25% of parenchyma affected) and tubulitis graded as follows: T2: Foci with 5 10 cells per tubular cross section T3: Foci with .10 cells per tubular cross section Arterial vessels may be injured by infiltrating T-cells. This lesion is called “endarteritis” or “endothelialitis.” Specifically, the infiltrating lymphocyte must be located underneath the endothelial cell, which itself must be swollen and uplifted. If these strict morphologic features are met, then the diagnosis of Acute Cell Mediated Vascular Rejection (Figs. 26.3 and 26.4) can be rendered by the pathologist.10 Further, such injury can be graded as follows: IIA. Cases with mild to moderate endarteritis and little to no interstitial inflammation and endarteritis graded as follows: V0: No arteritis V1: Intimal arteritis in ,25% of lumen (minimum of 1 lymphocyte involving 1 artery)

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FIGURE 26.3 Acute cell-mediated vascular rejection, Banff 2A: Lymphocytes underneath and lifting up swollen endothelial cells (i.e., endothelialitis). Note the surrounding dense infiltrate in the interstitium and accompanying tubulitis (H&E).

FIGURE 26.4 Acute vascular rejection, Banff 2B: Endothelialitis lesion with .25% luminal compromise. Also note the glomerulitis in the upper left corner suggestive of acute antibody-mediated rejection (ABMR) (Jones).

Vascular rejection with more severe arterial damage such as the IIB and III categories with the more significant V2 and V3 lesions (see below) likely driven by antibody-mediated mechanisms. Therefore, the term Acute Vascular Rejection can be used in this situation with the understanding that humoral immunity (antibodies) is playing more of a pathogenic role in the vascular damage than T-cells alone. IIB. Cases with severe endarteritis V2: Compromising $ 25% of lumen of $ 1 artery III Cases with severe endarteritis V3: Transmural arteritis 1 / 2 fibrinoid necrosis of muscular wall The smallest of the kidney’s vascular structures (the microcirculation) can also undergo endothelial injury by antibody-mediated mechanisms. The glomerular capillary loops and peritubular capillaries are in fact the primary locations for histologic evidence of Antibody-Mediated Rejection.11 Mononuclear inflammatory filling glomerular capillary loops (Fig. 26.5) is termed “glomerulitis”12 and the percentage of glomeruli involved assigned a (g) score as follows: G0: No glomerulitis G1: ,25% of glomeruli G2: 25% 75% of glomeruli G3: .75% of glomeruli Similarly, mononuclear inflammatory cells filling and dilating peritubular capillaries (Figs. 26.6 and 26.7) constitute “peritubular capillaritis”13 and can be assigned a (ptc) score as follows: Ptc0: ,10% of PTCs with intraluminal cells Ptc1: .10% of PTCs with ,5 cells per PTC Ptc2: .10% of PTCs with 5 10 cells per PTC Ptc3: .10% of PTCs with .10 cells per PTC

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FIGURE 26.5

Mononuclear inflammatory cells filling glomerular capillary lumina associated with swollen endothelial cells indicating glomerulitis and suggestive of antibody-mediated rejection (ABMR). Also note at 3 o’clock the presence of glomerular basement membrane splitting and cellular interposition, findings suggesting an element of chronic antibody-mediate rejection.

FIGURE 26.6 Dilated peritubular capillaries with increased intraluminal mononuclear inflammatory cells, interstitial edema and surrounding acute tubular injury comprise peritubular capillarities and suggest acute antibody-mediated rejection (ABMR) (Jones).

FIGURE 26.7 More exact localization of dilated peritubular capillaries, intraluminal inflammatory cells, tissue edema, and acute tubular injury suggestive of acute antibody-mediated rejection (ABMR) can be appreciated on this PAS stain.

Together, these findings of glomerulitis and peritubular capillaritis constitute “microcirculation inflammation” and are the classic histologic features of AMR.9 Neutrophils can also be part of the infiltrate affecting peritubular capillaries in AMR and the quantity (less or more than half of all inflammatory cells) should be specified.

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FIGURE 26.8

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Diffuse peritubular capillary positivity for C4d by Immunofluorescence microscopy in a case of acute antibody-mediated

rejection (ABMR).

In addition to the histologic findings described above, C4d staining by IF or IHC, and the donor specific antibody (DSA) status of the patient contribute to the overall diagnostic process. C4d is a breakdown product of C4b that is covalently bound to endothelial proteins serving as a longer lasting marker of complement activation than other complement components and surrogate for AMR.14 Immunofluorescence staining for more than 10% of peritubular capillaries (Fig. 26.8) is considered a positive result.9 DSA are serum antibodies against human leukocyte antigen class I and II that may appear or persist in the posttransplant period and result in injury to the endothelium of the microvasculature and graft loss.15,16 When all three conditions (microcirculation inflammation, C4d positivity in peritubular capillaries, and DSA positive in serum) are present, the pathologist may confidently diagnose the presence of AntibodyMediated Rejection (ABMR). Otherwise, a diagnosis of “suspicious for ABMR” is more appropriate. Other histologic findings that may suggest the presence of AMR include acute tubular injury with minimal inflammation and lack of other obvious cause, concomitant acute vascular rejection IIB or III, and thrombosis.8 Other aspects of the biopsy scored by the Banff system include: Total Inflammation (ti) score: Ti0: ,10% of cortex Ti1: 10% 25% Ti2: 26% 50% Ti3: .50% C4d Score in peritubular capillaries can be scored as the percent of C4d deposition in $ 5 high power fields, as seen in the immunofluorescence studies or from paraffin-embedded tissue, as follows: C4d0: 0% C4d1: 1% 9% C4d2: 10% 50% C4d3: .50%

26.4 CHRONIC REJECTION As acute cellular rejection (tubulitis) progresses from the acute to subacute and then on to chronic phases of injury, the afflicted tubules will atrophy and the interstitium will undergo fibrosis. The degree of interstitial fibrosis and tubular atrophy (IFTA) is a summation term to reflect overall kidney scarring and correlates well with declining graft function.17 Each component of IFTA can be assigned a score as follows: Interstitial fibrosis (ci) Ci0: # 5% of cortex with fibrosis Ci1: 6% 25% of cortex with fibrosis Ci2: 26% 50% of cortex with fibrosis Ci3: .50% of cortex with fibrosis

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Tubular atrophy (ct) Ct0: Ct1: Ct2: Ct3:

0% of cortex with tubular atrophy # 25% of cortex with tubular atrophy 26% 50% of cortex with tubular atrophy .50% of cortex with tubular atrophy

Acute vascular rejection, whether due to T-cell mediated mechanisms or antibody-mediated mechanisms, may also proceed to patterns of chronic scarring involving vessels; this chronic allograft vasculopathy is characterized by concentric arterial fibrointimal thickening.18 The degree of lumen narrowing by this reparative process assigned a score as follows: Cv0: 0% of lumen narrowing Cv1: # 25% of lumen narrowing Cv2: 26% 50% of lumen narrowing Cv3: .50% of lumen narrowing Of note, there is an intermediary stage between acute vascular rejection and chronic allograft arteriopathy, termed Chronic Active Vascular Rejection, in which there are inflammatory cells including “foam cells” within the thickened intima (Fig. 26.9). The lesions of AMR can also progress to states of chronicity and ongoing damage. Specifically, glomerulitis can eventually result in swollen endothelial cells, increased separation of endothelial cells from the GBM, lamina rara interna expansion, and new additional layers (duplication) of GBM material being laid down.19 By light microscopy, there is global splitting of GBMs (Fig. 26.10). These features together constitute “transplant

FIGURE 26.9 Interlobular to large artery with intimal fibrosis, lymphocytes, and subintimal foam cells, indicative of chronic active vascular rejection (CAVR) (H&E).

FIGURE 26.10 Global splitting of glomerular basement membranes in a patient with proteinuria in the late transplant period indicative of transplant glomerulopathy (TGP) (Jones).

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glomerulopathy”20 and may present with new or worsening proteinuria. The percent of the glomerular capillary loops with duplication of GBMs in the most affected glomerulus by light microscopy, TGP (cg) scored as follows: Cg0: ,10% Cg1: 10% 25% Cg2: 26% 50% Cg3: .50% Chronic damage of the peritubular capillaries may induce a similar mechanism of repair, including peritubular capillary multilamellation (PTCMML).13 This process is most easily seen by electron microscopy and only with much strain by light microscopy. A widely accepted scoring system has yet to be developed for this finding (Figs. 26.11 26.16). Additional aspects that are part of the Banff scoring system and ought to be included in the biopsy report are: Interstitial inflammation (i) in nonscarred cortex, away from areas of subcapsular and perivascular fibrous tissue scored as follows: I0: ,10% I2: 10% 25% I3: 26% 50% I4: .50%

FIGURE 26.11 Electron micrographic representation of glomerular basement membranes split by interposing cells, increased lamina rara interna, electron lucent material, endothelial cell injury and neo-membrane formation in a case of transplant glomerulopathy. Note the absence of immune complexes.

FIGURE 26.12 Electron micrograph showing peritubular capillary multilamellation (PTCMML) characterized by neo-tubular basement membrane formation due to repeated bouts of antibody-mediated injury.

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FIGURE 26.13 Viral cytopathic changes may include glassy intranuclear inclusions associated with acute tubular injury and a lymphoplasmacytic infiltrate characterizes polyomavirus nephropathy (PVN) (H&E).

FIGURE 26.14

Immunohistochemical staining of the same patient in Fig. 26.13 confirms the presence of polyoma virus Large T Antigen in

the tubular nuclei.

FIGURE 26.15 Frequently polyoma viral cytopathic changes, as in this case, may be quite bizarre and prompt consideration of other viral pathogens such as cytomegalovirus. Immunohistochemistry studies are very helpful in such instances (H&E).

Mesangial matrix increase (mm) is defined as matrix increase greater than 2 mesangial cells in width in $ 2 glomerular tufts: Mm0: 0% Mm1: # 25% Mm2: 26% 50% Mm3: .50% Arteriolar hyalinosis (ah) of arteries in a focal or circumferential pattern: Ah0: 0 arterioles with any hyaline Ah1: 1 arteriole with focal hyaline Ah2: $ 1 arteriole with focal hyaline Ah3: $ 1 arteriole with circumferential hyaline

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FIGURE 26.16 Immunofluorescence microscopy can also be helpful in cases of polyomavirus nephropathy by showing granular tubular basement membrane staining for IgG (shown) or C4d.

26.5 INFECTIONS 26.5.1 Polyomavirus Nephropathy Polyomavirus Nephropathy (PVN) was first described by Mackenzie in renal transplants nearly 30 years ago.21 Thirty to 60% of recipients develop viruria, 10% 20% develop viremia and 5% 10% go on to develop PVN. Up to 70% of patients with PVN undergo loss of the allograft within 2 3 years. The polymoma virus family includes numerous DNA viruses only a handful of which cause disease in humans. Simian virus 40 (SV40) rarely causes an interstitial nephritis in humans but is of enough homology to the more pathogenic members that immunohistochemistry testing for the large T antigen of SV40 is used routinely as a surrogate marker for BK and JC virus infections. JC virus, named after the initials of the first patients identified with infection, has a tropism for neural tissue and is the pathologic agent of Progressive Multifocal Leukoencephalopathy (PML), a white matter disease that rose to prominence during the early days of the AIDS epidemic, prior to the institution of highly active antiretroviral therapy (HAART). JC viral infection is responsible for about 15% of PVN, usually milder injury than that seen in BK infection. BK virus, also named after the first patient known to harbor the virus, is responsible for the remaining 85% of cases of PVN. The infection is thought to be acquired in childhood (seroprevalence of 80% in adults) and to lay dormant in the urothelium.22,23 For this reason, BK virus urothelial infection can lead to obstruction when urothelial damage occurs.24 Occasional shedding of virally infected tubular cells can be identified in urine cytology specimens as “decoy cells” because of their abnormal appearance, resembling precancerous or cancerous cells.25 Classic histologic features include a lymphoplasmacytic infiltrate, tubular injury, viral cytopathic changes including ground glass nuclei, smudgy hyperchromatic nuclei, and intranuclear inclusions.26 Occasionally, these viral cytopathic changes may extend to the parietal epithelial cells forming crescent-like proliferations.27 Additionally, the extent of tubular damage and inflammation brings acute cellular rejection into the differential diagnosis. Immunofluorescence staining may show granular tubular basement membrane staining for IgG, C3, or C4d.28 By EM, viral particles may form paracrystalline arrays of virions measuring 40 50 nm in diameter. Diagnosis is customarily confirmed by immunohistochemistry studies for SV40, as described previously. Polymerase-chain reaction (PCR) testing identifies virus in the blood and urine.

26.5.2 Adenovirus Adenovirus infection can be found in ,1% of kidney transplants with typical onset in the first 3 months post transplantation. Infection is most common in bone marrow and stem cell recipients. By light microscopy, the usual findings are a hemorrhagic interstitial nephritis that at times may also show granulomatous inflammation. Smudgy, basophilic cells with intranuclear inclusions, not unlike polyomavirus, are identified in tubular epithelial cells.29 Clinical suspicion coupled with IHC staining leads to the correct diagnosis.

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26.5.3 Cytomegalovirus Cytomegalovirus infection of the allograft occurs in about 20% of patients even with ganciclovir prophylaxis and up to 45% of those who do not receive prophylaxis.30 Morphologic findings include large intranuclear and cytoplasmic inclusions within tubular epithelial cells and endothelial cells (Fig. 26.17). The accompanying inflammation may be lymphocytic and/or granulomatous. If endothelial cells are the dominant target, the interstitial inflammation may be minimal. Further, endothelial cell damage by virus may lead to a thrombotic microangiopathy. Finally, glomerular capillary loop endothelial cell involvement may lead to a rare acute glomerulonephritis complete with endocapillary hypercellularity and crescent formation.31 IHC staining of paraffin-embedded tissue is the most common test performed by pathologists for confirmation of histologic findings (Fig. 26.18). EM may show nuclear and cytoplasmic virions measuring between 150 and 200 nm in diameter. Clinical testing may include serologies for IgM (acute infection) or IgG (chronic/latent infection). Indirect immunofluorescence studies may detect a CMV antigen (pp65 protein) in circulating leukocytes. Additional studies may be PCR or viral culture. Current immunosuppressive protocols have led to less graft loss than in the past.

FIGURE 26.17 Cytomegalovirus infections often result in large bizarre nuclei with intranuclear and cytoplasmic inclusions that are often difficult to differentiate from PVN. The infiltrate and acute tubular injury may be identical.

FIGURE 26.18

Immunohistochemistry is very helpful in specifically detecting the presence of cytomegaloviral proteins both in the nucleus and cytoplasm of infected cells, as in this case.

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26.5.4 Acute Pyelonephritis Acute pyelonephritis may be defined as acute (neutrophil rich) inflammation of the kidney parenchyma due to bacterial infection. Ascending infections from the lower urinary tract including urethra, bladder, and ureters are responsible for most (about 95%) of cases. Enteric gram-negative bacteria are most often the culprit and may be identified by urinalysis or grown in urine culture. Virulence factors such as pilli or fimbriae allow Escherichia coli attachment to urothelium. Serotypes H, K, and O are particularly prone to attachment via this mechanism. Hematogenous spread in septicemic patients and those with endocarditis account for most of the remaining cases. Staphylococcal species, enterics and fungal pathogens should be kept in mind especially in such clinical circumstances. Pathologic assessment may reveal yellow streaks on gross examination corresponding to medullary infection. There may be capsular scars suggesting previous infections/chronic infections. The diagnostic lesion seen by light microscopy is tubules filled and often dilated by neutrophilic plugs.32 Severe cases may have rupture of tubules with spillage of infected material into the renal interstitium and additional inflammatory reaction. Abscess formation with focal or multifocal collections of neutrophils displacing and destroying intervening parenchyma may also be identified. Severe cases may also be associated with papillary necrosis. Immunofluorescence and electron microscopic studies are of little diagnostic value but may be helpful in excluding other concomitant disease (e.g., C4d positive staining in PTCs, suggesting a component of AMR, immune complex deposits indicating an additional immune complex disease, etc.). As disheartening as it may be for the treating physician, there is no reason why infection and rejection cannot coexist. The transplanted organ is essentially a foreign body, so the natural response of the immune system is to reject it.33

26.5.5 Other Infectious Agents As stated earlier, the transplanted kidney is still a kidney with the possibility of all attendant pathologies. Thanks to the great immunosuppression of today’s regimens, grafts are more durable. However, this setting sets the perfect opportunity for opportunistic agents to flourish. Candida, Histoplasma, Cryptococcus, Zygomyctes, Coccidioidodes, Paracoccidioides, Microsporidia, Mycobacteria, and Norcardia, to name just a few, have been reported to take up residence and wreak havoc in the allograft.34

26.6 POSTTRANSPLANT LYMPHOPROLIFERATIVE DISORDERS (PTLD) In organ transplant recipients the risk of lymphoma is increased 20% 120% compared to the general population and develops as a consequence of immunosuppression. Posttransplant Lymphoproliferative Disorders (PTLD) comprises a spectrum of pathologies ranging from early, EBV driven polyclonal proliferations resembling infectious mononucleosis to EBV positive or negative, monoclonal or polyclonal B-cell or T-cell lymphomas. B cells are the origin of more than 85% of PTLD in organ transplant recipients. Early PTLD lesions show vague nodular proliferations with preservation of architecture and are more often found in children and seronegative adult recipients. Polymorphic and Monomorphic PTLD show abnormal lymphocyte morphology and disruption of renal architecture. Polymorphic PTLD is more frequent after primary EBV infection and more often involves the kidney allograft. Monomorphic PTLD may be B-cell ( . 60%) or T-cell (,5%) and present as a Diffuse Large B-cell Lymphoma (DLBCL) or T-cell Lymphoma. Less than 5% of cases present as Classic Hodgkin lymphoma with Reed-Sternberg cells and occur in renal allografts more often than other transplanted organs.35 The differential diagnosis includes acute cellular rejection and polyomavirus nephropathy. Immunohistochemical studies for polyoma virus help to distinguish the latter. PTLD can cause tubulitis and endarteritis, thus mimicking acute cellular or cell-mediated vascular rejection. IHC for CD20 (B-cell marker) and EBV can help highlight neoplastic B-cells invading tubules or vessels. However, this strategy will not work for the rare T-cell PTLD, which will stain for CD3 (T-cell marker) the same as acute rejection. In this case, assessment of clonality (kappa or lambda light chain restriction) may be of assistance. Therapy typically includes reducing immunosuppression, radiation, chemotherapy, antiviral drugs, or immune modulation with rituximab.36

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26.7 DRUG TOXICITIES 26.7.1 Calcineurin Inhibitor Toxicity The routine use of calcineurin inhibitors, beginning in the early 1980s, has been a major part of the success experienced by the field of organ transplantation. However, it should come as no surprise that with this success there will be some side effects. Briefly, calcineurin inhibitors, specifically cyclosporine (CsA) is a cyclic fungal peptide that complexes with cyclophilin (CyP) and inhibits calcium dependent calcineurin function.37 This process leads to decreased expression of various gene transcripts important for T-cell function. Interleukin-2, IL-3, IL-4, IL-12, tumor necrosis factor alpha and various growth factors are among the known products affected.38 It should be obvious that with this wide degree of mediator inhibition, the effects should be equally wide ranging. Pathologic findings may broadly be explained by ischemic injury. It is felt that the primary insult to the kidney is that of vasoconstriction of large arteries, usually of the size not seen on routine renal biopsy specimens.39 The result is a rising or persistently elevated creatinine with no morphologic abnormality identified on the biopsy material. The next step is acute tubular injury characterized by isometric vacuolization of tubular epithelial cells and eosinophilic cytoplasmic granules corresponding to megamitochondria (better evaluated by electron microscopy). Additionally, vacuolization is seen in other clinical settings such as renal ischemia or tubular epithelial injury caused by intravenous administration of hyperosmotic fluids (solutions of mannitol, inulin, glucose, sucrose, dextran, hydroxyethyl starch, urea, and radiocontrast agents.40 Vascular injury may be acute in the form of an acute thrombotic microangiopathy.39 Chronic vascular toxicity is characterized by nodular hyalinosis extending from the intima to the tunica media of arterial vessels, often in a “string of pearls” fashion (Figs. 26.19 and 26.20). Following the same theoretical explanation of ischemia, the layer of the arterial wall most dependent on intraluminal blood (oxygen) will be the most affected. Medial myocytes undergo cell death and are replaced with transudate plasma proteins that leak in from between the damaged endothelia. The clumps of muscle cells

FIGURE 26.19 Dense neutrophilic plugs filling distended tubules associated with surrounding neutrophilic infiltrate and acute tubular injury is diagnostic of acute pyelonephritis (PAS).

FIGURE 26.20 High power image of dense neutrophilic plugs filling, distending and leading to rupture of tubules with resultant neutrophilic interstitial inflammation (H&E).

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are replaced by nodular hyaline extending to the medial layer. Further chronic injury will lead to a striped pattern of interstitial fibrosis since the decreased blood flow follows the vasculature across medullary rays resulting in those areas most distant being the most affected.40 As with the acute thrombotic microangiopathy previously described, ongoing injury may result in a chronic microangiopathy with or without thrombosis. Calcineurin inhibition may also directly activate apoptosis genes and increase apoptosis in tubular and interstitial cells, thereby inducing tubular atrophy.41 Immunofluorescence study offers little assistance other than evaluation for additional disease processed. Electron microscopy can show the splitting of the GBM without immune complex deposition, adding credibility to the diagnosis of chronic TMA. The etiology, however, must be discerned by a complete clinicopathologic integrative evaluation (Figs. 26.21 and 26.22).

26.7.2 Mammalian Target of Rapamycin Inhibitor Toxicity This family of immunosuppressants consists of cyclic macrolide antibiotics that are isolated from the bacterium Streptomyces hygroscopicus that typically forms a complex that inhibits mammalian target of rapamycin (mTOR). Inhibition of this kinase results in the downregulation of ribosomal activity, numerous transcripts including hypoxia-inducible factor (HIF-1) and vascular endothelial growth factor (VEGF), and inhibition of various cell cycle regulators.42 Toxicities include severe acute tubular injury with epithelial cell necrosis as well as acute and chronic TMA.43 In addition, the necrotic and sloughed tubular epithelial cells may form peculiar intratubular casts44 with an appearance not unlike light chain cast nephropathy (LCCN, previously known as myeloma cast nephropathy or myeloma kidney). However, LCCN is by definition a monoclonal process by immunofluorescence study. Further, the ischemic damage can cause a pattern of segmental sclerosis45 leading to glomerulosclerosis similar to FSGS (even a collapsing FSGS). There may be some dysregulation of the podocytes themselves as proteinuria may occur without overt histologic findings.46

FIGURE 26.21 Nodular hyaline extending into the tunica medial of arterioles is suggestive but not necessarily diagnostic of calcineurin inhibitor (CNI) toxicity (Jones).

FIGURE 26.22

Nodular hyaline in the tunica media may also be seen with hypertensive injury as in this tortuous arteriole (PAS).

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References 1. Cornell LD, Smith RN, Colvin RB. Kidney transplantation: mechanisms of rejection and acceptance. Annu Rev Pathol 2008;3:189 220. 2. John R, Herzenberg AM. Our approach to a renal transplant biopsy. J Clin Pathol 2010;63(1):26 37. 3. Sis B, Mengel M, Haas M, Colvin RB, Halloran PF, Racusen LC, et al. Banff ‘09 meeting report: antibody mediated graft deterioration and implementation of Banff working groups. Am J Transplant 2010;10(3):464 71. 4. Waltzer WC, Miller F, Arnold A, Jao S, Anaise D, Rapaport FT. Value of percutaneous core needle biopsy in the differential diagnosis of renal transplant dysfunction. J Urol 1987;137(6):1117 21. 5. Herrera GA, Isaac J, Turbat-Herrera EA. Role of electron microscopy in transplant renal pathology. Ultrastruct Pathol 1997;21(6):481 98. 6. Racusen LC, Solez K, Colvin RB, Bonsib SM, Castro MC, Cavallo T, et al. The Banff 97 working classification of renal allograft pathology. Kidney Int 1999;55(2):713 23. 7. Solez K, Axelsen RA, Benediktsson H, Burdick JF, Cohen AH, Colvin RB, et al. International standardization of criteria for the histologic diagnosis of renal allograft rejection: the Banff working classification of kidney transplant pathology. Kidney Int 1993;44(2):411 22. 8. Solez K, Colvin RB, Racusen LC, Haas M, Sis B, Mengel M, et al. Banff 07 classification of renal allograft pathology: updates and future directions. Am J Transplant 2008;8(4):753 60. 9. Haas M, Sis B, Racusen LC, Solez K, Glotz D, Colvin RB. Banff 2013 meeting report: inclusion of c4d-negative antibody-mediated rejection and antibody-associated arterial lesions. Am J Transplant 2014;14(2):72 283. 10. Lefaucheur C, Loupy A, Vernerey D, Duong-Van-Huyen JP, Suberbielle C, Anglicheau D, et al. Antibody-mediated vascular rejection of kidney allografts: a population-based study. Lancet 2013;381(9863):313 19. 11. Mauiyyedi S, Crespo M, Collins AB, Schneeberger EE, Pascual MA, Saidman SL, et al. Acute humoral rejection in kidney transplantation: II. Morphology, immunopathology, and pathologic classification. J Am Soc Nephrol 2002;13(3):779 87. 12. Hara S, Matsushita H, Yamaguchi Y, Kawaminami K, Horita S, Furusawa M. Allograft glomerulitis: histologic characteristics to detect chronic humoral rejection. Transplant Proc 2005;37(2):714 16. 13. Liptak P, Kemeny E, Morvay Z, Szederkenyi E, Szenohradszky P, Marofka F, et al. Peritubular capillary damage in acute humoral rejection: an ultrastructural study on human renal allografts. Am J Transplant 2005;5(12):2870 6. 14. Lim BJ, Kim MS, Kim YS, Kim SI, Jeong HJ. C4d deposition and multilayering of peritubular capillary basement membrane in posttransplantation membranous nephropathy indicate its association with antibody-mediated injury. Transplant Proc 2012;44(3):19 620. 15. Einecke G, Sis B, Reeve J, Mengel M, Campbell PM, Hidalgo LG, et al. Antibody-mediated microcirculation injury is the major cause of late kidney transplant failure. Am J Transplant 2009;9(11):2520 31. 16. Delville M, Charreau B, Rabant M, Legendre C, Anglicheau D. Pathogenesis of non-HLA antibodies in solid organ transplantation: where do we stand? Hum Immunol 2016. In Press. http://dx.doi.org/10.1016/j.humimm.2016.05.021. 17. Solez K, Colvin RB, Racusen LC, Sis B, Halloran PF, Birk PE, et al. Banff ‘05 Meeting Report: differential diagnosis of chronic allograft injury and elimination of chronic allograft nephropathy (‘CAN’). Am J Transplant 2007;7(3):18 526. 18. Sibley RK. Morphologic features of chronic rejection in kidney and less commonly transplanted organs. Clin Transplant 1994;8(3 Pt 2):293 8. 19. Freese PM, Svalander CT, Molne J, Nyberg G. Renal allograft glomerulopathy and the value of immunohistochemistry. Clin Nephrol 2004;62(4):279 86. 20. Bhowmik DM, Dinda AK, Mahanta P, Agarwal SK. The evolution of the Banff classification schema for diagnosing renal allograft rejection and its implications for clinicians. Ind J Nephrol 2010;20(1):2 8. 21. Mackenzie EF, Poulding JM, Harrison PR, Amer B. Human polyoma virus (HPV)—a significant pathogen in renal transplantation. Proc Eur Dial Transplant Assoc 1978;15:352 60. 22. Hirsch HH, Randhawa P. BK polyomavirus in solid organ transplantation. Am J Transplant 2013;13(Suppl. 4):179 88. 23. Knowles WA. Discovery and epidemiology of the human polyomaviruses BK virus (BKV) and JC virus (JCV). Adv Exp Med Biol 2006;577:19 45. 24. Celik B, Randhawa PS. Glomerular changes in BK virus nephropathy. Hum Pathol 2004;35(3):367 70. 25. Cimbaluk D, Pitelka L, Kluskens L, Gattuso P. Update on human polyomavirus BK nephropathy. Diagn Cytopathol 2009;37(10):773 9. 26. Drachenberg CB, Papadimitriou JC, Hirsch HH, Wali R, Crowder C, Nogueira J, et al. Histological patterns of polyomavirus nephropathy: correlation with graft outcome and viral load. Am J Transplant 2004;4(12):2082 92. 27. Nair R, Katz DA, Thomas CP. Diffuse glomerular crescents and peritubular immune deposits in a transplant kidney. Am J Kidney Dis 2006;48(1):174 8. 28. Hever A, Nast CC. Polyoma virus nephropathy with simian virus 40 antigen-containing tubular basement membrane immune complex deposition. Hum Pathol 2008;39(1):73 9. 29. Storsley L, Gibson IW. Adenovirus interstitial nephritis and rejection in an allograft. J Am Soc Nephrol 2011;22(8):1423 7. 30. Liapis H, Storch GA, Hill DA, Rueda J, Brennan DC. CMV infection of the renal allograft is much more common than the pathology indicates: a retrospective analysis of qualitative and quantitative buffy coat CMV-PCR, renal biopsy pathology and tissue CMV-PCR. Nephrol Dial Transplant 2003;18(2):397 402. 31. Rane S, Nada R, Minz M, Sakhuja V, Joshi K. Spectrum of cytomegalovirus-induced renal pathology in renal allograft recipients. Transplant Proc 2012;44(3):713 16. 32. Gupta G, Shapiro R, Girnita A, Batal I, McCauley, Basu JA, et al. Neutrophilic tubulitis as a marker for urinary tract infection in renal allograft biopsies with C4d deposition. Transplantation 2009;87(7):1013 18. 33. Audard V, Amor M, Desvaux D, Pastural M, Baron C, Philippe R, et al. Acute graft pyelonephritis: a potential cause of acute rejection in renal transplant. Transplantation 2005;80(8):1128 30. 34. Badiee P, Alborzi A. Invasive fungal infections in renal transplant recipients. Exp Clin Transplant 2011;9(6):355 62. 35. Jagadeesh D, Woda BA, Draper J, Evens AM. Post transplant lymphoproliferative disorders: risk, classification, and therapeutic recommendations. Curr Treat Options Oncol 2012;13(1):122 36. 36. Oertel SH, Verschuuren E, Reinke P, Zeidler K, Papp-Vary M, Babel N, et al. Effect of anti-CD 20 antibody rituximab in patients with post-transplant lymphoproliferative disorder (PTLD). Am J Transplant 2005;5(12):2901 6.

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37. Emmel EA, Verweij CL, Durand DB, Higgins KM, Lacy E, Crabtree GR. Cyclosporin A specifically inhibits function of nuclear proteins involved in T cell activation. Science 1989;246(4937):1617 20. 38. Clipstone NA, Crabtree GR. Identification of calcineurin as a key signalling enzyme in T-lymphocyte activation. Nature 1992;357 (6380):695 7. 39. Mihatsch MJ, Thiel G, Basler V, Ryffel B, Landmann J, von Overbeck J, et al. Morphological patterns in cyclosporine-treated renal transplant recipients. Transplant Proc 1985;17(4 Suppl. 1):101 16. 40. Legendre C, Thervet E, Page B, Percheron A, Noel LH, Kreis H. Hydroxyethylstarch and osmotic-nephrosis-like lesions in kidney transplantation. Lancet 1993;342(8865):248 9. 41. Servais H, Ortiz A, Devuyst O, Denamur S, Tulkens PM, Mingeot-Leclercq MP. Renal cell apoptosis induced by nephrotoxic drugs: cellular and molecular mechanisms and potential approaches to modulation. Apoptosis 2008;13(1):11 32. 42. Roohi A, Hojjat-Farsangi M. Recent Advances in targeting mTOR signaling pathway using small molecule inhibitors. J Drug Target 2016;1 37. 43. Sartelet H, Toupance O, Lorenzato M, Fadel F, Noel LH, Lagonotte E, et al. Sirolimus-induced thrombotic microangiopathy is associated with decreased expression of vascular endothelial growth factor in kidneys. Am J Transplant 2005;5(10):2441 7. 44. Smith KD, Wrenshall LE, Nicosia RF, Pichler R, Marsh CL, Alpers CE, et al. Delayed graft function and cast nephropathy associated with tacrolimus plus rapamycin use. J Am Soc Nephrol 2003;14(4):1037 45. 45. Letavernier E, Bruneval P, Mandet C, Duong Van Huyen JP, Peraldi MN, Helal I, et al. High sirolimus levels may induce focal segmental glomerulosclerosis de novo. Clin J Am Soc Nephrol 2007;2(2):326 33. 46. Vollenbroker B, George B, Wolfgart M, Saleem MA, Pavenstadt H, Weide T. mTOR regulates expression of slit diaphragm proteins and cytoskeleton structure in podocytes. Am J Physiol Renal Physiol 2009;296(2):F418 26.

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27 Imaging-Based Monitoring of the Renal Graft Corinne Deurdulian and Hisham Tchelepi Keck School of Medicine of USC, Los Angeles, CA, United States

27.1 INTRODUCTION Renal transplantation is the ultimate treatment of end-stage renal disease. Over 100,000 individuals currently await renal transplants. In 2014, over 17,000 renal transplants were performed in the United States; about twothirds of the kidneys came from deceased donors and one-third from living donors.1 Comprehensive imaging of renal transplants is critical to ensure timely detection of abnormalities that may necessitate intervention. Ultrasonography (US) is the primary imaging tool for initial evaluation and follow-up of renal transplants. Specific indications for US are summarized in Table 27.1.2

27.2 RENAL TRANSPLANT ANATOMY The transplant kidney is usually placed extraperitoneally in either iliac fossa, more commonly on the right due to easier accessibility of the iliac vessels. A cadaveric kidney is typically harvested with the main renal artery (RA) intact and attached to a small patch of the aorta (Carrel patch), which is anastomosed end-to-side to the recipient external iliac artery (IA).3 In a living donor kidney transplant, an end-to-side anastomosis of the donor RA to the recipient external IA or an end-to-end anastomosis with the recipient internal IA is made. The donor renal vein (RV) is typically attached end-to-side with the recipient external iliac vein (IV).4 The transplant ureter is implanted superiorly along the bladder (ureteroneocystostomy) with the goal of preventing reflux to the transplant.5 In very small children, the allograft may be placed intraperitoneally.6 Additionally, in cases of pediatric deceased donors, both kidneys, aorta, and inferior vena cava may be transplanted to an adult recipient. Knowledge of the types of anastomoses is helpful for US assessment as multiple variations are possible; therefore it is recommended to review the operative report or discuss the procedure with the transplant surgeon.

27.3 ULTRASOUND IMAGING US imaging is performed with a curved 5 or 9 MHz transducer. Grayscale evaluation of the transplant kidney includes longitudinal and transverse images of the kidney (upper, mid, and lower) and urinary bladder. The presence of hydronephrosis and perinephric fluid collections should be thoroughly documented. Ureteral stent position, if present, should also be assessed. Color and spectral Doppler evaluation is paramount to thoroughly evaluate the renal transplant, including the following: • Renal parenchyma—Blood flow should be seen throughout the kidney on both color and power Doppler. Because power Doppler is more sensitive than conventional color Doppler, nearly the entire kidney should display robust flow (Fig. 27.1). Kidney Transplantation, Bioengineering, and Regeneration. DOI: http://dx.doi.org/10.1016/B978-0-12-801734-0.00027-8

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374 TABLE 27.1

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Indications for US Imaging of Renal Transplants

Baseline postoperative evaluation

Low or decreased urine output

Routine transplant surveillance

Persistent/refractory hypertension or bruit

Follow-up of abnormal findings (e.g., velocities, resistive indices, waveforms)

Assessment of hydronephrosis, hydroureter, or bladder abnormality

Assessment of vascular patency

Evaluation for complications after biopsy or surgery

Evaluation of fluid collections

Evaluation for malignancy (RCC, PTLD)

Pain, fever, sepsis

Hematuria

Abnormal laboratory values (creatinine)

Guidance for biopsy or drainage 83

Adapted from 2014 AIUM Practice Guidelines.

FIGURE 27.1 Transplant kidney. Normal gray scale (A, B), color Doppler (C), and power Doppler (D) appearances of transplant kidney obtained with curved 5 and 6 MHz transducers.

FIGURE 27.2 Arterial and venous anastomoses. Color Doppler images demonstrate widely patent arterial (A, arrow) and venous (B, arrow) anastomoses.

• Main RA • Patency and peak systolic velocity (PSV) of proximal, mid, and distal RA • Anastomosis patency (Fig. 27.2) • An additional RA, if present, should be independently evaluated and numbered (e.g., RA #1 and RA #2) (Fig. 27.3) I. KIDNEY TRANSPLANTATION

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FIGURE 27.3 Two main renal arteries two main renal arteries demonstrated by color and spectral Doppler (A) should be labeled as #1 and #2 (B) or superior and inferior (C) and consistently maintained on follow-up studies. Y-shaped appearance (D) of duplicate main renal arteries.

FIGURE 27.4

Normal intrarenal waveforms RIs. Normal intrarenal spectral waveforms (A and B). Note normal dichrotic notch (arrows).

• Intrarenal arteries • Resistive indices (RIs) and systolic acceleration times (SATs) of superior, mid, and inferior intrarenal arteries (Fig. 27.4) • IA—PSV should be obtained superior or cephalad to the RA anastomosis and is used to calculated the RA IA PSV ratio • Main RV • PSV • Venous patency evaluation of the RV at and distal to the anastomosis (Fig. 27.2) • IV Patency and PSV I. KIDNEY TRANSPLANTATION

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27.4 COMPLICATIONS: NONVASCULAR AND VASCULAR Nonvascular or urologic complications are estimated to occur in 1% 8% of transplant recipients.7 9 Examples include urinary obstruction, urine leak, fluid collections, strictures, and neoplasms. Early complications from obstruction, urine leak, or stricture typically occur within the first several months after transplantation and can usually be treated percutaneously. Treatment options include ureteral stents, Foley catheters, ureteral angioplasty, and percutaneous drainage.10 If surgery is indicated, especially in cases of adhesion and fibrosis related obstruction or chronic rejection, reconstruction of the ureteral anastomosis with the bladder decreases the incidence of these complications.11

27.4.1 Urinary Obstruction Ureteral stricture is the most common cause of urinary obstruction and usually occurs distally at or near its anastomosis with the bladder. Common causes of ureteral stricture include scarring, possibly from ischemia or rejection, surgical technique, and kinking. Urinary calculi, clots, sloughed papilla, pelvic fibrosis, fungus balls, and extrinsic compression are other causes of hydronephrosis.12,13 Patients who have undergone multiple procedures are at risk for remote complications from adhesions, fibrosis, or vascular insufficiency. Perirenal fluid collections such as lymphoceles, hematomas, and urinomas may also cause obstruction. Vesicoureteral reflux as a cause of hydronephrosis is more commonly seen in pediatric patients.14 Elevated serum creatinine and hydronephrosis (Fig. 27.5) on US are the most common findings in urinary obstruction but can also be seen with chronic rejection. Typically, renal colic symptoms do not occur as the transplant kidney is denervated. It should be noted that mild collecting system dilatation can be seen physiologically due to a full bladder, close proximity of the transplant kidney to the bladder, transient excretion, and accommodation of the urine output over time (Fig. 27.6). One pitfall from the lack of identifying hydronephrosis by US may be related to parenchymal edema and fibrosis.15 Radionuclide imaging shows retained radioactivity in cases of significant hydronephrosis. Additionally, diuretic renography can assess clearance times of the kidney to assess for delay. An antegrade nephrostogram can demonstrate the site of obstruction and provides information on an appropriate access route for urinary drainage.10

FIGURE 27.5 Mild hydronephrosis. US of renal transplant patient with new onset hyperkalemia demonstrates mild hydronephrosis (A) which is new compared with 1 year earlier (B).

FIGURE 27.6 Mild collecting system dilatation. Mild collecting system dilatation can be seen normally due to proximity of the transplant kidney to the bladder and due to reflux.

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FIGURE 27.7 Stent placement for hydronephrosis. Longitudinal (A) and transverse (B) images of the transplant kidney demonstrate mild hydronephrosis with a urinary stent, seen as a tubular structure with echogenic walls (arrows). Flow to the kidney is preserved (C). There is improved visualization of the stent on a subsequent study (D, arrow).

FIGURE 27.8 Stent on XR. A transplant ureteral stent is identified in the left lower quadrant. The proximal and distal ends should lie within the renal collecting system and bladder, respectively.

The treatment depends on the cause of urinary obstruction. Percutaneous nephrostomy, ureteral stent placement (Figs. 27.7 and 27.8), and balloon ureteroplasty may also be performed. Ureteral stents are commonly placed in the first month of transplant to help prevent sloughing of the urinary anastomosis. Percutaneous drainage of fluid collections and Foley catheters are additional treatment options.16

27.4.2 Urine Leaks and Urinomas Urine leaks or urinomas are simple fluid collections between the transplant kidney and bladder and typically occur within 2 weeks of surgery. Urine leaks usually arise from the renal pelvis, ureter, or ureteroneocystostomy site. Leakage from the calices is rare. Clinical signs include decreased urine output and fullness and tenderness along the operative bed.

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FIGURE 27.9 Urinoma. Between the transplant kidney and bladder is a 5.6 cm anechoic collection (arrows, A, B, and C). Percutaneous drainage revealed elevated creatinine consistent with a urinoma.

US images demonstrate a well-defined anechoic fluid collection that may increase in size over a short period of time (Fig. 27.8). Fluid sampled or drained under US guidance reveals a high creatinine level, differentiating it from seroma or lymphocele. Complications of urinomas include abscess formation and rupture, which can lead to urinary ascites (Fig. 27.9). Radionuclide imaging shows radiotracer activity around the transplant, outside of the urinary tract. Antegrade pyelography can pinpoint the extravasation site and thus assist in surgical planning. Prompt treatment of urine leaks is critical due to the risk of infection in immunocompromised patients.10 Percutaneous drainage of the urinoma should be performed. Percutaneous nephrostomy to divert urinary flow in conjunction with ureteral stent placement can treat a majority of cases.17,18 The ureter may be reimplanted if there is a persistent leak and incomplete healing. For caliceal leaks, percutaneous nephrostomy without stenting is performed. Foley catheters are used to decompress the bladder, especially in cases of suspected bladder leak, usually at the cystostomy or ureteroneocystostomy site.19

27.4.3 Peritransplant Fluid Collections Fluid collections surrounding the transplant kidney include seromas, lymphoceles, hematomas, urinomas (discussed above) and abscesses. Size, location, and change in size over time are evaluated to determine clinical importance. Small hematomas and seromas are common in the immediate posttransplant period, often manifesting as crescentic collections around the kidney. Although these collections often resolve, following the size of the collections is important, as increasing size may necessitate intervention.15 Progressively increasing collections may be seen with urinoma, abscess, and vascular injury. Urinomas are more common 1 week to 1 month after transplantation. Lymphoceles are more likely to develop 1 2 months after transplant. Percutaneous sampling is often necessary for diagnosis, if clinically indicated.20 Timing of transplant collections is included in Table 27.2. 27.4.3.1 Hematomas Hematomas are most common in the peritransplant period but also occur after biopsy, trauma, and spontaneously. Most of the time, hematomas that are small resolve spontaneously, but when large, they can cause mass effect on the transplant kidney and hydronephrosis.15 On US, hematomas typically appear as complex collections

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TABLE 27.2 Peritransplant Fluid Collections Timing of collections

Types of collections

Immediate, within 1 week of transplant

Perinephric hematoma Seroma

Early, 1 week to 1 month after transplant

Urine leak Urinoma

Late, .1 month after transplant

Lymphocele

FIGURE 27.10

Hematoma. US images 1 day after renal transplant demonstrate a complex perinephric collection consistent with an hematoma.

FIGURE 27.11

Perinephric hematoma on CT. Hyperattenuating fluid surrounding the allograft on CT (A and B) is consistent with

hematoma.

and can be echogenic (usually in the acute phase), isoechoic, or hypoechoic (Fig. 27.10). Over time, hematomas typically decrease in echogenicity and develop septations, consistent with liquefaction. On CT, hematomas may appear as complex collections with areas of high attenuation (Fig. 27.11). On MRI, hematomas often have heterogeneously increased T1 and T2 signal intensity. Occasionally, hematomas may be intraparenchymal (Fig. 27.12). Because hematomas are usually self-limited and multiloculated, drainage of the collection is generally not performed and not recommended, as these collections do not drain sufficient material that justifies an invasive procedure; however, if there is clinical concern for abscess, the fluid may be aspirated for diagnostic purposes. An infected hematoma can be drained percutaneously with 12 14F drains and periodic irrigation.10 Acutely enlarging hematomas in the immediate postoperative period may necessitate urgent surgical intervention.21 27.4.3.2 Lymphoceles Lymphocele is a common peritransplant fluid collection with a reported prevalence of up to 18%.22 Although they can develop at any time after transplant, they most commonly occur within 1 2 months after surgery (Fig. 27.13). Leakage from the lymphatic channels along the surgical bed result in lymphoceles. Risk factors

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FIGURE 27.12 Parenchymal hematoma. Longitudinal (A) and transverse (B) images of the transplant kidney demonstrate a mildly complex parenchymal collection that evolved over time and gradually decreased in size.

FIGURE 27.13 Lymphocele. A new perinephric fluid collection was seen 2 months after transplant (arrows, A and B). The fluid is mildly complex with a few internal echoes.

predisposing to lymphatic leakage include medications, rejection, diabetes, and retransplantation.23,24 Similar to urinomas, these collections occur between the transplant kidney and bladder. Lymphoceles on US are anechoic and may contain septations. They may have a more complex appearance if they become infected. On CT, lymphoceles usually demonstrate water-attenuation levels, differentiating them from hematomas and abscesses. If clinically indicated, MRI or nuclear scintigraphy may be performed to exclude the presence of blood or urine, respectively.15 While small lymphoceles are usually monitored by US, larger lymphoceles are often drained as they may cause mass effect and hydronephrosis. The creatinine level of the lymphocele is similar to the serum level, differentiating lymphocele from urinoma. Recurrent lymphoceles after drainage are common and can be treated with prolonged catheter drainage and sclerotherapy.25 Similar to other fluid collections, lymphoceles may become infected, necessitating drainage and antibiotics. Laparascopic drainage is the preferred method when surgical drainage is indicated.26 Open drainage may be performed in patients with wound infection or when a small lymphocele is adjacent to vital structures.27 27.4.3.3 Infection and abscess Infection in the first year after transplant is common and should be identified and treated early to prevent graft-related compromise and failure. Common infections that occur in surgical patients include pneumonia, wound infection, and urinary tract infection.15 Immunosuppressive therapy and catheters are general risk factors for infection. Peritransplant abscess is rare. Etiologies include infected hematoma, urinoma, and lymphocele, as well as pyelonephritis. Clinical signs include fever and pain.

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On US, abscess may be suspected when a simple collection becomes more complex and contains gas, especially in the clinical setting of infection (Fig. 27.14). Abscesses, depending on size, may be treated with percutaneous drainage (Fig. 27.15), antibiotics, or both.

27.4.4 Renal Parenchymal Abnormalities Acute tubular necrosis, acute and chronic rejection, pyelonephritis, and medication toxicity are parenchymal complications that may occur. The various etiologies may be considered depending on timing and clinical factors (Table 27.3). US findings are often nonspecific in many cases.28 Although RIs are used to monitor the transplant kidney over time, biopsy is the gold standard by which to diagnose the etiology of graft failure or compromise.

FIGURE 27.14

Peritransplant abscess. Longitudinal (A) and transverse (B) images of a renal transplant demonstrate complex perinephric fluid contain echogenic foci (arrows) with reverberation artifact compatible with air. The patient had clinical signs of infection.

FIGURE 27.15

TABLE 27.3

Abscess drainage by CT. CT images (A and B) of an abscess (arrows) surrounding the allograft with drain placement (B).

Timing of Transplant-Related Parenchymal Complications

Abnormality

Onset

Findings

ATN

Within 1 week

Normal, enlarged kidney,

Acute Rejection

1 3 weeks post op

↑ RIs

↑ size, globular kidney, abnormal echogenicity, ↑ RIs, absent/reversed diastolic flow, patent renal vein

Chronic Rejection .3 months post op Mild hydronephrosis, abnormal echogenicity, reduced vascularity Anytime

Normal or patchy

↑ echogenicity with

↑

Pyelonephritis

perfusion,

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27.4.4.1 Acute Tubular Necrosis Acute tubular necrosis is the most common cause of delayed graft function within the first week of transplant. Risk factors include deceased donor kidney with increased cold ischemia time, greater weight and atherosclerotic disease burden in donor and recipient, older donor age, American Society of Anesthesia (ASA) IV status of recipient, and right kidney transplant.29 Up to 30% of ATN patients may require dialysis, but most cases resolve in the first 2 weeks.30 US findings may be normal, but nonspecific findings include elevated RIs (Fig. 27.16), abnormal parenchymal echogenicity, loss of corticomedullary differentiation, and renal enlargement.31 27.4.4.2 Acute Rejection Acute rejection may affect up to 40% of renal transplant recipients, usually occurring 1 3 weeks posttransplant.30 It is often differentiated from ATN as ATN is more common in the first few days after transplant and acute rejection is more common after 1 week. High-dose steroid therapy is used to reverse acute rejection. Clinically, it may be difficult to isolate acute rejection from urinary obstruction, ATN, pyelonephritis, and medication toxicity; therefore, biopsy is indicated. US findings include elevated RIs, absent or reversed diastolic flow, and elongated or globular kidney (Fig. 27.17).31 A patent RV excludes RV thrombosis as an etiology of absent or reversed diastolic flow. Hyperacute rejection is not imaged as it occurs at the time of surgery and related to preformed recipient antibodies.16

FIGURE 27.16 Severe ATN. POD 3. Spectral Doppler image of the allograft 3 days after transplant (A) demonstrates absent diastolic flow and an RI of 1 in the super, mid (A), and inferior interlobar arteries. There was no renal vein thrombosis by Doppler. One month later (B), normal intrarenal waveforms are present with a normal RI of 0.6 in the mid interlobar artery. A urinary stent (arrow, C) had been placed in the interval.

FIGURE 27.17 Acute rejection. Reversal of diastolic flow and elevated PSVs within the main renal artery (A, 228 cm/s) and iliac artery (not shown). This finding can be seen with acute rejection, acute tubular necrosis, and impending main renal vein thrombosis as the main renal vein and anastomosis (B) are currently patent. Biopsy was consistent with acute rejection. I. KIDNEY TRANSPLANTATION

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27.4.4.3 Chronic Rejection Chronic rejection begins 3 months after transplantation and is the most common cause of allograft loss. The greatest risk factor is prior acute rejection. Again, US findings are nonspecific and include mild hydronephrosis, increased parenchymal echogenicity, loss of corticomedullary differentiation, cortical thinning, urothelial thickening, and decreased parenchymal vascularity.16 27.4.4.4 Medication Toxicity Medication toxicity related to immunosuppressive medication, especially cyclosporine, may lead to irreversible allograft dysfunction.32 The transplant kidney may be normal on US or may demonstrate mildly elevated RIs. 27.4.4.5 Pyelonephritis Similar to the native kidney, US findings in pyelonephritis may be normal or may include focal areas of increased parenchymal echogenicity, loss of corticomedullary differentiation, and increased renal size. Decreased perfusion of the affected regions may also be seen, along with elevated RIs. Biopsy may be necessary to differentiate pyelonephritis from acute rejection.30

27.4.5 Vascular Complications Vascular complications occur in less than 10% of renal transplants with the most common being renal artery stenosis (RAS).33 Other arterial complications include thrombosis of the iliac or RA and segmental infarction of the kidney. Post procedural complications include arteriovenous fistula (AVF) and pseudoaneurysm. Venous complications include RV thrombosis and RV stenosis. Compartment syndrome and allograft torsion may also occur. The more common complications are discussed below. 27.4.5.1 Renal Artery Stenosis RAS typically occurs within 1 year of transplant and more commonly in the first 3 months. The anastomosis is the most common site of stenosis.34 RAS is associated with refractory hypertension and hypertension associated with a bruit or graft dysfunction. On US, focal areas of color aliasing and increased flow are seen at the site of stenosis. Criteria to evaluate stenosis are variable. Peak systolic velocities of the main RA are considered abnormal when they exceed 300 cm/second (Fig. 27.18).35 This is the current notion as velocities above 200 cm/sec may often overestimate the underlying abnormality, resulting in more extensive or additional investigation such as CTA, MRA, and angiography.37,38 Attention should be paid when evaluating pediatric kidneys transplanted into adults, as concern for renal artery stenosis should not be raised unless the PSV is over 350 cm/sec. This is most likely related to the small size of the donor renal artery.39 The IA peak systolic velocity is obtained to calculate the RA/IA PSV ratio. Ratios .1.8 3.5 are considered elevated.40,41 This variation is based on multiple prior studies demonstrating stenosis. False positives may occur when there is a sharp bend in the vessel and proper angle adjustment is not possible. High-velocity flow is also commonly encountered in the perioperative period around the anastomosis (Fig. 27.19). Therefore, it is prudent to evaluate the PSV of the main RA in conjunction with the IA to assess for systemically increased flow.42 Signs of hemodynamically significant stenosis include aliasing, elevated velocities, spectral broadening, and a significant velocity gradient between the prestenotic and stenotic segments of .2:1. The intrarenal arteries may demonstrate tardus parvus waveforms and increase in SATs (Fig. 27.20). In addition to US, MR angiography (MRA) either without or with contrast can also demonstrate the narrowing of the RA. Percutaneous angioplasty is the treatment of choice to treat the narrowing and subsequently improve hypertension and creatinine levels.43 Repeat dilatation may be performed for refractory cases in up to 20% of cases.44 Stent placement is another option (Fig. 27.21). 27.4.5.2 Renal Artery Thrombosis RA thrombosis occurs in less than 1% of transplants, usually in the immediate postoperative period, and necessitates emergent revascularization.45 It is usually related to RA kinking, or torsion, or dissection. Other etiologies include rejection, ATN, and hypercoagulability.16 Clinical signs include decreased or absent urine output and swelling and tenderness over the graft site. On US, no arterial or venous flow is identified distal to the affected portion of the RA. The kidney may appear hypoechoic with a possible echogenic rim. To avoid a false positive diagnosis of thrombosis, patent vessels outside of the kidney at a similar or greater should be assessed.46 I. KIDNEY TRANSPLANTATION

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FIGURE 27.18 Renal artery stenosis. Markedly increased PSVs are noted in the proximal MRA at 506 cm/s (A), progressively increased over several prior studies (not shown). The iliac artery velocity is 220 cm/s (B). The ratio is 2.3. CDUS image (C) shows color aliasing at this level. Elevated PSV of the main renal artery of 295 cm/s (D) with color aliasing is noted. The iliac artery velocity is 128 cm/s (E). Findings are compatible with RAS.

FIGURE 27.19 Elevated perioperative velocities. Elevated peak systolic velocities (PSVs), postoperative day #1. Elevated PSVs of 245 cm/s in the iliac artery (A) and 266 cm/s in the main renal artery (B) 1 day after transplantation. Velocities began to normalize after 3 days. Elevated velocities were likely due to arterial spasm as there is normalization of flow on subsequent studies without intervention.

27.4.5.3 Infarction Segmental infarcts are more common than complete renal infarction from main RA occlusion. A completed infarcted kidney appears diffusely enlarged and hypoechoic. Segmental infarcts present as focal areas of decreased or absent flow, overlapping with pyelonephritis. The kidney may appear hypoechoic with a possible echogenic rim. Severe infarctions may be seen in cases of 2 renal arteries where 1 artery is compromised. This I. KIDNEY TRANSPLANTATION

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FIGURE 27.20

Signs of renal artery stenosis. Tardus parvus waveforms of the interlobar arteries in 2 different patients are demonstrated along with delayed systolic acceleration times of .0.08 s (A, 1.18 s, and B, 0.23 s).

FIGURE 27.21

Recurrent RAS after stent placement. Power Doppler Images demonstrate the presence of a stent within the main renal artery (A) and focal stenosis at the proximal margin of the stent (B, arrow).

may cause devascularization of up to half of the kidney.46 No arterial or venous blood flow is present if there is total vascular obstruction. There is overlap both with US and nuclear scintigraphy findings (photopenic area) with hyperacute or acute rejection.15 Although CT with contrast easily demonstrates perfusion defects in the transplant kidney, contrast is not administered in these patients due elevated creatinine levels and risk of graft compromise. Angiography and MRI/MRA may demonstrate evidence of decreased global and segmental perfusion. Additionally, contrastenhanced renal ultrasound may be performed. Angiography with possible thrombolytic therapy may be utilized for treatment and graft salvage. 27.4.5.4 Iliac Artery Stenosis and Dissection IA stenosis (Fig. 27.22) may cause tardus parvus waveforms in the ipsilateral main renal and intrarenal arteries. Elevated velocities may be present at the site of stenosis. IA dissection is a rare complication that may occur after clamping of the IA, especially in patients with atherosclerotic disease (Fig. 27.23).47 Symptoms include poorly controlled hypertension, loss of allograft perfusion, and impending allograft failure. An intimal flap is seen on US. Treatment options include stenting and surgery. 27.4.5.5 Post Biopsy Arteriovenous Fistulas and Pseudoaneurysms AV Fistulas can occur after biopsies and present as focally abnormal flow and abnormal spectral Doppler waveforms. The artifactual color is attributed to parenchymal tissue vibration, also referred to as a “color Doppler bruit.”48 AV fistulas usually occur along a segmental or interlobar artery and adjacent vein and presents with turbulent high-velocity flow with aliasing (Fig. 27.24). There is a high-velocity low resistance waveform of the feeding artery and arterialization of the draining vein. The size of the AVF is usually overestimated on CDUS due to reverberation. I. KIDNEY TRANSPLANTATION

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FIGURE 27.22

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Iliac artery stenosis. 3D MRA image shows focal narrowing of the iliac artery (arrow). Note the presence of 2 renal arteries.

FIGURE 27.23 Iliac Artery Dissection. Grayscale (A) and color Doppler (B) images of a renal transplant demonstrate the presence of an intimal flap (A, arrows) in the external iliac artery (EIA). Note adjacent external iliac vein (EIV). CDUS shows retrograde flow in the false lumen (blue arrow) and antegrade flow in the true lumen (red arrow).

FIGURE 27.24 Arteriovenous fistula post biopsy. CDUS (A) typically overestimates the size of the AVF due to tissue “humming” from reverberation of the arterial signal into the vein. This “humming” results in turbulent, high-velocity, low resistance waveforms (B and C).

Pseudoaneurysms can occur after biopsy and rarely after transplant at the anastomosis and can be mistaken for a simple or complex cyst on grayscale imaging. Color Doppler imaging demonstrates a markedly vascular structure with a characteristic “yin-yang” flow pattern. A neck with the communicating artery with the characteristic to-and-fro flow pattern may be identified (Fig. 27.25). Pseudoaneurysms can also be associated with AVFs. MRI/MRA can elucidate the anatomy when US imaging is limited by extensive perivascular vibration. I. KIDNEY TRANSPLANTATION

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FIGURE 27.25 Pseudoaneurysm of renal transplant. Grayscale (A and B) and color and power Doppler (C and D) images demonstrate a partially thrombosed 4.2 cm pseudoaneurysm that has gradually increased in size over several prior studies (not shown). CDUS (E) and spectral Doppler (F) images show high-velocity flow along the neck of the pseudoaneurysm (E, arrow) with aliasing and characteristic to-and-fro pattern.

Conservative management of AVFs and pseudoaneurysms is usually sufficient as most, especially small ones, resolve spontaneously. About 70% of all AVFs reportedly resolve within 1 2 years and 30% may persist or become symptomatic, resulting in persistent hematuria or allograft dysfunction.49 However, if a pseudoaneurysm is large (.2 cm) or progressively increases in size, it may rupture. Interventional embolization therapy with metallic coils may be employed to treat symptomatic AVFs and enlarging pseudoaneurysms (Fig. 27.26). 27.4.5.6 Renal Vein Thrombosis RV thrombosis is a rare complication of transplantation and typically occurs within the first week of surgery. Clinical signs include sudden onset of anuria and swelling and tenderness over the graft. Causes include hypovolemia, hypercoagulable state, propagation of upstream thrombus, venous compression from an adjacent fluid collection, anastomotic abnormality, and slow flow possibly due to rejection or other allograft issue. There may be a slightly higher prevalence of RVT in left lower quadrant transplants due to compression of the left common IA. The transplant kidney may be enlarged on US with reduced or absent venous flow. On spectral Doppler imaging, there is reversed U-shaped or plateau-like diastolic flow on the arterial waveforms.50 RV thrombosis may also be demonstrated on MR venography (Fig. 27.27). As noted previously, other causes of diastolic flow reversal include ATN, acute rejection; however, the flow reversal is usually not holodiastolic.46 RV thrombosis may be promptly treated with thrombectomy to salvage the transplant kidney. Infarction of the kidney may necessitate nephrectomy to prevent subsequent infection.51 27.4.5.7 Renal Vein Stenosis RV stenosis is extremely rare and may be related to compression by an adjacent collection, infection, narrowing at the venous anastomosis, and rejection.52 US findings include elevated intrarenal RIs and increased venous I. KIDNEY TRANSPLANTATION

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FIGURE 27.26 Pseudoaneurysm on angiography. Angiographic images of same patient in Fig. 27.23 demonstrate the neck of the pseudoaneurysm (A, arrow) and progressive filling with contrast (B, arrows). After embolization with metallic coils (C, arrow), no pseudoaneurysm is demonstrated.

FIGURE 27.27 Renal vein thrombosis. Complete diastolic flow reversal is noted in the renal arteries (A) with no flow in the renal vein on power Doppler imaging (B).

flow velocities and color aliasing (Fig. 27.28). Although color Doppler US will show a patent vein and venous anastomosis, the patient is at high risk for RV thrombosis.53 Treatment includes RV stenting (Fig. 27.29). One pitfall to be aware of is “overriding,” where proximity of the main RA to the main RV causes a similar appearance due to pulsation on the MRV. 27.4.5.8 Compartment Syndrome Increased abdominal pressures in the perioperative period may cause increased pressure in the extraperitoneal compartment causing allograft compression or decreased perfusion due to arterial kinking.54 Recognition of this entity necessitates urgent surgery with intraperitoneal placement of the allograft or retroperitoneal fasciotomy and decompression. US findings include diminished blood flow, intrarenal tardus parvus waveforms, and a patent RV.37

27.4.6 Transplant Kidney Torsion Torsion is a very rare complication that may occur within the first week to several months after surgery.55 On US, there is a change in the renal axis compared with baseline.16 The degree and duration of torsion affects the amount of vascular compromise.

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FIGURE 27.28 Main renal vein stenosis. Color Doppler images of the MRV at its anastomosis demonstrate narrowing and aliasing (A). Spectral Doppler images demonstrate elevated MRV velocities (B, 296 cm/s) but normal velocities in the iliac vein (C, 51 cm/s), consistent with MRV stenosis.

FIGURE 27.29 Renal vein stenosis, status post stenting Spectral Doppler images (same patient as Fig. 27.26) 3 months (A) and 5 months (B) after venous stenting demonstrate gradually improving velocities, decreasing to 160 cm/s and 59 cm/s, respectively.

27.4.7 Urinary Calculi Kidney transplant recipients are at higher risk of developing urinary calculi. Etiologies include persistent secondary hyperparathyroidism and hypercalcemia.56,57 As noted previously, because the transplant kidney is denervated, typical symptoms of renal colic are absent. US images may show echogenic foci with twinkle artifact. Small stones often may not demonstrate shadowing and often do not cause hydronephrosis. Hydronephrosis is present with obstructing stones. Antegrade pyelograms can also be used to locate the site and orientation of the stones. Percutaneous nephrostomy may be used to relieve renal obstruction.58 Lithotripsy is utilized to treat many cases of clinically significant stones. Percutaneous nephrostolithotomy may be used to treat upper tract stones.

27.4.8 Neoplasms Kidney transplant recipients are at significantly higher risk for developing carcinoma due to prolonged immunosuppression. Scott et al. reported a 6% prevalence of malignancy in these patients, predominantly skin cancers and lymphoma.59 Approximately 10% of all renal cell carcinomas occur in transplant kidneys.60 Patients with gross hematuria or glomerulopathy will undergo thorough evaluation including US and possible CT. The presence of a complex cystic mass is suggestive of renal cell carcinoma. However, posttransplant lymphoproliferative disorder (PTLD) and hemorrhagic cysts can have a similar appearance (Figs. 27.30 and 27.31). Percutaneous biopsy may be performed to determine the etiology of the mass. Non kidney-related malignancies are often treated conventionally. The recognition of PTLD is important as there are implications for immunosuppression. If the patient is diagnosed with PTLD, immunosuppressive therapy is either altered or ceased. PTLD is often associated with Epstein Barr virus (EBV). Other lesions occurring in the transplant kidney include cysts.

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FIGURE 27.30

Complex hypoechoic mass in transplant kidney. A new complex hypoechoic mass in the allograft was noted 7 years after transplantation. Biopsy revealed posttransplant lymphoproliferative disorder (PTLD).

FIGURE 27.31

Complex mass in a transplant kidney. A complex 3 cm mass is newly present in the upper pole of the transplant kidney (A and B) on longitudinal and transverse images. The presence of this mass is worrisome for renal cell carcinoma. The patient subsequently underwent a renal mass protocol CT scan.

27.4.9 Gastrointestinal and Herniation Complications The most common gastrointestinal (GI) complication of transplant patients is peptic ulcer-related GI tract hemorrhage.61 Adhesions may cause intestinal obstruction. A segment of bowel could herniate through a peritoneal defect and cause bowel or allograft compromise. Additionally, obturator herniation of the ureter may cause urinary obstruction.62 Patients receiving antibiotic therapy for C. difficile are at risk of developing pseudomembranous colitis.

27.5 OTHER IMAGING MODALITIES 27.5.1 Computed Tomography Computed tomography (CT) evaluation of the renal transplant is often performed for evaluation of peritransplant collections and for further evaluation of other complications detected by US. Hemorrhagic collections can often be differentiated from abscess and simple fluid collections due to differences in Hounsfield units and higher attenuating fluid layering in hemorrhagic collections (Fig. 27.32).63 CT urography is an excellent method to determine the site of urine leak in patients with urinomas. One downside to CT is the use of ionizing radiation and intravenous contrast. Because most clinicians prefer not to administer IV contrast, the density differences are key in trying to assess the findings. CT, if needed, is usually performed either after the US and/or nuclear medicine study.

27.5.2 Magnetic Resonance Imaging of Renal Transplant MRI in renal transplant can be utilized for the same indications as it is used for in native kidneys: Lesion characterization, evaluation of infection, assessment of peritransplant fluid collection, diagnosis of RAS, and evaluation of renal perfusion. Advantages of MRI include superior tissue differentiation when compared with other I. KIDNEY TRANSPLANTATION

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FIGURE 27.32 CT of transplant renal mass. Multiphase renal mass protocol CT in the same patient as Figure 27.31 demonstrates a partially hyperdense lesion with no definite enhancement on arterial (A), venous (B), and delayed (C) phases with no significant change in Hounsfield units compared with the noncontrast images (not shown). The lesion was thought to represent a hemorrhagic cyst, which was later confirmed at surgery.

FIGURE 27.33 MRA of transplant vessels. 3D volume rendered imaging from a follow-up MRA for suspected RAS on Doppler US demonstrates a widely patent renal artery and no significant stenosis.

modalities and the ability to image the surrounding tissues in greater detail.64 MR urography, MRA (Fig. 27.33), and MR renography offer a comprehensive transplant and peritransplant evaluation without the use of ionizing radiation.65 The MRI protocol in Table 27.4 is an example of types of sequences employed to evaluate the allograft. The last 2 rows address MRA and RAS. Cyst can be differentiated from a malignant renal neoplasm by lack of significant enhancement. However, in cases where gadolinium is not administered, differentiation of a complex cyst from malignancy may be difficult. Cysts may be simple or complex, which may contain hemorrhagic or proteinaceous debris. In renal transplant patients, lymphoproliferative disorder may also present as a focal renal neoplasm. If a renal lesion cannot be characterized by imaging alone, biopsy may be indicated. The diagnosis of RAS may be confirmed by MRA if indicated. Diffusion-weighted imaging (DWI) is another MRI technique that can be used to evaluate the renal parenchyma in suspected neoplasm, infection, or vascular compromise.66 DWI images may show increased signal along with corresponding low signal on apparent diffusion coefficient images with these entities.67 Although not routinely performed, renal perfusion techniques comparing transplant kidney enhancement and concentration of gadolinium in the aorta can be used to calculate renal blood flow.68,69 Gadolinium administration aids in detection of vascular and nonvascular complications related to transplant. Although gadolinium is generally considered safer than iodinated contrast given for CT scans, it carries a minute risk of nephrogenic systemic fibrosis and nephrogenic fibrosing dermopathy. These entities are more of a concern when the patient has acute and/or chronic renal failure or if the patient is on dialysis. The American College of Radiology (ACR) recommends avoiding gadolinium use in patients with an eGFR less than 30 mL/min/1.73 m2.70 I. KIDNEY TRANSPLANTATION

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MRI Sequences to Evaluate Renal Transplants

MRI Sequence

Purpose

Coronal T2 single-shot turbo spin echo

Assess of fluid collections and urinary tract obstruction; evaluate allograft parenchyma; characterize renal lesion

Axial T2 TSE without and with fat suppression

Same as coronal T2

Axial T1 GRE in-phase and opposed-phase

Evaluate allograft parenchyma; assess for corticomedullary differentiation; characterize fluid collections

Ultrafast single short EPI sequence with navigator (DWI); b-values (mm/s2): 0, 500, 100

Lesion detection; limited characterization

Axial and coronal T1-weighted fast spoiled gradient echo sequence for dynamic imaging pre contrast and post intravenous gadolinium axial images in the arterial, venous, and delayed phases

Evaluation allograft vasculature and perfusion; evaluate enhancement characteristics of renal lesions

Coronal 3D fast spoiled gradient echo with fat suppression, obtained after the dynamic series for delayed contrast-enhanced images

Evaluate allograft function by gadolinium excretion into urinary tract, assess for perfusion defects and lesions

Coronal static fluid T2 TSE

Evaluate excretory system

For suspected renal artery stenosis, do the following as the first post gadolinium sequence:Coronal 3D contrast-enhanced MR Angiography (3DMRA) followed by delayed T1 post contrast images as above

Evaluate allograft vasculature and perfusion; assess for renal artery stenosis

For noncontrast MRA:3D balanced steady-state free precession T2 with fat suppression

Evaluate allograft vasculature when gadolinium is contraindicated

FIGURE 27.34 Treatment of renal artery stenosis. High-grade stenosis of the proximal main renal artery (A, arrow), narrowed to 2 mm and subsequently dilated to 9 mm with angioplasty and stent placement (B, arrow).

27.5.3 Interventional Radiology Although the majority of the diagnostic work on transplant dysfunction is performed by US with MRI, CT, and nuclear scintigraphy as complimentary modalities, Interventional Radiology (IR) plays an important role in the diagnosis and treatment of many transplant-related complications. Endovascular therapy (Fig. 27.34), percutaneous urinary procedures, and drainage of fluid collections are some of the procedures that may provide definitive treatment or may stabilize the patient prior to surgical intervention as discussed previously.10 Angiography procedures are usually performed with low- or iso-osmolar contrast to minimize the risk of contrast-induced nephropathy. Carbon dioxide (CO2) angiography is often performed in cases of renal insufficiency to avoid potentially nephrotoxic iodinated contrast (Fig. 27.35).

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FIGURE 27.35 CO2 Angiogram. CO2 angiogram demonstrates focal narrowing of the proximal duplicated renal arteries (A). Post angioplasty (B), the renal artery narrowing is improved.

FIGURE 27.36 Biopsy set up. The upper or lower pole cortex should be targeted for biopsy with a tangential trajectory that avoids the renal hilum (A and B). Assessment of the distance to the cortex is important to determine appropriate needle length as well as identification of any overlying vessels (C).

There are special considerations that must be taken into account at the time of the interventional procedure. For suspected RAS, nonselective aortoiliac arteriography is performed prior to selective transplant arteriography to exclude an inflow-preanastomotic stenosis in the recipient arteries, which can mimic RAS.10 Other procedures performed include venogram for diagnosis of RV thrombosis and embolization of arteriovenous fistulas and pseudoaneurysms (Fig. 27.26). The type of anastomosis, e.g., end-to-side with the external IA or end-to-end with the internal IA, should be known ahead of time as it affects the angiographic approach. Percutaneous renal transplant biopsy remains the gold standard to diagnose renal transplant dysfunction. Indications for biopsy include signs of acute or chronic allograft rejection such as elevated creatinine, decreased urine output, proteinuria, unexplained fever, hypertension, and edema.71 Coagulation parameters should be assessed prior to biopsy. The biopsy is performed with real-time guidance under ultrasound. The upper or lower pole cortex should be targeted with the trajectory oriented in such a way that the needle only traverses the renal cortex (Fig. 27.36).72 Needle guidance utilization aids in keeping a steady trajectory during biopsy (Fig. 27.37). Biopsies can be safely performed with 18-gauge core biopsy needles, and the specimens can be assessed for glomeruli utilizing a dissecting microscope. At the author’s institution, two 18-gauge core specimens are adequate for electron microscopy and immunofluorescence. Complications from renal transplant biopsy are reported to range from 0.06% to 13%.71 As stated above, AVFs (Fig. 27.38) and pseudoaneurysms are the most common and can be treated with embolization if symptomatic or enlarging. Extrarenal pseudoaneurysm is a rare entity that may occur due to surgical technique or infection, usually at the site of arterial anastomosis. Due to the potential for rupture, this entity should be treated expeditiously.73 The pseudoaneurysm may be asymptomatic or present as a large, pulsating mass with abdominal pain. Extrinsic compression of the renal vessels may result in hypertension or allograft dysfunction. Although transplant nephrectomy is the treatment of choice to prevent rupture, coil embolization and US-guided thrombin injection have been reported

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FIGURE 27.37 Biopsy with needle guidance. The needle should be seen in its entirety and imaged as it enters the cortex (A) and after firing (B) to confirm appropriate placement. Needle guidance helps keep the biopsy needle on the intended trajectory (green dots).

FIGURE 27.38 Posttransplant angiogram. Angiogram performed after biopsy demonstrates a small AV fistula (red (gray) arrow). Because the AVF was small and did not demonstrate significant shunting, embolization was not performed.

in attempt to salvage the allograft.22,74,75 For patients with IA pseudoaneurysm after transplant nephrectomy, an endovascular stent graft is a treatment option.76

27.6 NEW DEVELOPMENTS IN TRANSPLANT ULTRASOUND 27.6.1 Contrast-Enhanced Ultrasonography Contrast-enhanced ultrasonography (CEUS) is currently an investigative tool in the United States but has been utilized successfully in Europe and Asia as a diagnostic tool for renal lesion assessment. CEUS can help differentiate cystic from solid lesions and benign complex cystic lesions from cystic lesions with enhancing components and more suspicious features (Figs. 27.39 and 27.40). Several studies have shown CEUS provides information about causes of graft dysfunction by quantitative microvascular perfusion assessment. Chronic allograft nephropathy can be diagnosed earlier with CEUS than with conventional color Doppler US by quantification of arterial inflow.77 CEUS has also been shown to help differentiate acute rejection from ATN in patients with delayed graft function. Patients with acute rejection I. KIDNEY TRANSPLANTATION

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FIGURE 27.39 Posttransplant lymphoproliferative disorder. CT performed for abdominal pain in a patient with a remote history of renal transplant revealed a hypoattenuating mass in the allograft (A), which was hypoechoic and hypovascular on US (B and C). Images 15 and 30 s after contrast administration (D and E) reveal a persistently hypoenhancing mass, biopsy proven PTLD. Courtesy of Mittul Gulati, MD.

FIGURE 27.40

RCC in renal transplant. Grayscale imaging of the transplant kidney suggests the presence of a hypoechoic mass in the medial upper pole of the kidney mass (A, arrows). Administration of contrast more clearly defines the mass (B, arrow) which turned out to be renal cell carcinoma.

demonstrated significantly longer inflow time of microbubbles into the renal cortex and pyramids than patients with ATN in a recent study.78

27.6.2 Elastography Ultrasound elastography is a noninvasive method to evaluate tissue stiffness. Although initially developed to assess liver stiffness, the applications of elastography have increased and now include assessment of renal allograft fibrosis.79,80 One method utilizes acoustic radiation force impulse (ARFI) quantification to measure stiffness of the allograft. The region of interest is mechanically excited by short acoustic pulses to cause tissue displacement; there is diminished tissue displacement in more fibrotic tissue. Factors affecting the results include the I. KIDNEY TRANSPLANTATION

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FIGURE 27.41 Transplant renal artery aneurysm. Aneurysm of the distal main renal artery is identified on both CT and on power Doppler US images. Fusing the CT images with ultrasound allows for improved visualization of the aneurysm by US.

FIGURE 27.42

B-Flow imaging of transplant renal artery. (A) Color and spectral Doppler of the mid-portion of a transplant renal artery. Elevated velocities around 250 cm/s raised concern for stenosis. Please note improper angle correction on the image. (B) B-Flow angiographic style image at the time of Doppler acquisition shows normal caliber of the proximal and mid renal artery and no stenosis.

region of interest, transducer pressure, angle of incidence, and target depth.81 Several studies have demonstrated that ARFI positively correlates with the grade of allograft fibrosis.82,83

27.6.3 Ultrasound With Fusion Ultrasound images can be fused with CT, MRI, PET, and PET-CT images to improve targeting lesions that may be suboptimally visualized on US. This may be helpful in detecting parenchymal lesions and vascular abnormalities on US (Fig. 27.41).

27.6.4 B-Flow Imaging B-Flow imaging is a useful adjunct to conventional Doppler to confirm or clarify Doppler findings or suggest the need for additional angiographic imaging (Fig. 27.42).84 It is a non-Doppler ultrasound technique of vascular imaging that directly displays of intravascular echoes during real-time grayscale imaging, similar to an angiogram. B-flow provides better visualization of the boundary between flowing blood and the vessel wall (Figs. 27.43 and 27.44) and can help distinguish true vascular stenosis from a false-positive finding on

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FIGURE 27.43 Presumed renal artery stenosis on US. (A) Color and spectral Doppler of a renal transplant demonstrates elevated peak systolic velocity in the renal artery at the anastomosis (.300 cm/s) suggestive of stenosis. (B) B-Flow image performed at the same time shows the artery at the anastomosis to be patent. The elevated velocity observed on spectral Doppler is most likely due to the angulated course of the renal artery (arrow) near the anastomosis.

FIGURE 27.44 Renal artery stenosis on B-Flow imaging. (A) Color and spectral Doppler image of the proximal transplant renal artery demonstrates elevated velocities (391 cm/s) and color aliasing. Incorrect angle correction is noted, but the Doppler findings of stenosis persisted even after optimization. (B) B-Flow image of the proximal transplant renal artery demonstrates a caliber change that was not apparent on the Doppler image. B-Flow is more helpful in identifying morphologic vascular abnormalities.

conventional Doppler. Additionally, availability of the technology on curvilinear transducers allows for evaluation of deeper vascular structures (Fig. 27.45). Advantages of B-Flow over conventional Doppler include: • • • • •

Prevention “blooming” or “bleeding” artifact encountered with color Doppler (Fig. 27.46) Improved resolution and definition of vessel wall Less angle-dependent Improved imaging of tortuous and small vessels Higher frame rate than conventional color Doppler and improved resolution

B-flow imaging can demonstrate the presence or absence of blood flow to the transplant kidney when CDUS is suggestive of pathology or equivocal (Figs. 27.47 and 27.48).

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FIGURE 27.45

Renal vein thrombosis. (A) Grayscale image of a right lower quadrant renal transplant demonstrates echogenic material filling and expanding the transplant renal vein (arrow). (B) No demonstrable flow is present on color Doppler imaging. (C) B-Flow image of the renal vein (arrow) shows no signal (dark lumen) supporting complete loss of flow. (D) MRA time of flight image of the RLQ renal transplant demonstrates central loss of flow related signal in the renal vein. Thrombus is also present in the visualized right iliac vein.

FIGURE 27.46 B-Flow imaging of AV Fistula. Spectral Doppler image of a kidney transplant (A) demonstrates an AVF as evidenced by color aliasing, tissue vibration artifact, and mixing of arterial and venous signal. B-Flow image (B) of same kidney more clearly depicts the true size of the AVF (arrows). CO2 angiogram (C) confirmed similar size of the AVF (arrow), which was successfully treated with coil embolization.

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FIGURE 27.47 B-Flow of renal perfusion. Color Doppler image (A) of a renal transplant with apparent absence of perfusion to periphery (bracket). Lack of signal to upper pole is due to technical factors. B-Flow image (B) of same transplant demonstrates improved visualization of vessels beyond the arcuate arteries (bracket).

FIGURE 27.48 Heterogeneous perfusion. Color Doppler image (A) of a renal transplant demonstrates patchy, very heterogeneous perfusion. B-Flow angiographic style image (B) confirms the paucity of intraparenchymal flow. Biopsy demonstrated underlying chronic rejection.

27.7 CONCLUSION US is the mainstay of renal transplant imaging as a majority of transplant and peritransplant pathologies can be visualized sonographically. MRI, nuclear scintigraphy, and CT are complimentary modalities that can confirm and demonstrate additional pathologies and can also be fused with US images for improved visualization of pathology. IR plays a vital role in diagnosis and treatment of transplant-related complications. New innovations such as contrast and elastography show promise as noninvasive methods of diagnosing rejection. B-flow imaging is a new sonographic imaging tool to provide superior resolution of the vasculature that can improve diagnostic confidence of transplant-related pathologies.

References 1. National Kidney Foundation. https://www.kidney.org/news/newsroom/factsheets/Organ-Donation-and-Transplantation-Stats. Accessed June 23, 2015. 2. American College of Radiology (ACR); Society for Pediatric Radiology (SPR); Society of Radiologists in Ultrasound (SRU); American Institute of Ultrasound in Medicine (AIUM). AIUM practice guideline for the performance of an ultrasound examination of solid-organ transplants. J Ultrasound Med 2014;33(7):1309 20. 3. Veale JL, Singer JS, Gritsch HA. In: Danovitch GM, editor. The transplant operation and its surgical complications in handbook of kidney transplantation. 5th ed Philadelphia, PA: Lippincott Williams & Wilkins; 2010. p. 181 97.

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4. Gritsch HA, Danovitch GM, Wilkinson A. Kidney transplantation. In: Hakim NS, Danovitch GM, editors. Transplantation surgery. London: Springer-Verlag; 2001. p. 135 63. 5. Kayler L, Kang D, Molmenti E, Howard R. Kidney transplant ureteroneocystostomy techniques and complications: review of the literature. Transplant Proc 2010;42:1413 20. 6. Tsai WE, Ettenger RB. Kidney Transplantation in Children, in Handbook of Kidney Transplantation Lippincott Williams & Wilkins. Danovitch GM, ed. Fifth ed. 2010. Philadelphia, PA. pp. 355 388. 7. Kocak T, Nane I, Ander H, Ziylan O, Oktar T, Ozsoy C. Urological and surgical complications in 362 consecutive living related donor kidney transplantations. Urol Int 2004;72:252 6. 8. Nie ZL, Zhang KQ, Li QS, et al. Urological complications in 1,223 kidney transplantations. Urol Int 2009;83:337 41. 9. Zavos G, Pappas P, Karatzas T, et al. Urological complications: analysis and management of 1525 consecutive renal transplantations. Transplant Proc 2008;40:1386 90. 10. Kobayashi K, Censullo ML, Rossman LL, et al. Interventional radiologic management of renal transplant dysfunction: indications, limitations, and technical considerations. Radiographics 2007;27:1009 130. 11. Gogus C, Yaman O, Soygur T, Beduk Y, Gogus O. Urological complications in renal transplantation: long-term follow-up of the Woodruff ureteroneocystostomy procedure in 433 patients. Urol Int 2002;69:99 101. 12. Bennett LN, Voegeli DR, Crummy AB, McDermott JC, Jensen SR, Sollinger HW. Urologic complications following renal transplantation: role of interventional radiologic procedures. Radiology 1986;160:531 6. 13. Mundy AR, Podesta ML, Bewick M, Rudge CJ, Ellis FG. The urologic complications of 1000 renal transplants. Br J Urol 1981;53:397 402. 14. Barrero R, Fijo J, Fernandez-Hurtado M, et al. Vesicoureteral reflux after kidney transplantation in children. Pediatr Transplant 2007;11:498 503. 15. Akbar SA, Jafri SZ, Amendola MA, Madrazo BL, Salem R, Bis KG. Complications of renal transplantation. RadioGraphics 2005;25 (5):1335 56. 16. Rodgers SK, Sereni CP, Horrow MM. Ultrasonographic evaluation of the renal transplant. Radiol Clin North Am 2014;52:1307 24. 17. Bhagat VJ, Gordon RL, Osorio RW, et al. Ureteral obstructions and leaks after renal transplantation: outcome of percutaneous antegrade ureteral stent placement in 44 patients. Radiology 1998;209:159 67. 18. Fontaine AB, Nijjar A, Rangaraj R. Update on the use of percutaneous nephrostomy/balloon dilation for the treatment of renal transplant leak/obstruc- tion. J Vasc Interv Radiol 1997;8:649 53. 19. Kahan BD. Surgical complications. In: Kahan BD, Ponticelli C, editors. Principles and practice of renal transplantation. London: Martin Dunitz; 2000. p. 219 50. 20. Pozniak MA, Dodd GD, Kelcz F. Ultrasonographic evaluation of renal transplantation. Radiol Clin North Am 1992;30:1053 66. 21. Gillenwater JY. Renal transplantation. In: Fletcher SM, Novick AC, editors. Adult and pediatric urology. 4th ed Baltimore, MD: Lippincott Williams & Wilkins; 2002. p. 941 54. 22. Greenberg BM, Perloff LJ, Grossman RA, Naji A, Barker CF. Treatment of lymphocele in renal allograft recipients. Arch Surg 1985;120:501 4. 23. Khauli RB, Stoff JS, Lovewell T, Ghavamian R, Baker S. Post-transplant lymphoceles: a critical look into the risk factors, pathophysiology and management. J Urol 1993;150:22 6. 24. Zietek Z, Sulikowski T, Tejchman K, et al. Lymphocele after kidney transplantation. Transplant Proc 2007;39:2744 7. 25. Amendola MA, Montalvo BM. Percutaneous management of lymphoceles. In: Banner MP, Mitchell CW, editors. Radiologic interventions: uroradiology. Philadelphia, PA: Lippincott Williams & Wilkins; 1998. p. 1487 93. 26. Ulrich F, Niedzwiecki S, Fikatas P, et al. Symptomatic lymphoceles after kidney transplantation - multivariate analysis of risk factors and outcome after laparoscopic fenestration. Clin Transplant 2010;24:273 80. 27. Fuller TF, Kang SM, Hirose R, Feng S, Stock PG, Freise CE. Management of lymphoceles after renal transplantation: laparoscopic versus open drainage. J Urol 2003;169(6):2022 5. 28. Brown ED, Chen MY, Wolfman NT, et al. Complications of renal transplantation: evaluation with US and radionuclide imaging. Radiographics 2000;20:607 22. 29. Lechevallier E, Dussol B, Luccioni A, et al. Posttransplantation acute tubular necrosis: risk factors and implications for graft survival. Am J Kidney Dis 1998;32(6):984 91. 30. Baxter GM. Ultrasound of renal transplantation. Clin Radiol 2001;56:802 18. 31. Pozniak MA, Kelcz F, D’Alessandro A, et al. Sonography of renal transplants in dogs: the effect of acute tubular necrosis, cyclosporine nephrotoxicity and acute rejection on resistive index and renal length. AJR Am J Roentgenol 1992;158:791 7. 32. Myers BD, Sibley R, Newton L, et al. The long term course of cyclosporine associated chronic nephropathy. Kidney Int 1988;33:590 600. 33. Dodd GD, Tublin AS, Sajko AB. Imaging of vascular complications associated with renal transplants. AJR Am J Roentgenol 1991;157:449 59. 34. Rees CR, Palmaz JC, Becker GJ, et al. Palmaz stent in artherosclerotic stenoses involving the ostia of the renal arteries: preliminary report of a multicenter study. Radiology 1991;181:507 14. 35. Snider JF, Hunter DW, Moradian GP, et al. Transplant renal artery stenosis: evaluation with duplex sonography. Radiology 1989;172:1027 30. 36. Thalhammer C, Aschwanden M, Mayr M, Koller M, Steiger J, Jaeger KA. Duplex sonography after living donor kidney transplantation: new insights in the early postoperative phase. Ultraschall Med 2006;27:141 5. 37. Horrow MM, Parsikia A, Zaki R, et al. Immediate postoperative sonography of renal transplants: vascular findings and outcomes. AJR Am J Roentgenol 2013;201:W479 86. 38. Brabrand K, Holdaas H, Gunther A, Midtvedt K. Spontaneous regression of initially elevated peak systolic velocity in renal transplant artery. Transpl Int 2011;24:555 9. 39. Gunther A, Foss A, Holdaas H, et al. Increased peak systolic velocity in the renal artery of paedi- atric kidneys transplanted to adult recipients. Nephrol Dial Transplant 2008;23:4041 3.

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40. De Morais RH, Muglia VF, Mamere AE, et al. Duplex Doppler sonography of transplant renal artery stenosis. J Clin Ultrasound 2003;31:135 41. 41. Gao J, Ng A, Shih G, et al. Intrarenal color duplex ultrasonography. J Ultrasound Med 2007;26:1403 18. 42. Gottlieb RH, Lieberman JL, Pabico RC, Waldman DL. Diagnosis of renal artery stenosis in transplanted kidneys: value of Doppler waveform analysis of the intrarenal arteries. AJR Am J Roentgenol 1995;165:1441 6. 43. Beecroft J, Rajan D, Clark T, Robinette M, Stavropoulos S. Transplant renal artery stenosis: outcome after percutaneous intervention. J Vasc Interv Radiol 2004;15:1407 13. 44. Raynaud A, Lucino S, de Almeida Augusto MC, Beyssen B, Gaux JC. Percutaneous endoluminal angioplasty of the transplanted kidney: long term follow up. J Radiol 1994;75:81 6. 45. Friedewald SM, Molmenti EP, Friedewald JJ, Dejong MR, Hamper UM. Vascular and nonvascular complications of renal transplants: sonographic evaluation and correlation with other imaging modalities, surgery, and pathology. J Clin Ultrasound 2005;33:127 9. 46. Tchelepi H, Grant EG, Ralls PW. Doppler ultrasound measurements in abdominal organ transplantation. In: Goldberg BB, McGahan JP, editors. Atlas of ultrasound measurements. 2nd edition Philadelphia, PA: Mosby; 2006. p. 403 12. 47. Delles C, Wittmann M, Renders L, et al. Restoration of renal allograft function by endovascular stenting of an iliac artery dissection. Nephrol Dial Transplant 2002;17(6):1116 18. 48. Middleton WD, Kellman GM, Melson GL, et al. Postbiopsy renal transplant arteriovenous fistulas: color Doppler US characteristics. Radiology 1989;171:253 7. 49. Martinez T, Palomares M, Bravo JA, et al. Biopsy-induced arteriovenous fistula and venous aneu- rysm in a renal transplant. Nephrol Dial Transplant 1998;13:2937 9. 50. Kaveggia LP, Parrella RR, Grant EG, et al. Duplex Doppler sonography in renal allografts: the significance of reversed flow in diastole. AJR Am J Roentgenol 1990;155:295 8. 51. Tublin ME, Dodd G. Imaging of organ transplantation. Radiol Clin North Am 1995;33:447 58. 52. Obed A, Uihlein DC, Zorger N, et al. Severe renal vein stenosis of a kidney transplant with beneficial clinical course after successful percutaneous stenting. Am J Transplant 2008;8:2173 7. 53. Gao J, Ng A, Shih G, et al. Intrarenal color duplex ultrasonography. J Ultrasound Med 2007;26:1403 18. 54. Ball CG, Kirkpatrick AW, Yilmaz S, et al. Renal allograft compartment syndrome: an underappreciated postoperative complication. Am J Surg 2006;191:619 24. 55. Wong-You-Cheong JJ, Grumbach K, Krebs TL, et al. Torsion of intraperitoneal renal transplants: imaging appearances. AJR Am J Roentgenol 1998;171:1355 9. 56. Cho DK, Zackson DA, Cheigh J, et al. Urinary calculi in renal transplant recipients. Transplantation 1988;45:899 902. 57. de Francisco AM, Riancho JA, Amado JA, et al. Calcium, hyperparathyroidism, and vitamin A metabolism after kidney transplantation. Transplant Proc 1987;19:3721 3. 58. Hefty T. Complications of renal transplantation: the practicing urologist’s role. AUA update series. Lesson 8, vol X. Linthicum, MD: American Urological Association; 1991. p. 58 61. 59. Scott MH, Sells RA. Primary adenocarcinoma in a transplanted cadaveric kidney. Transplantation 1988;46:157 8. 60. Penn I, Brunson ME. Cancers after cyclosporine therapy. Transplant Proc 1988;20(Suppl. 3):885 92. 61. Karakayali H, Emiroglu R, Sevmis S, et al. Post- operative surgical complications in renal transplant recipients: one center’s experience. Transplant Proc 2001;33:2683 4. 62. Weingarten K, D’Agostino H, Dunn J, et al. Obturator herniation of the ureter in a renal transplant recipient causing hydronephrosis: perioperative percutaneous management. J Vasc Interv Radiol 1996;7:939 41. 63. Sciascia N, Zompatori M, Di Scioscio V, et al. Multidetector CT-urography in the study of urological complications in renal transplant. Radiol Med (Torino) 2002;103(5-6):501 10. 64. Hohenwalter MD, Skowlund CJ, Erickson SJ, et al. Renal transplant evaluation with MR angiography and MR imaging. Radiographics 2001;21:1505 17. 65. Browne RFJ, Tuite DJ. Imaging of the renal transplant: comparison of MRI with duplex sonography. Abdom Imaging 2006;31:461 82. 66. Cova M, Squillaci E, Stacul F, et al. Diffusion-weighted MRI in theevaluation of renal lesions: preliminary results. Br J Radiol 2004;77:851 7. 67. Thoeny HC, De Keyzer F, Oyen RH, Peeters RR. Diffusion-weighted MR imaging of kidneys in healthy volunteers and patients with parenchymal diseases: initial experience. Radiology 2005;235:911 17. 68. Huang AJ, Lee VS, Rusinek H. Functional renal MR imaging. Magn Reson Imaging Clin N Am 2004;12:469 86. 69. Martirosian P, Klose U, Mader I, Schick F. FAIR true-FISP perfusion imaging of the kidneys. Magn Reson Med 2004;51:353 61. 70. ACR Manual on Contrast Media, Version 10, 2015. 71. Ahmad I. Biopsy of the transplanted kidney. Semin Interv Radiol 2004;21:275 81. 72. Deurdulian C, French N. Ultrasound-guided intervention in the abdomen and pelvis: review from A to Z. In: Chong WK, Dogra VS, editors. Abdominal ultrasound. Ultrasound clin, 9. Philadelphia, PA: Elsevier Science Publishers; 2014. p. 793 820. 4. 73. Donckier V, De Pauw L, Ferreira J, et al. False aneurysm after transplant nephrectomy: report of two cases. Transplantation 1995;60:303 4. 74. Koo CK, Rodger S, Baxter GM. Extra-renal pseudoaneurysm: an uncommon complication following renal transplantation. Clin Radiol 1999;54:755 8. 75. Reus M, Morales D, Vazquez V, Llorente S, Alonso J. Ultrasound-guided percutaneous thrombin injection for treatment of extrarenal pseudoaneurysm after renal transplantation. Transplantation 2002;74:882 4. 76. Peel RK, Patel J, Woodrow G. Iliac artery false aneurysm following renal allograft: presentation with non-specific inflammatory response and treatment by endovascular stent graft. Nephrol Dial Transplant 2003;18:1939 40. 77. Schwenger V, Hinkel UP, Nahm AM, et al. Real-time contrast-enhanced sonography in renal transplant recipients. Clin Transplant 2006;20 (Suppl. 17):51 4.

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78. Grzelak P, Szymczyk K, Strzelczyk J, et al. Perfusion of kidney graft pyramids and cortex in contrast-enhanced ultrasonography in the determination of the cause of delayed graft function. Ann Transplant 2011;16(1):48 53. 79. Zarzour JG, Lockhart ME. Ultrasonography of the renal transplant. In: Chong WK, Dogra VS, editors. Abdominal ultrasound. Ultrasound clin, 9. Philadelphia, PA: Elsevier Science Publishers; 2014 (4)683-655. 80. Sommerer C, Scharf M, Seitz C, et al. Assessment of renal allograft fibrosis by transient elastography. Transpl Int 2013;26(5):545 51. 81. He WY, Jin YJ, Wang WP, et al. Tissue elasticity quantification by acoustic radiation force impulse for the assessment of renal allograft function. Ultrasound Med Biol 2014;40(2):322 9. 82. Stock KF, Klein BS, Vo Cong MT, et al. ARFI-based tissue elasticity quantification in comparison to histology for the diagnosis of renal transplant fibrosis. Clin Hemorheol Microcirc 2010;46(2-3):139 48. 83. Syversveen T, Brabrand K, Midtvedt K, et al. Assessment of renal allograft fibrosis by acoustic radiation force impulse quantification a pilot study. Transpl Int 2011;24(1):100 5. 84. Wachsberg RH. B-flow imaging of the hepatic vasculature: correlation with color Doppler sonography. Am J Roentgenol 2007;188(6): W522 33.

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C H A P T E R

28 Immune Monitoring in Kidney Transplantation Mark Nguyen1, Anna Geraedts2 and Minnie Sarwal1 1

University of California, San Francisco, San Francisco, CA, United States 2Maastricht University, Maastricht, The Netherlands

28.1 INTRODUCTION In the modern age of immunosuppression, 1-year allograft survival has improved, but overall graft and patient survival have remained largely unchanged.1,2 With the recently revised kidney transplant allocation system, larger numbers of sensitized patients will be receiving transplants.3 Previously, patients with high levels of human leukocyte antigen (HLA) antibodies remained on the waitlist due low likelihood of negative crossmatching. Those patients are now given increased priority when a matching organ becomes available. Likewise, advances in desensitization and induction therapies have allowed for recipients with ABO incompatibility and high panel reactive antibody (PRA) levels to receive organs.4 These sensitized patients are at much higher risk for immune mediated graft failure; hence, it is essential to monitor the global immune response to prevent or diagnose graft injury. The etiology of long-term graft loss is multifactorial in nature but a large percentage is inherently related to immunosuppression.5 Calcineurin inhibitor (CNI) toxicity and BK virus nephropathy, both classic cases of overimmunosuppression, are serious posttransplant complications and associated with progression to dialysis dependence.6,7 On the contrary, alloimmunity, an issue with under-immunosuppression, has been implicated as a major contributor to graft failure in the setting of cellular rejection, acute and chronic antibody-mediated rejection (AMR), and transplant glomerulopathy.8 Distinguishing between the types of rejection is clinically difficult and generally requires histopathology (i.e., biopsy). Finding the right balance of immunosuppression may be the key to improved allograft outcomes. Therefore, immune monitoring is critical in preventing rejection and improvement may make invasive biopsies avoidable in the future. Successful monitoring for rejection requires a deep understanding of the immune response and the development of tools that take advantage of that knowledge. Immunity, whether innate or adaptive, is the culmination of complicated cellular and molecular processes resulting in protection from “foreign objects” or antigens. Ongoing investigation of this complex network of interactions has elucidated several important pathways and markers that provide insight into the current status of immunosuppression.8 11 In order to provide reliable information, assays that monitor these processes need to be sensitive, specific, reproducible, cost-effective, and readily available. Furthermore, results from these monitoring tools need to have quick turnaround and be easily interpretable so appropriate measures can be taken prior to graft injury or failure. Here we review the current offerings of post renal transplant monitoring, their shortcomings, and emerging assays that are shaping the future immune monitoring.

28.2 CURRENT STANDARDS OF IMMUNE MONITORING The universally accepted standards for monitoring renal function include routine clinic follow-up, serial serum creatinine measurements, and urine protein quantification; however, these current modalities lack sensitivity and Kidney Transplantation, Bioengineering, and Regeneration. DOI: http://dx.doi.org/10.1016/B978-0-12-801734-0.00028-X

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Markers predicting higher risk of graft injury Normal Allograft

Gene profile (blood and/or urine) indicating allograft injury Transcriptomic alteration

Histologic Alteration

Clinical Allograft

Graft Loss

Evidence of subclinical rejection or interstitial fibrosis/tubular atrophy on protocol biopsies

Loss of GFR Proteinuria Overt rejection

Dialysis dependence Retransplantation

FIGURE 28.1

Progression of allograft injury. Patients are thought to have genetic signatures that indicate a higher risk kidney injury prior to histologic or clinical evidence being apparent. Interventions prior to histologic or clinical alterations may prevent progression to allograft failure.

specificity with detection of injury in clinically stable patients (i.e., subclinical rejection (SCR)). Drug level monitoring and protocol biopsies are additional monitoring tools unique to kidney transplant patients. These modalities provide critical information needed to guide management and improve long-term outcomes. Despite clear clinical benefit, there are limitations which have led to the development of noninvasive assays with the aim of detecting subclinical injury prior to significant graft dysfunction (Fig. 28.1).

28.2.1 Drug Level Monitoring—Calcineurin Inhibitor Levels CNIs are currently the standard of care for immunosuppression in most kidney transplant recipients. Cyclosporine (CsA) and tacrolimus have dramatically improved short-term allograft outcomes since their adoption into clinical use. Under-dosing has been associated with higher rates of acute rejection and over-dosing increases the risk of electrolyte disturbances, metabolic derangements, and nephrotoxicity. Given the narrow therapeutic index, drug monitoring is vital with CNIs. The pharmacokinetics of CNI can be inconsistent and dependent on a number of variables such as presence of meals, variability of gastrointestinal motility (i.e., diarrhea), concurrent usage of medications affecting CYP450 3A4 activity, and decreased renal function.12 15 Due to inter- and intrapatient variability, drug monitoring allows for individualization of drug dosing in order to ensure efficacy and limit toxicity. Although there have not been randomized control trials comparing the outcomes for monitoring and not monitoring, it is generally accepted that drug monitoring is considered favorable.

28.2.2 Cyclosporine Monitoring Cyclosporine is a cyclic polypeptide that is highly insoluble in water, requiring suspension in emulsions for administration. Sandimmune, the first formulation of cyclosporine, had unpredictable bioavailability resulting in the development of Neoral.12,16 Neoral has been shown to be comparatively safe and tolerable in renal transplant patients, the majority of CsA dosing is based on pharmacokinetic trials using the improved formulation.17,18 Drug exposure of cyclosporine when measured after 4 hours (AUC0 4) has been shown to correlate well with the AUC over the entire 12-hour dosing interval.19 This is consistent with the thought that the highest variability in exposure occurs immediately after administration during the absorptive phase.20 Lower levels of AUC0 4 in

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TABLE 28.1

Target Cyclosporine Levels

Time (months)

Target C0 (ng/mL)

Target C2 (ng/mL)

0 3

200 300

1150 1550

3 6

125 250

800 1200

6 12

125 175

750 900

. 12

100 175

. 600

Target levels in the lower end of the range with use of thymoglobulin induction. C0, trough level; C2, level at 2 h after administration.

TABLE 28.2 Differences in Toxicity Profile Between Cyclosporine and Tacrolimus Adverse effect

Cyclosporine

Tacrolimus

New onset diabetes mellitus

m

mm

Dyslipidemia

m

Hypertension

mm

Osteopenia

m

Renal insufficiency

m

m

Neurotoxicity

m

mm

m

m

Diarrhea, nausea/vomiting Hyperkalemia

m

m

Hypomagnesemia

m

m

Hyperuricemia, gout

m

Gingival hyperplasia

m

Hirsutism

m

Malignancy

m

m

renal transplant patients have been shown to increase the risk of acute rejection. Conversely, the same study reported higher levels that are associated with nephrotoxicity and metabolic derangements. Compared to trough levels (C0), levels at 2 hours after drug administration (C2) correlate more closely to the AUC0 4 with a correlation (r2) of 0.80 versus 0.13 for C0.21 As such, it has been recommended that C2 be used for drug monitoring; however, most centers tend to use C0 due to ease of implementation in the outpatient setting. Furthermore, it has not been shown that using C2 or AUC0 4 over C0 offers a benefit in allograft outcomes. The risk of rejection is highest during the first 3 months following transplantation. Attaining goal cyclosporine levels immediately posttransplant has been shown to remarkably attenuate this risk, as demonstrated in a number of studies.22,23 As time passes, the risk of rejection decreases, so CNI target levels and doses should be lowered accordingly. The use of antibody therapy with induction agents have allowed for lower CNI goal levels reducing the risk of CNI toxicity.24,25 Goal cyclosporine levels are shown in Table 28.1.

28.2.3 Tacrolimus Monitoring Tacrolimus was first approved for use in transplant patients in 1994. Like cyclosporine, it is insoluble in water and has similar immunosuppressive benefits and monitoring characteristics. Although their mechanism of action affects the same pathway, their toxicity profiles are slightly different (Table 28.2). Trough levels are generally followed for the same reasons as those for cyclosporine—although C0 levels has been shown to better correlate with the AUC, with a correlation of up to 0.86.26 Higher levels should be targeted early after transplantation and similarly be lowered as time passes. As with cyclosporine, induction agents have also allowed for lower target goals.27 Goal tacrolimus trough levels are shown in Table 28.3.

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TABLE 28.3 Target Tacrolimus Levels Time (months)

Target C0 (ng/mL)

0 3

8 12

3 12

6 10

. 12

4 8

Target levels in the lower end of the range with use of thymoglobulin induction. C0 trough level.

Tacrolimus has compared favorably to cyclosporine in clinical outcomes leading to its preferential use by the majority of transplant centers.28 30 A randomized trial comprising of renal transplant patients receiving either tacrolimus or cyclosporine revealed a significantly higher occurrence (42.8% vs 25.9%; P , .001) of a composite endpoint consisting of biopsy-proven acute rejection, graft loss, and patient death in a 24-month period.31 The Efficacy Limiting Toxicity Elimination (ELITE-Symphony) study found, at 1 year, that patients receiving low dose tacrolimus compared to standard dose cyclosporine, low dose cyclosporine and sirolimus, had better eGFR, lower rates of biopsy-proven acute rejection, superior graft survival, and higher rates of posttransplant diabetes mellitus.32

28.3 PROTOCOL BIOPSIES Following serial serum creatinine measurements is the most widely implemented method of monitoring allograft function; however, it has limited sensitivity in detecting early rejection or other pathologic processes occurring in the allograft. Histologic evidence of rejection based on Banff criteria in kidneys with stable serum creatinine has been seen with the implementation of protocol biopsies.33,34 The main purpose of performing surveillance biopsies is to detect the presence of early rejection or chronic allograft nephropathy (CAN), two processes mediated by immune response. It is thought that early detection of these entities will allow for more timely therapies and improve allograft outcomes. The incidence of SCR in the literature has been reported in 3% 43% of allografts.35 39 Variability has been attributed to a number of factors including induction agent, quality of donor organ, delayed graft function (DGF) and immunosuppression regimen. This detection has proved critical, as SCR is predictive of poorer early and late outcomes. SCR at 3 months has been shown to significantly increase the incidence of CAN at 12 months, and early treatment improves rates of acute rejection episodes, eGFR decline, and interstitial fibrosis.37 Untreated SCR was found to correlate with higher degrees of interstitial fibrosis and tubular atrophy in subsequent biopsies. Likewise, evidence of CAN on protocol biopsies has been predictive of decreased renal function and allograft loss.40 43 High-risk patients may benefit the most from protocol biopsies. For instance, sensitized patients are at much greater risk of rejection than unsensitized patients. A study reviewing 116 surveillance biopsies performed at 1, 3, 6, and 12 months from 50 positive crossmatch patients revealed that 39.7% of biopsies performed at 1 month had subclinical cellular rejection.44 Biopsies at every time interval had a disconcerting 20% 30% positive staining for C4d which is concerning for AMR. DGF is another strong predictor of poor allograft function and the clinical diagnosis of rejection in this scenario is extremely challenging. In a study of 83 patients, 33 had DGF, of whom 18% had SCR on protocol biopsy (7 days posttransplant) compared to 4% of early functioning allografts.45 A large percentage of allograft failure in patients with DGF has been attributed to acute rejection.46 Transplant allograft biopsies are generally considered safe, and numerous studies have reported that major complications (i.e., hemorrhage, peritonitis, and graft loss) occur less than 1% of the time. Most of these procedures are done in an outpatient setting with high compliance from patients.47,48

28.4 DISADVANTAGES OF CURRENT STANDARDS The monitoring of serum creatinine and proteinuria lacks specificity and sensitivity. Once abnormalities are noted, there has already been significant injury, and treatment has been delayed. Immune monitoring using drug levels as a surrogate is challenging as plasma levels do not necessarily correlate well with AUC and, given CNI’s

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407

wide distribution in body, it may not accurately reflect pharmacodynamics.49 Moreover, pharmacokinetic monitoring does not take into account genetic variations or polymorphisms that can have a drastic influence on pharmacodynamics.50 The pharmacodynamic approach of monitoring of nuclear factor of activated T cell gene expression with CNI administration has shown promising results but may be difficult to interpret in the setting infection and has not been validated.51 High variability of SCR and CAN incidence on protocol biopsies in the literature has been attributed to differences in methodology. The dissimilarities in induction agents, immunosuppression regimen, HLA mismatches, and DGF incidence in between studies make generalizability and comparison difficult. More recent studies with modern immunosuppression have shown much-improved rates of SCR.39 The diagnosis of SCR and CAN is challenging to make, as the Banff criteria were originally intended for use in situations with high suspicion for rejection and clearer histologic findings.52 Frequently there are borderline changes that make distinguishing between SCR, CAN, or normal variation problematic. Certain histologic features of CAN are also seen with increasing age, so potentially many donor derived changes could be misclassified as CAN. Biopsies are ultimately operator dependent and subject to sampling error, leading to additional misdiagnosis. This variability can lead to both under- and over-treatment. Although biopsies are generally considered safe, they still expose patients, the majority of whom do not have rejection, to invasive procedures, discomfort, and unwarranted anxiety while adding to the costs of insurance payers.

28.5 NONINVASIVE MONITORING The limitations in current standards of immune monitoring have prompted the transplant community to develop new methods with higher sensitivity, specificity, and reproducibility. Monitoring has classically been separated into humoral and cellular (Fig. 28.2). Improved understanding of the immunity coupled with the advancement and increasing availability of molecular techniques have provided for novel methods of immune monitoring.

28.5.1 Humoral Immunity AMR, caused by circulating anti-HLA antibodies, can be categorized as hyperacute, acute, or chronic. Rates of hyperacute rejection have dramatically improved due to crossmatching, whereas acute and chronic rejections remain an obstacle to long-term allograft survival. Monitoring of donor-specific antibodies (DSA) remains the cornerstone of humoral immunity assessment; however novel assays are emerging with the promise to better characterize and understand B cells. Allograft Rejection Monitoring

Humoral

- DSA - B cell ELISPOT - C4d, C3d, C1q

Cellular - Urine proteomics - T cell cytokine assay - ImmuKnow - IFN-γ (T cell) ELISPOT - CRM - KSORT - CD8+ TEMRA

FIGURE 28.2 Strategies for noninvasive monitoring. DSA, donor-specific antibodies; ELISPOT, enzyme-linked immunospot; IFN-γ, interferon-gamma; CRM, common response module; KSORT, kidney solid organ response test.

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28.5.1.1 Anti-HLA: Donor Specific Antibodies The existence of antibodies to donor HLA antigens plays an important role in rejection and allograft failure. Preexisting antibodies have been known to result in poor outcomes since the 1960s when cytotoxic crossmatch positivity was first shown to result in immediate allograft failure.53 The crossmatch has since been a large determinant of organ allocation. Over the years the crossmatch has been refined with flow cytometry and ELISA for the detection of anti-HLA antibodies and more specifically, DSA.54 57 More recently, the Luminex assay has become the recognized standard of detecting DSA.58 60 It has become ubiquitous in transplant allocation, rejection risk assessment, AMR diagnosis, and immune monitoring. Preexisting DSA results in significantly poorer outcomes. A large observational study of 402 deceased donor kidney recipients revealed that 8-year graft survival was significantly worse in patients with preexisting DSA (61%) compared to nonsensitized (84%) and sensitized patients without DSA (93%). The peak DSA strength measured in mean fluorescence intensity correlated strongly with antibody-mediated rejection.61 Additional studies have reaffirmed preexisting DSA results in poorer allograft outcomes and higher rates of AMR.62 64 Better characterization of anti-HLA antibodies and understanding of AMR have led to the development of desensitization protocols. Those protocols aim to overcome the barrier of preexisting DSA and have been gaining wider acceptance. Desensitized patients have a survival benefit when compared to matched patients on remaining on the transplant waitlist.65 Despite acceptable short-term graft survival rates, AMR and SR occur more frequently in desensitized patients.66,67 Formation of de novo DSA following transplant is not uncommon, occurring in up to 20% in the first 5 years after transplant. Like preexisting DSA, de novo DSA portends higher rates of AMR and allograft failure.68,69 Additionally, DSA with the ability to activate the complement cascade is independently associated with graft failure. C4d binding in AMR has been well described but emerging evidence has shown complement split products, C1q and C3d, are predictive of AMR, graft loss, and transplant glomerulopathy.70 74 The presence of C1qpositive de novo DSA was associated with transplant glomerulopathy (sensitivity 81%, specificity 95%) and graft loss (sensitivity 79%, specificity 95%).73 Likewise, C3d-binding DSA at the time of AMR diagnosis was independently associated with allograft loss (hazard ratio 2.8; P 5 .03).74 It is clear complement activation plays a critical role in AMR and future studies are warranted to better refine immune profiling before and after transplantation. Current recommendations of DSA protocol monitoring are listed below: 1. High-risk patients (i.e., desensitized or DSA positive/XM negative) should be monitored by measurement of DSA and protocol biopsies in the first 3 months after transplantation. 2. Intermediate-risk patients (i.e., history of DSA but currently negative) should be monitored for DSA within the first month. If DSA is present, a biopsy should be performed. 3. Low-risk patients (i.e., nonsensitized first transplantation) should be screened for DSA at least once 3 12 months after transplantation. DSA monitoring should be considered with graft dysfunction, immunosuppression change or nonadherence, or suspicion of AMR. If DSA is detected, a biopsy should be performed and subsequent treatment should be administered based on the biopsy results. Early detection and reduction of DSA has led to improved outcomes so therapy should not be delayed.75 Additional work will be needed to understand the factors that contribute to de novo formation of DSA and long-term outcomes with treatment of DSA positivity without signs of rejection. 28.5.1.2 B Cell Anti-HLA Enzyme-Linked ImmunoSpot (ELISPOT) It is suggested an anamnestic response by memory B cells to antigens can drive acute rejection.76 Circulating levels of HLA antibodies in sensitized patients could be lower than the threshold of detection, resulting in a negative pretransplant screen. Upon reexposure to donor-derived antigens after transplant, memory B cells have the capability of producing antibodies, resulting in accelerated AMR.77 Memory B cells can also promote T cell mediated rejection by secretion of inflammatory cytokines and functioning as antigen presenting cells. Detection of HLA specific B cells from peripheral blood has been achieved with the implementation of HLA tetramer staining.78,79 ELISPOT has recently emerged as a modality to detect and quantify HLA specific B cells. Early assays have encouraging results for detecting both class I and II HLA specific B cells.80,81 Both class I and II donor specific memory B cells have been identified in sensitized patients and elevated donor-specific memory B cell responses were observed during AMR and prior to transplantation, independent of DSA levels. Higher levels were seen in more severe cases of AMR.82 These assays have not been implemented in the clinical setting but offer utility in improved risk stratification, understanding tolerance, and guidance of immunosuppression.

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28.5 NONINVASIVE MONITORING

28.5.2 Cellular Immunity Cellular rejection involves the alloactivation of antigen specific T cell lymphocytes. These helper T cells initiate a cascade of events resulting in the activation of cytotoxic T cells and, eventually, graft injury. Numerous modalities of cellular monitoring have resulted in improved detection of acute rejection. 28.5.2.1 Urine Transcriptomics and Proteomics Urine offers several advantages in clinical transcriptomics and proteomics. It can be easily obtained in large quantities without any invasive procedures. Proteins and peptides are also relatively stable in urine making processing and storage more reliable. A large body of literature has accumulated utilizing mass spectrometry, ELISA, multiplex beads and RT-PCR for the detection of numerous novel urinary biomarkers for allograft rejection (Table 28.4). The various biomarkers have been reviewed thoroughly elsewhere.100 Here we will focus on the recently reported studies from the Clinical Trials in Organ Transplantation (CTOT) consortium. CTOT-01 protocol was a prospective, multicenter observational study of noninvasive biomarkers in primary kidney allograft recipients.101 The study’s goal was to validate and determine the clinical utility of a panel of biomarkers in a cohort of 280 adult and pediatric patients. Mass spectrometry and ELISA were used to evaluate urinary markers that have been previously reported to be elevated in acute rejection (CCR1, CCR5, CXCR3, CCL5, CXCL9, CXCL10, IL-8, perforin and granzyme B). The major findings were urine granzyme B and CXCL9 were significantly elevated in patients with biopsy-proven rejection; however, their positive predictive values of 61% 67% were disappointing. In contrast, low levels of CXCL9 had a negative predictive value of 92% which may potentially be useful to rule out rejection. A low level of CXCL9 at 6 months was also associated with stable renal function and lower rates of AR. CTOT-17 will report on the much-anticipated 5-year outcomes of the CTOT-01 cohort. CTOT-04 protocol was a prospective, observational study of urine mRNA as potential markers for acute rejection in a cohort of 485 adult and pediatric patients.102 The urine mRNA of interest were CD3ε, perforin, TABLE 28.4

Urine Biomarkers Characteristics for Subclinical and Clinical Rejection Subclinical rejection

Biomarker 83 86

Perforin

Assay

Sensitivity (%)

Specificity (%)

Sensitivity (%)

Specificity (%)

Prognostic role

RT-PCR

80

90

55 100

79 95

87

RT-PCR

80

100

80

100

83 86

RT-PCR

60 88

77 92

RT-PCR

76 92

64 90

Predictive of graft function 6 months after AR

RT-PCR

100

100

Predictive of rejection reversal

RT-PCR

84 100

96 100

79.5 93

80 93.5

65 89

72 97

Level at 1 month predictive of graft function at 6 months

82

76.5

Predictive of steroid responsiveness

Granzyme A Granzyme B Serpin B9

Acute rejection

84,85,87

FOXP384,88 89,90

TIM-3

86,91 94

ELISA, multiplex beads

CXCL9

CXCL1092,93,95

97

86

64

ELISA, multiplex beads

CXCL1193

ELISA, multiplex beads

Fractalkine86

ELISA

SRM urine panel (35 peptides)98

SRM

uCRM score (11 gene panel)99

RT-PCR

45

87

AUC of 93% for acute rejection 88

94.1

RT-PCR, reverse transcriptase polymerase chain reaction; ELISA, enzyme-linked immunosorbent assay; SRM, Selective reaction monitoring; uCRM, urine common rejection module. Source: Adapted from Ho J., Wiebe C., Gibson I.W., Rush D.N., Nickerson P.W. Immune monitoring of kidney allografts. Am J Kidney Dis. 2012;60(4):629 640.

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granzyme B, proteinase inhibitor 9, CD103, CXCL10, CXCR3, TGF-β mRNA, and 18S rRNA. From the analysis, urinary CD3ε, CXCL10, perforin, and granzyme B mRNA were found to be elevated in acute cellular rejection (ACR). A signature three-gene set consisting of CD3ε mRNA, CXCL10 mRNA, and 18S rRNA was able to distinguish ACR with AUC 0.85 (P , .001). The gene set was externally validated (AUC 0.5; P , .001) in a cohort of 64 patients originally in the CTOT-01 study. It was also able to differentiate ACR from acute AMR and borderline rejection (AUC 0.78; P , .001). Both studies were limited by the yield of samples passing quality control. This is an inherent problem with extracting RNA from urine and has been an obstacle to clinical adoption. Prospective, interventional trials are needed to determine whether urinary proteomics monitoring protocols will lead to improved long-term allograft outcomes. 28.5.2.2 T Cell Activation Cytokine Assay Alloactivated T cells mediate cellular rejection with release of cytokines. In an observational, cross-sectional study of 64 transplant patients, blood was assayed for a panel of 21 cytokines secreted from peripheral blood monocytes cells.103 From a training cohort of acute rejecters, a panel of 6 cytokines (IL-1β, IL-6, TNF-α, IL-4, GM-CSF, and MCP-1) was found to differentiate AR. IL-6 was able to distinguish patients with acute rejection or borderline changes from patients with no rejection, with a sensitivity of 92%, and a specificity of 63%. A separate prospective study revealed elevated IL-10 and IFN-γ were elevated in acute rejection.104 IL-4, IL-6, and IL-10 were elevated in patients with chronic rejection, suggesting Th2 response may initiate or maintain chronic injury. Elevated pretransplant levels of IL-10 and IFN-γ have also been strongly associated with AR.105 The balance of proinflammatory and regulatory cytokines provides insight into global immunity and the understanding that complex interplay will be critically important in improving graft outcomes. 28.5.2.3 ImmuKnow Approved by the United States Food and Drug Administration in 2002, the ImmuKnow assay was designed to assess the global cellular immune function of transplant recipients.106 The assay measures intracellular ATP levels in stimulated CD41 T cells. Higher levels of ATP are thought to be a surrogate for CD41 activity and, thereby, net cellular immunity. The ImmuKnow assay was examined in a meta-analysis of 504 solid organ transplant recipients (kidney, heart, kidney-pancreas, liver, and small bowel) from 10 different centers.107 Data from the patients were pooled based on the clinical condition at the time of blood draw and up to 1 month prior to a clinical event. A total of 1833 ImmuKnow assays were performed. Clinical events consisted of stable (routine clinic visits), rejection (biopsyproven or medically treated due to clinical suspicion), or infection (positive culture, elevated PCR in the blood). Longitudinal samples from each patient were averaged during periods of clinical stability, whereas a sample taken during an adverse event was analyzed separately. Reduced ATP values (,225 ng/mL) were found to be associated with an increased risk of infections, and high values ( . 525 ng/mL) were found to be associated with an increased risk of acute rejection episodes. Similar results were found in a retrospective study of 42 kidney transplant patients and 25 healthy controls. A prospective, randomized in liver transplant recipients demonstrated outcomes were improved when ImmuKnow was used to guide immunosuppression.108 Most studies have used the Immuknow assay in the setting of known infection or rejection, limiting its application as a predictive tool. A single center study retrospectively analyzed 1330 assays from 583 patients.109 Assays were performed at protocol based screening at 0, 1, 6, and 12 months posttransplant, in the setting of clinical suspicion (rise in serum creatinine or infection), and testing of stable patients in the outpatient setting. Assays drawn within 90 days before a clinical event were selected for analysis. There was no association found between a single Immuknow assay and subsequent opportunistic infection or rejection within 90 days. In conjunction with other tests, the ImmuKnow assay could be useful marker for global immune status; however, given the variability of current studies, there is currently no clear consensus to its optimal utility in the clinical setting. 28.5.2.4 Interferon-Gamma ELISPOT Cytokines, as previously discussed, are major regulators of the immune response, and Interferon-Gamma (IFN-γ) has been a major focus of interest. The IFN-γ ELISPOT assay has been a powerful tool in detecting the frequency of alloantigen specific, activated or memory T cells, a marker for cellular immunity. A study of 23 kidney transplant recipients revealed that mean posttransplant IFN-γ frequencies were inversely correlated to eGFR at 6 and 12 months after transplant with a P-value of .007 and .033, respectively.110 Additional multivariate

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28.5 NONINVASIVE MONITORING

411

analysis revealed that low activity of donor-specific T cell alloreactivity was the most significant correlate to preserved eGFR at long-term follow-up (24 114 months).111 The use of pretransplant IFN-γ ELISPOT positivity has been shown to correlate with acute rejection and decreased renal function after transplant in various studies.112 115 In a cohort of 100 patients, positive pretransplant ELISPOT was associated with acute rejection (odds ratio 4.6, P 5 .009) independent of DGF, donor characteristics, HLA matching, African American ethnicity and dialysis vintage.116 Interestingly, the CTOT-01 study revealed patients with positive IFN-γ ELISPOT and received antithymoglobulin induction did not have reduced eGFR at 6 and 12 months following transplant.117 Similar to PRA, a panel reactive T cell (PRT) test has been developed as a screening tool. Frequency of IFN-γ spots against a panel of allogeneic cells provides a measure of patient sensitization. In a study of 30 subjects, subjects with a pretransplant positive IFN-γ ELISPOT to .75% of cell lines (i.e., PRT .75%) had a 54% incidence of acute rejection compared to an incidence of 5% with a lesser PRT score.118 In addition to acute rejection, positive IFN-γ ELISPOT ( . 25/300,000) cells are statistically higher in patients with CAN compared to control. In a study of 45 renal allograft recipients, 50% of subjects with CAN had a positive ELISPOT compared to 28.6% in control subjects—suggesting T cell mediated injury plays a role in CAN.119 There has been some conflicting data with ELISPOT suggesting it may be dependent on center specific protocols (i.e., induction agent, HLA matching, etc.). Regardless, larger trials will be needed to determine a reference range for positive results, validate the PRT, and, ultimately, guide immunosuppression management. 28.5.2.5 Common Rejection Module A microarray meta-analysis of eight independent transplants datasets, with 236 graft biopsy samples from four organs, identified a common immune response module of 11 genes (BASP1, CD6, CXCL10, CXCL9, INPP5D, ISG20, LCK, NKG7, PSMB9, RUNX3, TAP1) that distinguished acute rejection across different engrafted tissues.120 The common rejection module (CRM) genes correlated with degree of graft injury and predicted graft injury in two independent cohorts. Treatment of mouse HLA-mismatched cardiac transplants with atorvastatin and dasatinib via drug repositioning decreased CRM gene expression and cellular infiltrate during AR. A follow-up study evaluated tissue qPCR expression of CRM in 146 independent renal allografts from 122 unique patients with and without AR.121 A tissue CRM (tCRM) score was calculated by using the geometric mean of the fold changes of the respective genes. A tCRM score threshold of .2.24 was able to accurately distinguish AR from no-AR with high sensitivity (82.7%) and specificity (82.5%). When applied to an independent cohort, a PPV of 82.4% was obtained with the same threshold. A modified tCRM score (modeled 7 of 11 genes) at 6 months was able to predict interstitial fibrosis and tubular atrophy at 24 months (P 5 .037). In the urine, the CRM score (uCRM) was robust in distinguishing acute rejection (AUC 5 0.966; 95% CI 0.911 1), and was further validated in 87 independent serial samples (AUC 5 0.95; 95% CI 0.914 1).99 The tCRM and uCRM scores may provide a companion diagnostic for differentiating patients into high and low immune risk, for stratification into different investigative treatment arms, with an increased margin for patient and graft safety; however, validation will be needed against larger cohorts. These genes were derived from biopsy tissues but have the potential to be applied to serum making it noninvasive. 28.5.2.6 Kidney Solid Organ Response Test The Assessment of Acute Rejection in Renal Transplantation study was conducted across eight transplant centers and included both pediatric and adult subjects on diverse immunosuppression.122 Gene expression was measured by quantitative real-time PCR on peripheral blood samples to develop this noninvasive assay. From a training cohort of 143 samples, the Kidney Solid Organ Response Test (KSORT)—a 17-gene set—was established as discriminatory markers for acute rejection. KSORT was validated in 124 independent samples with an AUC of 0.95 and was able to robustly detect acute rejection in peripheral blood samples accurately (AUC 0.93) independent of age and time posttransplantation. KSORT was also able to predict AR in greater than 60% of samples up to 3 months prior to transplant. KSORT is limited by its reliance on histologic readings as the reference for its performance and inability to delineate T-cell and AMR. The study design was limited by the heterogeneity of the samples and lack of serially collected samples with validating biopsies. Despite these limitations, KSORT offers a promising method to detect and predict acute rejection and is being studied in a prospective, randomized, double blinded clinical trial.

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412 TABLE 28.5

28. IMMUNE MONITORING IN KIDNEY TRANSPLANTATION

Summary of Noninvasive Immune Monitoring Tests for Acute Rejection

Test

Analyte

DSA Monitoring

Donor specific anti-HLA antibodies

Sample source Blood

Assay

Type of rejection detected

Flow cytometry, ELISA, Luminex

Humoral Circulating DSA promote antibody-mediated rejection. Higher titers increase risk of rejection.

Biological basis of test

Performance at acute rejection detection Peak ELISA HLA-DSA PPV 41.3 Sensitivity 59.4% Specificity 92.7% Peak Luminex HLA-DSA PPV 34.9 Sensitivity 90.6% Specificity 85.4%

Current Luminex HLADSA PPV 31.6 Sensitivity 75.0% Specificity 86.2% Humoral Amnestic response by donor Donor specific memory B specific memory T cells when cell frequencies of .0.35 reexposed to donor derived predicted the presence of antigens. endarteritis in patients with AMRR (AUC 0.89)

B cell ElISPOT

Donor specific memory B cells

Blood

ELISPOT

T cell Alloactivation cytokine assay

Cytokines (IL-1β, IL-6, TNFα, IL-4, GM-CSF, and monocyte chemoattractant protein-1)

Blood

Flow cytometry, Luminex

Cellular

Cytokines are released from alloactivated T cells and mediate an inflammatory response.

tCRM (common rejection module)

11 Genes (BASP1, CD6, CXCL10, CXCL9, INPP5D, ISG20, LCK, NKG7, PSMB9, RUNX3, and TAP1)

Biopsy tissue, potential use in blood

qPCR

Cellular

Genes that were found to be significantly overexpressed (P , .0005) during acute rejection, irrespective of the transplanted organ.

Immuknow

ATP levels in CD4 T cells

Blood

ELISPOT

Cellular

Higher concentrations of ATP signify greater CD4 T cell activity and an overall increased global immune response.

IFN-γ (T cell) ELISPOT

IFN-γ

Blood

ELISPOT

Cellular

kSORT

Blood 17 Genes (FLAR, DUSP1, IFNGR1, ITGAX, MAPK9, NAMPT, NKTR, PSEN1, RNF130, RYBP, CEACAM4, EPOR, GZMK, RARA, RHEB, RXRA, SLC25A37)

qPCR

Cellular

Terminally differentiated CD81 TEMRA cells

CD81 TEMRA cells

Whole blood staining

Cellular

Blood

IL-6—AUC 0.79 TNF-α—AUC 0.86 MCP-1—AUC 0.73 IL-1β—AUC 0.81 GM-CSF—AUC 0.73 IL-4—AUC 0.76 Sensitivity—82.7% Specificity—82.5% PPV—82.4% (tCRM threshold of 2.24)

Detection limit of ATP 1 ng/mL For concentration .525 ng/mL Sensitivity—23.4% Specificity—80.9% IFN-γ is a cytokine marker Pretrasnplant cutoff level for T-cell activity. It activates of 12 spots per 200,000 macrophages and class II PBLs major histocompatibility Sensitivity—81.8% complex molecule expression. Specificity—64.7% NPV—89% PPV—46% Combination of significant Sensitivity—83% 92.3% genes in acute rejection Specificity—90.6% 99% identified by whole genome microarray analysis of biopsy and paired blood samples. Higher frequency of CD81 TEMRA cells decreases the diversity of T cells yielding lower frequencies of alloreactive T cells. TEMRA cells may also have immunosuppressive role.

Hazard ratio 0.96 for every percent increase in CD81 TEMRA cells TEMRA Hazard ratio 0.66 for every increase in tertile of CD81 TEMRA cells

ELISPOT, enzyme-linked immunospot; ELISA, enzyme-linked immunosorbent assay; qPCR, quantitative polymerase chain reaction; PPV, positive predictive value; NPV, negative predictive value.

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28.5.2.7 Terminally Differentiated CD81 TEMRA Cells Various types of T cells have been shown to contribute to transplant tolerance. These include the CD41CD251 regulatory T cells (Tregs) that express the transcription factor FOXP3, IL-10-producing Tr1 cells, CD81282 T cells, and anergic T cells. Progressive loss of renal function is associated with profound dysregulation of the T-cell system which may result in overall depressed T cell immunity and therefore, decreased risk of acute rejection.123 Patients with acute rejection have more naı¨ve T cells and lower levels of T cell dysregulation and differentiation.124 The percentage of CD81 TEMRA cells was shown to have a hazard ratio of 0.96 (P 5 .006) for rejection with every percent increase in CD81 TEMRA cells. It is hypothesized that a larger number of CD81 TEMRA cells decreases the diversity of T cells yielding lower frequencies of alloreactive T cells.125 CD81 TEMRA cells have also been implicated with suppressive functions as their presence may play a role in immunosenescence and decreased vaccine response.126 In contrast to short-term benefits, the accumulation of highly differentiate T cells has been implicated as a risk factor for long-term graft dysfunction.127 Larger prospective studies will be required to elucidate the role of TEMRA cells in allograft function. 28.5.2.8 Kidney Spontaneous Operational Tolerance Test Operation tolerance sporadically occurs in transplant patients allowing for total withdrawal of immunosuppression without any harm to the graft. Transcriptional profiling has allowed for better understanding of this unique state of immune quiescence. In a single study of 571 unique peripheral blood samples from 348 HLAmismatched renal transplant recipients and 101 nontransplant controls, microarray analysis was used to discover 141 genes differently expressed in operationally tolerant patients. Among those peripheral blood genes, a minimal set of 21 was able to accurately discriminate between tolerance and chronic rejection.128 High-throughput microfluidic qPCR for the 21 genes in a second independent sample revealed a set of three genes (KLF6, BNC2, and CYP1B1) was able to classify the tolerant samples with 84.6% sensitivity, 90.2% specificity, and an AUC of 0.95 (95% CI 0.97 0.92). The study design was limited due to small sample volumes, but Kidney Spontaneous Operational Tolerance Test (KSPOT) offers a potential means to monitor for graft accommodation and limit morbidity from immunosuppression.

28.6 CONCLUSION Current standards of immune monitoring have improved short-term outcomes, but rejection remains a significant barrier to long-term allograft survival. Therapeutic drug monitoring, serial serum creatinine measurements, and protocol biopsies lack the refinement and practicality needed to risk-stratify patients, guide immunosuppression therapy, and follow treatment responses. Novel modalities in immune monitoring offer the possibility of noninvasive prediction and detection of rejection, allograft survival, and tolerance. These new assays offer promising results thus far but need prospective, longitudinal studies in order to make the final transition from bench to bedside (Table 28.5).

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Mu¨ller-Steinhardt M, Fricke L, Kirchner H, Hoyer J, Klu¨ter H. Monitoring of anti-HLA class I and II antibodies by flow cytometry in patients after first cadaveric kidney transplantation. Clin Transplant 2000;14(1):85 9. 56. Moore SB, Ploeger NA, DeGoey SR. HLA antibody screening: comparison of a solid phase enzyme-linked immunoassay with antiglobulin-augmented lymphocytotoxicity. Transplantation 1997;64(11):1617 20. 57. Martin L, Guignier F, Bocrie O, et al. Detection of anti-HLA antibodies with flow cytometry in needle core biopsies of renal transplants recipients with chronic allograft nephropathy. Transplantation 2005;79(10):1459 61. 58. Pei R, Lee JH, Shih NJ, Chen M, Terasaki PI. Single human leukocyte antigen flow cytometry beads for accurate identification of human leukocyte antigen antibody specificities. Transplantation 2003;75(1):43 9. 59. Yang CW, Oh EJ, Lee SB, et al. Detection of donor-specific anti-HLA class I and II antibodies using antibody monitoring system. Transplant Proc 2006;38(9):2803 6. 60. Tait BD, Su¨sal C, Gebel HM, et al. Consensus guidelines on the testing and clinical management issues associated with HLA and nonHLA antibodies in transplantation. Transplantation 2013;95(1):19 47. 61. Lefaucheur C, Loupy A, Hill GS, et al. Preexisting donor-specific HLA antibodies predict outcome in kidney transplantation. J Am Soc Nephrol 2010;21(8):1398 406. 62. Amico P, Ho¨nger G, Mayr M, Steiger J, Hopfer H, Schaub S. Clinical relevance of pretransplant donor-specific HLA antibodies detected by single-antigen flow-beads. Transplantation 2009;87(11):1681 8. 63. Caro-Oleas JL, Gonza´lez-Escribano MF, Gonza´lez-Roncero FM, et al. Clinical relevance of HLA donor-specific antibodies detected by single antigen assay in kidney transplantation. Nephrol Dial Transplant 2012;27(3):1231 8. 64. Gloor JM, Winters JL, Cornell LD, et al. Baseline donor-specific antibody levels and outcomes in positive crossmatch kidney transplantation. Am J Transplant 2010;10:582 9. 65. Montgomery RA, Lonze BE, King KE, et al. Desensitization in HLA-incompatible kidney recipients and survival. N Engl J Med 2011;365 (4):318 26. 66. Marfo K, Lu A, Ling M, Akalin E. Desensitization protocols and their outcome. Clin J Am Soc Nephrol 2011;6(4):922 36. 67. Gloor J, Stegall MD. Sensitized renal transplant recipients: current protocols and future directions. Nat Rev Nephrol 2010;6(5):297 306. 68. Wiebe C, Gibson IW, Blydt-Hansen TD, et al. Evolution and clinical pathologic correlations of de novo donor-specific HLA antibody post kidney transplant. Am J Transplant 2012;12(5):1157 67. 69. Everly MJ, Rebellato LM, Haisch CE, et al. Incidence and impact of de novo donor-specific alloantibody in primary renal allografts. Transplantation 2013;95(3):410 17. 70. Loupy A, Lefaucheur C, Vernerey D, et al. Complement-binding anti-HLA antibodies and kidney-allograft survival. N Engl J Med 2013;369(13):1215 26. 71. Yell M, Muth BL, Kaufman DB, Djamali A. Ellis Tm. C1q binding activity of de novo donor-specific HLA antibodies in renal transplant recipients with and without antibody-mediated rejection. Transplantation 2015;99(6):1151 5. 72. Thammanichanond D, Mongkolsuk T, Rattanasiri S, et al. Significance of C1q-fixing donor-specific antibodies after kidney transplantation. Transplant Proc 2014;46(2):368 71. 73. Yabu JM, Higgins JP, Chen G, Sequeira F, Busque S, Tyan DB. C1q-fixing human leukocyte antigen antibodies are specific for predicting transplant glomerulopathy and late graft failure after kidney transplantation. Transplantation 2011;91(3):342 7. 74. Sicard A, Ducreux S, Rabeyrin M, et al. Detection of C3d-binding donor-specific anti-HLA antibodies at diagnosis of humoral rejection predicts renal graft loss. J Am Soc Nephrol 2015;26(2):457 67. 75. Everly MJ, Everly JJ, Arend LJ, et al. Reducing de novo donor-specific antibody levels during acute rejection diminishes renal allograft loss. Am J Transplant 2009;9(5):1063 71. 76. Zachary AA, Lucas DP, Montgomery RA, Leffell MS. Rituximab prevents an anamnestic response in patients with cryptic sensitization to HLA. Transplantation 2013;95(5):701 4. 77. Kurosaki T, Kometani K, Ise W. Memory B cells. Nature Rev Immunol 2015;15:149 59. 78. Mulder A, Eijsink C, Kardol MJ, et al. Identification, isolation, and culture of HLA-A2-specific B lymphocytes using MHC class I tetramers. J Immunol 2003;171(12):6599 603. 79. Zachary AA, Kopchaliiska D, Montgomery RA, Leffell MS. HLA-specific B cells: I. A method for their detection, quantification, and isolation using HLA tetramers. Transplantation 2007;83(7):982 8.

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80. Heidt S, Roelen DL, de Vaal YJ, et al. A novel ELISPOT assay to quantify HLA-specific B cells in HLA-immunized individuals. Am J Transplant 2012;12(6):1469 78. 81. Karahan GE, de Vaal YJ, Roelen DL, et al. Quantification of HLA class II-specific memory B cells in HLA-sensitized individuals. Hum Immunol 2015;76(2-3):129 36. 82. Lu´cia M, Luque S, Crespo E, et al. Preformed circulating HLA-specific memory B cells predict high risk of humoral rejection in kidney transplantation. Kidney Int 2015;88(4):874 87. 83. Li B, Hartono C, Ding R, et al. Noninvasive diagnosis of renal-allograft rejection by measurement of messenger RNA for perforin and granzyme B in urine. N Engl J Med 2001;344(13):947 54. 84. Aquino-Dias EC, Joelsons G, da Silva DM, et al. Non-invasive diagnosis of acute rejection in kidney transplants with delayed graft function. Kidney Int 2008;73(7):877 84. 85. Muthukumar T, Ding R, Dadhania D, et al. Serine proteinase inhibitor-9, an endogenous blocker of granzyme B/perforin lytic pathway, is hyperexpressed during acute rejection of renal allografts. Transplantation 2003;75(9):1565 70. 86. Peng W, Chen J, Jiang Y, et al. Urinary fractalkine is a marker of acute rejection. Kidney Int 2008;74(11):1454 60. 87. van Ham SM, Heutinck KM, Jorritsma T, et al. Urinary granzyme A mRNA is a biomarker to diagnose subclinical and acute cellular rejection in kidney transplant recipients. Kidney Int 2010;78(10):1033 40. 88. Muthukumar T, Dadhania D, Ding R, et al. Messenger RNA for FOXP3 in the urine of renal-allograft recipients. N Engl J Med 2005;353 (22):2342 51. 89. Renesto PG, Ponciano VC, Cenedeze MA, Saraiva Camara NO, Pacheco-Silva A. High expression of Tim-3 mRNAin urinary cells from kidney transplant recipients with acute rejection. Am J Transplant 2007;7(6):1661 5. 90. Manfro RC, Aquino-Dias EC, Joelsons G, Nogare AL, Carpio VN, Goncalves LF. Noninvasive Tim-3 messenger RNA evaluation in renal transplant recipients with graft dysfunction. Transplantation 2008;86(12):1869 74. 91. Schaub S, Nickerson P, Rush D, et al. Urinary CXCL9 and CXCL10 levels correlate with the extent of subclinical tubulitis. Am J Transplant 2009;9(6):1347 53. 92. Hu H, Kwun J, Aizenstein BD, Knechtle SJ. Noninvasive detection of acute and chronic injuries in human renal transplant by elevation of multiple cytokines/chemokines in urine. Transplantation 2009;87(12):1814 20. 93. Hu H, Aizenstein BD, Puchalski A, Burmania JA, Hamawy MM, Knechtle SJ. Elevation of CXCR3-binding chemokines in urine indicates acute renal-allograft dysfunction. Am J Transplant 2004;4(3):432 7. 94. Jackson JA, Kim EJ, Begley B, et al. Urinary chemokines CXCL9 and CXCL10 are noninvasive markers of renal allograft rejection and BK viral infection. Am J Transplant 2011;11(10):2228 34. 95. Tatapudi RR, Muthukumar T, Dadhania D, et al. Noninvasive detection of renal allograft inflammation by measurements of mRNA for IP-10 and CXCR3 in urine. Kidney Int 2004;65(6):2390 7. 96. Matz M, Beyer J, Wunsch D, et al. Early post-transplant urinary IP-10 expression after kidney transplantation is predictive of short- and long-term graft function. Kidney Int 2006;69(9):1683 90. 97. Ho J, Rush DN, Karpinski M, et al. Validation of urinary CXCL10 as a marker of borderline, subclinical and clinical tubulitis. Transplantation 2011;92(8):878 82. 98. Sigdel TK, Gao Y, Jintang H, et al. Mining the Human Urine Proteome for Monitoring Renal Transplant Injury. Manuscript submitted for publication. Kidney Int 2015;89:1244 52. 99. Sigdel TK et al. (2015). A non-invasive Urinary Common Rejection Module (uCRM) gene expression score enables accurate discrimination of Acute Rejection in kidney Transplant patients. Unpublished data (in submission, not yet submitted). 100. Ho J, Wiebe C, Gibson IW, Rush DN, Nickerson PW. Immune monitoring of kidney allografts. Am J Kidney Dis 2012;60(4):629 40. 101. Hricik DE, Nickerson P, Formica RN, et al. CTOT-01 consortium. Multicenter validation of urinary CXCL9 as a risk-stratifying biomarker for kidney transplant injury. Am J Transplant 2013;13(10):2634 44. 102. Suthanthiran M, Schwartz JE, Ding R, et al. Clinical trials in organ transplantation 04 (CTOT-04) study investigators. Urinary-cell mRNA profile and acute cellular rejection in kidney allografts. N Engl J Med 2013;369(1):20 31. 103. De Serres SA, Mfarrej BG, Grafals M, et al. Derivation and validation of a cytokine-based assay to screen for acute rejection in renal transplant recipients. Clin J Am Soc Nephrol 2012;7(6):1018 25. 104. Karczewski M, Karczewski J, Poniedzialek B, Wiktorowicz K, Smietanska M, Glyda M. Distinct cytokine patterns in different states of kidney allograft function. Transplant Proc 2009;41(10):4147 9. 105. Karczewski J, Karczewski M, Glyda M, Wiktorowicz K. Role of TH1/TH2 cytokines in kidney allograft rejection. Transplant Proc 2008;40 (10):3390 2. 106. Kowalski R, Post D, Schneider MC, et al. Immune cell function testing: an adjunct to therapeutic drug monitoring in transplant patient management. Clin Transplant 2003;17(2):77 88. 107. Kowalski RJ, Post DR, Mannon RB, et al. Assessing relative risks of infection and rejection: a meta-analysis using an immune function assay. Transplantation 2006;82(5):663 8. 108. Ravaioli M, Neri F, Lazzarotto T, Bertuzzo VR, et al. Immunosuppression modifications based on an immune response assay: results of a randomized, controlled trial. Transplantation 2015;99(8):1625 32. 109. Huskey J, Gralla J, Wiseman AC. Single time point immune function assay (ImmuKnowt) testing does not aid in the prediction of future opportunistic infections or acute rejection. Clin J Am Soc Nephrol 2011;6(2):423 9. 110. Na¨ther BJ, Nickel P, Bold G, Presber F, et al. Modified ELISPOT technique--highly significant inverse correlation of post-Tx donorreactive IFNgamma-producing cell frequencies with 6 and 12 months graft function in kidney transplant recipients. Transpl Immunol 2006;16(3-4):232 7. 111. Bestard O, Nickel P, Cruzado JM, et al. Circulating alloreactive T cells correlate with graft function in longstanding renal transplant recipients. J Am Soc Nephrol 2008;19:1419 29.

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112. Heeger PS, Greenspan NS, Kuhlenschmidt S, Dejelo C, Hricik DE, Schulak JA. Pretransplant frequency of donor-specific, IFN-gammaproducing lymphocytes is a manifestation of immunologic memory and correlates with the risk of posttransplant rejection episodes. J Immunol 1999;163:2267 75. 113. Tary-Lehmann M, Hricik DE, Justice AC, Potter NS, Heeger PS. Enzyme linked immunosorbent assay spot detection of interferongamma and interleukin 5-producing cells as a predictive marker for allograft failure. Transplantation 1998;66:219 24. 114. Augustine JJ, Siu DS, Clemente MJ, Schulak JA, Heeger PS, Hricik DE. Pre-transplant IFN-gamma ELISPOTs are associated with posttransplant renal function in African American renal transplant recipients. Am J Transplant 2005;5:1971 5. 115. Kim SH, Oh EJ, Kim MJ, Park YJ, Han K, Yang HJ. Pretransplant donor-specific interferon-gamma ELISPOT assay predicts acute rejection episodes in renal transplant recipients. Transplant Proc 2007;39:3057 60. 116. Augustine JJ, Poggio ED, Clemente M, et al. Hemodialysis vintage, black ethnicity and pretransplantation antidonor cellular immunity in kidney transplant recipients. J Am Soc Nephrol 2007;18:1602 6. 117. Hricik DE, Augustine J, Nickerson P, et al. CTOT-01 consortium. Interferon gamma ELISPOT testing as a risk-stratifying biomarker for kidney transplant injury: results from the CTOT-01 multicenter study. Am J Transplant 2015. Available from: http://dx.doi.org/10.1111/ ajt.13401. 118. Poggio ED, Augustine JJ, Clemente M, et al. Pretransplant cellular alloimmunity as assessed by a panel of reactive T cells assay correlates with acute renal graft rejection. Transplantation 2007;83:847 52. 119. Poggio ED, Clemente M, Riley J, et al. Alloreactivity in renal transplant recipients with and without chronic allograft nephropathy. J Am Soc Nephrol 2004;15:1952 60. 120. Khatri P, Roedder S, Kimura N, et al. A common rejection module (CRM) for acute rejection across multiple organs identifies novel therapeutics for organ transplantation. J Exp Med 2013;210(11):2205 21. 121. Sigdel TK, Bestard O, Tran TQ, Hsieh SC, Roedder S, Damm I, et al. A computational gene expression score for predicting immune injury in renal allografts. PLoS One 2015;10(9):e0138133. 122. Roedder S, Sigdel T, Salomonis N, et al. The kSORT assay to detect renal transplant patients at high risk for acute rejection: results of the multicenter AART study. PLoS Med 2014;11(11):e1001759. 123. Betjes MG, Huisman M, Weimar W, Litjens NH. Expansion of cytolytic CD4 1 CD28- T cells in end-stage renal disease. Kidney Int 2008;74(6):760 7. 124. Betjes MG, Meijers RW, de Wit EA, Weimar W, Litjens NH. Terminally differentiated CD8 1 Temra cells are associated with the risk for acute kidney allograft rejection. Transplantation 2012;94(1):63 9. 125. Hadrup SR, Strindhall J, Køllgaard T, et al. Longitudinal studies of clonally expanded CD8 T cells reveal a repertoire shrinkage predicting mortality and an increased number of dysfunctional cytomegalovirus-specific T cells in the very elderly. J Immunol 2006;176 (4):2645 53. 126. Effros RB. Role of T lymphocyte replicative senescence in vaccine efficacy. Vaccine 2007;25(4):599 604. 127. Yap M, Boeffard F, Clave E, et al. Expansion of highly differentiated cytotoxic terminally differentiated effector memory CD8 1 T cells in a subset of clinically stable kidney transplant recipients: a potential marker for late graft dysfunction. J Am Soc Nephrol 2014;25 (8):1856 68. 128. Roedder S, Li L, Alonso MN, et al. A three-gene assay for monitoring immune quiescence in kidney transplantation. J Am Soc Nephrol 2015;26(8):2042 53.

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C H A P T E R

29 Renal Function Measurements Esteban Porrini University of La Laguna, Tenerife, Spain

29.1 INTRODUCTION Renal transplantation is a procedure developed to restore renal function in patients with advanced renal disease. It is a major achievement in clinical medicine since it offers better survival and quality of life to patients with end stage renal disease.1 Accordingly, the main objective of clinical care in renal transplantation is to preserve adequate renal function of the graft. This is particularly relevant since transplant patients still have an increased prevalence of chronic kidney disease (CKD) with a high rate of graft loss; about 4% per year.2 Thus, the use of reliable methods to evaluate renal function is crucial in this population. In this chapter, the different methods (estimation and measurement) to evaluate renal function as well as their limitations in clinical practice will be described. Special focus will be given to the importance of measuring glomerular filtration rate (GFR) in renal transplant patients.

29.2 METHODS TO EVALUATE RENAL FUNCTION The evaluation of renal function requires the use of markers which ideally must be freely filtrated by the glomerulus, not reabsorbed, secreted or metabolized by renal tubuli, not bound to proteins, atoxic, metabolically inert, constantly produced and eliminated.3,4 Also, it is assumed that the rate of exclusion from the plasma by the kidneys equals the rate of its excretion into the urine.4 Markers of GFR can be endogenous or exogenous. Endogenous include creatinine and cystatin-C while exogenous includes inulin, radioisotopes like: el Tc99 DTPA, Cr51-EDTA and the nonradioactive agents like iohexol or iothalamate. The use of endogenous markers is considered as an estimation and the use of exogenous as measurement of GFR.

29.3 ESTIMATION OF GLOMERULAR FILTRATION RATE 29.3.1 Serum Creatinine-Creatinine Clearance In day-to-day clinical practice, renal function is evaluated by serum levels of creatinine. It is assumed that creatinine reflects properly renal function based on its inverse relationship with GFR. Creatinine shares many points with an ideal index of renal function: It is not bound to proteins, it is freely filtered by the glomerulus, and it is not metabolized by the kidney.3 However, creatinine has two major drawbacks: Tubular secretion and reabsorption. Tubular cells can secrete creatinine, which increases with the decrease of GFR.3,4 The magnitude of creatinine secretion is not irrelevant: It can increase up to 80% 100% in advanced renal disease and may equal creatinine filtration rate.3,4 Thus, creatinine does not have the necessary sensitivity to reflect changes in GFR: creatinine can remain stable while GFR decreases (Fig. 29.1—right panel). The magnitude of this lack of association is such that for the same value of creatinine, i.e., 1.5 mg/dL, measured GFR can range from 30 to 70 mL/minute Kidney Transplantation, Bioengineering, and Regeneration. DOI: http://dx.doi.org/10.1016/B978-0-12-801734-0.00029-1

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FIGURE 29.1 Relationship between serum creatinine (N 5 366) -right panel- and serum cystatin-C (N 5 192) -left panel- and measured GFR by iohexol plasma clearance in renal transplant patients.

(Fig. 29.1—right panel). This also shows clearly how creatinine overestimates real GFR, particularly in patients with CKD. It has to be indicated that these limitations of creatinine have been described decades ago.3 5 However, this has gone mostly unnoticed by the medical community. The plasma clearance of creatinine, which needs the collection of the urine output during 24 hours, is also used as an estimation of GFR. The limitations of creatinine indicated above are also pertinent to creatinine clearance, i.e., GFR overestimation due to increased tubular secretion of creatinine. Also, the recollection of urine can be cumbersome in some patients, which may limit this technique.

29.3.2 Cystatin-C More recently, cystatin-C has been proposed as a better marker than creatinine for the estimation of renal function. Cystatin-C is a small protein (122 aminoacid, 13-kDa), a cysteine proteinase inhibitor, product of a “housekeeping” gene expressed in all nuclear cells.6 Cystatine-C has many characteristics of an ideal marker of renal function, it has constant rate of production and is freely filtrated by the glomerulus.6,7 Although it is not secreted by tubular cells, cystatin-C is reabsorbed and metabolized by tubular epithelial cells.6,7 Thus, the reabsorbed cystatin-C does not return to the blood stream, which allows its use as a marker of renal function. Several formulas have been developed with cystatin-C to estimate renal function and have been tested in diverse populations, i.e., patients with CKD, renal transplantation, children with renal disease, cancer patients and renal transplant patients, etc. However, the reliability of this new marker, as well as its superiority compared with creatinine, is not clear.8 The major limitation of cystatin-C is the fact that it is influenced by factors related with renal function such as age, male gender, obesity, diabetes, hypertension, triglycerides, and inflammation.9 12 Thus, the levels of cystatine-c may reflect several conditions, not just renal function, which may be the cause of its lack of accuracy and precision in reflecting GFR. Also, as described with creatinine, the relationship between measured GFR and cystatin-C is not linear (Fig. 29.1—left panel). Moreover, cystatin-C can remain stable, i.e., 1.5 mg/L; measured GFR can range from 30 to 75 mL/minute.

29.3.3 Estimation Formulas Mainly due to the limitations of creatinine clearance to reflect GFR, and to avoid 24 hours urine collection, more than 50 formulas have been developed to estimate GFR.13 59 The first formula was described by Effersoe in 1957 to help physicians in the dosage of drugs according with renal function13 and the last formula was published in 2014.57 Formulas are, in short, mathematical algorithms applied to some variables with a known relationship with renal function. Mathematical procedures were designed to improve the capacity of these variables in the prediction of GFR. However, some limitations to this approach must be indicated. Formulas include very few variables, i.e., creatinine and/or cystatin-C, age, gender, and in some cases weight. No clear rationale for the inclusion

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of these variables has been provided. Gender and age are correlated with muscle mass, which is the source of creatinine. Thus, it might be argued that the prediction of GFR is mainly based on serum creatinine for the cases of creatinine-based formulas. Also, the mathematical analysis is seldom specified, which makes the understanding of the development of formulas very difficult. Finally, the original drawbacks of creatinine and cystatin as markers of GFR (see above) should be kept in mind when formulas are used as indicators of renal function.

29.3.4 Estimated GFR in Renal Transplantation Almost 30 studies evaluated the performance of formulas that estimate GFR in renal transplant patients compared with a gold standard method.60 A recent study evaluated the performance of 51 formulas, either creatinineor cystatin-based, in renal transplant patients compared with a gold standard procedure, the plasma clearance of iohexol.60 The majority of the equations have an error of 630% compared with a gold standard; the average of the estimations included in this 630% limit hardly exceeds 70%, indicating that in about one-third of the patients the error is even greater than 630%.60 This was observed in creatinine-based as well as in cystatin-based formulas and in those that combined both markers. Consequently, frequent misclassifications of CKD stages were observed. Creatinine-based formulas overestimated CKD stages, and cystatin-based formulas showed a similar rate of overestimation and underestimation of CKD stages. Other studies indicated a low performance of formulas in renal transplantation. Gaspari et al. analyzed 12 creatinine-based formulas and observed that at least 30% of the formulas were included within a 610% error compared with a gold standard.61 Masson et al. evaluated more than 800 patients and showed that the CKD-EPI creatinine equation did not offer a better GFR prediction compared with the modification of diet in renal disease (MDRD) equation, even in the earlier CKD stages.62 Moreover, at best 23% of the patients had an estimated GFR outside 30% of measured GFR. Finally, in terms of CKD classification, approximately one-third were misclassified. A logical consequence of this error is that formulas do not reflect properly GFR changes over time.61 Fauvel et al. observed in more than 600 patients that Cockroft-Gault, MDRD, and CKD-EPI formulas poorly reflected GFR changes, assessed by repeated measures of inulin clearance.63 Thus, formulas have a wide error in estimating GFR and importantly, this error is unpredictable. However, this problem has been underappreciated in the medical literature. This may be the consequence of using statistics not suitable for this type of analysis which leads to a misinterpretation in the accuracy and precision of GFR estimated by formulas.

29.3.5 How to Evaluate a Formula That Estimates GFR: Statistics of Agreement For the evaluation of estimated GFR, some methodological issues should be considered: (1) to define acceptable boundaries of error of estimated compared with measured GFR and (2) the percentage of estimations included within these boundaries; the use of appropriate agreement statistics, which evaluate precision and accuracy simultaneously and provide confidence intervals.64 67 (1) To define a priori acceptable boundaries of error and the percentage of estimations included within these boundaries is crucial. In other words, how much error is acceptable and how many cases must be included within these limits to accept that the estimation is reliable. Unfortunately, for the case of estimated GFR, this has not been established by consensus. An acceptable criterion may be that 90% of the estimations have to be included within a 610% error of measured GFR. The 610% criterion includes the overall reproducibility of a gold standard method (radioactive markers and nonradioactive markers). The 90% criterion indicates that the vast majority of the estimations should have this acceptable bias (,10%). Also, a reduced limit of error is necessary to reflect GFR decline over time. The cut-off criteria most frequently used in the literature is the percentage of estimations included within 630% error of measured GFR. This limit is clinically inappropriate, since according to this cut-off, for a real GFR of 60 mL/minute, a formula may estimate renal function ranging from 42 to 78 mL/minute and still be considered accurate. The question is whether this is acceptable or not. The majority of the studies considered this 630% boundary as a standard parameter to evaluate formulas without analyzing its clinical implications. It can lead to misclassification of patients according with the CKD stages. In our example the same patient can be classified as having CKD 2 or 3. Also, it can lead to an erroneous evaluation of GFR change over time. Finally, the average of the estimations included in this 630% limit hardly exceeds 70% and so, in one-third of the patients,

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the error of estimated GFR is even greater than 630%.60,68 Then, a wide limit of error and a reduced number of estimations included within these limits have been accepted as good agreement. (2) The use of appropriate statistics that provide confidence intervals is a relevant point in the evaluation of formulas. However, the statistics frequently used are not completely suitable for this analysis. For example, the mean error and mean percentage error, usually considered as bias, represent the difference between measurement and estimation for every patient. The statistics are shown as the mean value of all the differences. In the calculation of the mean, similar values of opposite sign cancel each other out, which reduces the real magnitude of the differences between estimated and measured GFR. So, mean or mean percentage errors do not represent real differences. Another aspect is that a test must consider both precision and accuracy simultaneously.67 Precision is defined as the closeness of agreement (degree of scatter) between a series of measurements obtained from multiple sampling of the same homogeneous sample under the prescribed conditions. Accuracy is defined as the deviation of mean from the true value (trueness). So, agreement can be considered as a function of the absolute difference between pair readings.67 Good precision or accuracy does not indicate a good agreement. The classical proof of this concept is the Pearsons correlation coefficient (a marker of precision) which may be excellent for two parallel curves (r . 0.9), but still the accuracy can be low if the difference between both curves is high. So, a crucial point in evaluating agreement is that the same statistic combines accuracy and precision. Lin and colleagues provide a comprehensive approach to agreement analysis with tests such as the concordance correlation coefficient (CCC), the total deviation index (TDI), and the coverage provability (CP).64 67 These tests combine accuracy and precision and provide a confidence interval for every result. CCC varies from 0 to 1 and combines meaningful components of accuracy and precision; a CCC greater than 0.90 indicates optimal concordance between measurements. TDI captures a large proportion of data within a prespecified boundary for allowed differences between two measurements. Coverage probability varies from 0 to 1 and it is a statistic that estimates whether a given TDI is less than a prespecified fixed percentage. For example, a TDI of 0.30 indicates that to include 90% of the estimations, the boundaries of error must be range from 230% to 30% of measured GFR. Finally, diversely from the 30% or 10% limits of accuracy, CCC, TDI, and CP are given with a 95% confidence interval, which favors generalizability of results. We acknowledge that this approach is not widely used in clinical studies. However, this is possibly the best way for studying the performance of estimated GFR. Table 29.1 shows an example of the results of these tests in more than 200 consecutive renal transplant patients, which represent an extension of a previous study.60 All patients underwent a plasma clearance of iohexol and the CCC, TDI, and CP were calculated for a representative group of formulas which include creatinine, cystatin, or both. The formula that showed the best agreement with the gold standard was the Hoeck equation with a TDI of 36.21% (95% CI 39.11). However, this “best agreement” meant that 90% of the estimations had an error range as wide as 636% compared with the gold standard. The remaining formulas: CKD-EPI-sc, Cockcroft-Gault, Rule, etc., and especially those that combine cystatin and creatinine, showed even wider limits of error. This indicates that the formulas have an average error from measured GFR greater than 30%, which makes them inappropriate to monitor renal function. Similar studies have been observed with all the formulas available in the literature.60 For statistical analyses our group developed a statistical package AGP (Agreement Program) v.1.0 (Geiko, SP) freely available at: http://investigacion.chuc.es/2011-09-10-20-17-00/area-de-metodologia. AGP is based on the R code originally developed by Lawrence Lin and Yu Yue64 and was developed to simplify the use of the tool given in the R agreement package.

29.3.6 Measured GFR: A Clinical Tool in Renal Transplantation Several methods have been described to measure GFR.69 The clearance of inulin, which requires continuous infusion and urine collection, is considered the gold standard method for the measurement of GFR. However, it is cumbersome and expensive, which has limited its use to clinical research.69 There are also other exogenous markers to measure GFR, like the clearance of radioactive agents chromium 51-labeled ethylenediaminetetraacetic acid, (51Cr-EDTA), diethylenetriaminepentaacetic acid (DTPA), and the nonionic radiocontrast agents iohexol and iothalamate.69 For all of them the plasma clearance and the renal clearance can be calculated. However, the plasma clearance, which is based on the measurement of the decrease in the concentration at several points after injection, is the most frequently used test. A recent systematic review compared these methods with the clearance of inulin indicated that renal clearance of 51Cr-EDTA or iothalamate and plasma clearance of 51Cr-EDTA or iohexol are sufficiently accurate to measure GFR.69 The use of methods that measure GFR is not as “popular” as formulas that estimate GFR, mainly because they are complex and time-consuming. However, these aspects need to be reconsidered and put into perspective, I. KIDNEY TRANSPLANTATION

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TABLE 29.1 Analysis of Agreement Between Estimated GFR Using Creatinine- and Cystatin-C-Based Formulas and Measured GFR (Iohexol Plasma Clearance) CCC

TDI

CP

CKD-EPI_sc

0.77 (0.73)

62.82 (67.59)

24 (22)

Cockcroft-Gault

0.68 (0.64)

80.83 (86.62)

17 (16)

Rule_CKD

0.77 (0.74)

62.01 (66.79)

24 (23)

aMDRD

0.79 (0.76)

55.58 (59.85)

27 (25)

Rule

0.64 (0.60)

102.30 (109.96)

14 (13)

LeBricon

0.77 (0.74)

54.34 (58.32)

24 (22)

Tan

0.88 (0.86)

39.21 (42.37)

36 (34)

Hoek

0.89 (0.87)

36.21 (39.11)

38 (36)

Larsson

0.88 (0.86)

40.57 (43.87)

35 (32)

Perkins

0.62 (0.58)

91.71 (96.97)

7 (5)

Grubb

0.77 (0.74)

79.00 (85.95)

20 (19)

Orebro

0.77 (0.74)

79.41 (86.50)

20 (19)

Rule_cc

0.80 (0.77)

60.63 (65.27)

23 (21)

Rule_txr

0.89 (0.87)

36.89 (39.85)

38 (35)

MacIsaac

0.85 (0.83)

43.57 (46.98)

32 (30)

ArnalDade

0.85 (0.83)

47.97 (51.90)

30 (28)

Jonsson

0.85 (0.82)

51.73 (56.07)

29 (27)

CKD_EPI_cc

0.87 (0.85)

45.15 (48.88)

32 (30)

CAPA

0.88 (0.86)

41.40 (44.77)

34 (32)

CREATININE-BASED FORMULAS

CYSTATIN-C-BASED-FORMULAS

CREATININE-CYSTATIN-C-BASED-FORMULAS Ma

0.81 (0.79)

54.29 (58.44)

25 (23)

Stevens

0.87 (0.85)

40.79 (44.11)

35 (32)

CKD_EPI_sc_cc

0.85 (0.83)

47.79 (51.78)

31 (29)

TDI, total deviation index; CCC, concordance correlation coefficient; CP, coverage probability. Results expressed for GFR values unadjusted by BSA (mL/min).

particularly after the amount of literature indicating a wide error with estimated GFR in renal transplantation60 63 and other clinical conditions like CKD, diabetes, liver transplantation, heart failure, cancer patients, etc.70 74 It is obvious that compared with a creatinine determination, the measurement of GFR is a complex and time-consuming procedure. However, the benefit obtained by their use, i.e., an accurate and precise value of renal function, clearly outweighs its limitations. Also, the technique is not different in terms of duration and complexity from other procedures that offer relevant information like kidney biopsy, colonoscopy, scanner, magnetic resonance, etc. Naturally, GFR measurement offers major benefits in a group of special clinical conditions in which a reliable evaluation of renal function is needed. These may include (1) dose calculation in chemotherapy or in drugs that are adjusted by renal function, i.e., carboplatin, ciclofosfamide; (2) evaluation of the risk of nephrotoxicity in patients with CKD who will undergo imaging tests with contrast agents; (3) diagnosis of renal disease in situations where creatinine is misleading; (4) follow-up evaluation in which missing a decline in renal function can be deleterious, i.e., chronic glomerulonephritis; (5) children with CKD, among others. In the renal transplant setting, the use of measured GFR offers reliable diagnosis of renal function and its changes over time.75,76 Also, measured GFR is helpful in the evaluation of living donors, which may help unmask occult CKD.77 I. KIDNEY TRANSPLANTATION

424

29. RENAL FUNCTION MEASUREMENTS

29.3.7 Use of the Plasma Clearance of Iohexol in Renal Transplant Patients Based on the previous studies, which showed the lack of reliability of estimated GFR, we wanted to use a gold standard method in the clinics and in research. In the Centre of Biomedical Research of the Canary Islands (CIBICAN) we developed and validated the technique of the plasma clearance of iohexol.78 This was done in collaboration with the Mario Negri Institute (Bergamo, Italy), a center with more than 25 years of experience with this procedure. Iohexol is a nonionic nonradioactive contrast media, easy to use, highly stable in plasma, and with an excellent safety profile. Centers with decades of experience have rarely observed side effects, which were always of only mild severity.61,70,76 The procedure is simple and reproducible, and the compound is highly stable in plasma, which allows safe transport of samples. Briefly, in the morning of renal function evaluation, 5 mL of iohexol solution (Omnipaque 300) is injected intravenously over 2 minutes. Blood samples are then taken at baseline (blank), 120, 180, 240, 300, 360, 420, and 480 minutes for patients with expected mGFR #40 mL/minute, and at 120, 150, 180, 210, and 240 minutes for those with expected mGFR .40 mL/minute. Iohexol plasma levels are measured by high-performance-liquid chromatography. The clearance of iohexol is calculated according to a one-compartment model (CL1) by the formula: CL1 5 Dose/AUC, where AUC is the area under the plasma concentration-time curve. According to Bro¨chner-Mortensen,79 plasma clearances are then corrected by using the formula CL 5 (0.990778 3 CL1) 2 (0.001218 3 CL12). TABLE 29.2 Clinical Examples of Renal Transplanted Patients and Living Donors. Overestimation or Underestimation When Comparing Estimated GFR by Diverse Formulas With Measured GFR (Iohexol Plasma Clearance) mGFR (mL/min)

Formulas (mL/min)

Comments

• • • • • • • • • • • • • • • • • • • • • • • •

20.21 (aMDRD) 20.94 (CKD-EPI) 20.85 (Cockorft-Gould) 37.63 (Cockorft-Gould) 20.0 (CKD EPI-cc) 17.05 (Jonsson-cys) 43.89 (aMDRD) 45.37 (CKD-EPI) 52.71 (Rule) 90.32 (Rule) 66.97 (Orebro-cys) 74.14 (Perkins-cys) 72.17 (aMDRD) 72.36 (CKD-EPI) 75.78 (C-G) 46.44 (Hoek-cys) 36.29 (Rule-cys) 86.24 (Rule) 55.35 (aMDRD) 38.33 (Rule-cys) 42.26 (CKD-EPI-cys) 142.40 (aMDRD) 147.99 (CKD-EPI) 157.45 (Rule-CKD)

100% overestimation

• • • • • • • • •

85.45 (CKD-EPI) 97.54 (Cockorft-Gould) 88.94 (Rule) 92.20 (Cockorft-Gould) 90.33 (CKD-EPI) 90.09 (Rule) 119.20 (Cockorft-Gould) 112.2 (CKD-EPI) 94.90 (aMDRD)

20% overestimation 40% overestimation 25% overestimation 25% overestimation

TRANSPLANTED PATIENTS 1

9.38

2

26.55

3

31.52

4

42.67

5

51.36

6

61.79

7

72.57

8

88.80

40% overestimation 30% underestimation 35% underestimation 40% overestimation 40% overestimation 70% overestimation .100% overestimation 60% overestimation 70% overestimation 40% overestimation

35% underestimation 40% underestimation 40% overestimation 35% underestimation 50% underestimation 40% underestimation 60% 70% overestimation

LIVING DONORS 1

70.30

2

72.57

3

74.72

Results expressed for GFR values unadjusted by BSA (mL/min).

I. KIDNEY TRANSPLANTATION

50% overestimation 50% overestimation 30% overestimation

REFERENCES

425

We are currently using the plasma clearance of iohexol in the renal transplant population followed in our center and in the evaluation of living kidney donors. An analysis of almost 200 patients showed that formulas are unreliable in reflecting GFR in this population (Table 29.1).60 Table 29.2 shows some real examples of our outpatient clinic in which GFR was measured with the plasma clearance of iohexol and estimated with several formulas. Patients were selected from GFR measurements ranging from 10 mL/minute to almost 90 mL/minute trying to reflect a complete spectrum of renal function in this population. The cases illustrate extreme overestimation of up to 100% and underestimation of 30% 40% of GFR with creatinine-based or cystatin-based equations respectively. Finally, similar errors were observed with all the available formulas.75 Another aspect of the use of measured GFR in clinical practice is the evaluation of living kidney donors. Table 29.2 shows three examples of living kidney donors in which estimated GFR overestimated renal function, while GFR gave results below the cut-off for accepting kidney donation: 80 mL/minute (Ana Gonza´lez Rinne, personal communication). Finally, kidney donation in these donors would have led to CKD, which was avoided by a measurement of GFR. So, our experience shows that measured GFR in renal transplant patients and in living donors is useful and gives relevant information to the clinician. After considering these results, the question is why the measurement of GFR is not more frequently performed in clinical practice. It is clear that not all the centers have the technology for its implementation. However, many tertiary referral centers have the necessary infrastructure, i.e., HPLC-UV. This might be the consequence of incomplete knowledge about the inaccuracies of estimated GFR and its consequences in clinical practice, or simply prejudices against techniques used in clinical research. In this regard, it has to be noticed that KDIGO guidelines suggest the use of gold standard methods “when GFR estimates based on creatinine are thought to be inaccurate and when decisions depend on more accurate knowledge of GFR, such as confirming a diagnosis of CKD, determining eligibility for kidney donation, or adjusting dosage of toxic drugs that are excreted by the kidneys.”80 In clinical research in renal transplantation is it clear that the evolution of GFR over time must be evaluated with gold standard methods. Estimated GFR is completely misleading as an outcome in clinical trials.61,70 Recently, the EMA suggested the use of gold standard methods in clinical trials aimed evaluating reno-protective measures that slow GFR decline over time.81

29.4 CONCLUSIONS In renal transplant patients, renal function cannot be monitored with formulas, which have a high error in reflecting renal function. When possible, measured GFR should be implemented. Of the several methods available, the plasma clearance of iohexol seems the more simple, safe, and relatively cheap. Otherwise, repeated creatinine measurements and clinical experience are more reliable than formulas.

Acknowledgments The author would like to acknowledge support from (1) the IMBRAIN project (FP7-RE6-POT-2012-CT2012-31637-IMBRAIN) funded under the 7th Frameworks Programme (capacities); (2) Fondos FEDER for the following grants: PI 07/0732, the REDINREN RD/0021/0008 and PI10/02428; and (3) funding of the Spanish Research Network for Kidney Diseases (REDINREN RD12/0021/0008). Finally, the author thanks Sergio Luis Lima for a critical review of the manuscript and Antonio Delgado for the design of Fig. 29.1.

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C H A P T E R

30 Pharmacokinetics and Genomics of Immunosuppressive Drugs Marek Drozdzik Pomeranian Medical University, Szczecin, Poland

30.1 INTRODUCTION Successful kidney transplantation became possible due to development of effective immunosuppressive drugs and treatment regimens. The first allogenic kidney transplants were protected from rejection by azathioprine, an antimetabolite. However, azathioprine monotherapy resulted in a limited number of transplant recipients with long-term graft function. Shortly afterwards, in the early 1960s, it was found that the combination of azathioprine with corticosteroids improved renal transplant recipient survival, as additive and synergistic effects were produced. The double medication strategy became the standard therapy by 1964, but still with unsatisfactory mortality and morbidity rates in cadaveric transplants, i.e., the 1-year graft function rate was approximately 50%. The next progression in immunosuppressive therapy was provided by discovery of cyclosporine (cyclosporin A), a calcineurin inhibitor, and its clinical application in 1976. For decades, the triple therapy of cyclosporine, azathioprine, and corticosteroids was the preferred regimen for allograft transplantation. The next advance came in 1994 with the introduction of mycophenolate mofetil, which replaced azathioprine almost universally. In the same year another calcineurin inhibitor, tacrolimus (given twice daily) was introduced, and in many centers it gradually replaced cyclosporine (in 2013 a once daily tacrolimus formulation was approved by FDA for maintenance therapy of kidney transplant recipients). Another therapeutic option appeared with development of m-TOR inhibitors: sirolimus (rapamycin), marketed in 1999, and everolimus, launched in 2010 to prevent organ rejection in adult kidney transplant recipients. These small-molecule drugs are mainly used for maintenance immunosuppression. The last drug approved by FDA in 2011 for maintenance therapy in kidney transplantation is belatacept, a fusion protein providing costimulation blockade (antagonist of the B7 ligands, B7-1 (CD80) and B7-2 (CD86)). Induction immunosuppression and acute rejection are mainly controlled by biological drugs. The first of them, polyclonal antibodies, antithymocyte globulin—equine and rabbit, became available for treatment of acute rejection in kidney transplantation in 1981 and 1998, respectively. More selective lymphocyte T targeting was possible with the development of monoclonal antibodies: muromonab-CD3 in 1987, basiliximab in 1998, and daclizumab in 1997. The list of currently FDA approved immunosuppressive drugs for maintenance therapy include: prednisone, tacrolimus, cyclosporine, mycophenolate mofetil, mycophenolate sodium, azathioprine, sirolimus, everolimus, and belatacept. These drugs are described in this chapter. Further information about immunosuppressive drugs and therapeutic regimens is presented in other chapters of the present book. Information about drug pharmacokinetics, i.e., mathematical description of the time course of drugs in the body, enables not only precise drug characteristics, but also provides clinicians useful therapeutic information on drug absorption, distribution, metabolism, and elimination, which can be used to optimize drug dosing. Further, measurement of drug blood concentration may be used to individualize medication. Many immunosuppressive agents are characterized by a narrow therapeutic index, significant inter- and intraindividual variability in blood concentrations, and substantial toxicity. The drug concentration variability can result from different factors such as sex,

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age, disease states, genetic variability, drug-drug and/or drug-nutrient interactions. The unpredictable drug concentrations affect clinical outcome of medication, especially for drugs characterized by the narrow therapeutic index. A drug is classified as a narrow therapeutic index agent when its dose that produces beneficial clinical effect is near to the dose that is likely to result in adverse effects. The therapeutic index is calculated as the ratio of the toxic and the therapeutic dose of a drug, and the closer to 1 it is, the more risky its clinical use. Based on blood concentration measurements and clinical observations, narrow therapeutic ratio is defined in FDA regulations at 21 CFR 320.33(c) as “there is less than a twofold difference in median lethal dose (LD50) and median effective dose (ED50) values, or have less than a twofold difference in the minimum toxic concentrations and minimum effective concentrations in the blood, and safe and effective use of the drug products requires careful dosage titration and patient monitoring.”1 Clinical use of drugs with a narrow therapeutic index produced the need for therapeutic drug monitoring (TDM), i.e., measurement of drug plasma concentration, in single or multiple time points, in order to achieve a desired therapeutic drug concentration. Therapeutic drug concentration is generally defined as the range of drug concentrations associated with maximal efficacy and minimal toxicity. Thus, TDM enables the individualization of dosing regimen to produce satisfactory clinical outcome and reduce a risk of side effects. However, the therapeutic range should be viewed with criticism, as other factors (e.g., disease states, electrolytes) could influence final effects of drug therapy. So, the therapeutic range provides information on drug concentrations, where the probability of desired clinical outcome is relatively high along with relatively low risk of unacceptable toxicity. TDM is widely practiced in immunosuppressive management, especially for cyclosporine, tacrolimus, everolimus, and sirolimus. Over the last few decades, it has become evident that genetic variability may contribute to the observed differences in drug responses. Pharmacogenetics/pharmacogenomics aims at identification of individual genetic predispositions to reduced clinical efficacy or an increased risk of adverse effects, in order to modify drug dosage or switch to alternative therapy, i.e., to individualize therapy. Genetic variation can influence response to drugs by affecting both pharmacokinetic factors (e.g., drug metabolism and transport) or pharmacodynamics (e.g., receptor functions). Influence of genetic factors, mostly drug metabolizing enzymes’, on immunosuppressive drug actions has been extensively studied. Defective variants, associated with reduced or complete absence of enzymatic activity (i.e., so-called poor metabolizers) may result in higher drug concentration upon administration of a standard dose, and thus elevated risk of toxicity. The presence of many active gene copies (i.e., so-called ultra-fast metabolizers), resulting in higher enzymatic activity, produce lower drug levels, that can be associated with reduced drug response (when an enzyme degrades the drug) or increased drug response/toxicity (when an enzyme activates the prodrug). Numerous single nucleotide polymorphisms (SNPs) were identified in genes encoding cytochrome P450 izoenzymes (e.g., CYP3A4, CYP2D6), and in some phase II enzymes (e.g., thiopurine S-methyltransferase), determining the observed differences in response to drugs principally metabolized by those enzymes. Variable drug transporter activity (e.g., P-glycoprotein), determined by genetic factors, may affect drug penetration through biologic barriers (e.g., blood brain barrier) and/or cell membranes, thus affecting drug concentration in target sites. Variable frequency of genetic variants (mostly produced by SNPs) determines differences in drug responses in ethnic cohorts. That issue may be of particular significance for SNPs not directly affecting protein expression/activity, which can be used as markers only in some ethnic groups, as a certain linkage between different loci can differentiate populations. In the case of some polymorphic enzymes, pharmacogenetically-guided protocols of immunosuppressive treatment have been established, providing recommended modifications of drug medication based on genetic testing results. In the case of immunosuppressive drugs the most complete and clinically useful information is available for tacrolimus and azathioprine. The pharmacokinetic data and pharmacogenetic/pharmacogemomic information are used in clinical practice in order to optimize drug dosing in a particular patient, leading to pharmacotherapy optimization, i.e., increasing its efficacy at the reduced risk of side effects. The available, clinically relevant data regarding immunosuppressive drugs is presented in the chapter.

30.2 TACROLIMUS 30.2.1 Pharmacokinetics Tacrolimus (TAC) is characterized by a narrow therapeutic index and large inter- and intraindividual pharmacokinetic variability.2 The drug has poor oral bioavailability, ranging from 5% to 93% (mean, 25%). Low TAC bioavailability (14%) was reported in patients awaiting renal transplantation.3 In general, oral doses of TAC should

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be 3 4 times higher than intravenous doses to produce comparable drug concentrations, since cytochrome P450 3A isoenzymes (CYP3A4/5) and transporter protein, P-glycoprotein expressed in gastrointestinal tract are responsible for presystemic metabolism and removal (drug transport back into the intestinal lumen) of the drug.4 Peak concentrations in whole blood occur at 1 2 hours after oral administration. After absorption, TAC is distributed to erythrocytes and is approximately 99% protein-bound to both α1-acid glycoprotein and albumin. Due to extensive binding to erythrocytes, TAC blood concentrations are on average about 15 times higher than serum or plasma concentrations.1,5 As a result, whole blood samples are used for TAC monitoring. Distribution into most tissues is extensive, and the drug also passes through the placenta and into breast milk.6 TAC is metabolized by cytochrome P450 3A (CYP3A) enzymes in liver and small intestine—in particular, CYP3A4 and CYP3A5. The drug undergoes O-demethylation, hydroxylation and/or oxidative metabolic reactions. There are at least 15 metabolites with the major metabolite being 13-O-demethyl-tacrolimus. Biliary excretion and fecal elimination are responsible for the major clearance of metabolites, whereas renal excretion plays only a small role (accounts for only 2.4% of TAC elimination).6,7 P-glycoprotein, expressed in hepatocytes, pumps the drug outside cells, and thus regulates access of TAC to CYP3A enzymes. Terminal half-life of TAC in kidney transplant recipients is approximately 8.7 hours. On the market, two formulas of TAC are available, i.e., once-daily formulation (QD) and twice-daily (BID) formulation of TAC. QD formulation of TAC has been developed to provide more convenient drug dosing and better patient compliance. Some studies have reported that the daily dose of TAC QD is higher than that of TAC BID required to produce similar trough concentrations, suggesting lower oral bioavailability of TAC QD than TAC BID.8,9 However, results of TAC QD trials and conversion trials from TAC BID to QD demonstrated similar safety and efficacy of both formulations as well as trough levels.10 12 Likewise, in pediatric renal recipients both TAC formulations are bioequivalent. However, a decrease in AUC0 24 and Cminute after the conversion, requiring close pharmacokinetic monitoring during the conversion period, was observed.13 A summary of product characteristics for Advagraf (QD) provides information that allograft transplant patients maintained on twice daily Prograf (BID) capsules dosing requiring conversion to once daily Advagraf should be converted on a 1:1 (mg:mg) total daily dose basis. TAC pharmacokinetics can be influenced by many factors. Kidney insufficiency reduces the drug absorption from gastrointestinal tract, but the drug clearance is not significantly correlated with creatinine clearance, and patients with severe renal dysfunction and patients on dialysis prior to kidney transplantation were characterized by comparable TAC clearance to healthy volunteers.2 However, the study of Undre et al. in renal transplant recipients revealed a decline in the dose-normalized trough concentration by 61.1% and TAC dose by 50% after 2 years from transplantation procedure.14 Those findings can be explained by a reduction in corticosteroid dosage and increased hematocrit and albumin concentrations observed in posttransplant period. It was also found that diarrhea caused a significant rise in TAC trough levels despite intake of stable doses, necessitating TAC dose reductions of 30% to obtain prediarrhea trough levels.15 Oral TAC absorption is greatly affected by food, particularly high-fat meals. It was found that mean area under the blood concentration-time curve (AUC0 N) values decreased by about 25% compared with fasting values (more prominent in high-fat than in low-fat meals).16 To avoid the possible effect of food on TAC bioavailability, the drug should be given at a constant time relation to meals. TAC elimination half-life ranges from 31.9 to 48.4 hours. The major determinant of TAC elimination is hepatic drug metabolism, mainly dependent on enzymatic metabolic activity (also to a minor extent on blood flow through liver). Therefore, liver failure as well as CYP3A4/5 enzymes inhibitors, e.g., aminodarone, azithromycin, clarithromycin, cyclosporine, delavirdine, diltiazem, entacapone (high dose), erythromycin, ethinyl estradiol, fluconazole, fluoxetine, fluvoaxamine, gestodene, indinavir, isoniazid, ketoconazole, metronidazole, nefazodone, nevirapine, norfloxacin, omeprazole, paroxetine (weak), propoxyphene, quinidine, ranitidine, ritonavir, saquinavir, sertraline, valproic acid and inducers, e.g., carbamazepine, dexamethasone (corticosteroids), phenytoin, progesterone, rifampin, oxcarbazepine, phenobarbital, phenylbutazone may affect the drug exposure. Products that can be used without medical supervision can also affect CYP3A4/5 enzymatic activity, resulting in TAC concentrations increase by St. John’s wort or decrease by grapefruit juice. St. John’s wort administration for 2 weeks increases twofold TAC dose-corrected AUC0212, which required TAC dose increase from a median 4.5 mg/day at baseline to 8.0 mg/day under St. John’s wort exposure. Two weeks after discontinuation of St. John’s wort, TAC doses were reduced to a median of 6.5 mg/day.17 Administration of grapefruit juice for 1 week results in significant increase in TAC blood concentration in liver transplant patients (effect depends on juice type), giving in a group of patients with significant increase in TAC concentrations a reduction of the drug administered dose by 2.3 6 1.3 mg/day.18

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TAC pharmacokinetics may be also affected by age, sex, and ethnicity. Female renal transplant recipients reach overall higher Cmax levels and AUC compared to males, but this effect seems to be seen rather in early posttransplant period.19,20 Young children (below 5 years) appear to need higher doses per kilogram body weight of TAC than older children and adults in order to maintain similar trough concentrations.21 Ethnicity may influence TAC pharmacokinetics. African Americans are characterized by significantly lower maximum drug concentration after oral (but not intravenous) administration than white and Latin Americans.22 These ethnic differences are most likely mediated via several nongenetic as well as genetic factors, including known genetic variations that impair transporter/enzyme activity in genes such as CYP3A4, CYP3A5, and ABCB1 (coding for P-glycoprotein).23

30.2.2 Therapeutic Drug Monitoring The interindividual variability of TAC pharmacokinetic parameters and a narrow therapeutic index makes TDM necessary to ensure appropriate immunosuppression levels and to minimize the risk of side effects. The drug trough concentrations (Cmin) have been found to correlate well with AUC, which reflects total body exposure to the drug.24 The drug levels are measured as predose trough concentrations (12 or 24 hours after previous dose for BID and QD formulations, respectively) in whole blood. TAC therapeutic ranges used by most centers at various time points after transplantation are as follow: 0 1 month—15 20 ng/mL; 1 3 months—10 15 ng/mL; and more than 3 months—5 12 ng/mL in blood.7 The product monograph recommends a trough level of 10 20 ng/mL in early posttransplantation and 5 20 ng/mL thereafter for BID (Prograf) and 10 20 ng/mL in early posttransplantation and 5 15 ng/mL during maintenance for QD (Advagraf). TAC blood concentrations are monitored 3 7 days a week for the first 2 weeks, followed by 3 evaluations for the next 2 weeks, and then at each outpatient visit. Unlike cyclosporine C2 levels, TAC peak concentration (Cmax) has not been routinely utilized for TDM. Two different TAC assay systems are used in clinical practice; the enzyme-linked immunosorbent assay (ELISA; Pro-Trac, IncStar) and microparticulate enzyme immunoassay (MEIA; IMx, Abbott Diagnostics). These two different assay systems produce similar results of TAC blood concentrations. However, both methods are based on the same monoclonal antibody for the TAC parent compound that often cross-react with metabolites. More recently, mass spectrometry (LC/MS/MS) methods were developed which enable specific TAC determination.25

30.2.3 Pharmacogenetics/Pharmacogemomics Polymorphisms in CYP3A4 and CYP3A5 have been reported to influence TAC dosing and serum concentrations in kidney transplant patients. The best-defined polymorphism affecting TAC concentrations is a common nonfunctional splicing-defect in CYP3A5*3 allele, determined by rs776746:G.A SNP. Most Caucasians lack enzyme activity and are defined as CYP3A5 nonexpressors. Dose-adjusted trough concentrations of TAC were in CYP3A5*3/*3 patients threefold higher than in CYP3A5*1/*3 patients, and 5.8-fold than in CYP3A5*1/*1.26 Another polymorphic loci, that influence TAC concentrations is 3A4*22 (rs35599367 C.T in intron 6), has been shown to affect TAC pharmacokinetics independently of CYP3A5 genotype. The first dose-adjusted concentrations of TAC were twofold higher in T-variant allele carriers compared with CC homozygotes. Moreover, it was found that combined CYP3A4/CYP3A5 genotype affected the concentration more strikingly, i.e., CYP3A poor metabolizer status was characterized by 1.6- and 4.1-fold higher TAC dose-adjusted concentrations than the intermediate and extensive metabolizers, respectively.27 Those observations also have clinical translation. Kuypers et al. demonstrated that carriers of CYP3A5*1 allele were at significantly higher risk of chronic irreversible druginduced nephrotoxicity, associated with higher drug exposure, than CYP3A5*3 allele subjects.28 Quteineh et al. reported significantly higher number of acute rejection episodes in CYP3A5*1 homozygotes compared to carriers of CYP3A5*1/*3 and CYP3A5*3/*3 genotypes (38% vs 10% and 9%).29 Effects of other polymorphisms evaluated in patients medicated with TAC seem to have less pronounced clinical impact than these described above. However, TAC dosing based on genotype has not reached routine patient care.

30.3 CYCLOSPORINE 30.3.1 Pharmacokinetics Cyclosporine (CsA), like tacrolimus, is characterized by a narrow therapeutic index and large inter- and intraindividual pharmacokinetic variability.30 On the market, two oral formulations of CsA are available, an

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oil-based formulation (Sandimmune) and a newer microemulsion formulation (Sandimmun Neoral), both provided as an oral solution or gelatine capsules. According to the manufacturer information the microemulsion oral solution and gelatine capsule formulations are bioequivalent.31 The absorption of the oil-based formulation of CsA in the gastrointestinal tract depends on bile acids, with the bile acid emulsification process being responsible for limited and variable absorption.32 The absolute oral bioavailability of the oil-based formulation ranges from 10% to 80% in adults, on average 30%, also with variability of the time to reach maximum concentration (tmax), on an average from 2 to 4 hours (range 1 8 hours).32 34 The CsA microemulsion formulation due to self-emulsifying properties is not markedly dependent on bile. Therefore, the bioavailability of the microemulsion formulation is generally about 30% higher (absolute bioavailability of about 40%) in comparison with the oil-based formulation, with 1 hour earlier tmax (1 2 hours after the dose).32,35 The microemulsified formulation is also characterized by lower intraindividual and comparable interindividual pharmacokinetic variability as compared with the oil-based formulation.36 Because microemulsion formulation seems not to be bioequivalent to oil-based formulation, conversion from microemulsion formulation to oil-based formulation using a 1:1 ratio (mg/kg/day) may result in lower CsA blood concentrations. Conversion from microemulsion formulation to oil-based formulation should be made with increased monitoring to avoid the potential of underdosing.37 After absorption, CsA is widely distributed, reaching values of the steady state volume of distribution (Vd) of 3 5 L/kg.33,38 In blood, 90% 98% of CsA is bound to red blood cells and plasma proteins, with about 20% 60% of the total CsA in plasma being bound to lipoproteins.39 As lipophilic compound, CsA widely distributes to tissues and accumulates mainly in leukocytes and fat-rich organs such as liver, adipose tissue, lymph nodes, and breast tissue and in kidney.40 In organ transplant patients due to binding to plasma lipids significantly lower CsA clearance was observed in patients with hyperlipidemia than in those with normal lipid levels.41 CsA crosses the placenta and is excreted in human milk. CsA is metabolized in the gastrointestinal tract and liver by P450 3A4/5 enzymes, resulting in the formation of up to 25 metabolites. The most prominent metabolites that are found in human blood are 2 hydroxylated products (AM1 and AM9).30 CsA is also a substrate for P-glycoprotein and multidrug resistance-associated protein 2 (MRP2, ABCC2) that play a role in transmembrane transport of the drug, thus determining its absorption in the gastrointestinal tract, enzymatic availability in hepatocytes and distribution to other cells.42 Most of the absorbed CsA (90%) is excreted in bile as metabolites, with less than 1% excreted as the unchanged drug. The rest is excreted in urine, with less than 0.1% as the unchanged drug.43 The terminal half-life of CsA in kidney transplant recipients is approximately 8.4 hours (range 5 18 hours). Pharmacokinetics of CsA is influenced by many factors. The drug absorption can be affected by food. However, the available data is not equivocal. An increase up to 45% 60% of CsA trough concentration, Cmax and AUC after oral administration of the oil-based formulation with a standard hospital breakfast as compared to fasting state drug application was reported.44 In contrast, other studies revealed delayed and impaired absorption by food or lack of influence of a low-fat meal or a high-fat meal on the absorption of the oil-based CsA formulation.45,46 Based on available information, patients should be instructed to take their CsA consistently in relation to the timing and composition of meals in order to minimize absorption variability. Contrary to TAC, trough levels of CsA remained stable without dose adjustments in kidney transplant patients with diarrhea.15 Likewise to TAC, increased CsA concentrations of more than 60% have been reported after intake of grapefruit juice. Administration of pomelo juice with CsA also increased AUC02t by about 20%, which can be considered clinically important. The observed effects are associated with inhibition of hepatic and intestinal CYP3A4/5 activity by grapefruit and pomelo juices.47 Herbal remedy, i.e., St. John’s wort, similarly to TAC, produces a decrease in CsA bioavailability, due to induction of CYP3A4/5 enzymatic activity and P-glycoprotein transporter function. In renal transplant patients, administration of 600 mg St. John’s wort for 2 weeks resulted in clinically relevant decrease in CsA concentration after 3 days in all treated patients, and 41% 46% reduction in the drug plasma concentration.48 Various case reports in the literature have described failed CsA treatment due to St. John’s wort intake. CsA is a substrate of CYP3A4/5 and P-glycorpotein, and in general shows the same drug drug interaction spectrum as tacrolimus (abbreviated drug list interacting with CYP3A4/5 and P-glycoprotein is presented in “Tacrolimus” section). CsA lowers mycophenolic acid concentrations by inhibiting MRP2 in liver.49 Liver pathologies resulting in reduced CYP3A4/5 activity affect CsA pharmacokinetics. In contrast to the effects of hepatic dysfunction, renal failure and hemodialysis do not seem to affect CsA clearance/exposure.50 However, CsA absorption is significantly lower among patients on maintenance hemodialysis who showed no predictive correlation with posttransplant levels. The C1 and C2 values were significantly higher posttransplant, suggesting a steeper absorption phase, which was consistent with the higher AUC024 in posttransplant period.

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The possible role of uremia in retarding absorption was proposed, which may have clinical significance for primary graft dysfunction.51 There is limited data on sex effects on CsA pharmacokinetics, but the available information suggests similar pharmacokinetics of the drug in kidney transplant male and female recipients.52 The difference in CsA pharmacokinetics was observed in children aged 2 5 years, where significant differences were noted in body weight-adjusted CsA dose, Vd, and apparent blood clearance (Cl) requiring higher doses, with a relative greater distribution, and exhibiting more rapid drug clearance than children over 10 years of age.53,54 Studies on pharmacokinetics of CsA in African Americans and white Americans revealed that the dose-adjusted AUC of intravenous and oral CsA was significantly lower in African Americans than in white Americans.55,56

30.3.2 Therapeutic Drug Monitoring The exposure to CsA is best characterized by AUC0212 (12-hour AUC), which is a good predictor of acute rejection and graft loss.57 However, this approach requires multiple blood sampling, which is a limitation of the method in clinical routine. In clinical practice single sampling methods are used, i.e., measurement of trough concentration (C0) or maximum concentration (C2). The 2-hour postdose level (C2) seems to be a more reliable tool to monitor CsA therapy, because C2 has a better correlation with AUC than trough level (C0). It better predicts both nephrotoxicity and acute rejection, whereas C0 is directly correlated with nephrotoxicity. CsA therapeutic level ranges are different at various posttransplant periods in renal transplant recipients. Within the first 2 weeks, recommended C0 and C2 values are: 200 400 ng/mL and 1.4 2.0 mg/L, respectively. Then, in 2 3 months after transplantation: C0 (good renal function) 125 275 ng/mL, C0 (poor renal function) 100 125 ng/mL, and C2—1.2 1.8 mg/L. From the 6th month to 1 year after transplantation CsA concentrations of C0 100 150 ng/mL and C2 0.7 1.0 mg/L are recommended. Beginning from 1 year and afterwards C0 of 75 100 ng/mL and C2 0.7 1.0 mg/L are set. Frequency of CsA blood levels determination should be at 2 3 days (in the first 4 weeks posttransplant), then monthly after 3 months.58 Conversion from microemulsion formulation to oil-based formulation should be made with increased monitoring to avoid the potential of underdosing.37 It is strongly recommended that CsA blood trough concentration be monitored every 4 7 days after the conversion. Data of CsA concentration should be analyzed with caution as many laboratory methods have been applied for the drug measurement. Parent-compound-specific assays correlate best with clinical events, and among them HPLC is the standard reference. The monoclonal antibody RIAs (monoclonal radioimmunoassay, various manufacturers) and the monoclonal antibody FPIA (monoclonal TDx assay, Abbott Diagnostics) methods offer sensitivity, reproducibility, and convenience.59

30.3.3 Pharmacogenetics/Genomics The available data on CsA pharmacogenetics in kidney transplant recipients are still debatable. As the drug is a substrate for CYP3A4/5, the respective polymorphisms may affect CsA metabolism. Some studies suggested that CYP3A5 expressors produce lower dose-adjusted trough blood concentrations of the drug than nonexpressors, with the most signicant effects observed in CYP3A poor metabolizers (combined poor metabolizer CYP3A4/ CYP3A5 genotype status) characterized by 1.5- and 2.2-fold higher CsA dose-adjusted concentrations than the intermediate and extensive metabolizers, respectively.26,27 However, the consensus for genetic effects on CsA pharmacokinetics is less convincible than for TAC.

30.4 SIROLIMUS/RAPAMYCIN 30.4.1 Pharmacokinetics Oral bioavailability of sirolimus (SRL) amounts to 15% in kidney transplant recipients. The drug is rapidly absorbed, reaching Cmax after about 2 hours after administration.60 SLR is widely distributed throughout the body (Vss/F 12 6 8 L/kg). In human whole blood, SRL is distributed among red blood cells (94.5%), plasma (3.1%), lymphocytes (1.0%), and granulocytes (1.0%), and is extensively bound (approx. 92%) to human plasma proteins, mainly serum albumin (97%), α1-acid glycoprotein, and lipoproteins. SRL is a cytochrome CYP3A substrate, and is transported by P-glycoprotein. More than 16 metabolites of SRL have been identified, some of them with low, not clinically significant immunosuppressive activity.61 SRL mean t1/2 is about 60 hours. Approximately 91% of the

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drug is eliminated through gastrointestinal tract and 2% through kidney. Dosage adjustments are not necessary for sex, age, body weight, ethnicity, or impaired renal function.62,63 SRL absorption is slower after a meal than in fasting state, a high-fat meal decreases AUC by 35%.64 It is recommended to administer the drug consistently, either with or without meals, in order to avoid intrasubject variability in whole-blood concentrations. SRL is metabolized through CYP3A4 and is a substrate of P-glycorpotein. Therefore inhibitors and inducers of the drug metabolism and transport (abbreviated drug list interacting with CYP3A4/5 and P-glycoprotein is presented in “Tacrolimus” section) may affect SRL exposure. Likewise, described for TAC, CsA and everolimus interaction with grapefruit juice and St. John’s wort may occur with SRL.65 CsA inhibits gastrointestinal and hepatic metabolism of SRL. Therefore, it should be administered separately with an interval of 4 hours in order to ensure that both drugs do not reach their maximum blood concentration at the same time. Administration of CsA and SIR increase the blood concentration of both drugs.66 The available data demonstrates a lack of any clinically important drug interaction between SIR and TAC in healthy subjects after single-dose administration.67

30.4.2 Therapeutic Drug Monitoring The immunosuppressive efficacy and adverse effects of SRL correlate with blood concentrations, and the drug displays large inter- and intrapatient variability. It is therefore a good candidate for TDM.62 SRL trough concentrations have shown good correlation with AUC curve and daily drug exposure, contrary to the drug daily dose.63 The drug levels for TDM are measured as predose trough concentrations in whole blood. Recommended SRL levels in regimens with calcineurin inhibitor are within 4 12 mg/mL in order to avoid both rejection episodes and drugrelated toxicity. In patients receiving CsA-sparing regimens, higher concentrations may be required, i.e., target through range of 12 20 mg/mL (16 24 ng/mL in the first year following transplantation) is recommended.68 According to Khan et al. SRL recommended levels without calcineurin inhibitors are 10 15 ng/mL in the first 3 months, afterwards between 8 and 12 ng/mL; and with calcineurin inhibitors between 5 and 10 ng/mL.63 Because of the time required to reach steady state, SRL dose adjustments would optimally be based on trough levels measured 5 7 days after a dose change. Various chromatographic and immunoassay methodologies are applied for SRL assays. Patient sample concentration values from different assays may not be interchangeable.

30.4.3 Pharmacogenetics/Pharmacogenomics A limited number of SRL pharmacogenetic studies have evaluated variables in the following genes: ABCB1, CYP3A4, CYP3A5 and IL-10 in patients after kidney transplantation.69,70 The results are inconsistent, and no recommendations for genotyping to individualize SRL therapy have been established.

30.5 EVEROLIMUS 30.5.1 Pharmacokinetics Everolimus (EVR) is a more water-soluble analog of SIR, and was developed to improve pharmacokinetic characteristics of SIR, particularly to increase its oral bioavailability. After oral administration the drug is rapidly absorbed with tmax after 30 minutes (range 0.5 1 hour).71 In blood, EVR is over 75% protein-bound, and mostly distributes (.75%) into blood cells.72 The average t1/2 of EVR is 18 35 hours (depending on the studied population), and steady state is reached within 7 days. EVR is extensively metabolized in the small intestine and liver, 98% of the drug is eliminated via bile in the form of metabolites. Cytochrome P450 (CYP) enzymes CYP3A4, CYP3A5, and CYP2C8 are involved in the drug metabolism, resulting in generation of 6 metabolites deprived of relevant clinical activity.71,73 As most of EVR (98%) is eliminated in the form of metabolites via bile, EVR pharmacokinetics does not seem to be affected by kidney function. Hemodialysis does not affect EVR exposure.74 EVR pharmacokinetics can be influenced by many factors. The drug absorption can be affected by food. High-fat meal delays tmax of EVR by a median 1.25 hours, reduce Cmax by 60%, and AUC by 16% in healthy volunteers. In the multiple-dose screening in patients with renal transplants, a high-fat meal delays tmax by a median 1.75 hours and reduces Cmax by 53% and AUC by 21%.75 Based on available information, patients should be instructed to take EVR consistently in relation to the timing and composition of meals in order to minimize absorption variability. EVR is metabolized predominantly via CYP3A4/5 and is the P-glycoprotein substrate. Therefore, the aforementioned (see “Tacrolimus” section) inducers and inhibitors of the enzymes can affect its metabolism, leading

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to increased concentrations of the drug. Likewise, grapefruit juice and St. John’s wort may affect EVR pharmacokinetics. In clinical settings, EVR can be comedicated with TAC or CsA. When EVR is used simultaneously with CsA, Cmax and AUC for both the compounds are increased.75 77 Thus, it is recommended that CsA and EVR are administered 4 hours apart. TAC effects on EVR pharmacokinetics depend on the dose. When TAC exposure is down-titrated within the trough concentration range of 1.5 7 ng/mL any significant interactions are observed.78 However, a starting EVR dose of 0.75 mg twice daily in combination with TAC result in mean trough EVR concentrations consistently greater than 3 ng/mL over 6 months.79 There is no detectable influence of sex, age, or weight on AUC of EVR.76 There is limited data published on EVR pharmacokinetics across racial or ethnic groups. It was found that African Americans had approximately a 20% higher drug clearance, but there is also a report showing EVR pharmacokinetics similar to Caucasians.80,81 As EVR is a substrate of CYP3A4/5, thus like other immunosuppressants being its substrate, the required dose in African Americans may be in general higher in order to achieve similar to white Americans trough concentrations.

30.5.2 Therapeutic Drug Monitoring EVR is characterized by a narrow therapeutic index and high interindividual and interoccasion variability in pharmacokinetics, such factors warranting TDM for pharmacotherapy individualization. The monitoring parameter most commonly used in clinical settings is EVR through concentration (C0), but some clinics use AUC.82,83 The C0 is a good surrogate marker of EVR exposure, and correlates with pharmacological response.84 The recommended therapeutic range for EVR is C0 of 3 8 ng/mL, as concentrations over 3 ng/mL have been associated with a decreased incidence of rejection, and concentrations .8 ng/mL with increased toxicity.85 Based on the available evidence, TDM for EVR may provide additional information on efficacy and safety compared with clinical judgment alone. C0 should be assessed approximately 2 weeks after commencing treatment (and approximately 2 weeks after any change in dose, or after an initiation or change in coadministration of CYP3A4 and/or P-glycoprotein inducers or inhibitors). Several analytic methods are available to quantify EVR concentrations. A standard for direct EVR measurement is LC-MS/MS (several protocols are available). The quantitative microsphere system (QMS) everolimus immunoassay (Thermo Fisher Scientific) is the only FDA-approved immunoassay. An FPIA (Seradyn Inc.) is available outside the United States. Other immunoassays are under development, but require final validation of their clinical utility.

30.5.3 Pharmacogenetics/Pharmacogenomics The available pharmacogenetic data suggests that EVR pharmacokinetics is not significantly influenced by polymorphisms located in genes coding for drug metabolizing enzymes CYP3A5 and CYP2C8 as well as drug transporter P-glycoprotein.86 Pharmacogenetic variables do not affect the drug pharmacokinetics in a clinically relevant manner, and therefore are not suitable to help improvement in prediction of EVR exposure.

30.6 MYCOPHENOLATE 30.6.1 Pharmacokinetics Mycophenolate is now the antimetabolite of choice in immunosuppressant regimens in kidney transplant recipients replacing azathioprine. Oral mycophenolate is available as the ester prodrug mycophenolate mofetil (MMF) or the enteric-coated sodium salt mycophenolate sodium (EC-MPS). MMF is deesterified in stomach and by tissue and plasma esterases to form mycophenolic acid (MPA), which is absorbed in the stomach and proximal small intestine. EC-MPS rapidly dissolves to MPA in gastrointestinal tract at pH .5.5, which is absorbed in the small intestine.87 Maximum MPA plasma concentrations usually occur within 0.5 2 hours of MMF oral intake, and after 2 3 hours after oral EC-MPS administration.88,89 Both MMF and EC-MPS are not bioequivalent. Bioavailability of MPA is also dose-dependent, decreasing with increased MMF dose.90 MPA C0 values were found to be 46% higher after EC-MPS than MMF in kidney transplant recipients.91 At clinically relevant concentrations, mycophenolic acid and MPAG are about 97% and 82% bound to albumin, respectively. MMF is activated by plasma and tissue esterases to the active MPA. MPA is metabolized in liver via UDP-glucuronosyltransferase isoforms, mainly 1A8, 1A9, and 2B7 to several metabolites. The 7-hydroxyglucuronide MPA (MPAG) is the major metabolite and is inactive. Two other minor metabolites are

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produced, inactive phenolic glucoside and acyl-glucuronide, the latter with some pharmacologic and toxicologic effects. These metabolites are excreted via bile into the intestine. MPAG undergoes enterohepatic recirculation via cleavage by bacterial β-glucuronidase to MPA, which is subsequently reabsorbed back into the systemic circulation, producing typical secondary MPA peak, usually from 6 to 8 hours after administration, in the concentration-time curve.92,93 Over 90% of the administered dose is excreted in the urine, mostly as MPAG. Renal impairment had no major effect on the pharmacokinetics of mycophenolic acid after single doses of MMF, but there was a progressive decrease in MPAG clearance as glomerular filtration rate (GFR) declined. Compared to individuals with a normal GFR, patients with severe renal impairment (GFR 1.5 L/hour/1.73 m2) showed three- to sixfold higher AUC values of MPAG. In rental transplant recipients during acute renal impairment in the early posttransplant period, the plasma MPA concentrations were comparable to those in patients without renal failure, whereas plasma MPAG concentrations were two- to threefold higher. Hemodialysis had no major effect on plasma mycophenolic acid or MPAG. Dosage adjustments appear to not be necessary either in renal impairment or during dialysis.94 The changes in MPA with both food and antacid are small in comparison with the interpatient variability and clinically not significant.95 Oral iron supplements do not produce significant effect of on MMF absorption.96 Sex, race, and body weight have little influence on MPA pharmacokinetic variability.97 However, one study suggests than MPA exposure is higher in African Americans than in Caucasians.98 Cyclosporine affects MPA pharmacokinetics reducing MPA exposure by 30% 40%, inhibiting biliary excretion of MPAG and hence enterohepatic recycling of MPA.99 Tacrolimus and sirolimus inhibit glucuronidation of MPA, which is associated with impaired MPA elimination.100 Corticosteroids were reported to reduce MPA exposure when given at high dosages.101 However, in the population pharmacokinetic model there was no correlation between corticosteroids dose and MPA pharmacokinetic parameters.102

30.6.2 Therapeutic Drug Monitoring In patients administered mycophenolate, a wide interindividual and intraoccasional variability in dosage requirements to produce target therapeutic MPA concentrations has been reported, demonstrating the need of TDM for both formulations.91 Although the current labeling information for MMF and EC-MPS does not indicate therapeutic monitoring of plasma MPA concentrations, there are clinical studies demonstrating a relationship between MPA pharmacokinetics and clinical outcome (summarized in Knight and Morris).103 MPA monitoring may be of importance in the early posttransplant period (the greatest variability in MPA pharmacokinetics is noted in the initial 2 months following transplantation), in high-immunological recipients, in patients on calcineurin-inhibitor-sparing regimens and in whom unexpected rejection or infections occurred. The parameter best describing MPA exposure is AUC, but its measurement is hardly feasible in clinical situations, and various limited sampling methods have been developed and validated, e.g., trough MPA concentration, single concentration-time points (e.g., C2 or C4), multiple concentration-time points (several specific timed points after dosing), single or multiple concentration-time points, for Bayesian analysis.99 HPLC is the analytical method available for MPA measurement.

30.6.3 Pharmacogenetics/Pharmacogenomics Pharmacogenetic factors can influence pharmacokinetics and pharmacodynamics of MPA. Among the potential candidate genes are those coding for UDP-glucuronosyltransferase and inosine monophosphate dehydrogenase (IMPDH) MPA target gene. Some studies suggest an association between polymorphisms in UGT1A9 and UGT1A8 as well as IMPDH1 and IMPDH2 genes and clinical outcomes.104 Currently, there are no guidelines for routine MPA pharmacogenetic assays, and pharmacogenetic information is not included in the drug labels.

30.7 AZATHIOPRINE 30.7.1 Pharmacokinetics Azathioprine (AZA) is readily absorbed after oral administration, with only 12.6% of the dose recovered from feces within 48 hours. It reaches Cmax 1 2 hours after administration, and is rapidly distributed throughout the body. Protein binding is not extensive (30%).105 The drug undergoes nonenzymatic conversion to

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6-mercaptopurine (6-MP) by compounds with sulfhydryl or amino groups such as cysteine, glutathione, and sulfide ion.106 In the next step, 6-MP is oxidized by xanthine oxidase to 6-thiouric acid or methylated to 6-methyl-mercaptopurine by thiopurine S-methyltransferase or converted to 6-thioguanine nucleotides (6-TGNs) by hypoxanthine guanine phosphoribosyltransferase. The 6-TGNs are active metabolites that inhibit de novo synthesis of purines (TGNs are responsible for both therapeutic and toxic effects).107 Proportions of metabolites are different in individual patients, and this presumably accounts for variable magnitude and duration of drug effects. The plasma t1/2 is 3 5 hours (decay rate for all S-containing metabolites of the drug). Up to 50% of a dose is excreted in urine over the first 24 hours after administration (10% as unchanged drug). In patients with deteriorated kidney function an accumulation of toxic TGNs is observed, which may require dose modification.107 Elimination during 8 hours of hemodialysis amounts to 45%, which is only slightly less than the elimination in the urine of normal persons over a period of 24 hours.108

30.7.2 Therapeutic Drug Monitoring The intraindividual variability in AZA pharmacokinetics is marked, but it is less pronounced than even larger interpatient variability.109 Therefore, TDM with drug concentration measurements is not implemented. It was attempted to monitor AZA therapy by 6-TGNs levels in a triple-drug regimen. However, there were no significant correlations between either white blood cell count and 6-TGNs concentrations or between 6-TGNs concentrations and 6-MP metabolites.110,111 Another approach for individualization, which is included in the FDA approved drug label, recommends assessment of TPMT activity in red blood cells (phenotyping). The enzymatic activity is measured with radiochemical and HPLC detection methods. Patients with low or absent TPMT activity are at an increased risk of developing severe, life-threatening myelotoxicity.112

30.7.3 Pharmacogenetics/Pharmacogenomics TPMT gene polymorphism is a model example in pharmacogenetics. Deficiency of the enzyme activity is associated with severe hematopoietic toxicity after administration of standard doses of thiopurine drugs (including AZA), usually several weeks from therapy initiation. TPMT-deficient renal transplant recipients (up to 0.3% in Caucasian populations), inheriting two variant alleles accumulate high concentrations of toxic TGNs in tissues during AZA treatment.113,114 Individuals heterozygous for one of the deficient alleles (most frequently TPMT*2, *3A or *3C) show intermediate enzyme activity and lower accumulation of TGNs, and are also at greater risk of AZA toxicity in comparison with normal TPMT activity subjects.115 The FDA approved statement in AZA label recommends patient genotyping for TPMT.

30.8 CORTICOSTEROIDS 30.8.1 Pharmacokinetics Prednisone (PN) is the FDA approved corticosteroid in maintenance therapy of kidney transplant patients. PN, an inactive compound, is metabolized in the liver, by the hepatic 11-β-hydroxydehydogenase, to its active metabolite prednisolone (PL), being the principal corticosteroid found in plasma in transplant recipients (76% of the corticosteroid dose is recycled). Interconversion is not a limiting factor, as it occurs even in patients with liver failure.116 PN and PL are rapidly absorbed after oral administration with Cmax within 1 3 hours of dosing in transplant recipients, with oral bioavailability of about 86% of PL and 93% of PN.117 PL and PN are characterized by a moderate Vd and penetrate quickly into the kidneys, intestine, skin, liver, and muscle. Tissue penetration of lipophilic PL is limited by P-glycoprotein.118 PL binds to both albumin, transcortin, and slightly to α1-acid glycoprotein in a nonlinear fashion, i.e., its binding decreases with increased concentrations of the drug. The nonlinear protein binding explains observed concentration dependent nonlinear pharmacokinetics.117,119 PL is metabolized mainly in liver, with resultant formation of at least 10 metabolites, which are partially conjugated. In its phase I metabolism, CYP3A4 and CYP3A5 are the best-defined participating enzymes. The conjugated and unconjugated metabolites have been found in urine.120,121 T1/2 of PN and PL is 2 4 hours in stable transplant recipients. Food intake is generally considered to prolong tmax, but not the extent of drug absorption.117 Increases in PL plasma concentrations were observed in liver and renal failure, in renal transplant patients, and in elderly patients.122,123 Hemodialysis (5 hours) was reported to remove 7% 17.5% of the total PL dose

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administered in kidney transplant recipients.124 In renal transplant recipients, PL renal and nonrenal clearances are decreased, with or without concomitant treatment with CsA.120,125 CsA does not change the dose-adjusted exposure of PL compared with TAC. Adult kidney transplant recipients can therefore continue on their usual PL dose when changing therapy between CsA and TAC.126 MMA and AZA did not appear to affect the pharmacokinetics of PL and PN.122

30.8.2 Therapeutic Drug Monitoring PL and PN are traditionally considered as wide therapeutic index drugs, and therefore not requiring TDM. Also their relatively long biological half-life in comparison to plasma half-life affects the direct relationship between corticosteroid concentration and efficacy or toxicity. Benefits from corticosteroid TDM may be seen in transplant recipients showing therapeutic failure, defined PL adverse effects, drug interactions or disease states affecting pharmacokinetics and/or pharmacodynamics.127,128

30.8.3 Pharmacogenetics/Pharmacogenomics In one study an association between AUC0 24 and Cmax of PL with NR1I2 polymorphism was reported. In the same study no significant differences in PL pharmacokinetics in patients stratified for CYP3A5 and ABCB1 genotypes were found.129 No recommendations for genotyping to individualize corticosterooid therapy have been established.

30.9 BELATACEPT 30.9.1 Pharmacokinetics Belatacept is administered intravenously; and its bioavailability is 100%. Patient exposure to the drug is proportional to dosage, with very little day-to-day variability. Vd is low: 0.11 0.12 L/kg, which is consistent with the physical property of belatacept as a large therapeutic protein, with distribution limited to the extracellular space.130 The metabolism of the drug has not been defined. Belatacept is not subjected to cytochrome P450 metabolism, and most probably is degraded through nonspecific mechanisms, i.e., via uptake and degradation by macrophages and Kupffer cells.130 The serum t1/2 of belatacept ranges from 8 to 8.5 days.130,131 In clinical trials, trough concentrations of belatacept were consistently maintained for up to 3 years posttransplant in kidney transplant patients.130 Belatacept concentrations are not increased in patients with kidney impairment.132,133 Hemodialysis does not affect belatacept elimination (due to its high molecular weight). However, the drug is removed via plasmapheresis.133 Body weight affects belatacept exposure, i.e., higher body weight results in faster drug elimination.133 Sex, age, and race do not affect the drug pharmacokinetics.133,134 Liver impairment with normal albumin level does not change belatacept elimination.133 However, there is no data available from patients with severe hepatic dysfunction. There is no effect of diabetes on belatacept clearance.133 The pharmacokinetic profile of balatacept in patients who had converted from calcineurin-based to belatacept-based treatment regimens is similar to that in treatment-naı¨ve patients receiving belatacept-based treatment regimens.135 The risk of drug drug interactions is very low, the drug is not metabolized by cytochrome P450 enzymes or UDP-glucuronosyltransferases.136 However, biologic drugs, through induced changes in cytokine levels, affect CYP enzymes activity, and thus interact with drugs being CYP substrates.137 A possible interaction between belatacept and MPA was described, with MPA Cmax and AUC decrease (but this observation was more likely associated with inhibitory effects of CsA on enterohepatic MPA recirculation).138

30.9.2 Therapeutic Drug Monitoring Belatacept pharmacokinetics and pharmacodynamics appear to vary little between individual patients, through concentration is consistently maintained during treatment, and therapeutic target range is not defined. Therefore, the rationale for belatacept TDM has not been established.134

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30.9.3 Pharmacogenetics/Pharmacogenomics There is no existing data on genetic variants associated with belatacept pharmacokinetics, and pharmacodynamic effects or clinical outcomes.

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71. Kirchner GI, Meier-Wiedenbach I, Manns MP. Clinical pharmacokinetics of everolimus. Clinical Pharmacokinetics 2004;43:83 95. 72. Kovarik JM, Hartmann S, Figueiredo J, Rouilly M, Port A, Rordorf C. Effect of rifampin on apparent clearance of everolimus. Ann Pharmacother 2002;36:981 5. 73. Jacobsen W, Serkova N, Hausen B, et al. Comparison of the in vitro metabolism of the macrolide immunosuppressants sirolimus and RAD. Transplant Proc 2001;33:514 15. 74. Thiery-Vuillemin A, Curtit E, Maurina T, et al. Hemodialysis does not affect everolimus pharmacokinetics: two cases of patients with metastatic renal cell cancer. Ann Oncol 2012;23:2992 3. 75. Kovarik JM, Hartmann S, Figueiredo J, et al. Effect of food on everolimus absorption: quantification in healthy subjects and a confirmatory screening in patients with renal transplants. Pharmacotherapy 2002;22:154 9. 76. Kovarik JM, Kaplan B, Silva HT, et al. 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Mudge DW, Atcheson B, Taylor PJ, et al. The effect of oral iron administration on mycophenolate mofetil absorption in renal transplant recipients: a randomized, controlled trial. Transplantation 2004;77:206 9. 97. Pescovitz MD, Guasch A, Gaston R, et al. Equivalent pharmacokinetics of mycophenolate mofetil in African-American and Caucasian male and female stable renal allograft recipients. Am J Transplant 2003;3:1581 6. 98. Tornatore KM, Sudchada P, Attwood K, et al. Race and drug formulation influence on mycophenolic acid pharmacokinetics in stable renal transplant recipients. J Clin Pharmacol 2013;53:285 93. 99. Tett SE, Saint-Marcoux F, Staatz CE, et al. Mycophenolate, clinical pharmacokinetics, formulations, and methods for assessing drug exposure. Transplant Rev (Orlando) 2011;25:47 57. 100. Grinyo´ JM, Ekberg H, Mamelok RD, et al. 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103. Knight SR, Morris PJ. Does the evidence support the use of mycophenolate mofetil therapeutic drug monitoring in clinical practice? A systematic review. Transplantation 2008;85:1675 85. 104. Kurzawski M, Dro´zdzik M. Pharmacogenetics in solid organ transplantation: genes involved in mechanism of action and pharmacokinetics of immunosuppressive drugs. Pharmacogenomics 2013;14:1099 118. 105. Elion GB. The significance of azathioprine metabolites. Proc Royal Soc Med 1972;65:257 60. 106. Elion GB. The George Hitchings and Gertrude Elion Lecture. The pharmacology of azathioprine. Ann NY Acad Sci 1993;685:400 7. 107. Chan GL, Erdmann GR, Gruber SA, Matas AJ, Canafax DM. Azathioprine metabolism: pharmacokinetics of 6-mercaptopurine, 6-thiouric acid and 6-thioguanine nucleotides in renal transplant patients. J Clin Pharmacol 1990;30:358 63. 108. Schusziarra V, Ziekursch V, Schlamp R, Siemensen HC. Pharmacokinetics of azathioprine under haemodialysis. Int J Clin Pharmacol Biopharm 1976;14:298 302. 109. Ohlman S, Albertioni F, Peterson C. Day-to-day variability in azathioprine pharmacokinetics in renal transplant recipients. Clin Transplant 1994;8:217 23. 110. Bergan S, Rugstad HE, Bentdal O, et al. Monitored high-dose azathioprine treatment reduces acute rejection episodes after renal transplantation. Transplantation 1998;66:334 9. 111. Chrzanowska M, Krzymanski M. Determination of 6-thioguanine and 6-methylmercaptopurine metabolites in renal transplantation recipients and patients with glomerulonephritis treated with azathioprine. Ther Drug Monit 1999;21:231 7. 112. Booth RA, Ansari MT, Tricco AC, et al. Assessment of thiopurine methyltransferase activity in patients prescribed azathioprine or other thiopurine-based drugs. Evid Rep Technol Assess 2010;196:1 282. 113. Kurzawski M, Dziewanowski K, Ciechanowski K, Drozdzik M. Severe azathioprine-induced myelotoxicity in a kidney transplant patient with thiopurine S-methyltransferase-deficient genotype (TPMT*3A/*3C). Transpl Int 2005;18:623 5. 114. Budhiraja P, Popovtzer M. Azathioprine-related myelosuppression in a patient homozygous for TPMT*3A. Nat Rev Nephrol 2011;7:478 84. 115. Yates CR, Krynetski EY, Loennechen T, et al. Molecular diagnosis of thiopurine S-methyltransferase deficiency: genetic basis for azathioprine and mercaptopurine intolerance. Ann Intern Med 1997;126:608 14. 116. Renner E, Horber FF, Jost G, Frey BM, Frey FJ. Effect of liver function on the metabolism of prednisone and prednisolone in humans. Gastroenterology 1986;90:819 28. 117. Pickup ME. Clinical pharmacokinetics of prednisone and prednisolone. Clin Pharmacokinet 1979;4:11 28. 118. Karssen AM, Meijer OC, van der Sandt IC, et al. The role of the efflux transporter P-glycoprotein in brain penetration of prednisolone. J Endocrinol 2002;175:251 60. 119. Czock D, Keller F, Rasche FM, et al. Pharmacokinetics and pharmacodynamics of systemically administered glucocorticoids. Clin Pharmacokinet 2005;44:61 98. 120. Frey FJ, Frey BM, Schnetzer A, Horber FF. Evidence that cyclosporin A does not affect the metabolism of prednisolone after renal transplantation. Transplantation 1987;43:494 8. 121. Garg V, Jusko WJ. Bioavailability and reversible metabolism of prednisone and prednisolone in man. Biopharm Drug Dispos 1994;15:163 72. 122. Bergmann TK, Barraclough KA, Lee KJ, Staatz CE. Clinical pharmacokinetics and pharmacodynamics of prednisolone and prednisone in solid organ transplantation. Clin Pharmacokinet 2012;51:711 41. 123. Gambertoglio JG, Amend Jr. WJ, Benet LZ. Pharmacokinetics and bioavailability of prednisone and prednisolone in healthy volunteers and patients: a review. J Pharmacokinet Biopharm 1986;8:1 52. 124. Frey FJ, Gambertoglio JG, Frey BM, et al. Nonlinear plasma protein binding and haemodialysis clearance of prednisolone. Eur J Clin Pharmacol 1982;23:65 74. 125. Frey FJ, Schaad HJ, Renner EL, Horber FF, Frey BM, Preisig R. Impaired liver function in stable renal allograft recipients. Hepatology 1989;9:606 13. 126. Bergmann TK, Isbel NM, Barraclough KA, Campbell SB, McWhinney BC, Staatz CE. Comparison of the influence of cyclosporine and tacrolimus on the pharmacokinetics of prednisolone in adult male kidney transplant recipients. Clin Drug Investig 2014;34:183 8. 127. Jusko WJ, Rose JQ. Monitoring prednisone and prednisolone. Ther Drug Monit 1980;2:169 76. 128. Potter JM, McWhinney BC, Sampson L, Hickman PE. Area-under-the-curve monitoring of prednisolone for dose optimization in a stable renal transplant population. Ther Drug Monit 2004;26:408 14. 129. Miura M, Satoh S, Inoue K, et al. Influence of CYP3A5, ABCB1 and NR1I2 polymorphisms on prednisolone pharmacokinetics in renal transplant recipients. Steroids 2008;73:1052 9. 130. Shen J, Townsend R, You X, et al. Pharmacokinetics, pharmacodynamics, and immunogenicity of belatacept in adult kidney transplant recipients. Clin Drug Investig 2014;34:117 26. 131. Vincenti F, Blancho G, Durrbach A, et al. Five-year safety and efficacy of belatacept in renal transplantation. J Am Soc Nephrol 2010;21:1587 96. 132. Rostaing L, Massari P, Garcia VD, et al. Switching from calcineurin inhibitor-based regimens to a belatacept-based regimen in renal transplant recipients: a randomized phase II study. Clin J Am Soc Nephrol 2011;6:430 9. 133. Zhou Z, Shen J, Hong Y, Kaul S, Pfister M, Roy A. Time-varying belatacept exposure and its relationship to efficacy/safety responses in kidney-transplant recipients. Clin Pharmacol Ther 2012;92:251 7. 134. de Raav GN, Bergan S, Baan CC, Weimar W, van Elder T, Hesselink DA. Therapeutic drug monitoring of belatacept in kidney transplantation. Ther Drug Monit (in press). 135. Garnock-Jones KP. Belatacept: in adult kidney transplant recipients. BioDrugs 2012;26:413 24. 136. Bestard O, Campistol JM, Morales JM, et al. Advances in immunosuppression for kidney transplantation: new strategies for preserving kidney function and reducing cardiovascular risk. Nefrologia 2012;32:374 84. 137. Lee J-I, Zhang L, Men AY, Kenna LA, Huang SM. CYP-mediated therapeutic protein drug interactions. Clin Pharmacokinet 2010;49:295 310. 138. Nujolixs (belatacept) package insert. Princeton: Bristol-Myers Squibb Co. 2013.

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31 Gene Expression Technology Applied to Kidney Transplantation Richard Danger1,2 and Sophie Brouard1,2,3 1

Universite´ de Nantes, Nantes, France 2Institut de Transplantation Urologie Ne´phrologie (ITUN), Nantes, France 3 CIC Biotherapy, Nantes, France

31.1 INTRODUCTION Renal transplantation is the treatment of choice for end-stage renal disease, improving patient’s life expectancy and quality of life. Sustained progress has been made in allograft acceptance, mainly thanks to immunosuppressive treatment from the 1980s and 1990s.1 In addition, with better management of patients and graft allocation, the early outcome survival rate is above 90%.2 However, long-term allograft survival is still low, with an allograft half-life around 10 years.2 In renal transplantation, physicians rely on a measure of graft function and histological analysis to manage allograft outcomes, and further tools would be useful. Impairment of renal function is an accurate marker of allograft injury but is poorly associated with allograft outcome.3 Histological biopsy analysis is the gold standard for diagnosis but biopsy is invasive, with a risk of rejection, and prone to errors in sampling and interpretation.3,4 Molecular profiling has improved our understanding of allograft response mechanisms and is also used to identify molecular-driven phenotypes and factors associated with graft damage in order to manage and prevent it.5,6 As molecular alterations can appear before cellular damage and organ injury, studies have aimed at using transcriptomic markers to accurately diagnose or predict early graft events. Ideally, such biomarkers would allow better patient follow up and earlier pharmacologic intervention, before damage occurs, in a personalized approach. As the literature is plethoric—a simple PubMed search with renal transplantation and gene expression terms provides more than 2500 articles—we decided to focus on recent, significant articles highlighting potential insights from gene expression in renal transplantation. Various events can shorten graft half-life: Mediocre allograft quality impairing its ability to cope with peritransplantation injury, acute rejection episodes (ARE) that can usually be resolved by immunosuppression but have a negative impact on graft outcome and chronic allograft dysfunction (CAD).2,3,7,8 This chapter looks at these events from a transcriptomic viewpoint, including messenger and micro RNAs (mRNAs, miRNAs) together, in order to summarize current knowledge of gene expression in renal transplantation.

31.2 GENE EXPRESSION MEASURE IN RENAL TRANSPLANTATION Analysis of gene expression generally pursues two central and interrelated goals, often performed sequentially: First the identification of differentially expressed genes or enriched gene sets to explain biological mechanisms, and second, the discovery of disease-associated genes, or gene sets associated with phenotypes, in order to define subgroups of clinical syndromes, and diagnose or predict diseases and outcomes. The identification of such biomarkers does not preclude the use of clinical parameters, as their association in a composite score can improve prediction,9,10 like scores combining demographic and clinical parameters to predict graft failure.11 Kidney Transplantation, Bioengineering, and Regeneration. DOI: http://dx.doi.org/10.1016/B978-0-12-801734-0.00031-X

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31.2.1 Overview of Gene Expression Technologies Used in Renal Transplantation The 21st century seen significant developments in various transcriptomic technologies with the possibility of profiling from individual single to thousands of genes in a single cell or an entire organism, and the renal transplantation field has benefited from these improvements.12 For more than 10 years, microarray technology and analysis methods have substantially improved, allowing the evaluation of numerous samples in a single run, reducing technical variability and experiment time, and the use of varied and small size samples and also giving high reproducibility while decreasing experiment price. With thousands of genes now evaluated and up to hundreds of samples, bioinformatic tools have also been developed to handle the huge amount of data for various purposes.13,14 Deep-sequencing RNA (RNA-Seq) technologies are providing an alternative to the use of microarrays by not only providing expression levels of virtually all expressed transcripts but also their sequence.15 As costs are rapidly decreasing and bioinformatic RNA-seq analysis tools are improving, RNA-Seq experiments will no doubt become more common in renal transplantation, though currently few studies have used this technique.16 19 Some studies suggest a similar performance between RNA-Seq and microarrays, others indicate a complementarity of the methods for low-expression genes.15,20,21 Both techniques are subject to the overfitting intrinsic to highthroughput technology and require additional validations using independent methods. The gold standard for measuring gene expression is quantitative, or real-time, PCR (qPCR).12,22 An absolute quantification of transcripts can be achieved using a standard curve, but the most common method is the relative quantification of transcript levels compared to reference genes. Since reliable measure of low-expression genes is problematic with microarrays and RNA-Seq, qPCR is recommended for its high sensitivity.23 Traditional qPCR methods, which measure a single gene, have been improved on with qPCR-based or multiplex methods that allow the measurement of up to several thousand genes in a single assay.12 However, to date, in renal transplantation qPCR has been used more as a validation method rather than for the discovery of gene signatures. Generated datasets are stored in public repositories, such as the Gene Expression Omnibus (GEO)24 or ArrayExpress,25 which allow peer review and reanalysis using different methodologies or looking at predetermined signatures. These datasets are also useful to the scientific community as they can be used in metaanalysis, by combining different datasets to increase sample numbers, potentiating statistical power and taking into account demographic and ethnic diversity. Such metaanalyses have been performed in renal transplantation and demonstrated their effectiveness by allowing cross-organ comparison in acute rejection26 and in operational tolerance.27

31.2.2 Biological Compartment Analysis Histological analysis of allograft biopsy remains the gold standard for assessing graft alterations and molecular analysis of biopsies has undeniably improved our understanding of mechanisms of allograft evolution.28 The concept of “molecular microscopy,” using microarray-based mRNA assessment, defines gene expression analysis of a biopsy, and aims to complement or to substitute for histological analysis, especially in cases where diagnosis is difficult to establish.10 However, because the biopsy procedure is invasive, can involve severe complications, is subject to sampling bias and subjective interpretation, and cannot be used for serial monitoring,4,29,30 urine would appear to be a useful source of biomarkers as it comes directly from the renal allograft. miRNAs’ resistance to enzymatic (RNases) and physical (freezing/defreezing) degradation make them good potential urine biomarkers.31 Peripheral blood is also easily available, though, in contact with the allograft, it contains immune cells, but considering the potential influence of other organs and its complex composition, its gene expression can be biased.32 Finally, whatever the peripheral source, careful methodology is required in collection, processing and analysis and further validation is needed.

31.3 GENE EXPRESSION ASSOCIATED WITH PRE- OR PERITRANSPLANTATION FACTORS As previously stated, organ quality is associated with the success of transplantation, but the allocation and the prediction of graft acceptance is a challenging task for physicians.33 The use of extended criteria reduces time on waiting lists but increases the risks of nonfunctional graft or delayed graft function (DGF) associated with

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diminished long-term outcome.33 The inability of some organs to recover from death stress and transplant injuries, including cold ischemia and ischemia/reperfusion (I/R),33 is certainly involved but the exact mechanisms are still unclear. As a result, the molecular profiling of preimplantation biopsy has been used to decipher such mechanisms and to develop potential tools to evaluate graft quality and predict its function in a new host after transplantation stress.

31.3.1 Factors Modulating Renal Molecular Profile at an Early Stage Donor age, as a major determinant of graft quality,34 affects the graft molecular profile.35 Supervised multiclass analysis has indicated 505 differential genes between young (8 weeks 8 years), adult (31 46 years), and old kidneys (71 88 years). In old kidneys, gene ontology analysis has shown a decrease in expression of genes related to energy metabolism and an increase in those related to inflammatory responses. 128 genes overlap with genes related to glomerular sclerosis, interstitial fibrosis, tubular atrophy, and fibrous intimal thickening. Evaluation of cellular senescence in preimplantation biopsy, notably using the expression of the cyclin-dependent kinase 2A (CDKN2A), has been described as a significant predictor of DGF and posttransplant renal allograft function.36,37 The combination of both chronological and biological ages increases the accuracy of renal function prediction. These results are consistent with the physiological process of aging, including cumulative environmental stress, which results in an inflammatory response linked to nephron loss and matrix remodeling. As renal transplant outcomes differ significantly between organs from living donors (LD) and deceased donors (DD),2 a number of studies have investigated their molecular differences. Clearly, an increased expression of proinflammatory genes is a common hallmark of DD compared to LD.38 40 Hauser et al. highlighted 132 genes that differentiate LD from DD in preimplantation biopsies, mainly genes with functional roles in cell communication, apoptosis and inflammation, including complement cascade.40 In zero-hour kidney biopsies, Kotsch et al. found greater induction of immune-related genes including CD69, chemokine (C-C motif) ligand 19 and 21 (CCL19, CCL21) and proteasome subunits 8, 9, and 10 (PSMB8, PSMB9, and PSMB10) in DD compared to LD.9 In postreperfusion biopsies, DD and LD grafts still exhibit different profiles, including a greater expression of complement protein C3 and chemokines (C-X-C motif) ligand 1 and 2 (CXCL1, CXCL2) in DD.38 According to Kusaka et al., 1-hour posttransplantation biopsies show downregulation of genes associated with metabolic pathways and overexpression of inflammation-related genes in DD.39 The difference in complement system between LD and DD is confirmed by Naesens et al., who demonstrate a significant correlation between longer cold ischemia time and higher expression of 6 complement genes in DD.41 Increased expression of complement-related genes in posttransplantation compared with implantation biopsies has also been observed irrespective of donor source, with the effect of peritransplantation phenomena including reperfusion. This I/R injury dynamic was also investigated by Hoffman et al., who examined LD kidneys before procurement, 30 60 minutes postreperfusion, and at least 1 month posttransplantation.42 I/R was characterized by increased levels of genes related to cellular adhesion, chemotaxis, apoptosis, monocyte recruitment, and activation. While T-cell-associated transcripts were not expressed postperfusion, their expression increased after I/R recovery. These results are consistent with monocyte infiltration and tubular injury postperfusion followed by T-cell infiltration in a stable posttransplant state observed by histological analysis. A multispecies metaanalysis identified 47 differential genes that may contribute to I/R injury.43 New candidate genes could be associated to new I/R mechanism while other, known, genes were found, such as immune-related genes like LCN2 (Lipocalin2), CCL2, CXCL1, and ICAM1 (intercellular adhesion molecule 1). As for mRNAs, several miRNAs have been described as being modulated in kidney after I/R injury in mice and/or patients after kidney transplantation, including miR-2444 and miR-21.45,46 Even surgical manipulation may influence the expression of ischemia and injury associated genes.47 DGF is associated with longer hospital stays, increased risk of rejection episodes, and poorer long-term graft outcomes and patient survival, and may thus be used as a surrogate marker of allograft outcome.33 DGF has also been found to be associated with elevated expression of inflammation-related genes48 and genes related to cell death.49 Comparing protocol biopsies, seven miRNAs are upregulated in DGF samples and miRNA targets are associated with pathways related to angiogenesis, proliferation, and apoptosis.50 These results show that factors influencing organ quality also modify gene expression, mainly with lower expression of metabolic pathway, cellular integrity, cell homeostasis genes, and higher levels of genes related to inflammation and apoptosis. In addition to preimplantation or at implantation responses, injury repair responses are apparent up to 6 weeks after transplantation.51 Mengel et al. found that modified gene expression correlates with DGF, but not with 3- and 6-month posttransplant graft injuries, suggesting that molecular events impacting allograft outcomes

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are initially hidden by tissue repair response. In a longitudinal analysis of renal allografts, an initial increase in adaptive immune-associated genes peaked 1 month after transplantation whereas sequential regulation of genes related to fibrosis peaked at 3 months.52 Wound healing remodeling and cell proliferation repair processes are observed between 3 and 12 months and macrophage function only after 12 months.52 These findings highlight the interest of strategies for preventing organ injury, such as pharmacologic preconditioning of the organ using cytoprotective molecules, targeting inflammation or complement activation.53 Gene expression analysis can also assess the validity of these methods that aim to reduce organ stress. Thus, use of pulsatile pumps has been associated with a decrease in inflammatory genes in preimplantation biopsies.54 However, while donor age and cold ischemic time was higher in the group with pulsatile pumps, there was no significant difference in the occurrence of DGF and graft function 2 years posttransplantation. Similarly, in biopsies, lower hypoxia-inducible factor-1α gene expression was observed in machine perfusion and living-related kidney donors compared to cold storage after reperfusion with a significantly higher 5-year graft survival.55

31.3.2 Prediction of Gene Expression at Transplantation Gene expression at the time of transplantation has also been shown to predict allograft function and graft outcome.40,41,56,57 Using an unsupervised clustering method, Mueller et al. showed that gene expression can identify three groups of patients, one LD and two DD, based on the patients with DGF who return to dialysis in the initial weeks after transplantation.56 Naesens et al. identified expression of complement-related genes of preimplantation biopsies, which correlate significantly with early graft function at days 2 and 3 and 2 3 years posttransplantation. Perco et al. identified three genes (NLRP2, IGJ, IGLA) that can explain 28% of the creatinine variability at 1 year after transplantation, whereas clinical and histological markers can only explain 14%.58 Similarly, Kotsch et al. found that gene expression in zero-hour biopsies, associated with clinical variables, can accurately predict DGF, development of acute cellular rejection and 6-month graft function.9 The combination of cell death related genes BAX (BCL2-associated X protein) and BCL2 (B-cell CLL/lymphoma 2) in preimplantation renal biopsies allow the prediction of DGF using multiple regression analysis and qPCR.49 Additional investigations are required to validate these potential biomarkers of organ quality but these studies show that gene expression at transplantation can be used to predict graft outcome, including DGF, ARE, and graft function.

31.4 GENE EXPRESSION TO ASSESS AND DIAGNOSE ACUTE RENAL REJECTION Diagnosis of acute rejection is based on histopathological findings from the biopsy after a rapid diminution in renal function. Clinicians need earlier diagnosis of AREs before irreversible allograft damage and other clinical manifestations. The search for blood biomarkers for ARE in transplantation was first emphasized in heart transplantation in a prospective study of 600 heart transplant recipients in the CARGO (Cardiac Allograft Rejection Gene Expression Observational) study.59 Of 97 candidate genes identified by microarrays, 11 genes identified patients with acute rejection (ISHLT grade 3A or higher) in two independent validation sets with quantitative PCR assays. These findings led to the development of a commercially available blood-based test, AlloMap, and represent a clinically relevant application for microarray in the field of cardiovascular medicine.

31.4.1 Gene Expression Profiling of Acute Renal Rejection in Biopsy The mechanisms of acute rejection are heterogeneous, including acute T-cell-mediated rejection (TCMR) and antibody-mediated rejection (ABMR), of which the latter could play a role in more than 30% of AREs.60 This heterogeneity is evidenced through molecular profiling.61 Using 26 biopsy samples from patients with ARE, unsupervised clustering showed three distinct subgroups. One exhibited T-cell and B-cell signatures, confirmed by immunohistochemical analyses. The second was associated with clinicopathological evidence of toxic drug effects or infection. The third was associated with molecular features of chronic allograft nephropathy, despite the biopsies finding histologic criteria for acute rejection. The CD201 B-cell infiltrate in the first group was significantly associated with both clinical glucocorticoid resistance and graft loss, confirming the usefulness and interest of gene microarrays in identifying molecular-based disease subgroups. A list of 70 intragraft genes correlate with acute rejection in kidney recipients in a metaanalysis.62 Most of these genes are associated with immune processes, antigen-presenting cells, cytotoxic T lymphocytes, and IFN-γ

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responses, which certainly indicate a predominant acute cellular rejection process. The Sarwal’s group also reported on 45 common upregulated genes,63 mainly implicated in inflammatory pathways, using 3 biopsy-based microarray datasets of AR from pediatric renal, adult renal,64 and adult cardiac65 transplant patients. In addition, they validated the overexpression of 3 proteins in the serum of cardiac allograft recipients with acute rejection: PECAM1 (platelet/endothelial cell adhesion molecule), CXCL9, and CD44. Performing a metaanalysis of 8 gene datasets in kidney, lung, heart, and liver recipients with ARE, Khatri et al. reported on 102 not-tissue-specific genes associated with ARE.26 A “leave-one-organ-out” metaanalysis followed by in silico validations using additional datasets focuses on 11 overexpressed genes in ARE in which the geometric mean constitutes a common rejection module (CRM) score. Since only a few genes in this CRM are described in others studies, the authors hypothesized that their cross-organ approach highlights genes related to common acute rejection mechanisms while single-organ studies are associated with tissue-specific genes.26 While validation is still awaited, this signature also provides rational drug design. The authors report on a murine model of cardiac transplant using atorvastatin and dasatinib, two drugs associated with regulation of CRM genes, a reduction in graft-infiltrating cells, and an increase in graft survival. Finally, a retrospective analysis of 2515 renal transplant patients validated the beneficial effects of atorvastatin on graft survival.26 These studies provide evidence that gene profiling in biopsies of acute rejection reflects complex biological mechanisms and corroborates with pathologic histological findings that show mixed TCMR and ABMR lesions. ARE is also associated with differential miRNA expression.19,50,66 68 Comparing normal and acute rejection biopsies, Anglicheau et al. identified the overexpression of miR-142-5p, miR-155, and miR-223 and the underexpression of miR-10b, miR-30a-3p, and let-7C respectively.67 Some of these were confirmed by Soltaninejad et al., who also reported on an overexpression of miR-142-5p, miR-142-3p, miR-155, and miR-223 in biopsy with acute TCMR.66 Using next-generation sequencing Liu et al. highlighted 75 differentially expressed miRNAs in 15 acutely rejected human renal allografts compared to normal ones, including previously described miRNAs (e.g., miR-145-5p, miR-146a, miR-155, and miR-10b).19 Focusing on miR-10b, they found that its downregulation contributes to the apoptosis of glomerular endothelial cells in which BCL2L11 (BCL2-like 11) is targeted. Thus, gene expression in the biopsy can participate towards accurate ARE diagnosis and, to some extent, help to determine the type of rejection.

31.4.2 Diagnosis of Acute Renal Rejection Through Urine Pioneers in urine profiling, Suthanthiran’s group described higher levels of various markers such as granzyme B (GZB) and perforin,69 CD103,70 serine proteinase inhibitor-9 (PI9),71 CXCL10 and chemokine (C-X-C motif) receptor 3 (CXCR3),72 and then of CD25, CD3E (CD3 Epsilon) and FOXP3 (forkhead box P3) in the urine of biopsy-proven acute rejection cases.73 In a multicenter study including more than 4000 urine specimens, the combined urinary mRNA levels of CD3E, CXCL10, and 18S rRNA could accurately diagnose and predict cellular ARE weeks before its appearance.74 Even if the modest specificity and sensitivity of this signature (72% and 71% respectively) limit its ARE diagnostic usefulness, its predictive power, independence to urinary tract infection, and noninvasive status may make it a useful screening tool in the clinic. As gene expression analysis has only been performed in a single center, upscaling this signature into a commercially available test represents the next challenge. The same group has complemented this signature with two 6-gene combinations able to distinguish acute rejection from acute tubular injury, and acute TCMR from acute ABMR with an AUC of 0.92 and 0.81, respectively.75 Another group showed that the urinary levels of CXCL10 can be used to diagnose TCMR (AUC 5 0.80), as previously described,72,74 and also ABMR (AUC 5 0.76) and are associated with the risk of graft loss.76 Furthermore, combining mean fluorescence intensity of the immunodominant donor-specific antibodies and urinary CXCL10 improves the diagnosis of ABMR (AUC 5 0.83; 95% CI 0.77 0.89; P , .001). Profiling miRNA expression in urine, Lorenzen et al. reported decreased levels of miR-10a and miR-210 and increased levels of miR-10b in patients with ARE.31 The association of miR-210 with 1-year eGFR and its independence of urinary tract infection suggest miR-210 as a promising biomarker for validation in a prospective and larger cohort. Thus, urine appears a promising compartment for the diagnosis of acute allograft rejection, allowing repeated measure. Furthermore, the combination of urinary gene expression with clinical parameters has been shown to enhance their diagnostic power.

31.4.3 Peripheral Blood in Acute Renal Rejection Diagnosis The first microarray study comparing the biopsies and peripheral blood leukocytes of ARE patients and controls showed only very little overlap between the two signatures.64 However, these are different compartments

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and this does not preclude the use of peripheral blood to identify potential biomarkers of allograft injury, and recent years have provided several promising blood signatures for ARE. Notably, a five-gene signature (DUSP1, NKTR, MAPK9, PSEN1, and PBEF1), with a useful sensitivity and specificity of 91% and 94% respectively, has been reported in peripheral blood from pediatric patients across three different microarray platforms.77 Interestingly, this signature was also able to identify 8 out of 12 samples from patients classified as borderline rejection by histology. The signature has been validated in an independent study on adult Asian kidney recipients.78 Sarwal’s group also recently proposed a gene sets of 17 transcripts, called kSORT (Kidney Solid Organ Response Test), able to predict AR in peripheral blood from pediatric and adult patients independent of age, time posttransplantation, and center.79 While such biomarkers seem promising, further validation is required on serially collected blood samples paired with confirmatory biopsies and adapted algorithms.80 Similarly, a signature of 200 highly significant probes has been described as discriminating patients with biopsy-proven ARE from those with excellent function and normal histology and from others with acute dysfunction with no biopsyproven rejection, attaining acceptable AUCs of respectively 0.88 and 0.950.81 A variety of gene signatures has been described as potentially diagnosing ARE, either in biopsy, urine, or blood. But the absence of a clear pattern highlights the need of further validation, notably in larger and nonselected or unbiased cohorts.

31.5 MICROARRAY ANALYSIS IN CHRONIC ALLOGRAFT INJURY CAD is a nonspecific pathology characterized by a slow and progressive deterioration of allograft function and constitutes the second most common cause of graft loss and return to dialysis.7,82,83

31.5.1 Genes Associated With Interstitial Fibrosis and Tubular Atrophy Interstitial fibrosis and tubular atrophy (IF/TA) is a common hallmark of CAD.84,85 Broad lists of genes— hundreds to a few thousands—have been associated with IF/TA or chronic allograft nephropathy (CAN), according to the 2005 Banff classification.84 The main biological functions of these genes are related to extracellular matrix remodeling, cell death/apoptosis, and immune response.86 91 Interestingly, modulation of gene expression associated with IF/TA is detectable as early as 3 months posttransplant.87,88 Comparing gene expression in 3-month biopsies from patients with and without IF/TA at 6 months posttransplantation, Scherer et al. show that significant changes in the transcriptome precede the histological phenotype of IF/TA.88 At 1 year posttransplantation, cases with biopsies with signs of fibrosis exhibited a higher number of differential genes that participate to inflammation, immunity, or response to injury compared to implantation biopsies (postreperfusion).92 As observed elsewhere,42 normal 1-year biopsies also exhibit changes of expression of inflammation-related genes compared to preimplantation biopsies. These genes are certainly associated with the engraftment of the kidney or a mechanism of preserving normal histology. In addition, different gene profiles are observed between 1-year biopsy with fibrosis alone and those with fibrosis with inflammation (IF 1 i), associated with reduced graft survival.85 Altered expression of a substantial proportion of acute rejection-associated genes62 was observed only in the IF 1 i group. The few differences observed at 4 months posttransplantation in the group with fibrosis seems to conflict with other studies88,89 but could also been explained by selection of patients with a LD transplant, no occurrence of DGF, ARE, and absence of other complications. In another study, Mengel et al. reported that inflammatory IF/TA (i-IF/TA) was associated with worse allograft survival and also infiltration in unscarred areas.93 IF/TA and i-IF/TA correlate with transcripts associated with B cells, plasma cells, and mast cells. Consistently, the same group reported that B-cell-associated transcripts (BATs) and immunoglobulin transcripts (IGTs) correlate with interstitial i-IF/TA in biopsies of more than 5 years posttransplantation.94 Thus, a major impact of IF/TA on molecular profile in the allograft is associated with the significance of the chronic inflammation process in graft losses due to IF/TA. In urine, among 56 differential miRNAs, Scian et al. found that the levels of three of them (miR-142-3p, miR-204, and miR-21) could be used to distinguish patients with histological evidence of IF/TA from those with a normal biopsy, despite no difference in graft function at the time of collection.95 Using multiplex miRNA sequencing, Ben-Dov et al. identify that the total miRNA content was 1.9-fold lower in total RNA from biopsies with IF/TA, hypothesizing that kidney injury might participate directly to the pool of circulating miRNA, by reduction of kidney mass, or because uremic toxins may inhibit miRNA biogenesis in vivo or miRNA detection in vitro.18

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They also identified 16 differential urinary miRNAs associated with IF/TA and validated eight of them, including miR-21 and the myeloid and lymphoid lineage related miR-142-3p/5p, in an independent cohort.

31.5.2 Diagnosis of Chronic Alloimmune Injuries As a leading cause of kidney transplant failure,96 accurate diagnosis of ABMR is a priority. Among the first studies, Scherer and colleagues identified a set of 10 genes in biopsies at 6 months (Banff 97 criteria) that prognose chronic rejection at 12 months after transplantation with 88% accuracy (Banff 97 criteria).97 Halloran’s team has contributed significantly to the identification of such molecular signatures.28,98 Using mice models, human cell lines and literature analysis, they built pathogenesis-based transcript sets (PBTs) reflecting biological mechanisms and associated with specific histological changes.99 The genes distinguishing rejection from nonrejection in 186 kidney transplant biopsies are composed mainly of the interferon gamma- and rejection-induced transcripts (GRIT), a PBT shared by TCMR and ABMR, which reflects common effector mechanisms with involvement of IFNγ.100 Consistent with this, a blood interferon-related signature has been associated with ABMR by Rascio’s team.101 Also a study by Petra et al. reported on the absence of a difference between chronic TCMR and ABMR when measuring “only” 376 selected genes associated with immune response.102 The mean expression of 119 endothelial-associated transcripts (ENDATs), extracted from the literature, was found to be higher in ABMR compared to TCMR and correlated with histopathologic lesions and alloantibodies.103,104 Only 40% of kidneys with high ENDAT expression and chronic ABMR or graft loss were C4d negative, reflecting the low sensitivity of C4d staining105 and highlighting the diagnostic interest of this ENDAT signature. Furthermore, 23 NK cell transcripts are differentially expressed in biopsies from DSA-positive versus DSA-negative patients and have been reported as DSA selective transcripts (DSASTs).106 NK cells were checked by immunostaining with increased CD561 cells in peritubular capillaries in ABMR, as well as CD681 macrophages.106,107 For diagnosis purposes, Edmonton’s group developed an ABMR score based on the expression of 20 probe sets related to endothelial and NK cells, consistent with previous reports, in 403 prospectively collected indication biopsies.103,106 108 This ABMR score was validated in the prospective INTERCOM study, including 300 biopsies for causes, with accuracy of 85%, which is a significant improvement on the conventional 3-year survival prediction.109 Recently, the ABMR score and the ENDAT signature showed independent association with risk of allograft loss in two early ABMR cohorts of 74 and 54 patients.10 The ability of this molecular score to diagnose ABMR was not assessed, but the authors show that among ABMR patients diagnosed by conventional diagnosis, the use of this molecular microscope improves the stratification of patients at high risk of graft loss. Recently, Halloran’s group presented quantitative cytotoxic T-cell-associated transcripts (QCATs)110 in association with a TCMR score based on the analysis of 403 kidney transplant biopsies composed of 61 probes with immune-related genes like CD69, CD28, and IFNG among the top 10.111 Discrepancies between the histological analysis and the TCMR score were mainly observed in situations where histology has known limitations, e.g., when severe scarring obscures TCMR.112 Molecular assessment of biopsy may offer an objective measure, especially when histological diagnosis is problematic and subject to subjectivity. In addition, the association of gene expression measure and clinical or histological parameters could outperform the use of these parameters alone. Finally, only two studies analyzed miRNA expression altered by chronic alloimmune injury.101,113 We identified the upregulation of miR-142-5p in PBMC and renal allograft of chronic ABMR compared to patients with a normal histology and reported a major gene network related to immune response including links with the NFκB complex, and T- and B-cell receptors among 41 potential miR-142-5p targets modulated in chronic ABMR patients.113 The second study reported on the downregulation of 16 miRNAs in chronic antibody-mediated rejection (CAMR) patients, with 13 involved in the inflammatory response.101 No common miRNA were found between the two studies, which could be due to different methodologies and cohorts used, suggesting the need for further investigation.

31.6 IMMUNOSUPPRESSIVE DRUGS IN TRANSPLANTATION 31.6.1 Immunosuppressive Drug Impact on Gene Expression As the use of immunosuppression (IS) is associated with a broad range of side effects,83,114 significant studies have been aimed at improving understanding of IS side effects in order to reduce them through individualization of immunosuppressive drug regime. Calcineurin inhibitor (CNI) toxicity has been associated with CAD.115

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108 genes linked to CNI toxicity have been identified, with functions related to macropinocytosis signaling, inhibition of matrix metalloproteases, and remodeling of epithelial adherens junctions.116 Patients exhibiting CAD at 24 months posttransplantation and histological evidence of IF/TA exhibit an overlap with genes of the CNI-toxicity signature of 7% and 22% at 3 and at 12 months post-KT, respectively, in contrast with patients with IF/TA but no CAD progression (overlap ,1% and 1%).116 These results evidence a nonimmunological factor involved in the progression to CAD in addition to immune-related genes associated with IF/TA. Early identification of the CNI toxicity signature may be a signal for a CNI conversion strategy. The effects of belatacept and cyclosporine A on 1-year allograft molecular profiles have been compared on matched preimplantation biopsies.52 Enriched gene sets from CNI biopsies correspond with genes previously described as associated with fibrosis, early tubulointerstitial damage and in vitro CNI toxicity on renal proximal tubular epithelia. However, this result is not surprising as these patients exhibited significantly higher histological levels of interstitial fibrosis (ci) and tubular atrophy (ct). As patients on belatacept exhibited much fewer signs of tubulointerstitial injury, these genes were not enriched but betalacept-treated patients exhibited a significant enrichment for genes previously associated with ARE61 and monocyte and NK cell-associated genes. As belatacept-treated patients exhibited a significantly higher incidence of ARE, the authors conclude that these enriched genes are linked to residual immune activation from earlier ARE or ongoing tissue remodeling and repair. Whereas these patients present no significant loss of kidney function over 3 years, the presence of NK-related transcripts, previously associated with ABMR,5 would indicate the need for additional follow up. Gene expression can also predict response to steroid treatment after ARE.117 While clinical and histological parameters are not associated to response to treatment, a combination of CD25:CD3e ratio and lymphocyte activation gene-3 (LAG3) expression can predict this response, despite relatively limited specificity and sensitivity of 78% and 60%, respectively. Similarly, the expression of CXCL10 and CXCL13 is upregulated in patients with ARE resistant to antirejection therapy.118

31.6.2 Gene Expression to Monitor Immunosuppressant Regime Changes The measurement of expression of nuclear factor of activated T cells (NFAT)-regulated genes (interleukin 2, interferon-gamma, and granulocyte-macrophage colony-stimulating factor in vitro in PBMC after stimulation (phorbol myristate acetate/ionomycin)) has been proposed to measure the biologic activity of CsA in blood.119 Two hours after CsA intake, NFAT-regulated gene expression decreases and is correlated with total drug exposure, while gene expression levels reach predose values 6 hours after oral CsA ingestion. Increased sensitivity of expression of NFAT-regulated genes to CsA was found in pediatric renal transplant recipients compared with adults.120 Interestingly, the same authors report that the levels of these NFAT-regulated genes is lower in stable older renal allograft recipients ($65 years) with opportunistic infections than in patients without infections, whereas the daily CsA dose and CsA blood concentrations were comparable.121 mRNA of the proliferating cell nuclear antigen gene (PCNA) has been proposed as one simple assay for monitoring the immune function.122 Interestingly, low PCNA expression is associated with viral reactivation after transplantation. The measure of interleukin expression (IL2, IL4, and IL6) in peripheral blood has also been proposed for assessing the pharmacodynamics of sirolimus.123 The authors found interindividual variations, independent of the concentration, which correlate with clinical efficacy. In addition, despite suppression of IL2 protein secretion and T-cell proliferation being generally induced, IL2 and IL4 mRNA repression was not always achieved, suggesting better accuracy of gene than protein expression. While IS side effects are well known, conversion to less toxic treatment, or minimization of IS, are appealing strategies.124,125 The molecular profile of patients who underwent tacrolimus (CNI) to sirolimus (mTOR inhibitor) conversion has been followed in the graft.126 Whereas patient and graft survival rates were similar, upregulated genes related to the activation of the immune system were clearly greater in number in the graft of sirolimusconverted patients, which is consistent with observed trends of increased macrophage infiltration and tubulitis. These results suggest that gene expression in the graft may predict a proinflammatory environment that may precede immune alterations in patients converted to sirolimus from tacrolimus. The intentional progressive withdrawal of IS can achieve up to 60% success rates in liver transplantation, confirming that a clinical state of operational tolerance to a mismatched graft, a specific situation where transplant patients maintain a well-functioning graft without rejection while immunosuppressive treatment is stopped, can been achieved in humans.127,128 In renal transplantation, spontaneous operational tolerance has also been described,129 131 but since these patients are usually noncompliant, refusing invasive procedures, gene expression

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studies have focused on peripheral blood.129 We have previously reported on a pattern of 49 blood genes associated with tolerance identified using microarray analysis.132 The transforming growth factor-β (TGFβ) regulates more than a quarter (27%) of the genes that differentiate operationally tolerant patients from others. A major B-cell-related signature is found in the blood of these tolerant patients, a discovery supported by phenotypic analyses.133 136 Consistent with this, we have also reported the overexpression of miR-142-3p in blood B cells from patients with operational tolerance and a possible implication of this miRNA in TGFβ signaling.137 A metaanalysis of these studies confirms the major contribution of B-cell genes in a tolerance signature27 and indicates a 20-gene minimal signature as biomarker of tolerance,129 which could be used to better track patients with standard IS. Whether these signatures can be used to identify patients receptive to progressive IS withdrawal is an important question. Attempts to identify such patients gave figures between 3.5% and 15% of the patients under IS.132,133,138 In 2014, a minimal tolerance signature composed of 2 genes IGKV1D-13 and IGKV4-1 was seen to be increased in CNI-treated patients and allowed the identification of a tolerance pattern in 0%, 7%, and up to 25% of patients with stable function under IS at 1, 5, and 10 years posttransplantation respectively139 suggesting an association between tolerance and time posttransplantation, as observed in liver allograft tolerance.127 Whereas this has been clinically tested in liver transplantation,127,128,140 attempts to minimize IS in renal transplantation have given more mixed results. Selecting low-risk patients using conventional clinical criteria, including a living donor and no DSA pretransplantation, tacrolimus withdrawal resulted in increased risk of ARE and/ or de novo DSA, despites 6 cases remaining off of tacrolimus for 24 months.141 Unfortunately, tolerance molecular signatures were not tested in this assay and their potential usefulness remains to be investigated.

31.7 CONCLUSION Since the 2000s, numerous studies have been published on gene expression in renal transplantation. While our understanding of the molecular mechanisms has undeniably improved, no commonly accepted biomarker has yet been identified. Despite a number of common genes, such as CXCL10, most gene signatures so far described are heterogenic. Differences in technology could be the explanation, but the use of small and highly selective cohorts certainly participates to the high diversity of proposed and nonvalidated biomarkers. Extensive, multicentric, and rigorous validations are now required, notably on nonselected populations for routine use. Molecular microscope use will certainly become an aid for histopathologists and it could be added in the Banff classification as discussed in 2014,105 if it is not associated with a more complex or slower decision-making process. However, its limited availability precludes its addition as a mandatory parameter, like electron microscopy for chronic glomerulopathy.105 Finally, these molecular signatures should be used to create composite scores, like the scores combining demographic and clinical parameters proposed to predict graft failure.11 The association of gene expression, demographic, clinical, and/or histological parameters would outperform the independent use of each parameter separately. Such composite scores could help clinicians in the selection of a graft with acceptable quality in a more methodical manner and stratify patients at risk. Similarly, such scores could help identify low-risk patients, potentially conducive to personalization of IS, as current parameters cannot do this with any great accuracy.141

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Transplantation 2007;83(11):1466 76. 93. Mengel M, Reeve J, Bunnag S, et al. Molecular correlates of scarring in kidney transplants: the emergence of mast cell transcripts. Am J Transplant 2009;9(1):169 78. 94. Einecke G, Reeve J, Mengel M, et al. Expression of B cell and immunoglobulin transcripts is a feature of inflammation in late allografts. Am J Transplant 2008;8(7):1434 43. 95. Scian MJ, Maluf DG, David KG, et al. MicroRNA profiles in allograft tissues and paired urines associate with chronic allograft dysfunction with IF/TA. Am J Transplant 2011;11(10):2110 22. 96. Lefaucheur C, Loupy A, Vernerey D, et al. Antibody-mediated vascular rejection of kidney allografts: a population-based study. Lancet 2013;381(9863):313 19. 97. Scherer A, Krause A, Walker JR, Korn A, Niese D, Raulf F. Early prognosis of the development of renal chronic allograft rejection by gene expression profiling of human protocol biopsies. Transplantation 2003;75(8):1323 30. 98. Halloran PF, Reeve JP, Pereira AB, Hidalgo LG, Famulski KS. Antibody-mediated rejection, T cell-mediated rejection, and the injuryrepair response: new insights from the Genome Canada studies of kidney transplant biopsies. Kidney Int 2014;85(2):258 64. 99. Mueller TF, Einecke G, Reeve J, et al. Microarray analysis of rejection in human kidney transplants using pathogenesis-based transcript sets. Am J Transplant 2007;7(12):2712 22. 100. Reeve J, Einecke G, Mengel M, et al. Diagnosing rejection in renal transplants: a comparison of molecular- and histopathology-based approaches. Am J Transplant 2009;9(8):1802 10. 101. Rascio F, Pontrelli P, Accetturo M, et al. A Type I Interferon signature characterizes chronic antibody-mediated rejection in kidney transplantation. The J Path 2015;(May). Available from: http://dx.doi.org/10.1002/path.4553. 102. Petra H, Eva H, Irena B, Petra H, Ondrej V. Molecular profiling of acute and chronic rejections of renal allografts. Clin Dev Immunol 2013;2013:509259. 103. Sis B, Jhangri GS, Bunnag S, Allanach K, Kaplan B, Halloran PF. Endothelial gene expression in kidney transplants with alloantibody indicates antibody-mediated damage despite lack of C4d staining. Am J Transplant 2009;9(10):2312 23. 104. Hayde N, Bao Y, Pullman J, et al. The clinical and genomic significance of donor-specific antibody-positive/C4d-negative and donorspecific antibody-negative/C4d-negative transplant glomerulopathy. ClinJ Am Soc Nephrol 2013;8(12):2141 8. 105. Haas M, Sis B, Racusen LC, et al. Banff 2013 meeting report: inclusion of c4d-negative antibody-mediated rejection and antibodyassociated arterial lesions. Am J Transplant 2014;14(2):272 83. 106. Hidalgo LG, Sis B, Sellares J, et al. NK cell transcripts and NK cells in kidney biopsies from patients with donor-specific antibodies: evidence for NK cell involvement in antibody-mediated rejection. Am J Transplant 2010;10(8):1812 22. 107. Venner JM, Hidalgo LG, Famulski KS, Chang J, Halloran PF. The molecular landscape of antibody-mediated kidney transplant rejection: evidence for NK involvement through CD16a Fc receptors. Am J Transplant 2015;15(5):1336 48. 108. Sellares J, Reeve J, Loupy A, et al. Molecular diagnosis of antibody-mediated rejection in human kidney transplants. Am J Transplant 2013;13(4):971 83. 109. Halloran PF, Pereira AB, Chang J, et al. Microarray diagnosis of antibody-mediated rejection in kidney transplant biopsies: an international prospective study (INTERCOM). Am J Transplant 2013;13(11):2865 74.

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110. Einecke G, Melk A, Ramassar V, et al. Expression of CTL associated transcripts precedes the development of tubulitis in T-cell mediated kidney graft rejection. Am J Transplant 2005;5(8):1827 36. 111. Reeve J, Sellares J, Mengel M, et al. Molecular diagnosis of T cell-mediated rejection in human kidney transplant biopsies. Am J Transplant 2013;13(3):645 55. 112. Halloran PF, Pereira AB, Chang J, et al. Potential impact of microarray diagnosis of T cell-mediated rejection in kidney transplants: the INTERCOM study. Am J Transplant 2013;13(9):2352 63. 113. Danger R, Paul C, Giral M, et al. Expression of miR-142-5p in peripheral blood mononuclear cells from renal transplant patients with chronic antibody-mediated rejection. PloS One 2013;8(4):e60702. 114. Dantal J, Godfrin Y, Soulillou JP. New insight into the pathogenesis of the ‘idiopathic nephrotic syndrome’. Nephrol Dial Transplant 1995;10(11):1979 82. 115. Nankivell BJ, Borrows RJ, Fung CL, O’Connell PJ, Chapman JR, Allen RD. Calcineurin inhibitor nephrotoxicity: longitudinal assessment by protocol histology. Transplantation 2004;78(4):557 65. 116. Maluf DG, Dumur CI, Suh JL, et al. Evaluation of molecular profiles in calcineurin inhibitor toxicity post-kidney transplant: input to chronic allograft dysfunction. Am J Transplant 2014;14(5):1152 63. 117. Rekers NV, Bajema IM, Mallat MJ, et al. Quantitative polymerase chain reaction profiling of immunomarkers in rejecting kidney allografts for predicting response to steroid treatment. Transplantation 2012;94(6):596 602. 118. Mao Y, Wang M, Zhou Q, et al. CXCL10 and CXCL13 Expression were highly up-regulated in peripheral blood mononuclear cells in acute rejection and poor response to anti-rejection therapy. J Clin Immunol 2011;31(3):414 18. 119. Giese T, Zeier M, Schemmer P, et al. Monitoring of NFAT-regulated gene expression in the peripheral blood of allograft recipients: a novel perspective toward individually optimized drug doses of cyclosporine A. Transplantation 2004;77(3):339 44. 120. Billing H, Sommerer C, Giese T, et al. Increased cyclosporin a sensitivity in vivo in pediatric renal transplant recipients compared with adults. Ther Drug Monit 2012;34(5):554 60. 121. Sommerer C, Schnitzler P, Meuer S, Zeier M, Giese T. Pharmacodynamic monitoring of cyclosporin A reveals risk of opportunistic infections and malignancies in renal transplant recipients 65 years and older. Ther Drug Monit 2011;33(6):694 8. 122. Niwa M, Miwa Y, Kuzuya T, et al. Stimulation index for PCNA mRNA in peripheral blood as immune function monitoring after renal transplantation. Transplantation 2009;87(9):1411 14. 123. Muller-Steinhardt M, Wortmeier K, Fricke L, Ebel B, Hartel C. The pharmacodynamic effect of sirolimus: individual variation of cytokine mRNA expression profiles in human whole blood samples. Immunobiology 2009;214(1):17 26. 124. Londono MC, Lopez MC, Sanchez-Fueyo A. Minimization of immunosuppression in adult liver transplantation: new strategies and tools. Curr Opin Organ Transplant 2010;15(6):685 90. 125. Londono MC, Rimola A, O’Grady J, Sanchez-Fueyo A. Immunosuppression minimization vs. complete drug withdrawal in liver transplantation. J Hepatol 2013;59(4):872 9. 126. Gallon L, Traitanon O, Sustento-Reodica N, et al. Cellular and molecular immune profiles in renal transplant recipients after conversion from tacrolimus to sirolimus. Kidney Intl 2015;87(4):828 38. 127. Benitez C, Londono MC, Miquel R, et al. Prospective multicenter clinical trial of immunosuppressive drug withdrawal in stable adult liver transplant recipients. Hepatology 2013;58(5):1824 35. 128. Whitehouse GP, Sanchez-Fueyo A. Immunosuppression withdrawal following liver transplantation. Clin Res Hepatol Gastroenterol 2014;38 (6):676 80. 129. Brouard S, Pallier A, Renaudin K, et al. The natural history of clinical operational tolerance after kidney transplantation through twentyseven cases. Am J Transplant 2012;12(12):3296 307. 130. Orlando G, Hematti P, Stratta RJ, et al. Clinical operational tolerance after renal transplantation: current status and future challenges. Ann Surg 2010;252(6):915 28. 131. Roussey-Kesler G, Giral M, Moreau A, et al. Clinical operational tolerance after kidney transplantation. Am J Transplant 2006;6(4):736 46. 132. Brouard S, Mansfield E, Braud C, et al. Identification of a peripheral blood transcriptional biomarker panel associated with operational renal allograft tolerance. Proc Nat Acad Sci USA 2007;104(39):15448 53. 133. Sagoo P, Perucha E, Sawitzki B, et al. Development of a cross-platform biomarker signature to detect renal transplant tolerance in humans. J Clin Invest 2010;120(6):1848 61. 134. Pallier A, Hillion S, Danger R, et al. Patients with drug-free long-term graft function display increased numbers of peripheral B cells with a memory and inhibitory phenotype. Kidney Int 2010;78(5):503 13. 135. Newell KA, Asare A, Kirk AD, et al. Identification of a B cell signature associated with renal transplant tolerance in humans. J Clin Invest 2010;120(6):1836 47. 136. Chesneau M, Michel L, Dugast E, et al. Tolerant kidney transplant patients produce B cells with regulatory properties. J Am Soc Nephrol 2015. Available from: http://dx.doi.org/10.1681/ASN.2014040404. 137. Danger R, Pallier A, Giral M, et al. Upregulation of miR-142-3p in peripheral blood mononuclear cells of operationally tolerant patients with a renal transplant. J Am Soc Nephrol 2012;23(4):597 606. 138. Brouard S, Le Bars A, Dufay A, et al. Identification of a gene expression profile associated with operational tolerance among a selected group of stable kidney transplant patients. Transplant Int 2011;24(6):536 47. 139. Moreso F, Torres IB, Martinez-Gallo M, et al. Gene expression signature of tolerance and lymphocyte subsets in stable renal transplants: results of a cross-sectional study. Transplant Immunol 2014;31(1):11 16. 140. Feng S, Ekong UD, Lobritto SJ, et al. Complete immunosuppression withdrawal and subsequent allograft function among pediatric recipients of parental living donor liver transplants. JAMA 2012;307(3):283 93. 141. Hricik DE, Formica RN, Nickerson P, et al. Adverse outcomes of tacrolimus withdrawal in immune-quiescent kidney transplant recipients. J Am Soc Nephrol 2015;26(12):3114 22, pii: ASN.2014121234.

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32 Acute Cellular Rejection Madhav C. Menon, Paolo Cravedi and Fadi El Salem Icahn School of Medicine at Mount Sinai, New York, NY, United States

32.1 INTRODUCTION Acute cellular rejection (ACR) remains an important event in kidney transplantation. Since Mitchinson’s early finding that adoptive transfer of primed T cells from one inbred mouse to another can induce accelerated acute rejection of a graft from the same donor, T cells have been recognized as the central players in its pathogenesis.1,2 However, data within the last decade that an initial innate immunity mediated allograft inflammation increases risk for ACR, along with more recent data on the capability of innate-immune cells to recognize nonself HLA molecules directly highlight the roles of other cells types in ACR.3,4 Defined simply, ACR in kidney transplantation is an alloimmune tubule-interstitial nephritis of varying severity and extent. The important hallmark of ACR is the cellular inflammatory reaction that, if left untreated, leads to irreversible histological damage that could result in graft loss.5 7 However, most ACR episodes are treatable with one or more currently available therapies and most treated patients with ACR fully recover their function, though ACR still represents a risk for shorter graft survival.8 10 Before the introduction of cyclosporine, ACR was common, occurring in up to 40% of kidney transplant patients.11 Thereafter, significant reduction has been achieved in the incidence of ACR with modern immunosuppression strategies.12 Recent estimates show rates around 10% 12% for living-donor and 12% 15% for deceaseddonor allografts (Fig. 32.1).11,13 The incidence of ACR reported clinically and defined by biopsy appears to decline markedly after the first 2 years posttransplantation, possibly because of the death of donor immune cells eliciting the alloimmune response. Most episodes of ACR are associated with a raise in serum creatinine. However, studies with surveillance biopsies found that histological features of ACR may occur subclinically (i.e., with a raise in serum creatinine #0.3 mg/dL), with an incidence ranging from 5% to 30%.10,14 17 The majority of these studies describe early subclinical rejection (SCR) episodes and hence later incidence of SCR is unclear. Furthermore, the impact of subclinically diagnosed rejection, and its treatment on allograft outcomes, has remained controversial.10,14,17 19

32.2 PATHOGENESIS 32.2.1 Alloreactive T-Cell Activation and Recruitment The immune system is frequently exposed to harmless (and sometimes beneficial) foreign antigens that do not require an aggressive effector response, such as gut flora. The context in which such foreign antigens are encountered is important in dictating the magnitude of the immune response. For example, the activation of leukocytes in an inflammatory environment augments the immune response. In transplantation, these inflammatory signals can be provided by brain death, the surgical trauma, and the oxidative stress of ischemia/reperfusion injury. Graft injured cells release damage-associated molecular patterns (DAMPs), which include several intracellular proteins, DNA, RNA, and nucleotides.20 DAMPs activate toll-like receptors on innate immune cells,20,21 inducing Kidney Transplantation, Bioengineering, and Regeneration. DOI: http://dx.doi.org/10.1016/B978-0-12-801734-0.00032-1

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20 Deceased donor

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15 Living donor 10

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12 24 36 48 Months posttransplant

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FIGURE 32.1 Incidence of first acute rejection among adult patients receiving a kidney transplant in 2005 09. Figure shows Kaplan Meier survival curves with the outcome of first episode of ACr as reported within the Scientific Registry of Transplant Recipients (SRTR) database including allografts transplanted between 2005 and 2009, for deceased-donor (upper line), and living-donor allografts. As shown, ACR incidence reduces after the initial 1 2 years posttransplant with deceased-donor organs maintaining a higher incidence.

the maturation and migration of antigen-presenting cells (APCs) to secondary lymphoid tissues where they trigger primary T-cell and B-cell responses.22,23 The principal alloantigens recognized by T cells, B cells, and antibodies are the human leukocyte antigens (HLAs) that differ between the donor and recipient. These are cell-surface proteins that are highly variable (polymorphic) between unrelated individuals. Two main classes of HLA proteins have been identified. Class I molecules (HLA-A, -B, and -C) are expressed on all nucleated cells, whereas class II molecules (HLA-DP, -DQ, and -DR) are present on APCs (including B cells, dendritic cells, macrophages, and other phagocytic cells), that are able to process and present foreign proteins to T cells. In the context of transplant alloreactive T cells are mainly activated by donor-derived APCs (direct pathway of allorecognition) or by recipient APCs presenting donor HLA peptides in the context of self-HLA (indirect pathway).24 Donor-derived “passenger” APCs residing in the donor organ and expressing large amounts of donor HLA antigens migrate out of the transplant into the draining lymph nodes, where they interact with host T cells via the direct pathway. As donor APCs decline in number over time, the contribution of the direct pathway also wanes.25 This may partly explain the higher incidence of ACR seen in the initial years after transplant. A newly recognized pathway of allorecognition is called the semidirect pathway. It occurs when intact donor HLA antigens are physically transferred to the membrane of host APCs and are then recognized by host T cells.24 Host APCs appear to acquire intact HLA molecules from exosomes secreted by donor APCs or through cell-to-cell contact. The relative contribution of this pathway to allograft rejection is not clear. Besides HLA molecules, non-HLA proteins that trigger an alloimmune response and are targeted during allograft rejection are referred to as “minor histocompatibility antigens.” It is likely that a large number of minor antigens exist, making it very difficult to match for them.26 Minor histocompatibility antigens alone cannot cause rapid rejection. However, when multiple minor antigens are mismatched, rejection can be as rapid as when HLA antigens are mismatched. Minor histocompatibility mismatches alone may be present in transplants between siblings with identical HLA molecules, leading to slow rejection of these transplants.26,27 The interaction between the HLA molecule and its cognate T-cell receptor (TCR), followed by subsequent T-cell signal transduction represents the initiating step that is necessary but not sufficient for T-cell rejection.3 In conjunction with the TCR-HLA engagement, costimulation must occur for full T-cell activation to be initiated. The second essential signal is costimulation, which is provided by the interaction of pairs of cell-surface molecules present on T cells and APCs.28 Absence or blockade of costimulatory signals typically results in T-cell unresponsiveness, or anergy. Costimulatory molecules are divided into two families: the B7 family, of which the prototype receptor ligand pair is CD28 (on the T cell) and CD80/86 (B7.1/B7.2, on the APC), and the TNF and TNF receptor (TNFR) family, best characterized by CD40 (on the APC) and CD154 (CD40L, on the T cell). The interface of a T cell with an APC in which both the TCR-HLA and costimulatory molecule interaction occurs is termed the immunological synapse. Formation of the immunological synapse leads to T-cell activation, which results in the transcription and translation of growth factors including cytokines (e.g., IL-2) and chemokines—key to amplifying the immune response. IL-2 has autocrine and paracrine effects on T cells. Chemokines released by T cells result in the recruitment of nondonor-specific T cells as well as other leukocytes into the ACR milieu.

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The process by which activated leukocytes migrate into the graft is termed leukocyte recruitment. Leukocyte recruitment is enhanced by vasodilation and endothelial activation in the proximity of the transplant. Chemokines that have been released from the allograft become tethered to the activated endothelium, providing a signal gradient recognized by passing leukocytes. When leukocytes bind to activated endothelium, further adhesions are made between integrin molecules on the leukocyte and endothelial adhesion molecules such as ICAM-1, which results in arrest of the leukocyte and extravasation into the transplanted organ.

32.2.2 Memory T Cells and Regulatory T Cells The primary immune response to a foreign antigen not previously encountered by the host is mediated by naive T lymphocytes. Naive T cells specific to the foreign antigen are present at a low precursor frequency, have a relatively high stimulation threshold (e.g., stringent dependence on costimulatory molecules), can only be activated within secondary lymphoid tissues (e.g., the spleen and lymph nodes),22 and are, therefore, slow to respond. In contrast, the secondary immune response to an antigen previously encountered by an individual (e.g., after vaccination or infection) is mediated by memory T cells and is significantly stronger and faster than a primary response. Antigen-specific memory T cells are long-lived lymphocytes that exist at a greater precursor frequency than their naive counterparts, have a low stimulation threshold and high proliferative capacity, and can be activated within secondary lymphoid tissues or at nonlymphoid sites—e.g., the site of infection or in the allograft itself.29 Humans for the most part are not exposed to alloantigens, with the exception of mothers who may have been sensitized to paternal antigens during pregnancy or individuals who had prior transfusions or organ transplants. Yet all humans, including those presumably never exposed to allogeneic cells or tissues, harbor alloreactive memory T cells. Accurate quantitation of alloreactive T cells has demonstrated that approximately 50% of the alloreactive T-cell repertoire in humans is made up of memory T lymphocytes.30,3,1 This finding can again be explained by the phenomenon of cross-reactivity, whereby memory T cells specific to microbial antigens also recognize alloantigens and contribute to the high precursor frequency of alloreactive T cells. Therefore, the extent of one’s alloreactivity is intimately shaped by one’s immunological memory to foreign antigens not necessarily related to the graft. Regulatory T cells (Tregs) are essential to maintain immune homeostasis, and are critical regulators for a variety of immune responses, including tolerance induction and maintenance for organ transplantation. There are two main types of Treg: thymus-derived CD41CD251FoxP31 natural Treg (nTreg) and adaptive/induced Treg (iTreg), which develop from naı¨ve T cells in the periphery under tolerogenic conditions. It has long been established that fork-head box P3 (FoxP3) is the major transcription factor that determines the fate, identity, and function of Tregs. Regulatory T cells capable of inhibiting effector T-cell activity are also often seen alongside effector T cells in the inflammatory response of ACR, but are not enough to control the alloimmune response.32

32.3 RISK FACTORS Better HLA matching, powerful immunosuppression, and improvement in overall patient management have changed the incidence and character of ACR.11 However, some identifiable risk factors for ACR remain. AfricanAmerican recipient race,33,34 deceased-donor kidneys, presence of delayed graft function, and younger age of recipients have all been associated with an increased risk of ACR. High HLA mismatches may also contribute an additive risk of ACR and ultimately impact graft survival.35 Presence of antibodies against HLA antigens, especially donor HLA, before transplantation36 38 is a wellrecognized risk for both antibody-mediated acute rejection (ABMR) and early ACR. These antibodies are detected by complement-dependent cytotoxicity testing (CDC-crossmatch), flow cytometric crossmatching, or by luminex method (using HLA antigen coated beads), and the individual relevance of these are discussed in more detail in Chapter 17, Histocompatibility Testing in the Transplant Setting.37 The chance of prior exposure to cross reactive antigens, and the development of antigen-experienced T cells from such an encounter (measured by the frequency of donor-reactive T cells), could also determine the risk of ACR before transplantation.39 Starting from this background, the preexisting Enzyme-Linked ImmunoSpot (ELISPOT)40 has been developed and standardized as an assay to quantify alloreactive memory T cells in humans. Importantly, numerous studies have found associations between ELISPOT positivity and risk of ACR, so that this assay has been proposed as a way to stratify patients’ risk.41,42

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32.4 CLINICAL FEATURES In the early 1960s, “classical” acute rejection syndrome, with fever and graft tenderness, was common, occurring in 40% 60% of renal transplants.11 At that time, immunosuppressive therapy for kidney allograft recipients consisted primarily of azathioprine and corticosteroids. Cases of unchecked ACR that progressed to allograft swelling and spontaneous rupture were frequently reported.43 45 These cases were beyond salvage and necessitated graft nephrectomy.43 This clinical picture has virtually disappeared in the calcineurin inhibitor era and been replaced by more insidious and silent renal graft dysfunction.11,46 The majority of ACR episodes are diagnosed nowadays by asymptomatic raise in serum creatinine. Mild graft tenderness may be elicitable, but is nonspecific and alternate differentials should be sought.47 Similarly, while gross hematuria has been reported as a manifestation of ACR (with or without ABMR)48,49 alternate causes such as genitourinary tract malignancy, adenoviral cystitis, BK viral ureteric disease, recurrent glomerular disease, or vascular events are more likely etiologies.50 53 Reduction in urine output may also occur, but it is generally unnoticeable.

32.5 PATHOLOGY Biopsy remains the gold standard technique to diagnose ACR. A group of renal pathologists, transplant surgeons, and nephrologists came together in 1991 to propose the first standardized diagnostic criteria for ACR.54 Several updates modifying different histological diagnostic criteria have been published since then.55 57 However, since the original Banff schema for ACR, lymphocytic tubulitis, and intimal arteritis have been regarded as the principal lesions indicative of ACR. Table 32.1 shows the Banff 2008 update for ACR and borderline lesions. Tubulitis is defined by the presence of lymphocytes inside the basement membrane of tubules. Lymphocytes are characterized by distinct hyperchromatic nuclei compared to adjacent tubular cells. One-to-four lymphocytes in a tubule are considered borderline or suspicious lesions for ACR (t1), while five or more (t2 and t3) define ACR in the presence of interstitial inflammation. Interstitial inflammation is predominantly lymphocytic and quantified as ,25% (i1), 25% 50% (i2), and .50% (i3) (Fig. 32.2). The presence of plasma cells and CD20-positive B cells may have prognostic impact and reflect steroid resistance.58,59 Similarly both peripheral eosinophilia and higher density of interstitial eosinophils in ACR have been reported to be associated with adverse prognosis.60,61 On the other hand, presence of neutrophilic infiltrates and specifically luminal neutrophils speaks against a diagnosis of ACR.62 However, peritubular neutrophilic capillaritis, a characteristic feature of ABMR, has been observed in up to 68% of biopsies with ACR, suggesting the frequent coexistence of ACR with ABMR.63 Tubules and interstitium in nonscarred areas should be evaluated to convincingly make a diagnosis of i- and t-lesions. Presence of arteritis in the presence of tubulitis denotes more severe ACR (Grades II and III in the Banff Schema). In arteritis, mononuclear cells are seen to progressively undermine the endothelium, with swelling and detachment of endothelial cells. Since arteritis needs to be ascertained, an adequate biopsy specimen should contain at least two representative arterioles. The presence of intimal TABLE 32.1

Modified Banff Schema for Acute Cellular Rejection55

1. T-CELL-MEDIATED REJECTION Acute T-cell-mediated rejection (type/grade): IA. Cases with significant interstitial infiltration (.25% of parenchyma affected, i2 or i3) and foci of moderate tubulitis (t2) IB. Cases with significant interstitial infiltration (.25% of parenchyma affected, i2 or i3) and foci of severe tubulitis (t3) IIA. Cases with mild-to-moderate intimal arteritis (v1) IIB. Cases with severe intimal arteritis comprising .25% of the luminal area (v2)III. Cases with “transmural” arteritis and/or arterial fibrinoid change and necrosis of medial smooth muscle cells with accompanying lymphocytic inflammation (v3) Chronic active T-cell-mediated rejection “Chronic allograft arteriopathy” (arterial intimal fibrosis with mononuclear cell infiltration in fibrosis, formation of neointima) 2. BORDERLINE CHANGES. “SUSPICIOUS” FOR ACUTE T-CELL-MEDIATED REJECTION This category is used when no intimal arteritis is present, but there are foci of tubulitis (t1, t2, or t3) with minor interstitial infiltration (i0 or i1) or interstitial infiltration (i2, i3) with mild (t1) tubulitis

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(A)

(B)

(C)

(D)

FIGURE 32.2 Histologic features of ACR: Panels (A D): Photomicrographs of varying grades of tubulitis and vascular intimal lesions. (A) Central tubule shows lymphocytes inside the lamina propria (arrow) numbering less than 4, i.e., t1, while adjacent panel (B) shows numerous lymphocytes .10, i.e., t3, by Banff criteria. (C) Photomicrograph of early intimal lesions with lymphocytes undermining the vascular intima of the blood vessel, v1 (arrow). (D) Severe vascular lesion, transmural arteritis with fibrinoid necrosis (arrow), and complete occlusion of vascular lumen, i.e., v3 lesion.

arteritis has been observed in isolation from tubulitis/interstitial inflammation—i.e., isolated v lesions—and may have prognostic connotation.64 Gene expression studies have suggested the presence of ABMR, especially if isolated v lesions are noted after 1-year posttransplantation.65

32.5.1 Monocyte-Mediated Acute Cellular Rejection The role of monocytes in animal models of acute rejection has been reported.66 In humans, approximately 34% (1000 4800 μL21) of white blood cells are lymphocytes, but only 4% (0 800 μL21) of white blood cells are monocytes in healthy adults. After conventional renal transplantation and postoperative immunosuppression, effects of monocytes during acute rejection may be overshadowed by the predominant presence of T lymphocytes in the grafts during acute rejection. Alemtuzumab (Campath-1H), a humanized monoclonal antibody against CD52, can cause more profound depletion of lymphocytes than monocytes. In 1998, Calne et al.67 reported that perioperative Campath-1H induction and postoperative cyclosporine maintenance resulted in a low rejection rate in 13 renal transplant recipients. Kirk et al.68 confirmed the effectiveness of pretreatment with Campath-1H, in suppressing acute rejection in 7 renal recipients of living-donor kidneys. However, administration of Campath-1H alone was associated with monocyte-associated acute rejection, which was treated with steroids.68 Monocytemediated acute rejection after treatment with Campath-1H alone has been reported also in bone marrow transplant recipients.69

32.5.2 Differential Diagnosis Acute interstitial nephritis may mimic ACR and antecedent suggestive history of implicated medications should be sought.70 Polyoma virus nephropathy causes interstitial nephritis with tubulitis. Distinct intranuclear basophilic viral inclusions without a surrounding halo, anisonucleosis, hyperchromasia, and chromatin clumping within infected cells may be observed.71 73 Confirmation of BK nephropathy is by positive SV40 antigen staining.

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Cytomegalovirus may also cause tubulointerstitial nephritis with cytoplasmic inclusions within infected cells.74 Both viruses cause viremia along with nephritis and provide clues to the diagnosis. The significance is the need to reduce immunosuppression and/or initiate specific antiviral therapy in these cases as opposed to ACR.

32.5.3 Limitation of Histology Analysis of Acute Cellular Rejection The principal rule for diagnosing T-cell-mediated ACR requires semiquantitative assessment of two lesions, interstitial inflammation (i-score) and tubulitis (t-score), using arbitrary thresholds.75,76 In protocol biopsies, both correlate with previous injury, but not with rejection,77 because the inflammatory response to injury can produce these lesions. Many biopsies with inflammation and tubulitis below the thresholds are called “borderline.” Furthermore, these lesions must be assessed in unscarred areas, rendering the diagnosis of ACR impossible in biopsies with advanced atrophy scarring. The second rule for diagnosing ACR relies on isolated v lesions: Intimal arteritis without sufficiently high i and t lesions (v . 0 and i , 2 or t , 2). This rule is suspect because v lesions can result from ABMR and AKI,56,78 and will probably change at the next Banff meeting.56 In addition, ACR diagnoses are poorly reproducible within the rules. Furness et al. found that agreement among pathologists using the same rules was not only limited but resistant to improvement.79,80 Therefore, despite its clear utility, other ways should be explored to reach a more reliable and reproducible way to diagnose ACR.

32.5.4 Molecular Diagnosis of Acute Cellular Rejection Transcriptional studies utilizing high-throughput (microarray, RNA sequencing) or low-throughput (quantitative PCR) technologies have suggested that simultaneous or antecedent gene expression changes in peripheral blood19,81 84 and allograft85 88 could have diagnostic, predictive, or prognostic value in ACR. These novel transcriptional data, while accurate in the research settings, have not translated into the clinical realm for various reasons. The transcriptional environment of ACR is influenced by numerous clinical factors that are prone to variation: Immunosuppressive agents used, severity of ACR (borderline vs grade I or II), the timing of the rejection episode posttransplantation, the extent of baseline disease in the organ and existing IF/TA, whether there is associated ABMR or infection, and the representativeness of the sample used for transcriptome analysis.89 91 Further, research studies rely on complex, expensive, and time-consuming analysis, and depend overly on “clean phenotypes” for diagnosis of ACR versus no ACR. Additionally, these studies tend to use highly selected patient samples, all of which have limited the generalizability of the data obtained.91 Most of these studies have depended exclusively on clinical episodes of ACR, and their role in diagnosing SCR is also uncertain. Nonetheless, substantial progress has been made in understanding the transcriptome of ACR in the preceding decade. Many transcriptional studies have attempted to identify gene expression profiles that can separate ACR from non-ACR. Akalin et al.85 initially used high-density oligonucleotide array to open-endedly analyze the allograft transcriptome of patients with ACR. After restricting their analysis to genes with fourfold or greater change between ACR and no ACR, they identified a panel of four transcripts that had discriminatory potential. Sarwal et al.58 used DNA microarrays in kidney allografts, and identified four histologic categories based on the molecular signature that corresponded well with the histologic phenotype. Based on a cohort of 403 allograft biopsies with ACR episodes varying from 6 months to 21 years, a group of investigators used microarray to identify a transcriptional signature in the allograft that would correlate consistently with ACR. They derived a genomic T-cellmediated rejection score (TCMR score) based on the top 20 differentially expressed genes between ACR and no ACR, which was able to differentiate these two conditions with high accuracy (AUC 5 89%).86,88 Subsequently, in a larger external validation cohort, applying the TCMR score in place of the histologic diagnosis, these authors observed a net correct reclassification in 26% biopsies.88 The authors point out that the reclassification occurred especially where scarring or non-ACR tubulitis limited the accuracy of histology. However, it must be noted that the biopsies were reclassified by using the TCMR score itself as the “gold-standard.” Interestingly, this group has reported repeatedly that a molecular TCMR score suggestive of ACR is not predictive of a poorer subsequent allograft outcome in their retrospective cohort, raising questions regarding the ultimate impact of ACR.

32.5.5 Noninvasive Diagnostic Tests for Acute Cellular Rejection Graft biopsy is universally regarded as the gold standard for diagnosing the presence of acute and chronic rejection, for characterizing the type of alloimmune response (whether T-cell-mediated or antibody-mediated) and for assessing the extent of immune-mediated graft injury.92 However, graft biopsy can recognize the immune

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TABLE 32.2 Year of studyRef.

Subclinical Cellular Rejection (SCR) and Variable Allograft Outcomes Observed in Clinical Studies CAN or IF/TA

eGFR decline

Subsequent ACR

Chronic rejection

Graft loss

30% (3 months) 15% (6 months)

Y

Y

Y

U

N

Sample size

SCR prevalence (months)

18

1998

72

200393

46

50% (2 years)

Y

Y

U

U

Y

94

126

30.8% (6 months)

N

N

U

N

U

15

248

4.6% (1 6 months)

N

N

N

U

N

95

2008

102

17% (1 month) 12% (3 months)

Y

Y

U

U

N

201196

164

11% (3 months) 23% (12 months)

Y

Y

U

U

Y

201210

520/119

29.4%/38.5% (1 4 months)

Y

U

U

Y

Y

2006

2007

Y, Yes; N, No; U, not reported/unknown; CAN, chronic allograft nephropathy; eGFR, estimated glomerular filtration rate; IF/TA, interstitial fibrosis/tubular atrophy.

reaction only at the latest stages, when allograft injury has already been established. Additionally, there is nothing that graft biopsy can do about identifying patients with ongoing over- or underimmunosuppression before the pathologic process takes place. The use of surveillance biopsies, namely biopsies performed in patients with stable graft function in order to diagnose subclinical forms of rejection before the onset of graft dysfunction, shares the same problem. Moreover, in renal transplant recipients, the use of surveillance biopsies did not improve graft outcome in some randomized clinical trials (Table 32.2).15 Numerous investigators have examined whether blood and/or urinary biomarkers could diagnose ACR with sufficient accuracy in order to avoid a biopsy. Several groups have identified peripheral blood gene expression changes that have correlated with ACR.19,81,84 In theory, a peripheral blood signature could obviate the need for biopsy, while an allograft signature still necessitates it. Further, a peripheral blood signature for SCR, or one that antedates the presence of ACR, has the potential to serve as an immune-monitoring tool. An interesting observation from simultaneous comparisons of blood and allograft transcriptomes of ACR has been the consistent identification of lack of overlap between the gene profiles in blood and biopsy that associate with ACR.97 Recently, a 17-gene qPCR-based assay in blood samples—the kidney solid organ response test (kSORT)—was validated in a multicenter cohort including 438 renal transplant patients as a diagnostic and possibly predictive noninvasive test for ACR.83 Recognizing the increased allograft infiltration and subsequent urinary appearance of lymphocytes during ACR episodes, preselected immune transcripts (T-cell/immune-related) in urinary cell pellets measured by qPCR have been reported to noninvasively differentiate ACR from non-ACR.32,98 100 One study used 18S-ribosomalRNA normalized, 3-gene signature (CD3ε, IP-10, and 18S-RNA) to identify ACR from non-ACR with high accuracy.99 However, urine handling and storage for accurate transcript assessment needs much rigor and most of these studies on urinary RNA have emerged from a small number of specialized research groups, and their performance in clinic settings is uncertain at this time. Similarly to RNA levels, urinary CXCL9 and CXCL10 protein levels have also shown to have diagnostic accuracy for ACR, suggesting a role for these proteins in noninvasive diagnosis of ACR.100,101 Measurement of the same chemokines in the urine by ELISA, a much easier and faster technique than qPCR on urinary cells, showed that these biomarkers correlate with the extent of subclinical tubulitis,102 and their increase is associated with ACR and urinary infection.100,103 By and large, despite substantial progress made, the optimal immune-monitoring tool to diagnose ACR without biopsy and risk-stratify patients with high accuracy has remained elusive. Molecular and cellular imaging techniques have shown significant potential in kidney transplantation. With more optimal tracers, which are constantly being developed, PET, MRI, and other devices may serve as valuable tools for the diagnosis and management of renal ACR.104 106 For instance, blood oxygen dependent MRI (BOLD-MRI) showed good discrimination between ACR, acute tubular necrosis (ATN), and stable graft function in a cohort of early posttransplant recipients.104 Similarly, dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI) differentiated ACR from stable graft function in a cohort of 50 recipients with 100% accuracy.106 In the future, these techniques may serve as ancillary strategies to aid in the noninvasive diagnosis of ACR, and

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may impact our ability to assess graft function and study response to therapy as well as progression of lesions, and ultimately relay information regarding graft prognosis.

32.6 TREATMENT Specific treatment for ACR episodes depends on the severity of ACR (i.e., histological grade), coexistence of ABMR lesions, likelihood of reversibility or stabilization of renal function in each case, weighed against the adverse effects of immunosuppression. There also exists considerable institutional variability in therapies chosen. In patients with advanced allograft dysfunction and irreversible histological damage, it is advisable to factor in the likelihood of retransplantation and the cumulative immunosuppression it would subsequently mandate. The major modalities for ACR treatment include pulse steroids and anti-T-cell therapies (antibody therapies including monoclonal anti-CD3 antibody, polyclonal equine antilymphocyte globulin (ATGAM), and rabbit antithymocyte globulin (ATG)). Several early trials have compared the efficacy of steroid therapy to antilymphocyte therapies in treating ACR.107 110 A metaanalysis of these and other studies including 956 patients concluded that antibody therapies were significantly more effective at reversing ACR episodes and reducing subsequent allograft loss than pulse steroids.111 This study found no significant differences in rates of infection and malignancy between these strategies, but the follow-up periods of the included studies were generally less than 1 year. Glucocorticoids bind to universally present cytoplasmic glucocorticoid receptors and inhibit the alloimmune response at multiple levels including inhibiting transcription factors for immune-related genes (e.g., activatorprotein 1, nuclear factor kappa B, and interleukin-1), inhibiting innate immune cells including APCs, preventing TH1-cell differentiation and T-cell proliferation.112 114 Based on early trials, pulse steroids are given intravenously at 3 5 mg/kg of methylprednisolone repeated over 3 days.115,116 Most centers use this regimen followed by a taper over 1 2 months as sole therapy for milder episodes of ACR (e.g., IA or less) or, where a contraindication exists for antibody therapy. In these cases steroids may be effective in reversing 60% 70% of ACR episodes. Use of steroids in high doses required for ACR treatment can be associated with substantial side effects—weight gain, glucose intolerance or new-onset diabetes, fluid retention and hypertension, acid-peptic disease/peptic ulceration, opportunistic infections (Pneumocystis pneumonia, CMV disease), and osteoporosis, or rarely avascular necrosis.117,118 Steroids are often used as adjuncts to antibody therapy in more severe cases of ACR (1B or greater). Rabbit antithymocyte globulin (ATG) is derived by immunizing rabbits with human thymocytes obtained during cardiac transplantation, whereas ATGAM is obtained from horses.119 While both ATGAM and ATG primarily deplete T lymphocytes by complement-dependent cytotoxicity, existence of antibodies in these polyclonal mixtures that target molecules on other cells types (B cells, innate immune cells, platelets) may contribute both to their efficacy and safety profiles. In a double-blind randomized trial comparing ATG to ATGAM, ATG therapy resulted in higher rates of reversal of ACR (88% vs 76%) and a lower rate of recurrent rejection at 3 months after antibody therapy (17% vs 36%), compared to ATGAM. For ACR treatment, ATG is generally dosed up to a total of 10 mg/kg administered as infusions with premedication in 3 5 divided doses over 3 5 days.120 OKT3 is not used in the United States, but was found to be similarly efficacious as ATGAM and ATG in ACR treatment in the metaanalysis by Webster et al.111 Important concerns with antibody-therapies are infusion related side effects (fever, rash), allergic reactions (rarely anaphylaxis), and hematological changes (thrombocytopenia and leukopenia), as well as opportunistic infections. Patients with prior exposure to leporine or equine products are especially at risk for allergic phenomena from ATG and ATGAM respectively. Small studies and series have suggested a role for alemtuzumab in more severe ACR (1B or greater) and steroid-resistant rejections with ACR response rates of 70% 75%.121 124 Allograft infiltrates containing CD-20positive B cells in ACR may be more refractory, and small studies have suggested benefit with rituximab in these cases.58,125 An early study tracing the response of serum creatinine to ACR treatment initiation suggested that improvement is delayed by 5 7 days (in responders), while a statistically significant difference in creatinine between responders and nonresponders was observed only by 14 days.126 Steroid-resistant rejection has therefore been defined on the basis of nonimprovement in urine output or the plasma creatinine concentration within 5 7 days of initiating pulse steroid therapy. ATG, alemtuzumab, and rituximab may have special roles in these cases. The occurrence of ACR episodes in modern transplantation should always elicit a search for underimmunosuppression relative to what is understood as a given recipient’s risk for ACR. The presence of risk factors such as those listed above may be observed. Recent episodes of immunization or infections may be elicitable, but

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469

whether a causative role exists is incompletely understood.127 129 Overzealous tapering of immune suppression based on time-bound institutional protocols or unrecognized pharmacokinetic interactions as well as patient nonadherence with therapy resulting in inadequate immunosuppressive agent levels are significant causes of ACR. These therapeutic decisions can be reversed or readdressed as a component of ACR treatment. For instance, higher tacrolimus trough level goals could be maintained after ACR episodes. Additionally, switching to a tacrolimus-based regimen from a cyclosporine-based regimen at the first ACR episode was associated with improved resolution of ACR episode and reduced ACR recurrences.130 Similarly, consideration may be given to increasing the dose of antimetabolite or opting for a steroid-continuation regimen as opposed to a steroidwithdrawal one as measures adjunctive to the specific treatment of the ACR episode.

32.6.1 Treatment of Subclinical Rejection The treatment of SCR identified incidentally on biopsy has proven more controversial. While some randomized studies have suggested that treatment of SCR in early biopsies (1, 3, or 6 months) is associated with improvements in later renal function (1 or 2 years),18,95 a large randomized trial in the tacrolimus era showed a significantly lower prevalence of SCR and did not identify a benefit at 2 years by treating these SCR episodes.15 The treatment strategy for borderline changes seen on allograft biopsies is also without consensus. Most centers, including ours, treat these episodes with short courses of oral steroids, especially if associated with renal dysfunction. Transcriptional data from some studies have suggested that borderline changes may be a part of a continuum between ACR and non-ACR,81 while other groups have identified that borderline changes can be grouped clearly into either ACR or non-ACR.131 Intriguingly, studies have suggested increased regulatory T-cell activity in blood or allograft of biopsies with borderline ACR, when compared to both ACR and non-ACR.132,133 The presence of ABMR along with ACR (i.e., mixed rejection) requires aggressive treatment, is associated with less complete response to therapy, and is discussed in Chapter 35.

32.6.2 Nonadherence as a Major Risk Factor for Acute Cellular Rejection Medical adherence, a reflection of the degree to which a patient follows directions regarding treatments, medications, and clinical surveillance as prescribed by their caregiver, is now a significant predictor of outcome, particularly among those for whom chronic therapies are required. Among transplant recipients, nonadherence is a major risk factor for rejection and allograft loss.134,135 A recent report from a “consensus conference” on nonadherence to immunosuppressive agents soberly concluded that nonadherence is more prevalent than previously assumed, is difficult to measure accurately, confers worse outcomes, occurs for a variety of reasons, and is hard to change from a behavioral perspective.136 Nevins et al., using electronic pill bottle monitoring, reported subclinical nonadherence ($7% missed doses by month 2 posttransplant) in 22.4% of patients, which correlated with increased rates of acute rejection and late graft loss.137 Along the same line, nonadherence has been shown as a key clinical parameter to predict development of de novo donor-specific antibodies, a major determinant of long-term graft loss. Time-dependent variability in tacrolimus trough blood levels (20.8% had a standard deviation .2.5) has been suggested as a reliable surrogate of nonadherence and/or suboptimal drug pharmacokinetics, as it correlates with late rejections, cg, and graft failure.136 Given the dominant role it plays in acute rejection and accelerating graft loss, effective interventions to address nonadherence are extremely important.135,138 Common reasons cited for nonadherence were oversleeping, work-related barriers and forgetfulness, forgetting to refill medications, changes in prescription, busyness, and traveling without medication.139,140 Greenstein and Siegal identified three groups of noncompliers: accidental noncompliers (47%), invulnerable noncompliers who had a belief of invincibility (28%), and decisive noncompliers (25%), each of which has different origins and will require different interventions.141 Successful intervention to improve adherence must be multidimensional. One approach is to use identified risk factors to avoid transplantation in high-risk candidates. As nonadherence to dialysis regimens may predict similar behavior posttransplantation, it is not unreasonable to delay transplantation until a patient demonstrates adherence to his or her dialysis regimen. Education regarding the importance of immunosuppressive agents in ensuring optimal outcomes must be an ongoing effort. Gordon et al. emphasize the importance of a medication schedule and use of cues, pillboxes, and reminders from others.139 Pinsky et al., finding that less-than-perfect adherence predicts allograft loss and

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increased costs, emphasize the need to maximize adherence rather than discourage low adherence.142 Many transplant centers use repetitive teaching to promote adherence. The reasons for nonadherence are complex and sometimes of a nature that makes assessment and intervention difficult. However, the result of these interventions should translate into reduced rates of ACR and ABMR, improved allograft, and patient survival.

32.7 IMPACT AND PROGNOSIS OF ACUTE CELLULAR REJECTION ACR is often treatable with current immunosuppressive agents. Further, when treated, ACR episodes are frequently reversible. Hence, some debate has been generated on the ultimate impact of ACR episodes on the allograft function and survival. Early interventional studies suggested that single ACR episodes before 6 months posttransplantation had minimal impact on subsequent allograft course.143 Late ACR (.1-year), ACR along with ABMR, and recurrent episodes of ACR, on the other hand, had greater impact on allograft survival and function.7 Newer transcriptional data have similarly suggested that ACR diagnosed by the molecular gene signature has minimal impact on long-term outcomes.65,87,88 However, key data on treatment of ACR, and therefore reversal of ACR, is not clearly defined in these retrospective cohorts. For instance, a recent retrospective study identified that SCR at 1 year had no impact on allograft survival, while subclinical antibody-mediated rejection impacted survival. However, in this cohort all patients with SCR were treated, which may explain the lack of impact since SCR episodes may have reversed.144 Both recent and older data have confirmed that ACR episodes with complete reversal of serum creatinine to pre-ACR baseline have minimal long-term impact.7,17 Analogously, more severe ACR episodes (grades Ib or more) are more likely to impact the allograft irreversibly. Careful histological studies have revealed that ongoing inflammation leads to progressive histological and functional decline,145 implying that ACR that remits incompletely leaving residual inflammation, or progresses subclinically, may cause longterm graft damage. Retrospective studies that have looked backward from the event of graft loss, have in turn identified ACR as responsible for 10% 12% of all graft losses.9 More recently studies have suggested an ACR-toantibody-mediated injury continuum, wherein early ACR (even subclinical) has been associated to later development of donor specific anti-HLA antibodies and chronic humoral rejection.10 In summary, a single, promptly treated, early episode of ACR may have minimal long-term impact, while recurrent and more severe episodes that may be more refractory to treatment may not be innocuous. The development of anti-HLA antibodies either subsequent or simultaneous to ACR episodes may need to be monitored, and could ultimately be of most consequence to the allograft.

References 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14.

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Gross hematuria as a sign of acute rejection. Pediatr Transplant 2014;18:E106 8. 50. Rastogi N, Williams G, Alencar H. Modality-specific occult intrarenal pseudoaneurysm in a renal allograft and the legacy of catheter angiography. Ann Vasc Surg 2013;27(1184):e7 11. 51. Lal A, Singhal M, Ramachandran R, Rathi M, Jha V, Khandelwal N. Percutaneous injection of acrylic glue into renal allograft pseudoaneurysm for control of intractable post-biopsy hematuria. Indian J Nephrol 2014;24:124 6.

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52. Drake KA, Najera L, Reed RC, Verghese PS. Unusual presentations of BK virus infections in pediatric renal transplant recipients. Pediatr Transplant 2013;17:E9 15. 53. Ison MG. Adenovirus infections in transplant recipients. Clin Inf Dis 2006;43:331 9. 54. Solez K, Axelsen RA, Benediktsson H, et al. International standardization of criteria for the histologic diagnosis of renal allograft rejection: the Banff working classification of kidney transplant pathology. Kidney Int 1993;44:411 22. 55. Solez K, Colvin RB, Racusen LC, et al. Banff 07 classification of renal allograft pathology: updates and future directions. Am J Transplant 2008;8:753 60. 56. Mengel M, Sis B, Haas M, et al. Banff 2011 Meeting report: new concepts in antibody-mediated rejection. Am J Transplant 2012;12:563 70. 57. Haas M, Sis B, Racusen LC, et al. Banff 2013 meeting report: inclusion of c4d-negative antibody-mediated rejection and antibodyassociated arterial lesions. Am J Transplant 2014;14:272 83. 58. Sarwal M, Chua MS, Kambham N, et al. Molecular heterogeneity in acute renal allograft rejection identified by DNA microarray profiling. New Engl J Med 2003;349:125 38. 59. Charney DA, Nadasdy T, Lo AW, Racusen LC. Plasma cell-rich acute renal allograft rejection. Transplantation 1999;68:791 7. 60. Hongwei W, Nanra RS, Stein A, Avis L, Price A, Hibberd AD. Eosinophils in acute renal allograft rejection. Transplant Immunol 1994;2:41 6. 61. Kormendi F, Amend WJ. The importance of eosinophil cells in kidney allograft rejection. Transplantation 1988;45:537 9. 62. Gupta G, Shapiro R, Girnita A, et al. Neutrophilic tubulitis as a marker for urinary tract infection in renal allograft biopsies with C4d deposition. Transplantation 2009;87:1013 18. 63. Gibson IW, Gwinner W, Brocker V, et al. Peritubular capillaritis in renal allografts: prevalence, scoring system, reproducibility and clinicopathological correlates. Am J Transplant 2008;8:819 25. 64. Sis B, Bagnasco SM, Cornell LD, et al. Isolated endarteritis and kidney transplant survival: a multicenter collaborative study. J Am Soc Nephrol 2015;26:1216 27. 65. Salazar ID, Lopez MM, Chang J, Halloran PF. Reassessing the significance of intimal arteritis in kidney transplant biopsy specimens. J Am Soc Nephrol 2015. pii: ASN.2014111064. 66. Grone HJ, Weber C, Weber KS, et al. Met-RANTES reduces vascular and tubular damage during acute renal transplant rejection: blocking monocyte arrest and recruitment. FASEB J 1999;13:1371 83. 67. Calne R, Friend P, Moffatt S, et al. Prope tolerance, perioperative campath 1H, and low-dose cyclosporin monotherapy in renal allograft recipients. Lancet 1998;351:1701 2. 68. Kirk AD, Hale DA, Mannon RB, et al. Results from a human renal allograft tolerance trial evaluating the humanized CD52-specific monoclonal antibody alemtuzumab (CAMPATH-1H). Transplantation 2003;76:120 9. 69. Buggins AG, Mufti GJ, Salisbury J, et al. Peripheral blood but not tissue dendritic cells express CD52 and are depleted by treatment with alemtuzumab. Blood 2002;100:1715 20. 70. Baradhi KM, Gohh R. A diagnostic conundrum: acute interstitial nephritis due to armodafinil versus acute cellular rejection in a renal transplant recipient a case report. Transplant Proc 2013;45:845 7. 71. Randhawa PS, Finkelstein S, Scantlebury V, et al. Human polyoma virus-associated interstitial nephritis in the allograft kidney. Transplantation 1999;67:103 9. 72. Drachenberg CB, Beskow CO, Cangro CB, et al. Human polyoma virus in renal allograft biopsies: morphological findings and correlation with urine cytology. Hum Pathol 1999;30:970 7. 73. Howell DN, Smith SR, Butterly DW, et al. Diagnosis and management of BK polyomavirus interstitial nephritis in renal transplant recipients. Transplantation 1999;68:1279 88. 74. Liapis H, Storch GA, Hill DA, Rueda J, Brennan DC. CMV infection of the renal allograft is much more common than the pathology indicates: a retrospective analysis of qualitative and quantitative buffy coat CMV-PCR, renal biopsy pathology and tissue CMV-PCR. Nephrol Dial Transplant 2003;18:397 402. 75. Mengel M, Sis B, Halloran PF. SWOT analysis of Banff: strengths, weaknesses, opportunities and threats of the international Banff consensus process and classification system for renal allograft pathology. Am J Transplant 2007;7:2221 6. 76. Racusen LC, Solez K. Nephrotoxic tubular and interstitial lesions: morphology and classification. Toxicol Pathol 1986;14:45 57. 77. Mengel M, Chang J, Kayser D, et al. The molecular phenotype of 6-week protocol biopsies from human renal allografts: reflections of prior injury but not future course. Am J Transplant 2011;11:708 18. 78. Reeve J, Einecke G, Mengel M, et al. Diagnosing rejection in renal transplants: a comparison of molecular- and histopathology-based approaches. Am J Transplant 2009;9:1802 10. 79. Furness PN, Taub N, Assmann KJ, et al. International variation in histologic grading is large, and persistent feedback does not improve reproducibility. Am J Surg Pathol 2003;27:805 10. 80. Furness PN, Taub N. Convergence of European Renal Transplant Pathology Assessment Procedures P. International variation in the interpretation of renal transplant biopsies: report of the CERTPAP Project. Kidney Int 2001;60:1998 2012. 81. Li L, Khatri P, Sigdel TK, et al. A peripheral blood diagnostic test for acute rejection in renal transplantation. Am J Transplant 2012;12:2710 18. 82. Khatri P, Roedder S, Kimura N, et al. A common rejection module (CRM) for acute rejection across multiple organs identifies novel therapeutics for organ transplantation. J Exper Med 2013;210:2205 21. 83. Roedder S, Sigdel T, Salomonis N, et al. The kSORT assay to detect renal transplant patients at high risk for acute rejection: results of the multicenter AART study. PLoS Med 2014;11:e1001759. 84. Kurian SM, Williams AN, Gelbart T, et al. Molecular classifiers for acute kidney transplant rejection in peripheral blood by whole genome gene expression profiling. Am J Transplant 2014;14:1164 72. 85. Akalin E, Hendrix RC, Polavarapu RG, et al. Gene expression analysis in human renal allograft biopsy samples using high-density oligoarray technology. Transplantation 2001;72:948 53. 86. Reeve J, Einecke G, Mengel M, et al. Diagnosing rejection in renal transplants: a comparison of molecular- and histopathology-based approaches. Am J Transplant 2009;9(8):1802 18.

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87. Reeve J, Sellares J, Mengel M, et al. Molecular diagnosis of T cell-mediated rejection in human kidney transplant biopsies. Am J Transplant 2013;13:645 55. 88. Halloran PF, Pereira AB, Chang J, et al. Potential impact of microarray diagnosis of T cell-mediated rejection in kidney transplants: the INTERCOM study. Am J Transplant 2013;13:2352 63. 89. Ong S, Mannon RB. Genomic and proteomic fingerprints of acute rejection in peripheral blood and urine. Transplant Rev 2015;29:60 7. 90. Ying L, Sarwal M. In praise of arrays. Pediatr Nephrol 2009;24(9):1643 59. 91. Reeve J, Halloran PF, Kaplan B. Common errors in the implementation and interpretation of microarray studies. Transplantation 2015;99:470 5. 92. Mehrotra A, Leventhal J, Purroy C, Cravedi P. Monitoring T cell alloreactivity. Transplant Rev 2015;29:53 9. 93. Shishido S, Asanuma H, Nakai H, et al. The impact of repeated subclinical acute rejection on the progression of chronic allograft nephropathy. J Am Soc Nephrol 2003;14:1046 52. 94. Scholten EM, Rowshani AT, Cremers S, et al. Untreated rejection in 6-month protocol biopsies is not associated with fibrosis in serial biopsies or with loss of graft function. J Am Soc Nephrol 2006;17:2622 32. 95. Kurtkoti J, Sakhuja V, Sud K, et al. The utility of 1- and 3-month protocol biopsies on renal allograft function: a randomized controlled study. Am J Transplant 2008;8:317 23. 96. Szederkenyi E, Ivanyi B, Morvay Z, et al. Treatment of subclinical injuries detected by protocol biopsy improves the long-term kidney allograft function: a single center prospective randomized clinical trial. Transplant Proc 2011;43:1239 43. 97. Flechner SM, Kurian SM, Head SR, et al. Kidney transplant rejection and tissue injury by gene profiling of biopsies and peripheral blood lymphocytes. Am J Transplantat 2004;4:1475 89. 98. Li B, Hartono C, Ding R, et al. Noninvasive diagnosis of renal-allograft rejection by measurement of messenger RNA for perforin and granzyme B in urine. New Engl J Med 2001;344:947 54. 99. Suthanthiran M, Schwartz JE, Ding R, et al. Urinary-cell mRNA profile and acute cellular rejection in kidney allografts. New Engl J Med 2013;369:20 31. 100. Hricik DE, Nickerson P, Formica RN, et al. Multicenter validation of urinary CXCL9 as a risk-stratifying biomarker for kidney transplant injury. Am J Transplant 2013;13:2634 44. 101. Rabant M, Amrouche L, Lebreton X, et al. Urinary C-X-C motif chemokine 10 independently improves the noninvasive diagnosis of antibody-mediated kidney allograft rejection. J Am Soc Nephrol 2015. Available from: http://dx.doi.org/10.1681/ASN.2014080797. 102. Schaub S, Nickerson P, Rush D, et al. Urinary CXCL9 and CXCL10 levels correlate with the extent of subclinical tubulitis. Am J Transplant 2009;9:1347 53. 103. Jackson JA, Kim EJ, Begley B, et al. Urinary chemokines CXCL9 and CXCL10 are noninvasive markers of renal allograft rejection and BK viral infection. Am J Transplant 2011;11:2228 34. 104. Han F, Xiao W, Xu Y, et al. The significance of BOLD MRI in differentiation between renal transplant rejection and acute tubular necrosis. Nephrol Dial Transplant 2008;23:2666 72. 105. Obeidat MA, Luyckx VA, Grebe SO, et al. Post-transplant nuclear renal scans correlate with renal injury biomarkers and early allograft outcomes. Nephrol Dial Transplant 2011;26:3038 45. 106. Khalifa F, Abou El-Ghar M, Abdollahi B, Frieboes HB, El-Diasty T, El-Baz A. A comprehensive non-invasive framework for automated evaluation of acute renal transplant rejection using DCE-MRI. NMR Biomed 2013;26:1460 70. 107. Shield 3rd CF, Cosimi AB, Tolkoff-Rubin N, Rubin RH, Herrin J, Russell PS. Use of antithymocyte globulin for reversal of acute allograft rejection. Transplantation 1979;28:461 4. 108. Filo RS, Smith EJ, Leapman SB. Therapy of acute cadaveric renal allograft rejection with adjunctive antithymocyte globulin. Transplantation 1980;30:445 9. 109. Hoitsma AJ, Reekers P, Kreeftenberg JG, van Lier HJ, Capel PJ, Koene RA. Treatment of acute rejection of cadaveric renal allografts with rabbit antithymocyte globulin. Transplantation 1982;33:12 16. 110. Theodorakis J, Schneeberger H, Illner WD, Stangl M, Zanker B, Land W. Aggressive treatment of the first acute rejection episode using first-line anti-lymphocytic preparation reduces further acute rejection episodes after human kidney transplantation. Transplant Int 1998;11(Suppl. 1):S86 9. 111. Webster AC, Pankhurst T, Rinaldi F, Chapman JR, Craig JC. Monoclonal and polyclonal antibody therapy for treating acute rejection in kidney transplant recipients: a systematic review of randomized trial data. Transplantation 2006;81:953 65. 112. Rhen T, Cidlowski JA. Antiinflammatory action of glucocorticoids new mechanisms for old drugs. New Engl J Med 2005;353:1711 23. 113. Halloran PF. Immunosuppressive drugs for kidney transplantation. New Engl J Med 2004;351:2715 29. 114. Menon MC, Murphy B. Maintenance immunosuppression in renal transplantation. Curr Opin Pharmacol 2013;13:662 71. 115. Gray D, Shepherd H, Daar A, Oliver DO, Morris PJ. Oral versus intravenous high-dose steroid treatment of renal allograft rejection. The big shot or not? Lancet 1978;1:117 18. 116. Vineyard GC, Fadem SZ, Dmochowski J, Carpenter CB, Wilson RE. Evaluation of corticosteroid therapy for acute renal allograft rejection. Surg Gyn Obst 1974;138:225 9. 117. Pichette V, Bonnardeaux A, Prudhomme L, Gagne M, Cardinal J, Ouimet D. Long-term bone loss in kidney transplant recipients: a cross-sectional and longitudinal study. Am J Kidney Dis 1996;28:105 14. 118. Weinstein RS. Clinical practice. Glucocorticoid-induced bone disease. New Engl J Med 2011;365:62 70. 119. Mohty M. Mechanisms of action of antithymocyte globulin: T-cell depletion and beyond. Leukemia 2007;21:1387 94. 120. Gaber AO, First MR, Tesi RJ, et al. Results of the double-blind, randomized, multicenter, phase III clinical trial of Thymoglobulin versus Atgam in the treatment of acute graft rejection episodes after renal transplantation. Transplantation 1998;66:29 37. 121. Friend PJ, Rebello P, Oliveira D, et al. Successful treatment of renal allograft rejection with a humanized antilymphocyte monoclonal antibody. Transplant Proc 1995;27:869 70. 122. Basu A, Ramkumar M, Tan HP, et al. Reversal of acute cellular rejection after renal transplantation with Campath-1H. Transplant Proc 2005;37:923 6. 123. Csapo Z, Benavides-Viveros C, Podder H, Pollard V, Kahan BD. Campath-1H as rescue therapy for the treatment of acute rejection in kidney transplant patients. Transplant Proc 2005;37:2032 6.

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124. van den Hoogen MW, Hesselink DA, van Son WJ, Weimar W, Hilbrands LB. Treatment of steroid-resistant acute renal allograft rejection with alemtuzumab. Am J Transplant 2013;13:192 6. 125. Becker YT, Becker BN, Pirsch JD, Sollinger HW. Rituximab as treatment for refractory kidney transplant rejection. Am J Transplant 2004;4:996 1001. 126. Shinn C, Malhotra D, Chan L, Cosby RL, Shapiro JI. Time course of response to pulse methylprednisolone therapy in renal transplant recipients with acute allograft rejection. Am J Kidney Dis 1999;34:304 7. 127. Muller V, Becker G, Delfs M, Albrecht KH, Philipp T, Heemann U. Do urinary tract infections trigger chronic kidney transplant rejection in man? J Urol 1998;159:1826 9. 128. Hurst FP, Lee JJ, Jindal RM, Agodoa LY, Abbott KC. Outcomes associated with influenza vaccination in the first year after kidney transplantation. Clin J Am Soc Nephrol 2011;6:1192 7. 129. Cainelli F, Vento S. Infections and solid organ transplant rejection: a cause-and-effect relationship? Lancet Infect Dis 2002;2:539 49. 130. Briggs D, Dudley C, Pattison J, et al. Effects of immediate switch from cyclosporine microemulsion to tacrolimus at first acute rejection in renal allograft recipients. Transplantation 2003;75:2058 63. 131. de Freitas DG, Sellares J, Mengel M, et al. The nature of biopsies with “borderline rejection” and prospects for eliminating this category. Am J Transplant 2012;12:191 201. 132. Nemeth D, Ovens J, Opelz G, et al. Does borderline kidney allograft rejection always require treatment? Transplantation 2010;90:427 32. 133. Bestard O, Cruzado JM, Rama I, et al. Presence of FoxP31 regulatory T Cells predicts outcome of subclinical rejection of renal allografts. J Am Soc Nephrol 2008;19:2020 6. 134. Kasiske BL, Gaston RS, Gourishankar S, et al. Long-term deterioration of kidney allograft function. Am J Transplant 2005;5:1405 14. 135. De Geest S, Borgermans L, Gemoets H, et al. Incidence, determinants, and consequences of subclinical noncompliance with immunosuppressive therapy in renal transplant recipients. Transplantation 1995;59:340 7. 136. Sapir-Pichhadze R, Wang Y, Famure O, Li Y, Kim SJ. Time-dependent variability in tacrolimus trough blood levels is a risk factor for late kidney transplant failure. Kidney Int 2014;85:1404 11. 137. Nevins TE, Robiner WN, Thomas W. Predictive patterns of early medication adherence in renal transplantation. Transplantation 2014;98:878 84. 138. Wiebe C, Gibson IW, Blydt-Hansen TD, et al. Evolution and clinical pathologic correlations of de novo donor-specific HLA antibody post kidney transplant. Am J Transplant 2012;12:1157 67. 139. Gordon EJ, Gallant M, Sehgal AR, Conti D, Siminoff LA. Medication-taking among adult renal transplant recipients: barriers and strategies. Transplant Int 2009;22:534 45. 140. Prendergast MB, Gaston RS. Optimizing medication adherence: an ongoing opportunity to improve outcomes after kidney transplantation. Clin J Am Soc Nephrol 2010;5:1305 11. 141. Greenstein S, Siegal B. Compliance and noncompliance in patients with a functioning renal transplant: a multicenter study. Transplantation 1998;66:1718 26. 142. Pinsky BW, Takemoto SK, Lentine KL, Burroughs TE, Schnitzler MA, Salvalaggio PR. Transplant outcomes and economic costs associated with patient noncompliance to immunosuppression. Am J Transplant 2009;9:2597 606. 143. Cecka JM. HLA matching for organ transplantation. . .why not? Int J Immunogenet 2010;37:323 7. 144. Loupy A, Vernerey D, Tinel C, et al. Subclinical rejection phenotypes at 1 year post-transplant and outcome of kidney allografts. J Am Soc Nephrol 2015;99(4):969 72. 145. Park WD, Griffin MD, Cornell LD, Cosio FG, Stegall MD. Fibrosis with inflammation at one year predicts transplant functional decline. J Am Soc Nephrol 2010;21:1987 97.

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C H A P T E R

33 Acute Antibody-Mediated Rejection Ivy A. Rosales and Robert B. Colvin Massachusetts General Hospital, Boston, MA, United States

33.1 INTRODUCTION Acute antibody-mediated rejection (AMR) or acute humoral rejection occurs in patients who are presensitized or who develop a threshold level of de novo donor specific antibody (DSA) at any point after transplantation. There are no specific clinical features that distinguish acute T-cell-mediated rejection (TCMR) from acute AMR. The histologic findings in acute AMR are often characteristic but quite variable and may resemble acute tubular injury or thrombotic microangiopathy (TMA) and may be obscured by features of TCMR. Thus, establishing the diagnosis of acute AMR entails careful correlation between clinical, histologic, and other laboratory findings. This chapter is excerpted and updated from a previous review.1

33.2 RISK FACTORS, PREVALENCE, AND CLINICAL PRESENTATION 33.2.1 Risk Factors The main risk factor for developing acute AMR is presensitization, i.e., history of blood transfusion, pregnancy, and prior transplantation(s). Solid phase assays using single antigen beads to determine DSA characteristics have expanded the knowledge on antibodies in AMR. Higher mean fluorescence intensities (MFIs) of pretransplant DSA detected by these assays are associated with an increased risk of acute AMR.2 The presence of C1q-fixing pretransplant DSA, indicative of the capacity for complement activation through the classical pathway, has also been shown to increase the risk for acute AMR.3 There is no dramatic difference in risk of developing acute AMR across different immunosuppressive regimens, including T-cell-depleting protocols.4 6

33.2.2 Prevalence Acute AMR has an overall frequency of about 6% in transplant recipients (Table 33.1) and occurs mostly in presensitized patients. The frequency is increased to about 28% in presensitized patients who have DSA (8% 43% among different centers).15

33.2.3 Clinical Presentation In the early posttransplant setting, acute AMR is seen in desensitized patients at around 1 3 weeks posttransplant. The mean day of onset has been observed at 151/2 11 days (earliest at 3 days), a feature that is similar to TCMR with a mean of 141/210 days.11,16 Similar clinical symptoms manifest in acute AMR and in TCMR. Acute AMR is typically characterized by a sudden rise in creatinine and oliguria, often requiring dialysis. In one study analyzing patients with antibodies to class I, rejection episodes in those with anti class I occurred earlier, more frequently developed oliguria (35% vs 10%), required dialysis (40% vs 10%) and had a higher rate of rise in Kidney Transplantation, Bioengineering, and Regeneration. DOI: http://dx.doi.org/10.1016/B978-0-12-801734-0.00033-3

475

© 2017 Elsevier Inc. All rights reserved.

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33. ACUTE ANTIBODY-MEDIATED REJECTION

Prevalence of Acute Antibody-Mediated Rejectiona

TABLE 33.1

N

%

Time (months)

232

8.2

#3

286

5.2

#3

833

6.2

# 12

Dorje, Oslo

1534

2.6

,3

Total

2885

4.4

67

29

265

35

93

37

377

21

Lorenz, Vienna

388

17

Total

1190

24

Patients 7

Crespo, Boston

8

Rocha, Durham

9

Matignon, New York 10

BIOPSIES FOR ACUTE REJECTION Mauiyyedi, Boston11 Nickeleit, Basel

12 13

Herzenberg, Vancouver 14

Mengel, Hanover 4

a

These data include all cases that were C4d1 (diffuse or focal), whether or not a concurrent component of acute cellular rejection was present, from a representative sample of case series.

serum creatinine.16 Acute AMR can also have a late onset. This is usually associated with lowering of immunosuppression or with patient noncompliance.17

33.3 PATHOGENESIS 33.3.1 DSA and C4d DSA are usually antibodies directed against HLA class I or II on the endothelium. DSA may also include antibodies against ABO blood group antigens in ABO-incompatible grafts, endothelial cell-specific antigens, antiendothelial cell antibodies, angiotensin II type 1 receptor, or other and unknown non-MHC antigens on the endothelium.18,19 The presence of DSA has been reported in 88% 95% of acute AMR with C4d deposition. It is detected in the serum by enzyme-linked immunoassays or the more recent and sensitive solid phase assays, which use single antigen beads. Interaction of DSA with the graft is demonstrated upon leaving a trail, C4d, an inactive fragment of C4b, which remains at the site of activation on the endothelium. With C4d, the action of DSA is thus reflected from activation of the classical pathway, thereafter depositing complement split-fragments onto the graft vascular bed. C4d was first detected in grafts by Feucht et al. in the early 1990s. They reported that complement splitfragments C4d and C3d are deposited in peritubular capillaries in transplanted kidneys with “cell-mediated rejection” and early dysfunctioning grafts.20,21 In general, antibodies bind to endothelial cell surface antigens but can be lost by modulation, shedding, or cell death, hence the paucity of immunoglobulins on tissue. As a result of antibody interaction, C4d covalently links to structural proteins on the endothelium. This linkage explains why C4d deposition remains for several days even when deposition of alloantibodies is not detectable. While C4d is a diagnostically useful immunologic marker of AMR, its clearance and relationship to DSA levels have not been documented in humans. Experimental animal studies on cardiac allografts show disappearance of C4d along with clearance of antibody a few days after treatment and as early as 7 8 days after a positive biopsy.22 The characteristics of C4d are further detailed in studies utilizing protocol biopsies. Some have shown that C4d deposition can precede histologic evidence of acute AMR. Haas and colleagues reported focal or diffuse C4d staining in 2 out of 82 one-hour postperfusion biopsies in recipients who had a positive pretransplant crossmatch and were treated with plasmapheresis. They also had a weakly positive flow crossmatch at the time of transplantation.23 Both patients later developed acute AMR at days 5 and 34 posttransplant.23 Peritubular capillary C4d

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staining in 1-week protocol biopsies has been observed in 30% of cases, of which 33% did not meet histologic criteria of acute rejection, but 82% developed rejection during further follow-up.24 In another study, C4d was positive in 13% of 48 1-week protocol biopsies.25 The criteria for establishing a diagnosis of acute AMR is therefore three-part: (1) histologic evidence of acute tissue injury, such as acute tubular injury, neutrophilic capillaritis, glomerulitis, fibrinoid necrosis, or arteritis as detailed in the following text; (2) immunopathologic evidence for antibody action, i.e., C4d deposition; and (3) serologic evidence of circulating DSA.26 Acute AMR also manifests differently depending on the timing and occurrence of DSA. Early acute AMR in positive-crossmatch kidney transplants present with high serum DSA levels, C4d deposition in peritubular capillaries, and often a “pure” acute AMR phenotype, i.e., neutrophilic glomerulitis and capillaritis, and not combined with features of TCMR (interstitial inflammation and tubulitis). Late posttransplant acute AMR occurs with de novo DSA, often combined with TCMR. Both acute and late acute AMR may develop into progressive chronic active AMR in positive crossmatch transplants and may manifest variably. C4d may be focal or negative. DSAs are often present in the peribiopsy period. Histologic features include mononuclear glomerulitis, capillaritis, and transplant glomerulopathy (glomerular basement membrane duplication).

33.4 PATHOLOGIC FINDINGS The distinguishing histologic features of acute AMR include neutrophilic capillaritis, neutrophilic glomerulitis, neutrophilic tubulitis, fibrinoid necrosis in glomeruli or arteries, and TMA. The pathology of C4d-positive but DSA-negative cases was not distinguishable from the C4d-positive DSA-positive cases.11

33.4.1 Glomeruli Intracapillary glomerular neutrophils are present in 10% 55% of cases (Figs. 33.1 and 33.2) and mononuclear glomerulitis is seen in 19% 90%.11,16,27,28 Glomerular intracapillary cells are mostly monocytes/macrophages (CD681).27 Fibrin thrombi are present in Fig. 33.3 about 20% of cases.11,16,27

33.4.2 Tubules Tubular injury is common in acute AMR and is characterized by loss of brush borders, thinning of cytoplasm, and paucity of nuclei. In one series, tubular injury was present in 75% of cases.11 Acute tubular injury may also be the only manifestation of acute AMR (Fig. 33.4).11 Neutrophilic tubulitis is a distinctive feature, found in 5% of cases of acute AMR11 while mononuclear tubulitis is found in 30% 80% of cases and is considered evidence of a concurrent cell-mediated component (Fig. 33.5).11,27

FIGURE 33.1 Glomerular capillary loops with neutrophils and apoptotic debris (arrow) at 1-year posttransplant. Peritubular capillaries also show neutrophils.

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FIGURE 33.2 (A) Peritubular capillary neutrophils and mononuclear cells and (B) hypercellular glomerulus with intracapillary neutrophils, mononuclear cells, and swollen endothelial cells in a 2-week posttransplant biopsy of a patient who had a second kidney transplant. The C4d was diffusely positive.

FIGURE 33.3 Glomerular thrombi and fragmented red cells within capillary loops in acute antibody-mediated rejection.

FIGURE 33.4 Day 10 posttransplant biopsy showing acute tubular injury with no inflammation. C4d was diffusely positive (inset).

33.4.3 Interstitium 33.4.3.1 Edema Edema, along with a mononuclear infiltrate, is often present in acute AMR. This feature is shared with acute cell-mediated rejection and may be seen with minimal and focal infiltrates. It is not known whether this histologic feature represents a component of cell-mediated rejection or is caused by antibody or complement interaction. 33.4.3.2 Infiltrate Macrophages infiltrate more in C4d-positive than in C4d-negative rejection.29 B cells can be present in aggregates as well as plasma cells, although an association with AMD has not been shown. Plasma cells can be seen in acute AMR. This has been described occurring together with massive edema and was associated with C4d, DSA, increased production of IFN gamma, and a poor prognosis.30 32 I. KIDNEY TRANSPLANTATION

33.4 PATHOLOGIC FINDINGS

FIGURE 33.5

479

Mitosis (arrow) in an endothelial cell (same patient as Fig. 33.2).

FIGURE 33.6

(A) Interstitial hemorrhage and (B) marked loss of capillaries in a patient who developed DSA 8 years posttransplant. The destruction of capillaries probably accounts for a negative C4d. Concurrent acute cellular rejection with many plasma cells is also present.

33.4.3.3 Hemorrhage/Infarct Interstitial hemorrhage (Figs. 33.6 and 33.7) can be prominent.11 Frank cortical infarction has been observed in a minority of cases (5%)11 and medullary necrosis can be seen (Figs. 33.8, 33.9, 33.10).

33.4.4 Vessels 33.4.4.1 Peritubular capillaries The presence of neutrophils in peritubular capillaries (PTCs) (Figs. 33.2, 33.11, and 33.7) is a feature that was observed in hyperacute rejection. It is one of the hallmarks of acute AMR cases and its presence has been associated with class I DSA.27 Monocytes or macrophages are also present in peritubular capillaries (Fig. 33.12). Marked dilation of peritubular capillaries is also a characteristic feature.27 33.4.4.2 Arteries and arterioles Acute AMR may manifest as acute TMA, myocyte necrosis, and, rarely, arterial thrombosis. Acute TMA is typically characterized by mucoid intimal thickening and trapped red cells. Myocyte necrosis can be seen in about 25% of cases. The arterial media shows necrosis with fragmentation of the elastic lamina. In one study, fibrinoid necrosis has been noted in 25% patients with class I antibody.27 About half of patients in another study had either fibrinoid necrosis (24%) or transmural arterial inflammation (18%), or both (12%).27 Similar lesions may be seen in arterioles. Endarteritis with mononuclear cells in the intima has been reported in 40% 50% of C4d-positive acute rejection cases. Whether the DSA is causing the endarteritis in this setting is uncertain, since TCMR also causes endarteritis. However, the presence of C4d or DSA in association with endarteritis are strong risk factors for graft loss.12,13,33

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FIGURE 33.7 (A) Neutrophils, in peritubular capillaries, and (B) interstitial hemorrhage in a 1.5-year posttransplant biopsy of a patient who had a preceding sore throat and a subsequent acute rise in serum creatinine to 5 mg/dl. (B) Features of acute cellular rejection, tubulitis and endothelialitis, were also present.

FIGURE 33.8 Early cortical necrosis in acute antibody-mediated rejection. Neutrophils are seen along peritubular capillaries and underneath the endothelium of an artery.

FIGURE 33.9 Medullary necrosis with C4d staining in a severe case of acute antibody-mediated rejection.

FIGURE 33.10 (A) Acute antibody-mediated rejection with infarction (right). Marked arteritis with subendothelial edema is present. (B) C4d of the same field showing positive staining in peritubular and glomerular peritubular capillaries in viable regions and negative in areas of infarction.

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481

FIGURE 33.11 Dilated peritubular capillaries with many neutrophils (arrows) and mononuclear cells in a 2.5-week posttransplant biopsy from a patient who received a second kidney transplant. A concurrent cellular rejection was also present.

FIGURE 33.12

Dilated peritubular capillaries with mononuclear cells and neutrophils in a patient who developed Class II DSA 3 years

posttransplant.

33.4.4.2.1 Immunofluorescence C4d staining in acute AMR is seen along peritubular capillaries in a diffuse, intense, linear, and circumferential fashion (Fig. 33.13). Staining can be seen in both cortex and medulla.11 Peritubular capillaries are usually dilated and can be easily seen on low-power magnification. Care is taken in evaluating dilated peritubular capillaries as they may mimic tubular cross sections (Fig. 33.14). Immunoglobulins are usually negative in peritubular capillaries. In some cases, IgM and IgG may be detectable.11 IgM is usually present in ABO-incompatible grafts with acute AMR.34 Glomerular C4d staining on immunofluorescence is considered nondiagnostic. Staining for immunoglobulins is not specific and mesangial IgM and IgG may be present. Segmental staining of tubular basement membranes for C4d can also be seen, a feature that has been noted for both acute AMR and cell-mediated rejection. 33.4.4.2.2 Immunohistochemistry C4d staining by immunoperoxidase on paraffin-embedded sections give a similar pattern and extent but the intensity is typically less and more variable (Fig. 33.15). Staining is also usually seen along glomerular capillary endothelium. In a study describing serial allograft biopsies of presensitized patients, the C4d was initially negative and later was noted to be positive. The positive C4d was preceded by capillaritis, which had been present for several days.35 Glomerular staining for C4d in fixed tissue is seen in about 30% of cases in addition to peritubular capillary staining.28 33.4.4.2.3 Electron Microscopy The value of electron microscopy in assessing allograft biopsies with acute AMR is less in comparison to light and immunofluorescence studies. Ultrastructural features nonetheless offer evaluation of extent of damage to the allograft, especially to glomerular and peritubular capillary endothelium. The glomeruli may show features

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33. ACUTE ANTIBODY-MEDIATED REJECTION

FIGURE 33.13

C4d by immunofluorescence shows diffuse strong staining along peritubular capillaries.

FIGURE 33.14

Tubular basement membranes also stain for C4d.

FIGURE 33.15

C4d by immunoperoxidase staining on paraffin-embedded sections shows diffuse strong and linear peritubular capillary

staining.

similar to TMA, with platelets, fibrin, and neutrophils in glomerular capillaries. The glomerular capillary endothelium is reactive and may show loss of fenestrations. In a series of mostly presensitized patients with C4dpositive biopsies in the first 3 months, there is endothelial cell swelling (88%), separated from the glomerular basement membrane (GBM) by a widened lucent space (100%), and early GBM duplication (76%).36 Similar ultrastructural changes were seen with little or no C4d deposition in patients with DSA and glomerulitis or capillaritis. These changes are more evident in allografts that later develop transplant glomerulopathy.37,38 Neutrophils and monocytes are found in peritubular capillaries with platelets and fibrin.38,39 Endothelial cell lysis, apoptosis, and fragmentation can be seen.40 Similar endothelial injury in small arteries plus fibrinoid necrosis, smooth muscle necrosis, and fibrin tactoids have also been observed.

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33.4 PATHOLOGIC FINDINGS

TABLE 33.2

483

Revised (Banff 2013) Classification of Acute/Active Antibody-Mediated Rejection (ABMR) in Renal Allografts

1. All three features must be present for diagnosisa,b • Histologic evidence of acute tissue injury, including one or more of the following: i) Microvascular inflammation (g . 0c and/or ptc . 0) ii) Intimal or transmural arteritis (v . 0)d iii) Acute thrombotic microangiopathy, in the absence of any other cause iv) Acute tubular injury, in the absence of any other apparent cause 2. Evidence of current/recent antibody interaction with vascular endothelium, including at least one of the following: • Linear C4d staining in peritubular capillaries (C4d2 or C4d3 by IF on frozen sections, or C4d . 0 by IHC on paraffin sections) • At least moderate microvascular inflammation ([g1ptc] $ 2)e • Increased expression of gene transcripts in the biopsy tissue indicative of endothelial injury, if thoroughly validatedf For all ABMR diagnoses, it should be specified in the report whether the lesion is C4d-positive (C4d2 or C4d3 by IF on frozen sections; C4d . 0 by IHC on paraffin sections) or without evident C4d deposition (C4d0 or C4d1 by IF on frozen sections; C4d0 by IHC on paraffin sections). b These lesions may be clinically acute, smoldering, or subclinical. Biopsies showing two of the three features, except those with DSA and C4d without histologic abnormalities potentially related to ABMR or TCMR (C4d staining without evidence of rejection. The clinical significance of these findings may be quite different in grafts exposed to antiblood-group antibodies [ABO-incompatible allografts], where they do not appear to be injurious to the graft34,41 and may represent accommodation. However, with anti-HLA antibodies such lesions may progress to chronic ABMR42 and more outcome data are needed). May be designated as ‘‘suspicious’’ for acute/active ABMR. c Recurrent/de novo glomerulonephritis should be excluded. d It should be noted that these arterial lesions may be indicative of ABMR, TCMR, or mixed ABMR/TCMR. ‘‘v’’ lesions are only scored in arteries having a continuous media with two or more smooth muscle layers. e In the presence of acute TCMR, borderline infiltrates or evidence of infection, ptc $ 2 alone is not sufficient to define moderate microvascular inflammation and g must be $ 1. f At present the only validated molecular marker meeting this criterion is ENDAT expression,43 and this has only been validated in a single center (University of Alberta). The use of ENDAT expression at other centers or other test(s) of gene expression within the biopsy as evidence of ABMR must first undergo independent validation as was done for ENDAT expression by Sis et al.43 cg, Banff chronic glomerulopathy score; EM, electron microscopy; ENDAT, endothelial activation and injury transcript; g, Banff glomerulitis score; GBM, glomerular basement membrane; IF, immunofluorescence; IHC, immunohistochemistry; ptc, peritubular capillary; TCMR, T-cell-mediated rejection; v, Banff arteritis score. a

33.4.4.2.4 Banff Classification of Acute/Active AMR Three histologic patterns of acute AMR are recognized. Type I shows acute tubular injury with minimal inflammation. Type II shows glomerular and peritubular capillary inflammation and/or thromboses. Type III is manifested by arterial lesions (fibrinoid necrosis or transmural inflammation; v3 lesion) in the original classification and now TMA and endarteritis have been added as arterial lesions (Table 33.2).44 In addition, biopsies should show evidence of C4d deposition (C4d2 or C4d3 by immunofluorescence or C4d . 0 by immunohistochemistry on paraffin sections) or at least moderate microvascular inflammation (g 1 ptc .2) or increased expression of gene transcripts in biopsy tissue indicative of endothelial injury.26,44 46 Serologic evidence of DSA, whether anti-HLA or against other antigens, must be present. All three parameters—histologic features, evidence of antibody interaction (any of C4d, microvascular inflammation, or gene transcripts), and serum DSA—are required for a diagnosis of acute/ active AMR. A diagnosis of “suspicious for acute/active AMR” may be given if only two are present. 33.4.4.3 Differential Diagnosis 33.4.4.3.1 Chronic active AMR Chronic active AMR (CAMR) usually presents with a slow decline in allograft function, with varying degrees of proteinuria. On biopsy, similar features of glomerulitis and capillaritis are present; however, the inflammatory cells are mostly mononuclear rather than neutrophils, which are characteristic of acute AMR, and acute lesions are absent (e.g., thrombosis, necrosis, neutrophils, TMA). 33.4.4.4 Acute cellular rejection TCMR characteristically shows interstitial inflammation and tubulitis. When concurrent acute AMR is present, glomerulitis and capillaritis may be equally prominent and C4d is positive. The presence of C4d has been observed in 20% 30% of TCMR cases. 33.4.4.5 Accommodation Accommodation has been observed in protocol biopsies in which there is C4d deposition without histologic evidence of graft injury or inflammation (Fig. 33.16) and without clinical allograft dysfunction. This state is commonly seen in protocol biopsies in ABO-incompatible grafts and appears to be relatively stable. In HLAincompatible grafts, the finding has been associated with later development of chronic AMR.

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484

FIGURE 33.16

33. ACUTE ANTIBODY-MEDIATED REJECTION

C4d deposition Day 16 posttransplant. This is most likely a state of accommodation. There was no inflammation in the

biopsy.

33.4.4.6 Acute pyelonephritis Neutrophils in the interstitium and in peritubular capillaries are features that are in common with acute AMR. In pyelonephritis, other characteristic features such as neutrophilic casts and neutrophilic tubulitis are also present. These features are usually patchy and often correlate with positive urine cultures. C4d is negative in pyelonephritis. 33.4.4.6.1 Clinical Course and Prognosis In comparison to acute TCMR, graft outcome is worse for acute AMR. It is much poorer in combined acute TCMR and AMR. C4d deposition is also an independent predictor of graft outcome in patients with acute AMR.33,47 Among various reports, 16% 50% of C4d-positive acute AMR grafts were lost in a year, compared with 3% 7% loss after a C4d-negative acute rejection episode.5,11,16,48 50 The risk of acute AMR and poorer outcome correlate with MFI of DSA in Luminex assays.51 Another risk factor for acute AMR is the capacity of DSA to fix complement as noted on C4d or C1q uptake by HLA-coated beads in pretransplant36 or posttransplant assays.3,47 Early graft loss in nonpresensitized patients who develop acute AMR is also notable. In a series of 469 nonpresensitized patients treated with alemtuzumab, 10% developed acute AMR and 20% of these lost their graft within a year.5 In another large series of 402 crossmatch-negative patients, 8% developed acute AMR; 1- and 5-year graft survival rates were 87% and 54%, respectively.51 Graft loss can be minimized through close monitoring and aggressive treatment. In a study of 51 presensitized patients treated with desensitization, only 4% of the grafts were lost within a year.52 Long-term effects of acute AMR include a fivefold higher risk of chronic AMR with transplant glomerulopathy (TG).53 The histologic types of acute AMR also show different graft survival rates and outcomes. Type III acute AMR generally has a lower graft survival than type I (tubular injury) or type II (capillaritis). One series showed an improved 1-year graft survival of type III acute AMR at 75%.5 Fibrinoid necrosis in type III AMR is a histologic feature that correlated with increased graft loss, with about 25% graft survival at 1 year.54 33.4.4.6.2 Treatment There are only a few randomized controlled trials of therapies for acute AMR.55 Treatment protocols are commonly designed to reduce antibody titers with plasma exchange and intravenous immunoglobulin (IVIG). Thymoglobulin is added to treat any concurrent TCMR.55,56 Desensitization studies show the best evidences for the utility of plasmapheresis and IVIG.56,57 Splenectomy has also been used.58 Immunoadsorption with protein A, which binds mainly to IgG, was subject to one randomized trial although this was prematurely terminated after noting a dramatically better outcome in the treated group.59 The use of eculizumab, an antibody to C5 that blocks the terminal complement pathway, reveals promising results. Its efficacy has been demonstrated in nonrandomized pilot trials60 and in isolated cases.61,62 Other common therapies include anti-B-cell agents, such as anti-CD20 (rituximab),63,64 which acts on pre- and mature B cells. This leads to transient B-cell depletion with Bcell recovery after 6 9 months. Off-label drugs that modulate B-cell and plasma cell function have also been used in the treatment of AMR. Bortezomib, a proteasome inhibitor used for plasma cell depletion in multiple myeloma, has been tried in a small cohort of acute AMR with evidence of success.63,64 Inhibitors of B-cell and plasma cell growth factors are potential agents to be tried in transplantation. These include belimumab, a human monoclonal antibody that inhibits B-cell activating factor (BAFF).

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485

33.4.4.6.3 ABO-Incompatible Grafts The removal of circulating ABO antibodies by plasmapheresis prior to transplantation has led to successful transplantation of ABO-incompatible (ABOi) kidneys without hyperacute rejection. Many studies on ABOi transplantation are from Japan, where they represent about 30% of the living donor grafts.65 Studies in Japan and in the United States have reported excellent results.65 70 These protocols also initially included splenectomy but this has been replaced by rituximab.66 In a 2011 study, standard immunosuppression has also shown successful results.71 Another method is by extracorporeal immunoabsorption,72 which reduces IgM and IgG antibodies but does not remove all of the ABO specificities.73 The risk for acute rejection in ABOi grafts is 2.3 times that of ABO-compatible grafts.68 Clinical acute AMR has been observed in a few desensitized recipients of ABOi grafts.66,74 Similar features are present as in DSA-associated acute AMR. IgM staining can be seen in peritubular capillaries.75 One-hour postperfusion biopsies have shown C4d in 57% of ABOi grafts, usually also with IgM (88%) and occasionally with IgG (40%).75 Of these cases, 37% developed acute rejection in the first month.75 In one study, 12% of patients were diagnosed with subclinical acute AMR on protocol biopsies.74 In another study, 28% of protocol biopsies showed subclinical rejection at 6 12 months.41 Only a few of these studies report simultaneous occurrence of de novo anti-HLA antibodies. Recovery from acute AMR in ABOi grafts correlates with a decrease in ABO antibody titers. C4d deposition, however, does not concurrently disappear after reduction of ABO antibodies, an opposite pattern to acute AMR caused by HLA antibodies.76 Protocol biopsies also show persistent C4d deposition in 94% of patients at 1 year.41 The presence of C4d in ABOi grafts is similar to C4d deposition in “accommodation” where there is absence of inflammation and graft dysfunction. This may be due to the lack of Fc receptors for the dominant IgM isotype of blood group antigens. This also suggests that accommodation to ABO antibodies may be more easily achieved than to HLA antibodies and may protect against the effects of HLA antibodies.67,77 The frequency of transplant glomerulopathy is higher in ABOi grafts compared with ABO compatible grafts (13% 15% vs 7% 8%, respectively) (P , .001).41 The presence of HLA DSA is one of the risk factors for transplant glomerulopathy69,78 in ABOi grafts, which highlights the importance of detecting concomitant anti-HLA DSA in ABOi patients. Graft survival rates for ABOi grafts are almost similar to ABO-compatible grafts, with 1-year graft survival rate of 95% and 5-year graft survival rate of 91%.65,67,68 Major risk factors that lead to lower 5-year graft survival rates include acute AMR within the first 3 months (84% with early AMR episode vs 95% without). The risk of developing AMR is increased with higher levels of ABO antibodies. Graft survival however has been achieved despite high titers of recurrent IgG and IgM antibodies after 1 year.70

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Capillary deposition of complement split product C4d in renal allografts is associated with basement membrane injury in peritubular and glomerular capillaries: a contribution of humoral immunity to chronic allograft rejection. J Am Soc Nephrol 2002;13(9):2371 80. 36. Lawrence C, Willicombe M, Brookes PA, Santos-Nunez E, Bajaj R, Cook T, et al. Preformed complement-activating low-level donorspecific antibody predicts early antibody-mediated rejection in renal allografts. Transplantation 2013;95(2):341 6. 37. Wavamunno MD, O’Connell PJ, Vitalone M, Fung CL, Allen RD, Chapman JR, et al. Transplant glomerulopathy: ultrastructural abnormalities occur early in longitudinal analysis of protocol biopsies. Am J Transplant 2007;7(12):2757 68. 38. Liptak P, Kemeny E, Morvay Z, Szederkenyi E, Szenohradszky P, Marofka F, et al. Peritubular capillary damage in acute humoral rejection: an ultrastructural study on human renal allografts. Am J Transplant 2005;5(12):2870 6. 39. Lajoie G. Antibody-mediated rejection of human renal allografts: an electron microscopic study of peritubular capillaries. Ultrastruct Pathol 1997;21(3):235 42. 40. Lerut E, Kuypers D, Van Damme B. C4d deposition in the peritubular capillaries of native renal biopsies. Histopathology 2005;47(4):430 2. 41. Setoguchi K, Ishida H, Shimmura H, Shimizu T, Shirakawa H, Omoto K, et al. Analysis of renal transplant protocol biopsies in ABOincompatible kidney transplantation. Am J Transplant 2008;8(1):86 94. 42. Bravou V, Galliford J, McLean A, Willicombe M, Taube D, Cook HT, et al. A case of chronic antibody-mediated rejection in the making. Clin Nephrol 2013;80(4):306 9. 43. Sis B, Jhangri GS, Bunnag S, Allanach K, Kaplan B, Halloran PF. Endothelial gene expression in kidney transplants with alloantibody indicates antibody-mediated damage despite lack of C4d staining. Am J Transplant 2009;9(10):2312 23. 44. Haas M, Sis B, Racusen LC, Solez K, Glotz D, Colvin RB, et al. Banff 2013 meeting report: inclusion of c4d-negative antibody-mediated rejection and antibody-associated arterial lesions. Am J Transplant 2014;14(2):272 83. 45. Solez K, Colvin RB, Racusen LC, Sis B, Halloran PF, Birk PE, et al. Banff ’05 Meeting Report: differential diagnosis of chronic allograft injury and elimination of chronic allograft nephropathy (’CAN’). Am J Transplant 2007;7(3):518 26. 46. Solez K, Colvin RB, Racusen LC, Haas M, Sis B, Mengel M, et al. Banff 07 classification of renal allograft pathology: updates and future directions. Am J Transplant 2008;8(4):753 60.

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47. Yabu JM, Higgins JP, Chen G, Sequeira F, Busque S, Tyan DB. C1q-fixing human leukocyte antigen antibodies are specific for predicting transplant glomerulopathy and late graft failure after kidney transplantation. Transplantation 2011;91(3):342 7. 48. Tyden G, Kumlien G, Fehrman I. Successful ABO-incompatible kidney transplantations without splenectomy using antigen-specific immunoadsorption and rituximab. Transplantation 2003;76(4):730 1. 49. McCalmon Jr. RT, Tardif GN, Sheehan MA, Fitting K, Kortz W, Kam I. IgM antibodies in renal transplantation. Clin Transplant 1997;11 (6):558 64. 50. Lederer SR, Kluth-Pepper B, Schneeberger H, Albert E, Land W, Feucht HE. Impact of humoral alloreactivity early after transplantation on the long-term survival of renal allografts. Kidney Int 2001;59(1):334 41. 51. Lefaucheur C, Loupy A, Hill GS, Andrade J, Nochy D, Antoine C, et al. Preexisting donor-specific HLA antibodies predict outcome in kidney transplantation. 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Plasma exchange and tacrolimus-mycophenolate rescue for acute humoral rejection in kidney transplantation. Transplantation 1998;66(11):1460 4. 57. Vo AA, Lukovsky M, Toyoda M, Wang J, Reinsmoen NL, Lai CH, et al. Rituximab and intravenous immune globulin for desensitization during renal transplantation. N Engl J Med 2008;359(3):242 51. 58. Kaplan B, Jie T, Diana R, Renz J, Whinery A, Stubbs N, et al. Histopathology and immunophenotype of the spleen during acute antibodymediated rejection. Am J Transplant 2010;10(5):1316 20. 59. Bohmig GA, Wahrmann M, Regele H, Exner M, Robl B, Derfler K, et al. Immunoadsorption in severe C4d-positive acute kidney allograft rejection: a randomized controlled trial. Am J Transplant 2007;7(1):117 21. 60. Wang H, Rollins SA, Gao Z, Garcia B, Zhang Z, Xing J, et al. Complement inhibition with an anti-C5 monoclonal antibody prevents hyperacute rejection in a xenograft heart transplantation model. Transplantation 1999;68(11):1643 51. 61. Noone D, Al-Matrafi J, Tinckam K, Zipfel PF, Herzenberg AM, Thorner PS, et al. Antibody mediated rejection associated with complement factor h-related protein 3/1 deficiency successfully treated with eculizumab. Am J Transplant 2012;12(9):2546 53. 62. Chehade H, Rotman S, Matter M, Girardin E, Aubert V, Pascual M. Eculizumab to treat antibody-mediated rejection in a 7-year-old kidney transplant recipient. Pediatrics 2015;135(2):e551 5. 63. Schmidt N, Alloway RR, Walsh RC, Sadaka B, Shields AR, Girnita AL, et al. Prospective evaluation of the toxicity profile of proteasome inhibitor-based therapy in renal transplant candidates and recipients. Transplantation 2012;94(4):352 61. 64. Walsh RC, Everly JJ, Brailey P, Rike AH, Arend LJ, Mogilishetty G, et al. Proteasome inhibitor-based primary therapy for antibodymediated renal allograft rejection. Transplantation 2010;89(3):277 84. 65. Takahashi K, Saito K. ABO-incompatible kidney transplantation. Transplant Rev (Orlando) 2013;27(1):1 8. 66. Fuchinoue S, Ishii Y, Sawada T, Murakami T, Iwadoh K, Sannomiya A, et al. The 5-year outcome of ABO-incompatible kidney transplantation with rituximab induction. Transplantation 2011;91(8):853 7. 67. Ichimaru N, Takahara S. Japan’s experience with living-donor kidney transplantation across ABO barriers. Nat Clin Pract Nephrol 2008;4(12): 682 92. 68. Montgomery JR, Berger JC, Warren DS, James NT, Montgomery RA, Segev DL. Outcomes of ABO-incompatible kidney transplantation in the United States. Transplantation 2012;93(6):603 9. 69. Gloor JM, Cosio FG, Rea DJ, Wadei HM, Winters JL, Moore SB, et al. Histologic findings one year after positive crossmatch or ABO blood group incompatible living donor kidney transplantation. Am J Transplant 2006;6(8):1841 7. 70. Tobian AA, Shirey RS, Montgomery RA, Cai W, Haas M, Ness PM, et al. ABO antibody titer and risk of antibody-mediated rejection in ABO-incompatible renal transplantation. Am J Transplant 2010;10(5):1247 53. 71. Flint SM, Walker RG, Hogan C, Haeusler MN, Robertson A, Francis DM, et al. Successful ABO-incompatible kidney transplantation with antibody removal and standard immunosuppression. Am J Transplant 2011;11(5):1016 24. 72. Biglarnia AR, Nilsson B, Nilsson Ekdahl K, Tufveson G, Nilsson T, Larsson E, et al. Desensitization with antigen-specific immunoadsorption interferes with complement in ABO-incompatible kidney transplantation. Transplantation 2012;93(1):87 92. 73. Genberg H, Kumlien G, Wennberg L, Tyden G. The efficacy of antigen-specific immunoadsorption and rebound of anti-A/B antibodies in ABO-incompatible kidney transplantation. Nephrol Dial Transplant 2011;26(7):2394 400. 74. Fidler ME, Gloor JM, Lager DJ, Larson TS, Griffin MD, Textor SC, et al. Histologic findings of antibody-mediated rejection in ABO bloodgroup-incompatible living-donor kidney transplantation. Am J Transplant 2004;4(1):101 7. 75. Kanetsuna Y, Yamaguchi Y, Horita S, Tanabe K, Toma H. C4d and/or immunoglobulins deposition in peritubular capillaries in perioperative graft biopsies in ABO-incompatible renal transplantation. Clin Transplant 2004;18(Suppl 11):13 17. 76. Abe M, Sawada T, Horita S, Toma H, Yamaguchi Y, Teraoka S. C4d deposition in peritubular capillary and alloantibody in the allografted kidney suffering severe acute rejection. Clin Transplant 2003;17(Suppl 10):14 19. 77. Haas M, Segev DL, Racusen LC, Bagnasco SM, Locke JE, Warren DS, et al. C4d deposition without rejection correlates with reduced early scarring in ABO-incompatible renal allografts. J Am Soc Nephrol 2009;20(1):197 204. 78. Toki D, Ishida H, Setoguchi K, Shimizu T, Omoto K, Shirakawa H, et al. Acute antibody-mediated rejection in living ABO-incompatible kidney transplantation: long-term impact and risk factors. Am J Transplant 2009;9(3):567 77.

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34 Vascular Complications in Renal Transplantation Hany El-Hennawy, Christian C. Morrill, Giuseppe Orlando and Alan C. Farney Wake Forest Baptist Health, Winston Salem, NC, United States

34.1 BACKGROUND Numerous technical complications may occur during and following renal transplant that may impact graft survival and patient morbidity, but renal arterial thrombosis is perhaps the most devastating, as it is often difficult to identify vascular occlusion in sufficient time to prevent infarction and loss of the allograft. This chapter will focus on arterial thrombosis because the subject is also germane to the field of regenerative medicine and attempts to develop organs—a major hurdle in generating a new organ will be an intact vascular supply that is low risk for thrombosis.1

34.2 ARTERIAL COMPLICATIONS AFTER RENAL TRANSPLANTATION 34.2.1 Thrombosis 34.2.1.1 Introduction Arterial thrombosis occurs upon erosion or rupture of an atherosclerotic plaque and, through plateletmediated thrombi, can cause ischemic injuries, especially in tissues with a terminal vascular bed.2,3 Clinical examples include thrombotic cerebrovascular accident and coronary thrombosis related to ruptured plaque, and together these account for a significant number of lives lost. Arterial thrombosis may also occur in the setting of traumatic injury, e.g., in arterial stretch that occurs with rapid deceleration. Stretch injuries of the renal artery, caused by motor vehicle accident, may result in thrombosis and ipsilateral renal ischemia or infarction. Studies of patients who undergo rapid revascularization of such kidneys generally show little to no function in the involved kidney in the long term, indicating that successful intervention might only occur if the period of ischemia is shorter than what can generally be achieved clinically.4 For many of the same reasons that thrombosis occurs naturally, arterial thrombosis may occur following surgical manipulation such as an anastomosis. Obviously, solid organ transplantation requires vascular anastomosis, and is therefore prone to this adverse outcome. Similar to other clinical scenarios, vascular thrombosis of a transplanted organ is a devastating complication. Renal arterial or venous thrombosis accounts for as much as a third of early graft loss at 1 month5,6 and up to 45%
47% within the first 2
3 months.7 Arterial thrombosis is reported to occur at 0.2%
7.5% and venous thrombosis 0.1%
8.2%, with the highest incidence among children and infants, and the lowest in living donors.8 Clinically, graft thrombosis occurs either “early” or “late,” with technical factors predominating early and immunological causes as the likely explanation for late thrombosis. In a series of pancreas grafts explanted for thrombosis, a University of Maryland study found that grafts lost at Kidney Transplantation, Bioengineering, and Regeneration. DOI: http://dx.doi.org/10.1016/B978-0-12-801734-0.00034-5

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2 weeks and beyond universally showed evidence of acute rejection.9 Grafts lost early, within 48
72 hours, are much more likely to be associated with purely technical reasons for vascular thrombosis in the broad sense. Technical loss does not necessarily imply a surgical misadventure. Indeed, the term is variably used to describe almost all factors that might increase the risk for vascular thrombosis short of alloimmunity. Clinically, renal artery thrombosis has been associated with hypotension during the surgery or postoperatively, “rough handling” of the organ, the administration of antilymphocyte globulin, early postoperative treatment with cyclosporine, disparity in donor and recipient vessel size, certain types of anastomoses, multiple (small) vessels, renal-artery stenosis, and compartment syndrome.10 Fallahzadeh et al. reported a case of acute renal artery thrombosis that developed early posttransplantation due to distal renal artery stenosis.11 Groggel reported a case of acute renal artery thrombosis in a well-functioning, long-term renal allograft in which there was clear angiographic evidence of a normal renal artery 2 years earlier.12 In another study, the most significant risk factors for developing thrombosis were donor age below 6 or above 60 years, or recipient age below 5
6 years, peri- or postoperative hemodynamic instability, peritoneal dialysis (PD), diabetic nephropathy, a history of thrombosis, deceased donor, no use of aspirin, delayed graft function (DGF), or .24 hours cold ischemia.8 Amezquita et al. reported that the right renal graft has increased thrombosis risk compared to the left.13 However, a few centers, with both high and low thrombosis incidence, have found no correlation.14 Female donor gender doubled the risk of thrombosis in one study,5 probably due to smaller vessel size, as suggested by the authors. However, this has not been confirmed in other studies.15 Moreover, renal grafts with multiple arteries (multiple renal arteries would tend to be smaller) have not been associated with an increased risk of thrombosis.15 However, in case of multiple renal arteries, there might be a failure to detect thrombosis of smaller renal arteries. There is no doubt that at the extreme of size small renal arteries pose technical challenges and that the risk of arterial thrombosis increases. Organ preservation (or lack of it) might potentially increase the risk of arterial thrombosis either through decreased organ perfusion (increased resistance associated with reperfusion injury to the parenchyma) or direct ischemic injury to the vascular endothelium. At least within the limits of current clinical practice, warm ischemia time (WIT) has not been found to be a risk factor for thrombosis development.16 For a living donor, a kidney may be exposed to several minutes of WIT before flush with cold preservation solution (so-called extraction time). Clinically this seems to be of little consequence. In contrast, kidneys procured from deceased cardiac donors (DCD) are exposed to a variable period of warm ischemia depending upon the agonal phase of death and surgical recovery. Adequate clinical definitions to measure WIT in DCD donors are lacking, making correlation to outcomes such as arterial thrombosis questionable. Compared to WIT, cold ischemia time (CIT) has relatively clear beginning and endpoints. Measurement of CIT begins at the time of cross clamp and organ flush (with cold preservation solution) in the deceased donor and ends with organ reperfusion. Sometimes a difficult anastomosis, such as that in an obese recipient or a recipient with vascular disease, may blur the definition of CIT, as the graft may warm just prior to release of clamps. Although most renal transplants are completed within 30 hours of CIT, some successful kidney transplants have been accomplished with CIT exceeding 50 hours. Amezquita et al. reported that CIT less than 24 hours (mean of 20
21) has no increased risk of thrombosis.13 Other reports stated a higher thrombosis incidence with longer CIT,17 especially when CIT exceeds 24 hours.14 In and of itself, CIT appears to have little or no impact on the risk of arterial thrombosis, at least within the limits of current clinical practice. However, DGF (which might be a consequence of prolonged CIT) is correlated to thrombosis and a vascular cause for impaired initial graft function should always be considered.16 Intra- and postoperative hemodynamic instability probably increases the risk of thrombosis7 as it is a surrogate measure of graft perfusion. In one study, instability was defined as period(s) of mean arterial blood pressure (MAP) # 70 mmHg, for more than 10 minutes intraoperatively or during the first 48 hours postoperatively. As defined, 32.5% of the patients who later suffered from acute vascular thrombosis had initial hemodynamic instability, while only 4.5% of recipients without vascular complications had hypotensive period(s). 33.3% (11/33) of hemodynamic unstable patients developed thrombosis and lost their graft.18 Some clinicians might note that MAPs of 65
70 should not really be considered hypotension, but the point may be that a transplanted graft may fare better with even some “elevation” of BP (or perfusion) following transplantation. Certain disease states may be associated with vascular thrombosis, both arterial and venous. Patients with diabetic nephropathy have an increased thrombotic risk.15 However, Penny et al. found no association with diabetes mellitus.5 Systemic lupus erythematosus increases the thrombosis risk compared to hypertensive nephropathy,19 which is most probable due to the antiphospholipid antibody syndrome.20

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Inherited or acquired forms of thrombophilia increase the risk of thrombosis in general and hence also in a newly received renal graft and anticoagulation may be a sound consideration. Guirguis et al. reported a patient with acute renal artery and vein thrombosis associated with abnormally short activated partial thromboplastin time and factor V Leiden mutation.21 Screening for thrombophilia has shown to decrease the number of thrombotic events.22 Patients with thrombophilia have an increased risk of repeated thrombosis in successive transplants unless they receive anticoagulation therapy. Vega et al. reported a patient who developed renal artery thrombosis after withdrawal from anticoagulation therapy.23 Preemptive transplantation has been found to be associated with increased thrombotic risk compared to transplantation after initiation of hemodialysis (HD).6 A high native urine production in combination with dialysis is a risk factor.8 Stated in another way, uremia, which may induce a platelet defect (see the following section, “Bleeding vs thrombosis in uremic patients”), may reduce the risk of thrombosis. Several studies have found more thrombotic graft loss among patients who have been on PD prior to transplantation compared to HD.6,24 Compared to HD, PD is associated with protein loss, which may explain the thrombotic diathesis. When strategies to reduce the risk for thrombosis of the renal graft artery are considered, aspirin has been suggested to be associated with a lower risk of thrombosis.25 Also, administration of rabbit antithymocyte globulin in a pediatric population decreased the platelet count and reduced thrombosis risk significantly.26 The distribution of thrombotic risk factors was similar in the groups, but it was not a randomized study. 34.2.1.2 Bleeding Versus Thrombosis in Uremic Patients In the renal failure patient, complex situations involving both hypo- and hypercoagulable states can arise, leading to a precarious balance of bleeding and thrombotic risk. These opposing risks make prevention and management of vascular complications difficult.27 1. Platelet dysfunction in uremic patients. Ineffective adhesion to the subendothelium arises from reduced expression of Gp1b receptors and reduced affinity for von Willebrand factor (VWF). Conformational changes occur in platelet fibrinogen receptors, affecting platelet activation, and in vitro platelet aggregation responses to agonists, such as adenosine diphosphate (ADP), collagen, and epinephrine, are impaired. Uremic patients have raised levels of nitric oxide and prostacyclin. Together, in increased amounts, they are potent inhibitors of platelet activation, mediated through increases in platelet cGMP28 and cAMP29 respectively. Relaxation of vascular tone limits platelet adhesion and platelet
platelet interaction. There is a reduction in ADP and serotonin (5HT), giving an acquired storage pool defect. Cyclooxygenase activity may also be defective, with consequent deficiency of thromboxane A2 production. This, in turn, may be due to the increased cAMP levels. Abnormalities in platelet calcium metabolism have also been reported.27 2. Cryofibrinogenemia in patients with renal disorders. Cryofibrinogenemia (CryoFg) is an underrecognized cryoprotein that is detected in up to 11% of patients with renal disorders and can be life-threatening when untreated. Symptoms are mainly thrombotic cutaneous manifestations, but other thrombotic localizations may occur. In patients with end-stage renal disease, thromboses are common and may manifest in the form of more or less severe thrombotic events including renal artery thrombosis, recurrent arteriovenous fistula thrombosis, recurrent dialysis catheter thrombosis, and/or obstruction of deep veins.29 3. Role of platelets and VWF. Hemostasis and pathological thrombus formation are dynamic processes that require multiple adhesive receptor
ligand interactions, with blood platelets at the heart of such events.30 Platelets have the capability to adhere to the injured vessel wall, to be activated by contact with various substrates and soluble activators, and to form aggregates stabilized by a fibrin network.3 Platelet adhesion, activation, and aggregation on the exposed subendothelial extracellular matrix (ECM) are essential for hemostasis. Binding of the glycoprotein (GP)Ib-V-IX complex to immobilized VWF initiates adhesion of platelets to the ECM, and enables the collagen receptor GPVI to interact with its ligand and to mediate platelet activation. This process is reinforced by local thrombin and platelet-derived secondary mediators. Formed platelet aggregates are stabilized by fibrin formation and signaling events between adjacent platelets involving multiple platelet receptors, such as the newly discovered C-type lectin-like receptor 2 (CLEC-2).30,31 4. A two-step mechanism of arterial thrombus formation. Reininger et al. hypothesized that plaques induce thrombus formation by two discrete steps. The rapid first phase of GPVI-mediated platelet adhesion and aggregation onto plaque collagen occurred within 1 minute. The second phase of coagulation started after a delay of 3 minutes with the formation of thrombin and fibrin, and was driven entirely by plaque TF. Coagulation occurred only in flow niches provided by platelet aggregates, with no evidence for a role of blood-borne TF and FXIIa. Inhibition of GPVI but not plaque TF inhibited plaque-induced thrombus formation.32

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5. FeCl3-induced arterial thrombosis. FeCl3 may alter the ability of adhesive proteins, including collagen, fibrinogen, and VWF, to support platelet adhesion. GPVI and β1 integrins, known to be involved in initial platelet adhesion and activation, do not play a critical role in FeCl3-induced thrombus formation.33 34.2.1.3 Diagnosis Clinical suspicion for vascular thrombosis alone may be sufficient to prompt immediate surgical intervention. For example, a renal graft that made urine in the operating room but suddenly ceases to do so in recovery should raise warning bells; an example of this is the recipient of a living donor kidney who suddenly stops making urine posttransplant. Usually by the time confirmatory testing can be obtained it is too late to salvage complete thrombosis and achieve any meaningful graft function. In a reported series of thrombotic complications Srivastava et al. reported that cases of arterial thrombosis had sudden onset anuria with minimal or no abdominal discomfort, while venous thrombosis presented as severe oliguria associated with intense graft site pain and tenderness.34 Often, however, the clinical scenario is complicated: Patients with residual renal function may make normal amounts of urine prior to transplantation, hematuria (which might indicate venous thrombosis) commonly results from surgical manipulation of the bladder, and oliguria or even anuria may result from reperfusion injury in the setting of prolonged CIT or other donor factors that are associated with DGF. Doppler ultrasound is suited as a screening method for the detection of impaired graft perfusion. Magnetic resonance (MR) imaging may also be used for an accurate diagnosis of vascular complications and to support decisions for appropriate surgical or interventional treatment,35 although it is more expensive than ultrasound and cannot be used in all patients. Sainz-Esteban et al. reported the use of radionuclide renography with Tc-99m MAG3 in the diagnosis of RA thrombosis, the study showing no flow across the artery and no graft vascularization.36 However, despite occasional reports of graft salvage (see the following text), a renal graft that lacks arterial inflow (complete occlusion) typically cannot be salvaged in the amount of time it takes to perform and interpret diagnostic tests. Imaging that suggests a vascular complication should generally prompt surgical exploration, but if the imaging findings suggesting lack of perfusion are confirmed, explant is often necessary. The diagnostic and clinical dilemma is the differentiation between low flow and no flow states. 34.2.1.4 Treatment Depending on the pathophysiology and clinical presentation, treatment of the thrombosis of the renal artery of a transplanted kidney may vary. 1. Thrombectomy. Lee et al. reported management of five cases of RA thrombosis by thrombectomy; two patients recovered moderate renal function after thrombectomy in spite of the estimated occlusion time of 4 and 12 hours, respectively, under normothermic conditions. Three cases of occlusion were associated with preexisting renal artery stenosis.37 Garcia et al. reported successful treatment of RA thrombosis with operative thrombectomy and intraarterial infusion of recombinant tissue-type plasminogen activator.38 2. Immediate retransplantation. Phelan et al. reported their experience with nine cases of immediate retransplantation following early kidney transplant thrombosis. All of the retransplants worked immediately. Four allografts failed after a mean of 52.5 months (2
155 months). Two of these died with a functioning allograft, one failed owing to chronic allograft nephropathy and one owing to persistent acute cellular rejection. The remaining five patients still had a functioning allograft after a mean of 101.8 months (7
187 months).39 3. Ex vivo thrombectomy. Iwami et al. reported a case of renal artery thrombosis while using a metallic coil stent; the rapid formation of thrombus occluded the graft artery completely. In an emergent surgical operation, the graft was explanted and irrigated after extensive thrombectomy. The graft was reimplanted by using an internal iliac artery graft. The patient was in good health with stable graft function for 3 years after the operation.40 4. Interventional radiologic management. Open surgery for correction of early vascular complications in allogenic kidney transplantation carries the risk for increased morbidity and graft loss. Humke et al. reported the management of three patients with percutaneous transluminal angiography and placement of a vascular stent. Anticoagulation was started with low-dose aspirin 100 mg/day. In all three patients, an intimal dissection of the renal artery with formation of a relevant stenosis was found. Stenosis was corrected by angioplasty and stenting. In one case an incidentally found renal vein thrombosis (RVT) was additionally treated by

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transcatheter thromboaspiration. No complications related to the interventions occurred. In two patients diuresis returned immediately and renal function remained stable in the long term (follow-up 6 months).41 5. Intraarterial fibrinolysis. Rouvere et al. treated four patients with acute transplant artery thrombosis using intraarterial fibrinolysis. Fibrinolysis was achieved with tissue plasminogen activator (n 5 1) or urokinase (n 5 3). They concluded that fibrinolysis seems an efficient treatment that may save transplants after up to 24 hours of the arterial occlusion.10 6. Percutaneous thrombus aspiration. Dores et al. treated transplant renal artery graft thrombosis with percutaneous thrombus aspiration and subsequent balloon angioplasty of the entire artery, followed by stent implantation in a second procedure.42 Klepanec et al. treated acute transplant renal artery thrombosis with adjunctive catheterdirected thrombolysis and stent placement. The graft was salvaged with restoration of renal function and renal artery patency at the 3-year follow-up point.43 7. Prevention. Smith et al. observed that the use of IL-2 receptor antibodies as induction therapy is associated with a significantly decreased risk of graft failure due to thrombosis.44 Whether the observed effect of inhibition of the IL-2 receptor is related to an immunosuppressive property or some other effect is not clear. Induction immunosuppression, regardless of type, might reduce late arterial thrombosis by reducing alloimmune vascular injury. Avoidance of early arterial thrombosis is far more effective than attempts at graft salvage once thrombosis has occurred. Once a clot has formed, graft ischemia rapidly ensues and thrombus is naturally resistant to rapid dissolution (for evolutionary reasons this is clear: We would bleed to death from minor injury). Clinical suspicion for thrombophilia (such as prior thrombotic events and/or disease states associated with thrombophilia in the recipient, donor ischemic factors, and hypoperfusion of the graft) should be recognized and if tolerated the recipient should be managed with anticoagulation. As a default, all renal transplant recipients should receive low-dose aspirin unless there is a history of intolerance or the risk of bleeding is prohibitive. Fortunately, the incidence of arterial thrombosis in clinical renal transplantation is low. However, attempts to develop or regenerate organs may encounter increased risk of thrombosis depending on the strategy employed.

34.2.2 Stenosis of the Renal Artery Transplant renal artery stenosis (TRAS), a narrowing of the artery to the kidneys, is a recognized complication in renal transplantation often causing posttransplant arterial hypertension, allograft dysfunction, and loss of graft.45 Occurrence of TRAS varies widely from 1% to 23% in different series.45 This wide variance may be attributed to heterogeneous criteria used to establish diagnosis, graft preservation methods, and surgical expertise. In the United States, Hurst et al. found an overall incidence rate of 8.3/1000 patient-years with the highest incidence rate in the first 6 months and in patients aged 65 or older.2 Mean time to diagnosis of TRAS is 0.83 6 0.81 years after transplant.46 Renal artery stenosis frequently presents with worsening or refractory hypertension and/or graft dysfunction in the absence of rejection, ureteric obstruction, or infection. TRAS may also cause fluid retention which, combined with hypertension, may cause edema, congestive heart failure, or recurrent bouts of pulmonary edema that may ensue abruptly and resolve rapidly. Asymptomatic TRAS is not uncommon and has increased since initiation of routine imaging. In one study, up to 12.4% of patients were found to have TRAS using Doppler imaging during routine screening in otherwise asymptomatic patients in contrast to 2.4% of symptomatic patients in whom Doppler was used to confirm clinical suspicion.47 Patients diagnosed with TRAS are typically older and more likely to have diabetes mellitus, ischemic heart disease, atherosclerosis, or hypertension. Transplant factors associated with TRAS include donor age over 50 years, expanded criteria donor, DGF, cytomegalovirus match status including recipient positivity, year of transplant, and induction immunosuppression. In a study by Hurst et al. deceased-donor transplants showed a higher rate of TRAS than living donor transplants.46 Surgical causes of TRAS include techniques in organ retrieval and suturing leading to intimal tears that may progress to stenosis, trauma to the renal artery from vascular clamps, and kinking of the artery. A few reports indicate that end-to-side anastomoses are more prone to develop TRAS than end-to-end anastomosis; however, existing data are unable to prove this relationship.48 Treatment modalities for TRAS include conservative therapy, angioplasty, and surgery. When renal function is stable and hemodynamically significant stenosis is excluded, no specific intervention is indicated. In this case, pharmacologic treatment is adequate to control BP with consistent Doppler monitoring. When renal function progressively deteriorates, BP cannot be controlled, or noninvasive procedures suggest progression of the stenosis,

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the suggested treatment is diagnostic arteriography with angioplasty and stenting where indicated (Fig. 34.1). Percutaneous transluminal renal angioplasty (PTA) has been successful in restoring perfusion in 70%
90% of cases.49 PTA with stent placement has stenosis recurrence rates less than 10%.50 In contrast, recurrence rates for PTA alone range from 10% to 33% of cases over 6
8 months.51 When PTA is unsuccessful, or severe stenosis indicates high risks of complications, open surgery may be necessary as a rescue therapy.1 Surgical success rates range from 63% to 92% with a recurrence rate of approximately 12%.52

34.2.3 Pseudoaneurysms Pseudoaneurysms are rare among renal transplant patients, with an occurrence rate below one percent.53 In the event of a pseudoaneurysm, blood will leak through a tear in the vessel wall collecting in the area between the vessel and the surrounding tissues. In renal transplant patients, the pseudoaneurysm often occurs at the sight of anastomosis.54 Etiology for pseudoaneurysms includes surgical technical difficulty, degeneration, and infection.55 In cases of infection, multiple pseudoaneurysm-causing pathogens have been identified, however, the most common pathogens are either Candida albicans or Staphylococcus aureus.56 Many patients are asymptomatic and present as incidental findings on computed tomographic (CT) scans or ultrasound while others present with a pulsatile mass and abdominal pain. In rare cases, a pseudoaneurysm may cause decreased graft function due to compression of the aneurysm itself. Additionally, some cases of lumbosacral plexopathy have been attributed to compression from a transplant pseudoaneurysm.57 Pseudoaneurysm diagnosis is confirmed using Doppler ultrasound or CT scan. Treatment options include open surgery, endovascular aneurysm repair, ultrasound-guided percutaneous thrombin injection, or coil embolization.58 Open surgery treatment may include transplant nephrectomy or ligation of pseudoaneurysm with bypass from a vein graft. Less invasive treatments such as endovascular aneurysm repair should be considered first where patient’s renal function is normal and etiology is noninfective. Endovascular aneurysm treatment has shown more positive early outcomes with shorter hospitalization time compared to surgical intervention.58 Surgery is indicated for symptomatic patients with pseudoaneurysms .2.5 cm or continuing to expand, or if etiology can be traced to an infectious process.59

34.3 RENAL VEIN COMPLICATIONS AFTER KIDNEY TRANSPLANTATION 34.3.1 Thrombosis RVT is amongst the most frequent events of graft damage/loss in the first month posttransplantation. In a study conducted by Giustacchini et al., RVT was the causal factor in 20% of renal graft loss cases.60 Overall, the prevalence of RVT ranges from 0.1% to 6% in renal transplant patients with 80% of transplant RVT occurring within the first month.61 Patients with transplant RVT often present with swelling of the leg and of the kidney, anuria or oliguria, hematuria, increased creatinine, and occasionally graft pain and tenderness. RVT has also been associated with FIGURE 34.1 Angiograms show RAS (left panel) occurring 15 months after a deceased-donor renal transplant. RAS was successfully treated with a 6 3 18 mm Palmaz Blue balloon expandable stent (right panel).

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deep vein thrombosis (DVT) extending into iliac veins. Transplant RVT can be visualized and diagnosed using Doppler ultrasound. In many cases the cause of RVT remains unknown; however, its etiology is often connected to surgical technique or hypercoagulation of the patient. Hypercoagulation has many causes, either acquired or congenital, one of which is a deficiency of factor V due to genetic point mutations often leading to serious thromboembolic complications. Surgical difficulties leading to RVT are graft vein torsion or kinking, a long vein, and vascular endothelium injury inflicted during transplantation and surgical benching of kidney. In most cases, treatment of RVT is immediate surgical exploration. If the graft is still viable, a surgical thrombectomy is performed; otherwise the kidney is removed. Prior to surgery, catheterized thrombolytics may be helpful in reducing periprocedural morbidity and treating possible underlying DVT. When a RVT is not isolated or a complete blockage exists, some success has been had with anticoagulation treatment alone.62

34.3.2 Stenosis Presently, there are no prospective studies or retrospective reviews of post
kidney transplantation renal vein stenosis (RVS), possibly because RVS after renal transplantation is extremely rare.63 Causes for RVS include acute graft rejection, local infection, high-pressure turbulent flow in the presence of arteriovenous fistulae, external pressure/compression from crossing iliac artery, lymphoceles, hematomas, kinking of renal vein, or surgical clamping.64,65 RVS typically occurs early postoperative and involves a relatively short segment of vein. Clinical symptoms and signs are not specific in transplant renal venous stenosis. However, the presence of unexplained graft dysfunction and an enlarged kidney should lead a physician to suspect transplant RVS. Indication of RVS by color Doppler ultrasound is a significant reduction in the color signal of the main renal vein and a high-velocity jet followed by distal turbulence.66 In some cases color Doppler ultrasound may not be sufficient in diagnosing RVS due to minimally impaired venous flow. In these cases, further radiologic examination is indicated such as MR or CT angiography. However, CT angiography should be used with care on renal transplants due to nephrotoxicity of iodine contrast material. Treating RVS requires surgical venous reconstruction or venoplasty with or without the use of an endovascular stent. Many studies have shown that minimally invasive endovascular venoplasty with stent placement significantly reduces complications, morbidity, and mortality in comparison to open surgery.62
65,67 In addition, endovascular venoplasty with stenting has lower recurrence of restenosis compared to venoplasty alone.

34.4 LYMPHOCELES Lymphocele formation post
kidney transplantation is a common and well-known complication affecting 1%
26% of patients. It is believed that lymphoceles are caused by transection of the lymphatic vessels accompanying external iliac vessels during transplantation surgery and subsequent lymph accumulation in a nonepithelialized cavity in the extraperitoneal plane adjacent to the transplanted kidney.68 Factors affecting lymphocele occurrence include mammalian target of rapamycin inhibitors, high-dose corticosteroids, and delayed graft function.69 Lymphoceles are normally found incidentally during routine ultrasonography 2 weeks to 6 months after transplantation, with peak incidence occurring at 6 weeks.70 Notwithstanding, lymphoceles have been reported 3.7 years after transplantation.69 Depending on the number and size of lymphoceles, kidney function may be affected by mass effect or compression to ureter or vasculature. Additionally, lymphoceles may manifest with edema of inguinal region, abdominal discomfort, urgency, graft function deterioration, and DVT after compression of external iliac vein. Diagnosis of lymphoceles normally includes confirmation through ultrasound, intravenous pyelography, CT or lymphangiography.71 Biochemical analysis is equally important to differentiate lymphoceles from urine leakage (urinoma) or sera (seroma). Prior to ultrasound examination lymphocele incidence ranged from 0.6% to 18.1%.72 After the advent of ultrasound, lymphocele incidence actually varied between 0.6% and 33.9%. Additionally, incidence of symptomatic lymphocele ranges from 0.3% to 26% with a mean of 5.2%.69 Many risk factors, both surgical and medical, attribute to lymphocele formation. Surgical causes include dissection of the lymphatic vessels around the iliac vasculature of the recipient and dissection of the renal lymphatic tissues of the donor either at the time of organ procurement or during benching of the organ. When lymphatic tissues from the organ or patient are not sufficiently sutured or clipped they become open sources of

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retroperitoneal lymph and often progress into lymphoceles. Surgical techniques are crucial to minimize lymphatic derangement of the recipient. For example, one approach indicates implantation of allograft in the omolateral iliac fossa with anastomoses of the renal artery and vein on the common iliac vessels. This technique has reduced lymphocele rates in patients from 8.5% to 2.1%.73 In some cases, higher incidence of lymphoceles has been linked to laparoscopic procurement of grafts from live donors. Donor grafts with multiple arteries have been speculated as having more abundant lymphatics and thus a greater risk of lymphocele formation.74 However, other studies have found no difference in rates of lymphocele formation between surgical techniques or between surgeons with varying experience in renal transplantation.71 Lymphocele treatment modalities include aspiration, sclerotherapy, drain placement, laparoscopic surgery, and open surgery. Laparoscopic fenestration has been noted as the preferred treatment by Lucewitz et al., citing lower rates of recurrence when compared to open surgery, aspiration, or sclerotherapy. Laparoscopic treatment has also exhibited the lowest complication rate, 14% compared to 30% and 16% for open surgery and aspiration. Furthermore, average length of stay of laparoscopic patients is half the time of patients following open surgery treatment.

34.5 VASCULAR COMPLICATIONS AT THE BACKTABLE Bench preparation of the renal graft is the second step of a successful kidney transplant (the first being its procurement), requires surgical skill and finesse, as well as graceful manipulation. The preparation of the vascular pedicle is probably the most critical step. Unexpected vascular abnormalities or damage may become apparent only during benching and can result very vexing, but require immediate attention and careful management. Clearly, organ shortage is forcing transplant surgeons to use also marginal grafts, including those presenting important vascular alterations or damage. In such cases, in-depth knowledge of the vascular anatomy and its variants, the correct identification and preservation of the arterial supply, and familiarity with microsurgical techniques for vascular reconstruction are mandatory.

34.5.1 Renal Artery Aneurysms Renal artery aneurysms (RAAs) are an uncommon entity whose incidence is approximately 0.01%
0.09% in the general population and 0.7%
0.9% in subjects undergoing angiographic investigation. RAAs can be addressed with direct resection and immediate reconstruction. The preferred treatment is the arterial reconstruction, but this procedure is difficult to perform when the RAA is located at the renal artery bifurcation or its branches. Endovascular therapy may have a role in the treatment of distal RAA (involving renal artery branches) and strategies may adopt embolization, stent-graft exclusion, simple coil embolization, stent-coiling, and coil occlusion with positive results. However, when RAA is detected in a kidney graft, backtable resection and repair with venous patches has proved to be a valuable treatment option, thus permitting the utilization of the organ that would otherwise be wasted, without adversely impacting the transplant outcome (Fig. 34.2).

34.5.2 Severe Atherosclerosis of the Renal Artery Donors with longstanding hypertension and/or diabetes, with or without a history of smoke, may present systemic atherosclerosis. In these cases, the abdominal aorta shows scattered hard plaques that may extend to and well beyond the ostium of the renal artery. However, even if plaques are severe and hard, or extend into the renal artery for several millimeters or centimeters, they can be safely removed in order to allow safe implantation with negligible risk for thrombosis. Plaques are removed with a maneuver called endoarterectomy, whereby the walls of the renal artery are everted until the distal end of thrombus (Fig. 34.3).

34.5.3 Iatrogenic Damage It is not uncommon to receive grafts whose vessels have been damaged at the time of procurement. Vessel lacerations may be due to a myriad of accidental factors, among which lack of experience, rough manipulation by the surgeon, poor technical finesse, or inadequate characterization of the vascular anatomy for which accessory arteries or veins are misdiagnosed. In some circumstances, the inadequate length of a given vessel is

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FIGURE 34.2 The upper panel shows an aneurysm (green or open arrow) of the renal artery accidentally found at benching. Black or solid arrow indicates the kidney graft. The mid panel shows the opening of the aneurysm. In particular, the open arrow points to the aorta, and the solid arrow indicates the aneurysm. The lower panel shows the renal artery after repair of the aneurysm consisting in a standard aneurysmectomy, namely, the resection of the cul-de-sac of the aneurysm, followed by suturing of borders with separate prolene 7/0 stitches transversally to the major axis of the vessel. Adapted from Gravante G, Pisani F, D’Angelo M, Iaria G, Orlando G. Renal artery aneurysms in kidney grafts. Am J Surg 2008;196(5):e46-7, with permission.

FIGURE 34.3 The left panel shows a severe hard plaque of a left renal graft, as it appeared after endoarterectomy by avulsion. The right panel shows the appearance of the renal artery after the removal of the plaque. Transplant’s postoperative course was uneventful and Doppler ultrasound showed patent renal artery.

deliberately determined, like when the liver team sections the subhepatic vena cava very far from the liver in order to grant abundant length of the cava for the liver transplant. In this case, the vena cava may be cut at the ostium of the right renal vein, so impairing the creation of the vena cava conduit. Alternatively, the renal vein or the vena cava conduit may present lacerations, whereby repair options depend on the extent of the damage, on the presence of sufficient vascular tissue that may allow adequate repair of the defect, or on whether or not donor iliac vessels are available for reconstruction. Fig. 34.4 shows the reconstruction of a large defect of the vena cava

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FIGURE 34.4 Reconstruction of a large defect of the subhepatic vena cava. The right kidney was received with an atrium patch that was used to reconstruct the defect consisting in the total absence of the vena cava wall medial to the ostium of the renal vein (upper left panel). The patch was flipped caudally in order to cover the defect (lower left panel). 6/0 prolene sutures were run to secure the newly constructed vena cava conduit (upper right panel). The lower right panel shows the vena cava conduit in situ after reperfusion.

conduit. The liver team had sectioned the subhepatic vena cava very distal from the liver, at the level of the right renal vein. The defect could be repaired by flipping over a flap of vena cava that had been left at procurement. In other circumstances, damage may occur while benching, for the same reasons highlighted previously. In the case, e.g., of the preparation of en bloc pediatric kidneys, attention should be paid to the numerous vessels that may arise from the vena cava conduit as well as from the abdominal aorta, and dissection should be extremely meticulous. Inadvertent ligation of one of the renal arteries may lead to a catastrophic outcome with acute necrosis of the kidney.

References 1. Peloso A, Katari R, Patel T, et al. Considerations on the development of a model of kidney bioengineering and regeneration in rats. Expert Rev Med Devices 2013;10(5):597
601. 2. Previtali E, Bucciarelli P, Passamonti SM, Martinelli I. Risk factors for venous and arterial thrombosis. Blood Transfus 2011;9:120
38. 3. Gachet C. Molecular mechanisms of platelet activation. Bull Acad Natl Med 2013;197(2):361
73. 4. Haas CA, Dinchman KH, Nasrallah PF, Spirnak JP. Traumatic renal artery occlusion: a 15-Year review. J Trauma 1998;45(3):557
61. 5. Penny MJ, Nankivell BJ, Disney AP, Byth K, Chapman JR. Renal graft thrombosis: a survey of 134 consecutive cases. Transplantation 1994;58:565
9. 6. McDonald RA, Smith JM, Stablein D, Harmon WE. Pretransplant peritoneal dialysis and graft thrombosis following pediatric kidney transplantation: a NAPRTCS report. Pediatr Transplant 2003;7:204
8. 7. Bakir N, Sluiter WJ, Ploeg RJ, van Son WJ, Tegzess AM. Primary renal graft thrombosis. Nephrol Dial Transplant 1996;11(1):140
7. 8. Keller AK, Jorgensen TM, Jespersen B. Identification of risk factors for vascular thrombosis may reduce early renal graft loss: a review of recent literature. J Transplant 2012;2012:793461. 9. Drachenberg CB, Papadimitriou JC, Farney A, et al. Pancreas transplantation: the histologic morphology of graft loss and clinical correlations. Transplantation 2001;71(12):1784
91. 10. Rouvie`re O, Berger P, Be´ziat C, et al. Acute thrombosis of renal transplant artery: graft salvage by means of intra-arterial fibrinolysis. Transplantation 2002;73(3):403
9. 11. Fallahzadeh MK, Yatavelli RK, Kumar A, Singh N. Acute transplant renal artery thrombosis due to distal renal artery stenosis: a case report and review of the literature. J Nephropathol 2014;3(3):105
8. 12. Groggel GC. Acute thrombosis of the renal transplant artery: a case report and review of the literature. Clin Nephrol 1991;36(1):42
5. 13. Amezquita Y, Mendez C, Fernandez A, et al. Risk factors for early renal graft thrombosis: a case-controlled study in grafts from the same donor. Transplant Proc 2008;40(9):2891
3. 14. Nagra A, Trompeter RS, Fernando ON, et al. The effect of heparin on graft thrombosis in pediatric renal allografts. Pediatr Nephrol 2004;19 (5):531
5. 15. Englesbe MJ, Punch JD, Armstrong DR, Arenas JD, Sung RS, Magee JC. Single-center study of technical graft loss in 714 consecutive renal transplants. Transplantation 2004;78(4):623
6. 16. Hernandez D, Rufino M, Armas S, et al. Retrospective analysis of surgical complications following cadaveric kidney transplantation in the modern transplant era. Nephrol Dial Transplant 2006;21(10):2908
15. 17. Perez FM, Rodrguez-Carmona A, Garcia FT, Tresancos C, Bouza P, Valdes F. Peritoneal dialysis is not a risk factor for primary vascular graft thrombosis after renal transplantation. Perit Dial Int 1998;18(3):311
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18. Jordan ML, Cook GT, Cardella CJ. Ten years of experience with vascular complications in renal transplantation. J Urol 1982;128(4):689
92. 19. Ojo AO, Hanson JA, Wolfe RA, et al. Dialysis modality and the risk of allograft thrombosis in adult renal transplant recipients. Kidney Int 1999;55(5):1952
60. 20. Vaidya S, Wang CC, Gugliuzza C, Fish JC. Relative risk of post-transplant renal thrombosis in patients with antiphospholipid antibodies. Clin Transplant 1998;12(5):439
44. 21. Guirguis N, Budisavljevic MN, Self S, Rajagopalan PR, Lazarchick J. Acute renal artery and vein thrombosis after renal transplant, associated with a short partial thromboplastin time and factor V Leiden mutation. Ann Clin Lab Sci 2000;30(1):75
8. 22. Kranz B, Vester U, Nadalin S, Paul A, Broelsch CE, Hoyer PF. Outcome after kidney transplantation in children with thrombotic risk factors. Pediatr Transplant 2006;10(7):788
93. 23. Vega SJ, Goecke SH, Va´squez GA, Pen˜a MA. Renal artery thrombosis after withdrawal from anticoagulation therapy in a kidney transplant recipient with thrombophilia: report of one case. Rev Med Chil 2007;135(1):98
102. 24. Snyder JJ, Kasiske BL, Gilbertson DT, Collins AJ. A comparison of transplant outcomes in peritoneal and hemodialysis patients. Kidney Int 2002;62(4):1423
30. 25. Stechman MJ, Charlwood N, Gray DW, Handa A. Administration of 75mg of aspirin daily for 28 days is sufficient prophylaxis against renal transplant vein thrombosis. Phlebology 2007;22(2):83
5. 26. Kamel MH, Mohan P, Conlon PJ, Little DM, O’Kelly P, Hickey DP. Rabbit antithymocyte globulin related decrease in platelet count reduced risk of pediatric renal transplant graft thrombosis. Pediatr Transplant 2006;10(7):816
21. 27. Pavord S, Myers B. Bleeding and thrombotic complications of kidney disease. Blood Rev 2011;25(6):271
8. 28. Mellion BT, Ignarro LJ, Ohlstein EH, Pontecorvo EG, Hyman AL, Kadowitz PJ. Evidence for the inhibitory role of guanosine 30 ,50 monophosphate in ADP-induced human platelet aggregation in the presence of nitric oxide and related vasodilators. Blood 1981;57:946
55. 29. Terrier B, Izzedine H, Musset L, et al. Prevalence and clinical significance of cryofibrinogenaemia in patients with renal disorders. Nephrol Dial Transplant 2011;26(11):3577
81. 30. Bryckaert M, Rosa JP, Denis CV, Lenting PJ. Of von Willebrand factor and platelets. Cell Mol Life Sci 2015;72(2):307
26. 31. Nieswandt B, Pleines I, Bender M. Platelet adhesion and activation mechanisms in arterial thrombosis and ischaemic stroke. J Thromb Haemost 2011;9(Suppl. 1):92
104. 32. Reininger AJ, Bernlochner I, Penz SM, et al. A 2-step mechanism of arterial thrombus formation induced by human atherosclerotic plaques. J Am Coll Cardiol 2010;55(11):1147
58. 33. Eckly A, Hechler B, Freund M, et al. Mechanisms underlying FeCl3-induced arterial thrombosis. J Thromb Haemost 2011;9(4):779
89. 34. Srivastava A, Kumar J, Sharma S, Abhishek Ansari MS, Kapoor R. Vascular complication in live related renal transplant: an experience of 1945 cases. Indian J Urol 2013;29(1):42
7. 35. Libicher M, Radeleff B, Grenacher L, et al. Interventional therapy of vascular complications following renal transplantation. Clin Transplant 2006;20(Suppl. 17):55
9. 36. Sainz-Esteban A, Banzo I, Quirce R, et al. Diagnosis of common iliac artery thrombosis and renal artery thrombosis in a kidney transplant by radionuclide renography. Clin Nucl Med 2007;32(12):944
6. 37. Lee HM, Mendez-Picon G, Pierce JC, Hume DM. Renal artery occlusion in transplant recipients. Am Surg 1977;43(3):186
92. 38. Garcia A, Vedula G, Nowygrod R, Ratner LE, Goldstein MJ. Recombinant tissue-type plasminogen activator in the treatment of acute renal artery thrombosis after kidney transplantation. Am J Transplant 2010;10(8):1931
3. 39. Phelan PJ, Magee C, O’Kelly P, O’Brien F, Little D, Conlon PJ. Immediate re-transplantation following early kidney transplant thrombosis. Nephrology 2011;16(6):607
11. 40. Iwami D, Harada H, Miura M, Seki T, Togashi M, Hirano T. Successfully rescued renal graft artery thrombosis by ex vivo thrombectomy: a case report. Transplant Proc 2009;41(5):1951
3. 41. Humke U, Takahashi M, Siemer S, Uder M. Interventional radiologic management for early post-transplant perfusion failure of renal allografts. Aktuelle Urol 2004;35(4):320
5. 42. Dores H, Campante Teles R, Nogueira A, et al. Percutaneous thrombus aspiration in renal artery stenosis after renal transplantation. Rev Port Cardiol 2012;31(12):803
8. 43. Klepanec A, Balazs T, Bazik R, Madaric J, Zilinska Z, Vulev I. Pharmacomechanical thrombectomy for treatment of acute transplant renal artery thrombosis. Ann Vasc Surg 2014;28(5):1314.e11
4. 44. Smith JM, Stablein D, Singh A, Harmon W, McDonald RA. Decreased risk of renal allograft thrombosis associated with interleukin-2 receptor antagonists: a report of the NAPRTCS. Am J Transplant 2006;6(3):585
8. 45. Bruno S, Remuzzi G, Ruggenenti P. Transplant renal artery stenosis. JASN 2004;15(1):134
41. 46. Hurst FP, Abbott KC, Neff RT, et al. Incidence, predictors and outcomes of transplant renal artery stenosis after kidney transplantation: analysis of USRDS. Am J Nephrol 2009;30(5):459
67. 47. Wong W, Fynn SP, Higgins RM, et al. Transplant renal artery stenosis in 77 patients - Does it have an immunological cause? Transplantation 1996;61(2):215
19. 48. Morris PJ, Yadav RV, Kincaid-Smith P, et al. Renal artey stenosis in renal transplantation. Med J Aust 1971;1(24):1255
7. 49. Greenstein SM, Verstandig A, McLean GK, et al. Percutaneous transluminal angioplasty. The procedure of choice in the hypertensive renal allograft recipient with renal artery stenosis. Transplantation 1987;43(1):29
32. 50. Leertouwer TC, Gussenhoven EJ, Bosch JL, et al. Stent placement for renal arterial stenosis: where do we stand? A meta-analysis. Radiology 2000;216(1):78
85. 51. Raynaud A, Bedrossian J, Remy P, et al. Percutaneous transluminal angioplasty of renal transplant arterial stenoses. AJR Am J Roentgenol 1986;146(4):853
7. 52. Roberts JP, Ascher NL, Fryd DS, et al. Transplant renal artery stenosis. Transplantation 1989;48(4):580
3. 53. Goldman MH, Tilney NL, Vineyard GC, et al. 20-year survey of the arterial complications of renal transplantation. Surg Gyn Obst 1975;141 (5):758
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54. Dimitroulis D, Bokos J, Zavos G, et al. Vascular complications in renal transplantation: a single-center experience in 1367 renal transplantations and review of the literature. Transpl Proc 2009;41(5):1609
14. 55. Smeds MR, Ofstein R, Peterson GJ, et al. Endovascular repair of a para-anastomotic pseudoaneurysm after renal autotransplantation: an alternative to open reconstruction. Ann Vasc Surg 2013;27(1):110.e5
8. 56. Osman I, Barrero R, Leon E, et al. Mycotic pseudoaneurysm following a kidney transplant: a case report and review of the literature. Ped Transpl 2009;13(5):615
19. 57. Luzzio CC, Waclawik AJ, Gallagher CL, et al. Iliac artery pseudoaneurysm following renal transplantation presenting: as lumbosacral plexopathy. Transplantation 1999;67(7):1077
8. 58. Bracale UM, Carbone F, del Guercio L, et al. External iliac artery pseudoaneurysm complicating renal transplantation. Int Cardiovasc Thor Surg 2009;8(6):654
60. 59. Al-Wahaibi KN, Aquil S, Al-Sukaiti R, et al. Transplant renal artery false aneurysm: case report and literature review. Oman Med J 2010;25 (4):306
10. 60. Giustacchini P, Pisanti F, Citterio F, et al. Renal vein thrombosis after renal transplantation: an important cause of graft loss. Transpl Proc 2002;34(6):2126
7. 61. Khaja MS, Matsumoto AH, Saad WE. Complications of transplantation. Part 1: renal transplants. Cardiov Int Rad 2014;37(5):1137
48. 62. Cercueil JP, Chevet D, Mousson C, et al. Acquired vein stenosis of renal allograft - percutaneous treatment with self-expanding metallic stent. Nephr Dial Transpl 1997;12(4):825
6. 63. Pan MS, Wu RH, Sun DP, et al. Renal vein stenosis with transudative ascites from graft after renal transplantation with good response after percutaneous stent placement. Transpl Proc 2014;46(2):598
601. 64. Obed A, Uihlein DC, Zorger N, et al. Severe renal vein stenosis of a kidney transplant with beneficial clinical course after successful percutaneous stenting. Am J Transpl 2008;8(10):2173
6. 65. Mei Q, He X, Lu W, et al. Renal vein stenosis after renal transplantation: treatment with stent placement. J Vasc Interv Radiol 2010;21:756
8. 66. Kim JK, Han DJ, Cho KS. Post-infectious diffuse venous stenosis after renal transplantation: duplex ultrasonography and CT angiography. Eur Radiol 2002;12:S118
20. 67. Diehm N, Baumgartner I, Mohaupt M, et al. Endovascular stenting of a renal transplant vein. VASA 2006;35(3):195
7. 68. Lucewicz A, Wong G, Lam VWT, et al. Management of primary symptomatic lymphocele after kidney transplantation: a systematic review. Transplantation 2011;92(6):663
73. 69. Khauli RB, Stoff JS, Lovewell T, et al. Post-transplant lymphoceles
a critical look into the risk factors, pathophysiology and management. J Urol 1993;150(1):22
6. 70. Pollak R, Veremis SA, Maddux MS, et al. The natural history and therapy of perirenal fluid collections following renal transplantation. J Urol 1988;140(4):716
20. 71. Ranghino A, Segoloni GP, Lasaponara F, et al. Lymphatic disorders after renal transplantation: new insights for an old complication. Clin Kid J 2015;8(5):615
22. 72. Braun WE, Banowsky LH, Straffon RA, et al. Lymphocytes associated with renal transplantation. Report of 15 cases and review of the literature. Am J Med 1974;57(5):714
29. 73. Sansalone CV, Aseni P, Minetti E, et al. Is lymphocele in renal transplantation an avoidable complication? Am J Surg 2000;179(3):182
5. 74. Mazzucchi E, Souza AA, Nahas WC, et al. Surgical complications after renal transplantation in grafts with multiple arteries. Int Braz J Urol 2005;31(2):125
30.

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35 Infections in Kidney Transplant Recipients Aynaa Alsharidi, Deepali Kumar and Atul Humar University Health Network, Toronto, ON, Canada

35.1 TIMELINE OF INFECTIONS Kidney transplantation has become a common procedure worldwide and has been shown to be associated with lower mortality rate and is more cost-effective than long-term dialysis.1,2 However, infections are a major cause of morbidity and mortality in kidney transplant recipients.3,4 In the first month posttransplant, patients are at risk for bacterial nosocomial infections including those related to the wound, central intravenous access, urinary catheterization, and pneumonia. Donor-derived infections also need to be considered in the first few weeks posttransplant. Between months 1 and 6 posttransplant, opportunistic infections may start to occur. Following 6 months, the risk of late opportunistic infections such as cytomegalovirus (CMV) remains. Fig. 35.1 shows a typical timeline of infectious complications postkidney transplant. The typical timeline however has been modified by many factors. Current prophylaxis and prevention strategies specifically alter the presentation of common pathogens. For example, CMV prophylaxis for 3 6 months typically means that onset of viremia and disease is pushed beyond these time points. Similar, other herpes virus infections may be prevented and delayed by use of CMV prophylaxis. Modifications of immunosuppression, e.g., with treatment of acute rejection and use of different types of immunosuppression, e.g., agents such as alemtuzumab, or belatacept5,6 affect the occurrence of infection as well. Table 35.1 shows an example of how different immunosuppression regimens may alter the risk of specific infectious complications.5 9 Timeline of infections postkidney transplant Early (6 months ● Respiratory viruses ● Urinary tract infection ● BK nephropathy ● Late CMV (post prophylaxis) ● PTLD ● Other opportunistic infection based on risk factors ● Late PCP (depends on prophylaxis)

Timeline of infections postkidney transplant.

Kidney Transplantation, Bioengineering, and Regeneration. DOI: http://dx.doi.org/10.1016/B978-0-12-801734-0.00035-7

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© 2017 Elsevier Inc. All rights reserved.

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TABLE 35.1

Immunosuppressive Agents and the Variable Risk of Infection

Immunosuppressive agent

Pathogen

Comment (see references below)

Thymoglobulin

Diverse risk of OI increase herpes viruses

Nonspecific targeting

mTOR inhibitor

Decrease CMV and possibly BK virus Treat KSHV

mTOR important in viral replication cycle

Eculizumab

Meningococcal disease

Inhibition of terminal complement pathway

Mycophenolate

Possible less risk for PTLD versus other agents

Inhibits B-cell proliferation

Belatacept

Possible increase in CNS PTLD in EBV donor-positive/recipient negative patients

Mechanism unknown

35.2 PRETRANSPLANT SCREENING OF THE KIDNEY DONOR AND RECIPIENT Routine pretransplant screening should exclude any active infections and evaluate for latent ones in either the recipient or donor.10 Examples of active infections in the recipient may include chronic infections such as diabetic foot osteomyelitis, or acute infections such as pneumonia. Generally any consideration for transplant should be delayed until such infections have resolved or are adequately controlled. In terms of latent viruses, screening is useful for either risk stratification or direct management. Patients should be screened for CMV, Epstein Barr virus (EBV), varicella zoster virus (VZV) antibodies as well as HIV, hepatitis B, and hepatitis C viruses.10 Syphilis screening is also recommended. Recipients from endemic areas should be screened for malaria, Strongyloides and Chagas. TB screening using a TB skin test or interferon-gamma release assay is also recommended for recipient screening.11 Living donor screening follows a similar pathway. In terms of deceased donors it is currently impractical to screen deceased donors for TB due to the nature of the testing and the urgency of transplant. Deceased donors should undergo testing with urine and blood cultures as well as serology for CMV, EBV, VZV, hepatitis B (including antiHBc and HBsAg), hepatitis C (HCV), and HIV. The Public Health Service (PHS) now recommends universal HCV NAT testing for deceased donors and HIV ribonucleic acid (RNA) by NAT or HIV antigen (e.g., HIV Ag/Ab combination assay) for all potential organ donors identified as being at increased risk for HIV infection. West Nile virus (WNV) testing in donors should be done in areas with established WNV during the risk period.

35.3 DONOR-DERIVED INFECTIONS A donor-derived source of infection is important to consider in any infection that occurs in the first few weeks following transplant. Transmissions have been documented for several viruses including WNV, rabies, and lymphocytic choriomeningitis virus.12 14 For these reasons, donors with encephalitis or meningitis of unknown etiology should not be used for the purposes of kidney transplantation. HIV and HCV transmissions have also occurred despite negative serologies.15 In most of these cases, transmission has been attributed to window period infection. The use of NAT, especially in donors with known behavioral risk factors for acquiring HIV, HCV, or HBV, likely diminishes (but does not eliminate) the risk of such transmission events and improves the likelihood of organ utilization in this setting.16 Bacterial transmission of pathogens may occur as well, specifically if the donor is bacteremic17 or has an active pyelonephritis. In such instances it is important to give recipients adequate antibiotic coverage in the immediate postoperative period based on cultures obtained from the deceased donor.

35.4 SPECIFIC INFECTIONS 35.4.1 Technical and Postoperative Complications Technical and other postoperative complications tend to be observed less frequently after kidney transplantation compared with liver or pancreas transplantation. Surgical site complications may include wound infection, either superficial or deep. Anastomotic urinary leaks and strictures can also occur and peritransplant hematomas, urinomas, or lymphoceles may become secondarily infected.

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35.4.2 Urinary Tract Infections Urinary tract infection (UTI) is the most common bacterial infection postkidney transplant.18 The prevalence of UTI in kidney transplant recipients varies widely, from 23% to 75%. Interestingly, it is associated with higher mortality rate, as high as 41% as compared to noninfected recipients in one study.19 In addition, healthcare marginal costs were increased by $17,691 for UTI alone. These costs are even higher when UTIs are combined with other site infections. The risk factors for UTIs include a combination of host, technical and posttransplant factors. Female gender, age of 45 years and older, obesity, diabetes mellitus, cardiovascular diseases, and pretransplant dialysis are the most significant host factors. Deceased and expanded criteria donors are also associated with higher risk of UTI. Any instrumentation such as indwelling catheters and routine ureteric stenting at the time of transplantation is associated with a significantly increased rate of posttransplant infections.19,20 Posttransplant, use of immunosuppressive and induction therapy with cell-depleting antibodies, such as antithymocyte globulin, are linked to a higher incidence of UTI. Diagnosis of UTI is based on clinical, laboratory, and radiological parameters. Symptomatic UTI classically presents with burning micturition, dysuria, urgency, frequency, and change in urine appearance or smell. However, in kidney transplant recipients, clinical presentations may vary significantly and traditional signs and symptoms may be absent. The presence of allograft tenderness is a significant sign of graft pyelonephritis. Laboratory tests include urine culture and analysis. Blood culture testing may help, especially if the patient is febrile. A significant level of pyuria is usually observed with a WBCs count .10 per high power field. Growth of .105 colony forming units/mL of single organism from the urine indicates UTI. Nevertheless, lower degree of pyuria or bacterial colony counts does not exclude UTIs in organ transplant recipients. Kidney transplant recipients in particular may develop allograft infection that usually presents with transplant site tenderness, fever, chills, and rigors, with or without bacteremia. Atypical forms of presentations are not uncommon and have to be taken seriously until active infection is excluded. These include isolated fever, graft discomfort, or tenderness alone. Prompt management of UTIs in a kidney transplant recipient is important not only to eradicate the infection, but also to prevent allograft injury and to decrease the risk of morbidity and mortality related to sepsis in the setting of an immunocompromised host. Antibiotic therapy can be started empirically to cover both gram-positive and gram-negative bacteria. In addition, selection of initial antibiotic therapy can be based on the previous culture isolates or severity of current illness; however, it should be modified according to the isolated organism once culture results are available. There is insufficient data regarding the optimal duration of antibiotics for therapy of UTIs in renal transplant recipients. However, the current recommendation is to treat mild symptomatic UTI (e.g., cystitis) for 5 7 days.3,21 In patients with early UTIs, i.e., in the first 6 months posttransplant, even mild cases should be treated for 7 10 days. Pyelonephritis or urosepsis, however, should be treated for longer duration, usually 14 21 days. The goal is to optimize antimicrobial therapy since early UTIs are associated with an increased risk of allograft loss and late infections (6 months posttransplant) are linked to death and graft failure in many studies.22 25 Prevention is one of the most important management strategies in posttransplant care that encompasses both donor and recipient factors. For example, all donors should be screened by testing their urine and blood cultures and where possible, any active bacterial infections should be treated before organ donation. In deceased donors, cultures results may become available after proceeding with the transplantation, in which case recipients should be treated with antimicrobial therapy accordingly. In recipients, prophylaxis with 1 year of trimethoprimsulfamethoxazole (TMP/SMX) was found to be effective in the prevention of UTIs after renal transplant.26 28 In a prospective, double-blind, randomized clinical trial, Khosroshahi et al. found that high dose of TMP/SXT (320/1600 mg, daily in two divided doses) had significantly reduced the UTI occurrence within 1 month postkidney transplantation compared to low- or moderate-dose TMP/SXT, 80/400 or 160/800 mg, daily, respectively.29 A recent study done by Singh et al. found that a daily 480 mg dose of TMP/SMX as Pneumocystis jiroveci pneumonia (PJP) prophylaxis was not associated with reduced prevalence of asymptomatic bacteriuria (ASB) and UTIs, either cystitis or allograft pyelonephritis.30 Indeed, this protocol was associated with an increase of both amoxicillin and TMP/SMX resistance, which appeared within the first 30 days after TMP/SMX exposure. Quinolones are an alternative prophylaxis choice although there is insufficient data to recommend their routine usage as prophylaxis postkidney transplant. Only one randomized study compared the two drugs (6 months of ciprofloxacin 250 mg daily vs TMP/SMX 80/400 mg daily) and showed that ciprofloxacin was at least as effective as TMP/SMX in the prevention of UTI in renal transplant recipients, and is better tolerated.31 Ciprofloxacin prophylaxis was associated with a higher incidence of PJP than is TMP/SMX therapy. In a more recent retrospective study, the addition of 30 days of ciprofloxacin to 6 months of 800/160 mg TMP/SMX daily prophylaxis after

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kidney transplantation was associated with a 54% reduction in the 1-year incidence of UTI when compared with 6 months of TMP/SMX alone.32 Nevertheless, fluoroquinolones use is limited by ongoing increase in the incidence of multidrug-resistant organisms and the potential concern about Clostridium difficile related colitis.33 35.4.2.1 Asymptomatic Bacteriuria There is no consensus whether ASB should be treated in renal transplant recipients.18 Therefore, many physicians tend to treat rather than observe, especially in the first 3 6 months posttransplant. This decision to treat may be influenced by other risk factors such as the use of induction therapy, indwelling urinary catheter, urinary stent, and patients with early graft dysfunction. However, small clinical trials have not demonstrated that treatment of ASB postkidney transplant prevents symptomatic UTIs.34,35 A prospective randomized control study of 88 renal transplant recipients did not show significant difference in rate of bacteriuric episodes or symptomatic UTIs between treated and untreated groups.36 In addition, ASB and its recurrence does not appear to adversely affect allograft function at least early posttransplant. Another retrospective study of 334 patients with asymptomatic Escherichia coli or Enterococcus faecalis bacteriuria demonstrated that both treated and untreated groups were similar in developing symptomatic UTI.37 35.4.2.2 Recurrent Urinary Tract Infections Recurrent UTI can be defined as two or more episodes of UTI in 6 months or three or more episodes in 12 months. It occurs in 2.9% 72% of postkidney transplant recipients, commonly in the first year posttransplant.38 41 Anatomical and functional abnormalities play major roles in recurrent UTI postrenal transplant, e.g., ureteric stricture, urolithiasis, or vesicoureteral reflux. It can also develop from native kidneys or underlying genitourinary diseases like polycystic kidney disease, neurogenic bladder, or prostatitis. Postoperatively, perinephric collections can be infected or evolve to abscesses. Therefore, patients with recurrent UTI have to be investigated to rule out any of these contributing factors. Initial work-up should include pelvic imaging, either ultrasound (US) or computerized tomography (CT). In addition, postvoid residual volume, voiding cystourethrogram, and bladder urodynamic studies may be helpful for delineating any functional abnormalities. Cystoscopy may be required to evaluate and manage structural abnormalities of the urethra or bladder. If no secondary cause can be found, antibiotic therapy can be given for 2 weeks. In case of relapse, a prolonged course of antimicrobial therapy is an option for 4 weeks or more. If further UTIs reoccur within the defined period, the use of prophylaxis for 3 6 months can be offered with close follow up. Selection of the specific antibiotic for prophylaxis should be guided by prior susceptibility tests, specifically, last culture isolate if any. TMP/SMX is the most common recommended prophylaxis agent. It is an attractive option being the prophylaxis choice for PJP as well. Other oral options are fluoroquinolones, cephalosporins, and nitrofurantoin. Repeated urinalysis and urine cultures while taking the prophylaxis therapy are necessary to ensure clearance of the infection. However, if it persists, patients have to be reevaluated for their compliance, evolution of bacterial resistance, or newly developed secondary causes. A few studies have demonstrated that multidrug-resistant bacteria are associated with significant higher rate of recurrent UTI in renal transplant recipients.42,43 Other cofactors such as diabetes mellitus should be controlled to decrease the risk of recurrent UTI. In addition, a judicious reduction in immunosuppressive therapy may help.

35.4.3 Candiduria No clear consensus definition for significant candiduria exists. In trials it is often defined as the presence of more than 105 Candida organisms in midstream urine. Candiduria is common postkidney transplant. However, there is a significant lack of prospective data on this important problem and so the optimal approach to such infections is mostly based on small studies and expert opinion and data from nontransplant studies. In a case control study, candiduria occurred in 11% of the renal transplant recipients.44 In the same study, female sex, intensive care unit (ICU) admission, antibiotic use in the month before candiduria, presence of an indwelling bladder catheter, diabetes, neurogenic bladder, and malnutrition were independent predictors of candiduria. For symptomatic Candida cystitis and fluconazole-susceptible organisms, fluconazole for 2 weeks is the recommended therapy.45 The optimal therapy for fluconazole-resistant candiduria is unclear but a amphotericin B deoxycholate derivative (e.g., AmBisome) for 1 7 days or oral flucytosine for 7 10 days is recommended. Removal of an indwelling bladder catheter, if feasible, is strongly recommended to decrease risk of candiduria. If it persists, intraabdominal complications should be ruled out by abdominal imaging. Significantly, candiduria was associated with lower survival rate in critically ill patients, which could be related to the severity of illness, comorbidities, and the increased use of invasive devices rather than directly affected by the infection per se.44

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35.4.4 Cytomegalovirus CMV remains one of the most common viral opportunistic infections after kidney transplantation.46,47 Reactivation of latent virus is a complex, poorly understood process. Virus may be of donor origin, recipient origin, or commonly both donor and recipient strains may reactivate concurrently.48 Risk factors for CMV reactivation include the degree and type of exogenous immunosuppression, the host immune response, and poorly defined viral virulence factors.47,49 Symptomatic patients are classified as having CMV disease, which presents as a viral syndrome (fever, malaise) or as tissue-invasive disease, such as hepatitis or pneumonitis. CMV can also have immunomodulatory effects related to viral replication, resulting in other opportunistic infections and acute and chronic allograft injury. The risk of CMV disease is highest in donor-positive, recipient-seronegative (D 1 R-) patients who lack cellular and humoral immunity.46 Specific immunosuppression drugs are more likely to result in CMV, especially the use of induction agents such as ATG and alemtuzimab.6 As noted in Table 35.1, certain types of immunosuppression, specifically the use of mTOR inhibitors9,50 may actually be beneficial in terms of limiting CMV replication, although the exact mechanisms by which this occurs have not been well defined. 35.4.4.1 Pathogenesis of Cytomegalovirus After Transplant The precise factors leading to CMV reactivation in transplant recipients are only partially understood. CMV is a remarkably complex virus and its genome encodes a large number of immunomodulatory proteins. In an individual patient, control of viral replication is likely due to a combination of host and viral factors that influence reactivation from latency. CMV is able to trigger robust responses from virtually every arm of the immune system. However, the T-cell-mediated adaptive immune response is predominant in the protection against CMV.51 In particular, interferon (IFN)-γ producing CMV-specific CD8 1 T cells play a critical role in limiting CMV viremia during the initial phase of primary infection; the CD4 1 T-cell subset seems to be more relevant in establishing long-term immune control. Although cytotoxic CD8 1 T cells may target a great variety of viral proteins (approximately 70% of the viral proteome), the responses against tegument phosphoprotein pp65 and immediate early-1 (IE-1) antigens are essential. Natural killer and regulatory T cells (Tregs) also contribute to the immune response to CMV.52 The enumeration and ex vivo assessment of the functionality of CMV-specific cell subsets is being increasingly advocated to categorize the actual risk of developing CMV disease in a given patient. Such an approach should allow clinicians to at least in part individualize the prevention and treatment strategy against CMV. In one study, predictive value of CD8 1 T-cell response to CMV was assessed.53 A total of 127 CMV D 1 / R- patients were assessed with serial CD8 1 T-cell measurements using the Quantiferon-CMV assay. The incidence of CMV disease was significantly higher in patients with undetectable IFN-γ responses at the end of prophylaxis versus those with positive responses. A number of viral factors may also play a role in determining the risk of CMV disease. The virus commits a large percentage of its total genome-coding capacity to the tasks of modulating host cell behavior. These include CMV gene products aimed at escaping host defense mechanisms through alterations in MHC class 1 protein expression.54 It has also been shown that mixed infections with multiple strains of CMV are common posttransplant and contribute to pathogenesis.55 35.4.4.2 Cytomegalovirus Prevention and Treatment A prevention strategy for CMV is critical postkidney transplant. Typically this involves either prophylaxis or preemptive therapy.46 Prophylaxis involves giving antiviral therapy early posttransplant for a defined duration. In preemptive therapy, patients are monitored at regular intervals for early evidence of CMV replication by use of a laboratory assay (usually polymerase chain reaction (PCR)). Patients with replication above a certain threshold are then treated with antiviral therapy to prevent symptomatic disease. Both approaches are successful in preventing CMV, but neither is perfect. Recently the use of increasing durations of prophylaxis (6 or even 12 months) has been advocated for high-risk patients.56,57 In the IMPACT trial CMV D 1 /R- kidney transplant patients were randomized to receive either 100 days or 200 days of valganciclovir prophylaxis.56 The longer duration significantly decreased the incidence of CMV disease but was associated with a higher incidence of leukopenia. Valganciclovir is the most common agent used either in a prophylaxis or preemptive strategy and is generally well tolerated postkidney transplant with the major dose limiting side effect related to bone marrow suppression.58 Treatment of CMV viremia with oral or intravenous antiviral therapy has good efficacy and generally low rates of resistance.59 61 However, recurrent CMV viremia is very common, e.g., with a 1-month course of secondary prophylaxis the recurrence rate is 30%.59 Multiple episodes of CMV viremia may lead to the development of drug resistance.

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35.4.5 BK Virus BK virus (BKV) is one of the most common but challenging infections postkidney transplant. Serologic studies indicate that exposure to BKV initially occurs during childhood and results in asymptomatic or mildly symptomatic infections. It was isolated for the first time in 1971 from a urine sample of a renal transplant recipient.62 64 The virus remains latent within the kidney tubular epithelium and genitourinary tract. After transplantation, reactivation may present as BK viruria, viremia, or interstitial nephritis known as BKV-associated nephropathy (BKVAN). Declining renal allograft function is the most common presentation in asymptomatic patients with BKVAN leading to a progressive rise in serum creatinine. Postkidney transplant, the main triggers to reactive dormant BKV are immunosuppressive therapy. Tacrolimus, mycophenolate mofetil, and steroids are the most reported immunosuppressive therapy associated with BK viremia.65,66 For this reason, reduction of immunosuppressants is believed to be the cornerstone in BKVAN management. Antiviral therapies such as cidofovir and leflunomide have been tried in few studies but have not been well evaluated in large randomized-controlled trials.67,68 Recent studies evaluating quinolones for either prophylaxis or treatment have not shown any benefit to this class of agents.69,70 The gold standard diagnostic tool for BKVAN is a histopathological evidence of viral cytopathic changes in the epithelium of tubules, glomeruli, and collecting ducts with interstitial inflammation. Screening tests include urine for decoy cells and PCR testing of urine or blood (plasma). Decoy cells, which originate from infected renal tubular cells with nuclei altered by viral inclusions, is very sensitive (100%) but has a low positive predictive value of 29% for the diagnosis of BKVAN.71 Quantification of viral load in blood and urine has also been used to diagnose BKVAN.72 Testing of BKV DNA in plasma by PCR assay has sensitivity and specificity of 100% and 88%, respectively, depending on the threshold used.71 Currently, the optimal approach to BK is a prevention strategy. Typically patients are monitored by blood/plasma PCR for evidence of early viral replication prior to development of BKVAN. In these patients immunosuppression is reduced in an effort to clear viremia and prevent the development of BKVAN.

35.4.6 Influenza The epidemiology of influenza in transplant recipients has only been partially defined. Reported attack rates have varied considerably, probably due to differences in transplant populations, immunosuppression protocols, exposures, and type and virulence of circulating influenza viruses. Complications of influenza infection appear to be common in transplant patients. In some reports, a relatively high rate of progression to viral pneumonia has been observed, especially in lung transplant recipients and hematopoietic stem cell transplant recipients.73 75 The largest review of influenza in the solid organ transplant population was done during the 2009 10 outbreak of influenza A(H1N1)pdm09.76 This was a retrospective study of 237 transplant patients with pandemic H1N1 infection and included both adults and pediatric patients. The majority of the patients reported in the study were hospitalized (71%) and 16% were admitted to the ICU. Approximately one-third of patients had pneumonia at presentation. Death occurred in 4% of patients. Early initiation of antiviral therapy (within 48 hours of symptom onset) was associated with decreased hospitalization, ICU admission, and a lower risk of death. Prolonged viral shedding and enhanced viral replication are hypothesized to be important aspects of influenza infection in immunocompromised patients.77 The main classes of antivirals for influenza are the neuraminidase inhibitors (oseltamivir, zanamivir) and M2 inhibitors (amantadine, rimantadine).78 The neuraminidase protein is responsible for release of progeny virus; M2 protein is an ion channel that is important for uncoating of virus inside the cell. Early initiation of antiviral therapy for immunocompromised patients is recommended by many experts and is supported by observational data. Resistance to a specific antiviral may be present at baseline in specific strains of influenza or may emerge during the course of therapy, often referred to as emergent resistance. It has been suggested that influenza (and other respiratory viral infections) may lead to important immunological sequelae, resulting in graft rejection and/or graft dysfunction.79 This may be secondary to activation of immunological mechanisms, including the upregulation of alloreactive antibodies or of proinflammatory cytokines such as TNF-α, IL-6, and IL-8. Some studies in kidney and liver recipients have reported a high incidence of acute rejection following infection with influenza.79 However, a causal relationship has not been established, and several studies are contradictory. The most suggestive data are in lung transplant recipients, in whom community-acquired respiratory viruses have been implicated as triggers for acute rejection and for the development of bronchiolitis obliterans syndrome.80,81 Early influenza vaccination is an important part of any influenza prevention strategy. However, vaccines may have suboptimal immunogenicity in this patient population.82 85

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35.4.7 Kidney Transplantation in the HIV-Positive Recipient The development of very potent antiretrovirals for the treatment of HIV has dramatically changed the face of this once fatal infection. Now, a prolonged lifespan is achievable in patients with HIV infection, and more specifically it is possible to limit viral replication to a threshold below the limit of detection in peripheral blood by standard viral load assays. End-stage kidney disease is not an uncommon occurrence in HIV-infected patients, reaching up to 5%.86 The causes of renal disease are varied but HIV-associated nephropathy (HIVAN) is the most common cause, accounting for around 50% of the HIV patients with end-stage kidney disease.87 The collapsing form of focal segmental glomerulosclerosis has been considered the primary form of HIVAN, which is found in 60% of renal biopsies.44 Other causes of renal disease include drug induced toxicity from the antiretrovirals, sepsis, or HCV coinfection.86 Kidney transplantation is highly feasible in HIV-infected recipients. The rates of patient and graft survival among HIV-infected recipients are similar to HIV-negative recipients.88,89 In these studies, progression of HIV disease was not seen posttransplant, but the major challenge was a significantly increased risk of graft rejection among HIV-infected recipients. Interestingly, patient and allograft outcomes are superior in HIV monoinfected than HCV monoinfected recipients.90 Indeed, HIV/HCV coinfected patients have worse outcomes than their monoinfected counterparts. HCV infection was associated with higher rate of nonopportunistic infections but the mechanisms by which HCV infection increases risk of serious infections, particularly sepsis, are not well understood.89 However, with the availability of very potent direct-acting antivirals for HCV, this will likely be less of a problem. Selection of the appropriate recipient is essential prior to considering transplantation. For kidney transplant recipients, relevant criteria include CD4 .200 cells/mm3, HIV viral load ,50 copies/mL, stable antiretroviral regimen, and no active opportunistic infections.91 Similarly, in a research setting, these criteria may be applied for HIV-infected donors. In 2013, the HIV Organ Policy Equity (HOPE) Act revised the Organ Transplant Amendments Act of 1988, an old law that banned patients from receiving organs from HIV-infected donors. The HOPE Act enables HIV-infected patients to receive organ donation from another HIV-infected patient under a research umbrella. One potential risk of using an HIV-positive donor organ into an HIV-positive recipient is the transmission of a drug-resistant virus strain to the recipient. The type and proportion of antiretroviral therapy (ART) drug resistance may be tested to decrease risk of HIV superinfection with a donor drug resistant virus that would not be suppressed by the recipient’s current ART regimen. However, HIV tropism and resistance tests are time consuming and would normally not be possible when a deceased donor is available. Another issue after transplantation is drug drug interactions between antiretrovirals and immunosuppressive therapies. Specifically, between calcineurin inhibitors (CNIs) and protease inhibitors (PIs) or nonnucleoside reverse transcriptase inhibitors (NNRTIs). Patients using PI-containing regimen require lower dose of CNI while those on NNRTI-containing regimen need higher doses.92 In addition, the cumulative side effects by using ART, prophylactic antimicrobials, and immunosuppression agents in the same patient may be significant. Opportunistic infections are major sources of morbidity and mortality in both posttransplant and HIV-infected patients, which necessitate further research studies in the HIV/AIDS setting to optimize current antimicrobial prophylaxis regimens. In a large, prospective, nonrandomized trial, Stock et al. found that 38% of kidney transplant recipients with HIV had developed infections, and 60% were serious infections within the first 6 months after transplantation.93 In the same study, 2 out of 150 total patients had Kaposi’s sarcoma but other neoplasms were observed at rates consistent with non-HIV-infected recipients. Until further data is available, most centers are following current recommendations and guidelines that are used for non-HIV transplant recipients. The only exception is using lifelong PJP prophylaxis rather than 6 12 months, which is the usual duration in nonHIV-infected kidney transplant recipients.88

35.5 CONCLUSIONS The challenges posed by infections will continue to evolve as transplantation evolves. This is due to changing technologies, and changing immunosuppression regimens. It is unknown how new therapies related to regenerative medicine will further affect the development of infectious complications. In addition, pathogens constantly evolve, and may develop drug resistance or new virulence patterns, resulting in changes to current management strategies. Vigilance and continued research are important aspects of preventing infectious complications posttransplant.

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A phase III study of belatacept versus cyclosporine in kidney transplants from extended criteria donors (BENEFIT-EXT study). Am J Transplant 2010;10(3):547 57. 6. LaMattina JC, Mezrich JD, Hofmann RM, Foley DP, D’Alessandro AM, Sollinger HW, et al. Alemtuzumab as compared to alternative contemporary induction regimens. Transplant Int 2012;25(5):518 26. 7. Struijk GH, Bouts AH, Rijkers GT, Kuin EA, ten Berge IJ, Bemelman FJ. Meningococcal sepsis complicating eculizumab treatment despite prior vaccination. Am J Transplant 2013;13(3):819 20. 8. Sampaio MS, Cho YW, Shah T, Bunnapradist S, Hutchinson IV. Association of immunosuppressive maintenance regimens with posttransplant lymphoproliferative disorder in kidney transplant recipients. Transplantation 2012;93(1):73 81. 9. Brennan DC, Legendre C, Patel D, Mange K, Wiland A, McCague K, et al. Cytomegalovirus incidence between everolimus versus mycophenolate in de novo renal transplants: pooled analysis of three clinical trials. 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63. Goudsmit J, Wertheim-van Dillen P, van Strien A, van der Noordaa J. The role of BK virus in acute respiratory tract disease and the presence of BKV DNA in tonsils. J Med Virol 1982;10(2):91 9. 64. Gardner SD, Field AM, Coleman DV, Hulme B. New human papovavirus (B.K.) isolated from urine after renal transplantation. Lancet 1971;1(7712):1253 7. 65. Borni-Duval C, Caillard S, Olagne J, Perrin P, Braun-Parvez L, Heibel F, et al. Risk factors for BK virus infection in the era of therapeutic drug monitoring. Transplantation 2013;95(12):1498 505. 66. Hirsch HH, Vincenti F, Friman S, Tuncer M, Citterio F, Wiecek A, et al. Polyomavirus BK replication in de novo kidney transplant patients receiving tacrolimus or cyclosporine: a prospective, randomized, multicenter study. Am J Transplant 2013;13(1):136 45. 67. Lamoth F, Pascual M, Erard V, Venetz JP, Nseir G, Meylan P. Low-dose cidofovir for the treatment of polyomavirus-associated nephropathy: two case reports and review of the literature. Antiviral Ther 2008;13(8):1001 9. 68. Josephson MA, Gillen D, Javaid B, Kadambi P, Meehan S, Foster P, et al. Treatment of renal allograft polyoma BK virus infection with leflunomide. Transplantation 2006;81(5):704 10. 69. Knoll GA, Humar A, Fergusson D, Johnston O, House AA, Kim SJ, et al. Levofloxacin for BK virus prophylaxis following kidney transplantation: a randomized clinical trial. JAMA 2014;312(20):2106 14. 70. Lee BT, Gabardi S, Grafals M, Hofmann RM, Akalin E, Aljanabi A, et al. Efficacy of levofloxacin in the treatment of BK viremia: a multicenter, double-blinded, randomized, placebo-controlled trial. Clin J Am Soc Nephrol 2014;9(3):583 9. 71. Hirsch HH, Knowles W, Dickenmann M, Passweg J, Klimkait T, Mihatsch MJ, et al. Prospective study of polyomavirus type BK replication and nephropathy in renal-transplant recipients. N Engl J Med 2002;347(7):488 96. 72. Nickeleit V, Klimkait T, Binet IF, Dalquen P, Del Zenero V, Thiel G, et al. Testing for polyomavirus type BK DNA in plasma to identify renal-allograft recipients with viral nephropathy. N Engl J Med 2000;342(18):1309 15. 73. Boudreault AA, Xie H, Leisenring W, Englund J, Corey L, Boeckh M. Impact of corticosteroid treatment and antiviral therapy on clinical outcomes in hematopoietic cell transplant patients infected with influenza virus. Biol Blood Marrow Transplant 2011;17(7):979 86. 74. Gainer SM, Patel SJ, Seethamraju H, Moore LW, Knight RJ, Gaber AO. Increased mortality of solid organ transplant recipients with H1N1 infection: a single center experience. Clin Transplant 2012;26(2):229 37. 75. Khanna N, Steffen I, Studt JD, Schreiber A, Lehmann T, Weisser M, et al. Outcome of influenza infections in outpatients after allogeneic hematopoietic stem cell transplantation. Transplant Infect Dis 2009;11(2):100 5. 76. Kumar D, Michaels MG, Morris MI, Green M, Avery RK, Liu C, et al. Outcomes from pandemic influenza A H1N1 infection in recipients of solid-organ transplants: a multicentre cohort study. Lancet Infect Dis 2010;10(8):521 6. 77. Memoli MJ, Athota R, Reed S, Czajkowski L, Bristol T, Proudfoot K, et al. The natural history of influenza infection in the severely immunocompromised vs nonimmunocompromised hosts. Clin Infect Dis 2014;58(2):214 24. 78. Ison MG. Influenza prevention and treatment in transplant recipients and immunocompromised hosts. Influenza Other Respir Viruses 2013;7(Suppl. 3):60 6. 79. Vilchez RA, McCurry K, Dauber J, Lacono A, Griffith B, Fung J, et al. Influenza virus infection in adult solid organ transplant recipients. Am J Transplant 2002;2(3):287 91. 80. Fisher CE, Preiksaitis CM, Lease ED, Edelman J, Kirby KA, Leisenring WM, et al. Symptomatic respiratory virus infection and chronic lung allograft dysfunction. Clin Infect Dis 2016;62(3):313 19. 81. Kumar D, Husain S, Chen MH, Moussa G, Himsworth D, Manuel O, et al. A prospective molecular surveillance study evaluating the clinical impact of community-acquired respiratory viruses in lung transplant recipients. Transplantation 2010;89(8):1028 33. 82. Baluch A, Humar A, Eurich D, Egli A, Liacini A, Hoschler K, et al. Randomized controlled trial of high-dose intradermal versus standard-dose intramuscular influenza vaccine in organ transplant recipients. Am J Transplant 2013;13(4):1026 33. 83. Fairhead T, Hendren E, Tinckam K, Rose C, Sherlock CH, Shi L, et al. Poor seroprotection but allosensitization after adjuvanted pandemic influenza H1N1 vaccine in kidney transplant recipients. Transplant Infect Dis 2012;14(6):575 83. 84. Manuel O, Humar A, Berutto C, Ely L, Giulieri S, Lien D, et al. Low-dose intradermal versus intramuscular trivalent inactivated seasonal influenza vaccine in lung transplant recipients. J Heart Lung Transplant 2011;30(6):679 84. 85. Kumar D, Danziger-Isakov L. Immunization against influenza: a balancing act. Am J Transplant 2011;11(8):1561 2. 86. Wyatt CM, Morgello S, Katz-Malamed R, Wei C, Klotman ME, Klotman PE, et al. The spectrum of kidney disease in patients with AIDS in the era of antiretroviral therapy. Kidney Int 2009;75(4):428 34. 87. Laradi A, Mallet A, Beaufils H, Allouache M, Martinez F. HIV-associated nephropathy: outcome and prognosis factors. Groupe d’ Etudes Nephrologiques d’Ile de France. J Am Soc Nephrol 1998;9(12):2327 35. 88. Muller E, Barday Z, Mendelson M, Kahn D. HIV-positive-to-HIV-positive kidney transplantation--results at 3 to 5 years. N Engl J Med 2015;372(7):613 20. 89. Roland ME, Barin B, Huprikar S, Murphy B, Hanto DW, Blumberg E, et al. Survival in HIV-positive transplant recipients compared with transplant candidates and with HIV-negative controls. Aids 2016;30(3):435 44. 90. Sawinski D, Forde KA, Eddinger K, Troxel AB, Blumberg E, Tebas P, et al. Superior outcomes in HIV-positive kidney transplant patients compared with HCV-infected or HIV/HCV-coinfected recipients. Kidney Int 2015;88(2):341 9. 91. Boyarsky BJ, Durand CM, Palella Jr. FJ, Segev DL. Challenges and clinical decision-making in HIV-to-HIV transplantation: Insights from the HIV literature. Am J Transplant 2015;15(8):2023 30. 92. Frassetto LA, Browne M, Cheng A, Wolfe AR, Roland ME, Stock PG, et al. Immunosuppressant pharmacokinetics and dosing modifications in HIV-1 infected liver and kidney transplant recipients. Am J Transplant 2007;7(12):2816 20. 93. Stock PG, Barin B, Murphy B, Hanto D, Diego JM, Light J, et al. Outcomes of kidney transplantation in HIV-infected recipients. N Engl J Med 2010;363(21):2004 14.

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C H A P T E R

36 Metabolic Disorders Following Kidney Transplantation Quirino Lai and Francesco Pisani University of L’Aquila, L’Aquila, Italy

36.1 INTRODUCTION Several metabolic processes are altered by kidney transplantation (KT), mainly due to a mechanism of overlapping between preexisting favoring conditions and side effects of immunosuppressive (IS) agents. Among the metabolic alterations commonly observed after KT, diabetes mellitus (DM), dyslipidemias, and uric acid metabolism represent the most important ones, mainly due to the clinical impact they may present not only in relation to graft function but also in terms of patient survival.

36.2 DIABETES MELLITUS According to the WHO and the American Diabetes Association, DM is defined as: (1) fasting plasma glucose $ 126 mg/dL (7.0 mmol/L) (fasting is defined as no caloric intake for at least 8 hours); or (2) symptoms of hyperglycemia and a casual plasma glucose $ 200 mg/dL (11.1 mmol/L) (casual is defined as any time of day without regard to time since last meal: classic symptoms of hyperglycemia include polyuria, polydipsia and unexplained weight loss); or (3) 2-hour plasma glucose $ 200 mg/dL (11.1 mmol/L) during an oral glucose tolerance test (the test should be performed as described by the WHO, using a glucose load containing the equivalent of 75 g anhydrous glucose dissolved in water).1 In the specific context of KT, DM (1) can be already present at the moment of transplant; or (2) it can develop for the first time after KT. In this latter condition, DM is defined as new-onset diabetes after transplantation (NODAT).

36.2.1 NODAT: Definition, Risk Factors, and Diagnosis Typically, DM is already known at the moment of transplant, due to its insurgence before or during the waiting time period. On the other hand, in the specific case of NODAT the diagnosis is done immediately after KT. As a consequence, early detection and intervention are mandatory, with the intent to improve the changes or reversing or ameliorating them. In the case of DM already known before KT, it ranges from 10% to 20% of recipients. NODAT incidence widely varies according to the definition of diabetes and to the adopted IS. Typically, the highest incidence of NODAT is observed within the first 3 months after transplantation.2 The cumulative incidence of NODAT by the end of the first post-KT year has generally been found to be 2% 13% in children, with up to one quarter of cases presenting at least an impaired glucose tolerance (Table 36.1).3 9 In adults, the incidence of NODAT is higher, reaching 10% 30% of cases (Table 36.2).10 35 Kidney Transplantation, Bioengineering, and Regeneration. DOI: http://dx.doi.org/10.1016/B978-0-12-801734-0.00036-9

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TABLE 36.1

Incidence and Risk Factors for NODAT in Pediatric Population of KT Patients

Author

Year

NODAT incidence (%)

2001

36/1365 (2.6)

Greenspan

2002

16/229 (7.0)

Prokai6

2008

6/45 (13.3) NODAT 7/45 (15.6) with IGT

Burroughs7

2009

154/2168 (7.1)

Buyan8

2010

Kuo9

2010

6/54 (11.1) NODAT 7/54 (13.0) IGT 125/2726 (4.6)

4

Al-Uzri

5

NODAT risk factors

Family history of DM Tacrolimus use Peri-KT hyperglycemia Tacrolimus use Tacrolimus trough level Steroid pulse therapy Family history of DM CMV D 1/R 2 status Recipient age 13 18 years Recipient age 19 21 years BMI $ 30 Tacrolimus use

Recipient age .10 years Abnormal BMI percentile (,5% or .85%) Steroid use at discharge

BMI, body mass index; CMV, cytomegalovirus; D, donor; DM, diabetes mellitus; IGT, impaired glucose tolerance; KT, kidney transplantation; NODAT, new-onset diabetes after transplantation; R, recipient.

TABLE 36.2

Incidence and Risk Factors for NODAT in Adult Population of KT Patients

Author

Year

NODAT incidence (%)

11

1994

57/748 (7.6)

Recipient age .50 years

Nonwhite race

2001

436/2078 (21.0)

Recipient age .45 years KT after 1995

African American race BMI

Kasiske

2003

2768/11,659 (24.0)

Gourishankar15

2004

38/386 (9.8)

HCV infection BMI $ 30 Tacrolimus use High HLA mismatches Rejection episode(s) Tacrolimus use

Romagnoli27

2005

36/538 (6.7)

Recipient age African American race Hispanic race Male donor Older recipient age Deceased donor HCV infection Family history of DM

16

Araki

2006

39/528 (7.4)

Shah28

2006

1581/15,309 (10.3)

Porrini29

2008

31/154 (20.0)

Roland30

2008

182/828 (22.0)

2010

215/2168 (9.9)

2011

31/120 (25.8)

Recipient age Tacrolimus use Triglycerides Pretransplant IGT ADPKD

2012

73/218 (33.5)

Recipient age HCV infection

Lv34

2014

87/428 (20.3)

Gaynor35

2015

108/481 (22.5)

Fasting plasma glucose Recipient age BMI BMI Sirolimus use Nonwhite race

Fryer

13

Cosio

14

31

Marrero

Caillard32 Al-Ghareeb

33

NODAT risk factors

Tacrolimus use Rejection episode(s) Recipient age BMI Tacrolimus use Recipient age Pre-KT BMI Early low-grade proteinuria

BMI Recipient age BMI HCV infection African American race Triglyceride/HDL-cholesterol ratio Pulse pressure HCV infection BMI Rejection episode(s) Family history of DM Tacrolimus use HCV infection Deceased donor kidney Older recipient age Rejection episode(s)

ADPKD, autosomic dominant polycystic kidney disease; BMI, body mass index; DM, diabetes mellitus; HCV, hepatitis C virus; HDL, high-density lipoprotein; HLA, human leukocyte antigen; IGT, Impaired glucose tolerance; KT, kidney transplantation; NODAT, new-onset diabetes after transplantation.

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36.2 DIABETES MELLITUS

515

Several risk factors increase the incidence of NODAT. Patients having one or more of these additional risk factors may be more frequently screened. Use of IS drugs is strongly connected with the development of NODAT; increasing the dose of one of these medications may also improve the risk of its development. Among the IS drugs, calcineurin inhibitors (CNIs) (tacrolimus and cyclosporine),5 7,14 16,28,31 corticosteroids,6,9 and mTOR inhibitors (mTORi) (sirolimus and everolimus)35 are the most commonly connected with NODAT. Tacrolimus and cyclosporine cause NODAT mainly by directly reducing insulin secretion from pancreatic beta cells.17,18 NODAT reversion has been reported after reduction, replacing, or discontinuation of these drugs, thanks to the limitation of beta insulae damage: However, the clinical evidence of this approach is anecdotally reported only in case reports or monocenter series.19 Due to its higher effect on the pancreatic cells, the risk of NODAT with tacrolimus is greater than with cyclosporine. Only small experiences exist on the switch from tacrolimus to cyclosporine with the intent to ameliorate the symptoms of NODAT: Up to now, no more than 200 cases are reported in the literature, with remarkable rates of diabetes resolution (40% 80%).20 Also in the case of steroids, few data exist on the effects of their reduction or withdrawal on reversing NODAT once it has occurred.21,22 In a recent prospective randomized trial, corticosteroid withdrawal has shown a limited impact in reducing NODAT when compared to low-dose prednisone (5 mg/day from month 6 to 5 years) (22.5% vs 21.5%).23 In many studies, rejection episodes are directly connected with NODAT development, probably due to the use of high-dose steroids.15,16,32,35 As a consequence, in case of acute rejection treatment with high-dose corticosteroids, screening for NODAT should be promptly done. Sirolimus has not been as well studied as the previously reported drugs. Some observational studies have found that sirolimus use was associated with an increased incidence of NODAT;24 however, randomized trials have produced conflicting results.25,26 Also in this case, few or even no data are available on whether discontinuing sirolimus will reverse NODAT. There is no evidence that azathioprine or mycophenolic acid cause NODAT. NODAT incidence is no doubt higher in individuals having the contemporaneous presence of IS and other risk factors. As a consequence, an individualized therapy should be accordingly taken into account, choosing the best IS drugs able to balance the risk of NODAT with the risk of rejection. In all the studies reported on this field, the risk of NODAT is universally increased by obesity.7,9,13,14,16,27 29,31,34,35 Also African Americans and Hispanic individuals commonly develop NODAT after KT.11,13,14,28,35 Older recipient age is a risk factor that shows a linear relationship with the risk of NODAT development.11,13 16,28,29,31,33 35 Hepatitis C infection has also been connected with the risk of NODAT.14,15,28,31,33,34 Several other risk factors have been reported in the literature: among them, we report family history of DM,5,6,27,33 impaired glucose tolerance,32 and dyslipidemia (Tables 36.1 and 36.2).29,31 Diagnosis of NODAT is done similarly to DM: Fasting plasma glucose, 2-hour glucose tolerance testing (after a 75-g glucose load), and hemoglobin A1c (HbA1c) are the screening tests typically used for NODAT detection. Timing of screening is controversial; Kidney Disease: Improving Global Outcomes (KDIGO) recommendations report at least (1) one screening weekly for the first post-KT 4 weeks; then (2) every 3 months for the first posttransplant year; and (3) annually, thereafter.36 Screening must be also done after starting, or substantially increasing the dose of CNIs, mTORi, or corticosteroids.

36.2.2 Management of NODAT or Diabetes Already Present at the Moment of Transplantation Both the groups of patients having NODAT or DM already present at the time of transplantation are associated with worse outcomes, including increased graft failure, cardiovascular diseases, and mortality.37 Untreated diabetes may increase the risk of metabolic complications, including hyperkalemia, and even ketoacidosis.38 Thus, a correct therapeutic strategy is required. Due to the presence of advanced autonomic neuropathy causing diabetic gastroparesis and hypoglycemic unawareness, it may be difficult to maintain under “tight” control a DM already present at the time of transplantation. Although in the general DM population it has been demonstrated that mortality rates may be decreased by targeting HbA1c levels ,6.0%,39 the attempt to achieve this value in KT patients may be difficult, eventually resulting in a higher rate of complications like severe hypoglycemia: In a randomized controlled trial comparing intensive glucose control with usual care in 99 KTs, the incidence of severe hypoglycemia was significantly higher in the intensive glucose control arm.40 In addition, some medications commonly used to treat diabetes may need dose reduction, or should be avoided in patients with reduced kidney function.41 As a consequence, it is typically recommended to target HbA1c levels 7.0% 7.5% in KT patients with DM. In the specific case of NODAT, longstanding complications are typically not present;

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36. METABOLIC DISORDERS AFTER KIDNEY TRANSPLANT

consequently, unclear data exist on the effective blood glucose and HbA1c targets to be achieved in the clinical practice.36 Looking at the possible strategies to adopt with the intent to minimize the risk of diabetes after KT, three different aspects can be investigated: prediction, prevention, and treatment. 36.2.2.1 Prediction A score has been developed with the intent to predict NODAT before KT. Seven simple pretransplant clinical and laboratory measurements have been used, including older recipient age, planned corticosteroid therapy after transplant, prescription for gout medicine, higher BMI, higher fasting glucose, higher triglycerides, and family history of type 2 DM.42 Recently, genetic polymorphisms have been investigated with the intent to identify biological markers able to predict NODAT. Two studies observed the ability in predicting NODAT of the G allele at position 2174 of the IL-6 gene promoter and of interleukin (IL)-7R, IL-17E, IL-17R, and IL-17RB.43,44 36.2.2.2 Prevention Lifestyle interventions aimed at promoting fat reduction are extremely efficacious in reducing the incidence of type 2 DM in the general population.45 Although it has somewhat presumably a similar effect on NODAT development, such a strategy has not already been tested in patients after KT. 36.2.2.3 Treatment Several drugs like metformin and pioglitazone are commonly used in patients without renal failure to control glycemia in type 2 DM. In case of post-KT good allograft function, these drugs may be prescribed for the treatment of preexisting DM or NODAT.46 No study has investigated the role of either of these oral agents in the prevention of NODAT. Moreover, due to their adverse effects (lactic acidosis and volume retention), their use is restricted in case of chronic kidney disease. A recent randomized clinical trial assigned 50 nondiabetic patients to two groups in the immediate post-KT period: A treatment group (isophane insulin for evening blood glucose $ 140 mg/dL) and a control group (short-acting insulin and/or oral antidiabetic agents for blood glucose $ 180 250 mg/dL). In the treatment group, lower odds of NODAT and hemoglobin A1c were observed. One year after transplantation, all patients in the treatment group were insulin-independent, whereas 7 (28%) of 25 controls required antidiabetic agents.47 Due to the role played by IS in determining the development of NODAT, “tailored” changes in IS medications are also potentially able to completely reverse or at least ameliorate NODAT. Among them, we can include (1) reducing the dose of tacrolimus, cyclosporine, or corticosteroids; (2) discontinuing tacrolimus, cyclosporine, or corticosteroids; (3) replacing tacrolimus with cyclosporine, mycophenolic acid, or azathioprine; and (4) replacing cyclosporine with mycophenolic acid or azathioprine. In a 2015 study performed on 628 KT cases, a progressive improvement was observed across the time in terms of glycemic values control (HbA1c decrease: 7.3%, 6.9%, 6.9%, 6.6%): No higher risk of impaired graft function, graft loss, or death were consequently observed also in patients with NODAT or preexisting DM, underlying the possibility to obtain excellent results under the current climate of a “more rationale” post-KT glucose monitoring.48

36.3 DYSLIPIDEMIAS Dyslipidemias are abnormalities in circulating lipoproteins, commonly associated with an increased risk of cardiovascular events. According to the Kidney Disease Outcomes Quality Initiative (KDOQI) Dyslipidemia Guidelines, the threshold measures used for defining a state of dyslipidemia in the clinical practice are total cholesterol $ 240 mg/dL; LDL cholesterol (LDH-C) $ 130 mg/dL; triglycerides $ 200 mg/dL (very high $ 500 mg/ dL). In case of HDL cholesterol (HDL-C), it is defined low when ,40 mg/dL.49

36.3.1 Dyslipidemias in the Setting of Kidney Transplantation Unfortunately, dyslipidemias are very common in transplanted patients, occurring in up to 90% of adult cases50 57 and in more than a half of adolescent KT recipients58 60 (Table 36.3). A possible explanation for the high incidence of dyslipidemias in transplanted patients can be found in the side effects of some IS agents. Steroids,

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36.3 DYSLIPIDEMIAS

TABLE 36.3

Incidence of Dyslipidemias in Adult and Adolescent Populations of KT Patients Year

No. of patients

Total cholesterol .200 mg/dL

LDL-C .100 mg/dL

HDL-C ,40 mg/dL

1992

100

83%

90%

21%

1993

214

90%

97%

48%

1996

403

94%

1997

50 5 Cya

83%

93%

30%

163

50 5 AZA

87%

92%

14%

159

2003

280 5 SIR

80%

322

Ramezani

2007

687

60%

196

56

2011

103

36%

52%

2011

474

64%

68%

52%

250

1994

69

66%

81%

19%

142

2000

62

52%

157

2011

71

27%

147

Author

TG mg/dL

ADULTS Gonyea50 51

Moore

52

Aakhus

53

Brown

54

Chueh

55

Razeghi

57

Spinelli

195

18%

ADOLESCENTS Milliner58 59

Silverstein

60

Derakhshan

AZA, azathioprine; Cya, cyclosporine; HDL-C, high-density lipoprotein cholesterol; LDL-C, low-density lipoprotein cholesterol; SIR, sirolimus; TG, triglycerides.

TABLE 36.4 After KT

Randomized Controlled Trials Focused on the Role of IS Regimens in Relation to the Modification of Dyslipidemia Change compared to baseline (%)

Author

Year

No. of patients

IS treatment

John61

1999

208

High-dose Cya

13

19

Low-dose Cya

19

13

Vanrenterghem62

63

Johnson

64

Budde

2000

2000

2011

500

223

503

Total C

TG

MMF 1 Cya 1 STER (stop)

16

3

MMF 1 Cya 1 STER

1 29

1 10

TAC 1 AZA

1 14

Cya 1 MMF

1 25

TAC 1 MMF

1 12

Cya 1 EC-MPS 1 STER Switch from Cya to EVE

AZA, azathioprine; C, cholesterol; Cya, cyclosporine; EC-MPS, enteric-coated mycophenolate sodium; EVE, everolimus; IS, immunosuppression; MMF, mycophenolate mofetil; STER, steroids; TAC, tacrolimus; TG, triglycerides.

cyclosporine, and mTORi everolimus and sirolimus represent the most important factors among the several potential remediable causes of dyslipidemia after KT (Table 36.4). A randomized controlled trial investigating the role of high versus low levels of cyclosporine showed that patients receiving higher doses of this drug presented increased cholesterol and triglycerides levels.61 Another multicenter randomized double-blind controlled study looking at the role of steroid withdrawal showed a significant reduction of steroid-related adverse events in patients stopping the drug: Of particular interest, both cholesterol and triglycerides decreased.62 A study comparing tacrolimus and cyclosporine reported a marked increase of cholesterol in patients taking cyclosporine.63 A randomized controlled study focused on the late (4.5 months) switch from cyclosporine to everolimus reported higher mean lipid concentrations observed in the switched group with respect to the patients who remained with cyclosporine.64 A systematic review of randomized controlled trials focused on the use of mTORi regimens after

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36. METABOLIC DISORDERS AFTER KIDNEY TRANSPLANT

KT reported in all but one of the 17 investigated trials higher levels of cholesterol and triglycerides in patients taking mTORi. Moreover, an increased prevalence of treatment with lipid-lowering agents (approximately 60% of patients) was observed in patients under therapy with mTORi, with no substantial difference between sirolimus or everolimus use.65 As a consequence, a screening of lipid status is recommended every time after the introduction of drugs known to cause dyslipidemias. Typically, it is recommended to perform a screening of lipid status in all the patients undergoing KT every 2 3 months after transplantation and then annually.36

36.3.2 Peculiarities of Treatment of Dyslipidemias After Kidney Transplantation Several studies have reported a positive association between dyslipidemias and acute cardiovascular diseases in the KT population.66 70 A multivariable Cox regression analysis performed on 427 KT recipients identified as risk factors for cardiovascular adverse events the following parameters: DM (relative risk 4.3), age .50 years (RR 5 2.7), body mass index .25 kg/m2 (RR 5 2.6), smoking (RR 5 2.5), LDL cholesterol .180 mg/dL (RR 5 2.3), and uric acid .6.5 mg/dL.67 Another study performed on 406 KTs directly correlated ischemic heart disease, besides age and gender, with total cholesterol.68 Moreover, several observational studies performed in the general population and in patients with chronic kidney disease reported that various dyslipidemias are directly associated with decreased kidney function.71 74 Although no study exists in the specific setting of KT, a metaanalysis based on the studies performed in the general population showed that a better lipidic control performed using statins may preserve renal function, also reducing the risk of proteinuria; the mechanisms for such a phenomenon are unclear.75 Evidence from the general population indicates that treatment of dyslipidemias reduces the risk of cardiovascular accidents.76 Until now, only one prospective randomized trial (the ALERT study) based on the effects of dyslipidemia management in KT patients has been published. Also the ALERT study strongly supported the previous statement that dyslipidemia management is associated with a significant reduction in the incidence of cardiac death and myocardial infarction: 2102 KT patients with total cholesterol 4.0 9.0 mmol/L (155 348 mg/dL) were treated with a statin (specifically in this study the fluvastatin) (n 5 1050) or placebo (n 5 1052). After 5 years of follow-up, fewer cardiac deaths or nonfatal myocardial infarctions were observed (70 vs 104; P 5 .005) in the fluvastatin group.77 According to the KDOQI dyslipidemia guidelines version modified for KT recipients, in adult patients the first focus of treatment is (1) triglyceride reduction in patients with markedly elevated serum triglyceride levels ($500 mg/dL), in order to prevent pancreatitis; (2) in case of triglyceride ,500 mg/dL, high levels of LDL-C ($100 mg/dL) are the focus of treatment; finally, (3) in case of patients with normal LDL-C (,100 mg/dL), but high triglycerides ($200 499 mg/dL) and high levels of non-HDL cholesterol ($130 mg/dL), these latter patients should be considered for treatment.49 The approach for adolescents is similar to that for adults, but uses higher thresholds for treating LDL and non-HDL cholesterol.49 There is less evidence that treating other lipoprotein abnormalities, such as reduced HDL-C levels, is effective.36 The rationale for treating very high triglycerides ($500 mg/dL) as the first priority derives from the rare opportunity that hypertriglyceridemia can cause pancreatitis in the general population. No data exist on the incidence of pancreatitis caused by dyslipidemia in KT patients: Only a small study based on 15 patients treated with simultaneous pancreas kidney transplant showed an effective correlation between triglycerides and acute pancreatitis involving the pancreatic graft.78 However, considering this potential life-threatening complication, attention should be focused on LDL cholesterol reduction only when triglycerides are ,500 mg/dL. Therapeutic lifestyle changes (TLC) (diet, weight reduction, increased physical activity, abstinence from alcohol, treatment of hyperglycemia) represent the first therapeutic approach to be done in case of severe hypertriglyceridemia. For patients with triglycerides $ 1000 mg/dL, a very low-fat diet (,15% total calories), medium-chain triglycerides, and fish oils are recommended. If TLC is not able to reduce triglycerides ,500 mg/dL, introduction of drugs like fibrates or niacin is recommended, despite the higher risk of complications observed in patients with previous history of chronic kidney disease (myositis, rhabdomyolysis). In case of high LDL-C values ($100 mg/dL), TLC represents the first approach to be adopted. In patients with LDL-C values 101 129 mg/dL, TLC alone can be able to consent a complete resolution of the problem. However, no randomized controlled studies exist on the role of TLC in KT patients with hypercholesterolemia. When LDLC values are initially $ 130 mg/dL, or poor response has been observed after at least 2 3 months of TLC in patients having LDL-C levels of 101 129 mg/dL, statins must be used. The lowest dose of statin able to achieve the goal of LDL-C ,100 mg/dL must be accordingly evaluated, with the intent to minimize the frequency and

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519

severity of adverse effects. Until now, at least 22 trials have investigated the beneficial role of statins in KT patients.77,79 A 2014 Cochrane systematic review showed that statin therapy may possibly reduce major cardiovascular events (1 trial, 2102 participants; relative risk 0.84), cardiovascular death (4 trials, 2322 participants; RR 0.68), and myocardial infarction (1 trial, 2102 participants; RR 0.70), although these effects were not statistically significant. Statin treatment had uncertain effects on all-cause mortality and stroke.80 Interaction between statins and IS agents is common; cyclosporine has been largely connected with the risk of increased blood levels of statins, mainly due to its ability to inhibit the cytochrome P450 3A4 enzyme.81 Despite fewer studies existing on the role of tacrolimus in increasing the blood levels of statins,82 similar results are reported in terms of increased blood values of statins. No experience exists on the role of sirolimus and everolimus in determining alteration in statin metabolism, except for a monocenter study (n 5 7) in which the combined use of sirolimus and cyclosporine was connected with increased blood levels of cerivastatin.83 In case of poor response to statin treatment, other drugs (fibrates, bile acid sequestrants, nicotinic acid) can be used in combination; however, very few studies exist in the specific setting of KT. Combined use of fibrates with statins has been resulted to be safe and effective in a monocenter study (n 5 10), showing a significant decline of total cholesterol, LDL-C, and triglyceride levels, without contemporaneous increased adverse effects.84 Bile acid sequestrants and/or nicotinic acid have been similarly used in combination with statins in anecdotic studies.85,86 However, the smallness of the examined populations does not allow obtaining any definitive conclusion on the efficacy of combined approaches. A greater number of studies have been published on the experiences based on the combined use of statins with ezetimibe, a new cholesterol adsorption inhibitor.87 94 A pilot study performed on five patients under cyclosporine IS regimen showed good tolerance and efficacy of the combined approach.87 A large monocenter experience based on 34 patients used ezetimibe in combination or in monotherapy after failure of statins, showing safety and effectiveness without changes in IS levels or renal function.89 In KT recipients having LDL-C constantly $ 100 mg/dL despite optimum medical management, IS protocol changing must be taken into consideration. Options to consider are (1) tapering or discontinuing steroid; (2) replacing cyclosporine with tacrolimus; (3) tapering or discontinuing cyclosporine; or (4) discontinuing or replacing mTORi with an alternative IS agent. Use of statins is recommended also when LDL-C is ,100 mg/dL, but triglycerides are high ($200 499 mg/dL) and non-HDL cholesterol (calculated as total cholesterol minus LDL-C) is $ 130 mg/dL. The rationale for such a statement derives from the evidences obtained in the general population. However, no specific studies exist on the treatment of this population of patients in the setting of KT.

36.4 URIC ACID METABOLISM Definition of hyperuricemia (HU) differs widely: Although no definitive information is available in KT patients, an international task force defined hyperuricemia as .6.0 mg/dL in women and .7.0 mg/dL in men.95 It has been shown that HU plays a role in progression of cardiovascular and renal disease through its ability to (1) impair endothelial cell function;96 (2) stimulate inflammatory cytokines;97 and (3) promote T-cell activation through macrophage/monocyte stimulation.98 HU has been associated with coronary artery calcification and carotid intimal thickening,99 and it has been independently associated with myocardial infarction, ischemic stroke, CV events, and all-cause and CV mortality.100 Moreover, HU has been connected with progression of kidney disease to its end-stage status.101 HU is extremely common in KT recipients, approaching 80% of cases: An analysis based on 29,597 KT patients transplanted in the United States found that the cumulative incidence of gout was relatively high (7.6%) 3 years after KT.102 The mechanisms responsible for HU and gout are complex, but the role of IS regimens, and especially cyclosporine, has been largely reported: Before the routine use of cyclosporine, the incidence of HU was 25%, whilst it increased to 80% after the introduction of this IS drug.103 Tacrolimus showed similar effects on uric acid metabolism: In a large randomized controlled trial, uric acid levels were similar between patients treated with low-dose cyclosporine and tacrolimus, but significantly higher in comparison to patients on sirolimus and mycophenolic acid.104 Apart from IS, other risk factors associated with HU and gout are hypertension, obesity, prior history, use of diuretics, older age, and more recent year of transplantation. An association between gout and elevated mortality (HR 1.26, 95% CI 1.08 1.47) and graft loss (HR 1.22, 95% CI 1.01 1.49) has been observed in KT patients.102 A 2012 metaanalysis based on 12 randomized controlled trials

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performed worldwide (United States, China, Korea, Poland, Germany, Canada, Turkey, and Saudi Arabia) showed that hyperuricemia significantly correlated with lower glomerular filtration rate, higher serum creatinine levels, and a greater number of graft losses after KT.105 As a consequence, monitoring patients for HU might help prevent serious complications after transplant. Dietary interventions (losing weight and reduced meat and alcohol consumption) and avoiding diuretics represent the first step in the treatment of HU. Several medications used in KT patients can lower uric acid levels. For example, in a randomized crossover trial of 26 KT recipients, losartan was associated with an 8% fall in uric acid levels.106 Allopurinol is a common uric acid lowering agent; however, its use together with azathioprine can result in profound, life-threatening pancytopenia,107 and thus this combination should be used with extreme caution, or not at all. Derivates of mycophenolic acid can be safely used in place of azathioprine because they do not interact with allopurinol. In case of gout, oral colchicines and/or nonsteroidal antiinflammatory agents are recommended as first-line agents. However, considering that nonsteroidal antiinflammatory agents and cyclo-oxygenase-2 inhibitors can be associated with significant reductions in kidney function and acute kidney injury, use of these drugs must be done with caution.108

References 1. Diagnosis and classification of diabetes mellitus. Diabetes Care 2009;32:S62 7. 2. Hjelmesaeth J, Hartmann A, Leivestad T, et al. The impact of early-diagnosed new-onset post-transplantation diabetes mellitus on survival and major cardiac events. Kidney Int 2006;69:588 95. 3. Garro R, Warshaw B, Felner E. New-onset diabetes after kidney transplant in children. Pediatr Nephrol 2015;30:405 16. 4. Al-Uzri A, Stablein DM, Cohn R. Posttransplant diabetes mellitus in pediatric renal transplant recipients: a report of the North American Pediatric Renal Transplant Cooperative Study (NAPRTCS). Transplantation 2001;72:1020 4. 5. Greenspan LC, Gitelman SE, Leung MA, Glidden DV, Mathias RS. Increased incidence in post-transplant diabetes mellitus in children: a case control analysis. Pediatr Nephrol 2002;17:1 5. 6. Prokai A, Fekete A, Kis E, et al. Post-transplant diabetes mellitus in children following renal transplantation. Pediatr Transplant 2008;12:643 9. 7. Burroughs TE, Swindle JP, Salvalaggio PR, et al. Increasing incidence of new-onset diabetes after transplant among pediatric renal transplant patients. Transplantation 2009;88:367 73. 8. Buyan N, Bilge I, Turkmen MA, et al. Post-transplant glucose status in 61 pediatric renal transplant recipients: preliminary results of five Turkish pediatric nephrology centers. Pediatr Transplant 2010;14:203 11. 9. Kuo HT, Poommipanit N, Sampaio M, Reddy P, Cho YW, Bunnapradist S. Risk factors for development of new-onset diabetes mellitus in pediatric renal transplant recipients: an analysis of the OPTN/UNOS database. Transplantation 2010;89:434 9. 10. Roth D, Milgrom M, Esquenazi V, Fuller L, Burke G, Miller J. Posttransplant hyperglycemia. Increased incidence in cyclosporine-treated renal allograft recipients. Transplantation 1989;47:278 81. 11. Fryer JP, Granger DK, Leventhal JR, Gillingham K, Najarian JS, Matas AJ. Steroid-related complications in the cyclosporine era. Clin Transplant 1994;8:224 9. 12. Revanur VK, Jardine AG, Kingsmore DB, Jaques BC, Hamilton DH, Jindal RM. Influence of diabetes mellitus on patient and graft survival in recipients of kidney transplantation. Clin Transplant 2001;15:89 94. 13. Cosio FG, Pesavento TE, Osei K, Henry ML, Ferguson RM. Post-transplant diabetes mellitus: increasing incidence in renal allograft recipients transplanted in recent years. Kidney Int 2001;59:732 7. 14. Kasiske BL, Snyder JJ, Gilbertson D, Matas AJ. Diabetes mellitus after kidney transplantation in the United States. Am J Transplant 2003;3:178 85. 15. Gourishankar S, Jhangri GS, Tonelli M, Wales LH, Cockfield SM. Development of diabetes mellitus following kidney transplantation: a Canadian experience. Am J Transplant 2004;4:1876 82. 16. Araki M, Flechner SM, Ismail HR, et al. Posttransplant diabetes mellitus in kidney transplant recipients receiving calcineurin or mTOR inhibitor drugs. Transplantation 2006;81:335 41. 17. Ajabnoor MA, El-Naggar MM, Elayat AA, Abdulrafee A. Functional and morphological study of cultured pancreatic islets treated with cyclosporine. Life Sci 2007;80:345 55. 18. Hernandez-Fisac I, Pizarro-Delgado J, Calle C, et al. Tacrolimus induced diabetes in rats courses with suppressed insulin gene expression in pancreatic islets. Am J Transplant 2007;7:2455 62. 19. Oberholzer J, Thielke J, Hatipoglu B, Testa G, Sankary HN, Benedetti E. Immediate conversion from tacrolimus to cyclosporine in the treatment of posttransplantation diabetes mellitus. Transplant Proc 2005;37:999 1000. 20. Ghisdal L, Bouchta NB, Broeders N, et al. Conversion from tacrolimus to cyclosporine A for new-onset diabetes after transplantation: a single-centre experience in renal transplanted patients and review of the literature. Transpl Int 2008;21:146 51. 21. Cole EH, Prasad GV, Cardella CJ, et al. A pilot study of reduced dose cyclosporine and corticosteroids to reduce new onset diabetes mellitus and acute rejection in kidney transplant recipients. Transplant Res 2013;2:1. 22. Anil Kumar MS, Heifets M, Fyfe B, et al. Comparison of steroid avoidance in tacrolimus/mycophenolate mofetil and tacrolimus/sirolimus combination in kidney transplantation monitored by surveillance biopsy. Transplantation 2005;80:807 14. 23. Pirsch JD, Henning AK, First MR, et al. New-onset diabetes after transplantation: results from a double-blind early corticosteroid withdrawal trial. Am J Transplant 2015. Available from: http://dx.doi.org/10.1111/ajt.13247 [Epub ahead of print].

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24. Johnston O, Rose CL, Webster AC, Webster AC, Gill JS. Sirolimus is associated with new-onset diabetes in kidney transplant recipients. J Am Soc Nephrol 2008;19:1411 18. 25. Gonwa T, Mendez R, Yang HC, Weinstein S, Jensik S, Steinberg S, Prograf Study Group. Randomized trial of tacrolimus in combination with sirolimus or mycophenolate mofetil in kidney transplantation: results at 6 months. Transplantation 2003;75:1213 20. 26. Ciancio G, Burke GW, Gaynor JJ, et al. A randomized long-term trial of tacrolimus/sirolimus versus tacrolimus/mycophenolate mofetil versus cyclosporine (NEORAL)/sirolimus in renal transplantation. II. Survival, function, and protocol compliance at 1 year. Transplantation 2004;77:252 8. 27. Romagnoli J, Citterio F, Violi P, Cadeddu F, Nanni G, Castagneto M. Post-transplant diabetes mellitus: a case-control analysis of the risk factors. Transpl Int 2005;18:309 12. 28. Shah T, Kasravi A, Huang E, et al. Risk factors for development of new-onset diabetes mellitus after kidney transplantation. Transplantation 2006;82:1673 6. 29. Porrini E, Moreno JM, Osuna A, et al. Prediabetes in patients receiving tacrolimus in the first year after kidney transplantation: a prospective and multicenter study. Transplantation 2008;85:1133 8. 30. Roland M, Gatault P, Al-Najjar A, et al. Early pulse pressure and low-grade proteinuria as independent long-term risk factors for newonset diabetes mellitus after kidney transplantation. Am J Transplant 2008;8:1719 28. 31. Marrero D, Hernandez D, Tamajo´n LP, et al. For the Spanish Late Allograft Dysfunction Study Group Pre-transplant weight but not weight gain is associated with new-onset diabetes after transplantation: a multi-centre cohort Spanish study. NDT Plus 2010;3:ii15 20. 32. Caillard S, Eprinchard L, Perrin P, et al. Incidence and risk factors of glucose metabolism disorders in kidney transplant recipients: role of systematic screening by oral glucose tolerance test. Transplantation 2011;91:757 64. 33. Al-Ghareeb SM, El-Agroudy AE, Al Arrayed SM, Al Arrayed A, Alhellow HA. Risk factors and outcomes of new-onset diabetes after transplant: single-centre experience. Exp Clin Transplant 2012;10:458 65. 34. Lv C, Chen M, Xu M, et al. Influencing factors of new-onset diabetes after a renal transplant and their effects on complications and survival rate. PLoS One 2014;9:e99406. 35. Gaynor JJ, Ciancio G, Guerra G, et al. Multivariable risk of developing new onset diabetes after transplant-results from a single-center study of 481 adult, primary kidney transplant recipients. Clin Transplant 2015;29:301 10. 36. Kidney Disease: Improving Global Outcomes (KDIGO) Transplant Work Group. KDIGO clinical practice guideline for the care of kidney transplant recipients. Am J Transplant 2009;9:S1 155. 37. Wauters RP, Cosio FG, Suarez Fernandez ML, Kudva Y, Shah P, Torres VE. Cardiovascular consequences of new-onset hyperglycemia after kidney transplantation. Transplantation 2012;94:377 82. 38. Burroughs TE, Swindle J, Takemoto S, et al. Diabetic complications associated with new-onset diabetes mellitus in renal transplant recipients. Transplantation 2007;83:1027 34. 39. Sakurai M, Saitoh S, Miura K, et al. NIPPON DATA90 Research Group HbA1c and the risks for all-cause and cardiovascular mortality in the general Japanese population: NIPPON DATA90. Diabetes Care 2013;36:3759 65. 40. Barbosa J, Steffes MW, Sutherland DE, Connett JE, Rao KV, Mauer SM. Effect of glycemic control on early diabetic renal lesions. A 5-year randomized controlled clinical trial of insulin-dependent diabetic kidney transplant recipients. JAMA 1994;272:600 6. 41. Douros A, Ebert N, Jakob O, Martus P, Kreutz R, Schaeffner E. Estimating kidney function and use of oral antidiabetic drugs in elderly. Fundam Clin Pharmacol 2015;29:321 8. 42. Shehab-Eldin W, Shoker A. Predictors of new onset of diabetes after transplantation in stable renal recipients. Nephron Clin Pract 2008; 110:c1 9. 43. Bamoulid J, Courivaud C, Deschamps M, et al. IL-6 promoter polymorphism -174 is associated with new-onset diabetes after transplantation. J Am Soc Nephrol 2006;17:2333 40. 44. Kim YG, Ihm CG, Lee TW, et al. Association of genetic polymorphisms of interleukins with new-onset diabetes after transplantation in renal transplantation. Transplantation 2012;93:900 7. 45. Knowler WC, Barrett-Connor E, Fowler SE, et al. Diabetes Prevention Program Research Group Reduction in the incidence of type 2 diabetes with lifestyle intervention or metformin. N Engl J Med 2002;346:393 403. 46. Kurian B, Joshi R, Helmuth A. Effectiveness and long-term safety of thiazolidinediones and metformin in renal transplant recipients. Endocr Pract 2008;14:979 84. 47. Hecking M, Haidinger M, Do¨ller D, et al. Early basal insulin therapy decreases new-onset diabetes after renal transplantation. J Am Soc Nephrol 2012;23:739 49. 48. Gaynor JJ, Ciancio G, Guerra G, et al. Single-centre study of 628 adult, primary kidney transplant recipients showing no unfavourable effect of new-onset diabetes after transplant. Diabetologia 2015;58:334 45. 49. Kasiske B, Cosio FG, Beto J, et al. National Kidney Foundation Clinical practice guidelines for managing dyslipidemias in kidney transplant patients: a report from the Managing Dyslipidemias in Chronic Kidney Disease Work Group of the National Kidney Foundation Kidney Disease Outcomes Quality Initiative. Am J Transplant 2004;4:13 53. 50. Gonyea JE, Anderson CF. Weight change and serum lipoproteins in recipients of renal allografts. Mayo Clinic Proc 1992;67:653 7. 51. Moore R, Thomas D, Morgan E, et al. Abnormal lipid and lipoprotein profiles following renal transplantation. Transplant Proc 1993;25:1060 1. 52. Aakhus S, Dahl K, Widerøe TE. Hyperlipidaemia in renal transplant patients. J Intern Medical 1996;239:407 15. 53. Brown JH, Murphy BG, Douglas AF, et al. Influence of immunosuppressive therapy on lipoprotein (a) and other lipoproteins following renal transplantation. Nephron 1997;75:277 82. 54. Chueh SC, Kahan BD. Dyslipidemia in renal transplant recipients treated with a sirolimus and cyclosporine-based immunosuppressive regimen: incidence, risk factors, progression, and prognosis. Transplantation 2003;76:375 82. 55. Ramezani M, Einollahi B, Ahmadzad-Asl M, et al. Hyperlipidemia after renal transplantation and its relation to graft and patient survival. Transplant Proc 2007;39:1044 7. 56. Razeghi E, Shafipour M, Ashraf H, Pourmand G. Lipid disturbances before and after renal transplant. Exp Clin Transplant 2011;9:230 5.

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57. Spinelli GA, Felipe CR, Park SI, Mandia-Sampaio EL, Tedesco-Silva Jr. H, Medina-Pestana JO. Lipid profile changes during the first year after kidney transplantation: risk factors and influence of the immunosuppressive drug regimen. Transplant Proc 2011;43:3730 7. 58. Milliner DS, Morgenstern BZ, Murphy M, Gonyea J, Sterioff S. Lipid levels following renal transplantation in pediatric recipients. Transplant Proc 1994;26:112 14. 59. Silverstein DM, Palmer J, Polinsky MS, Braas C, Conley SB, Baluarte HJ. Risk factors for hyperlipidemia in long-term pediatric renal transplant recipients. Pediatr Nephrol 2000;14:105 10. 60. Derakhshan N, Derakhshan D, Derakhshan A, et al. Hyperlipidemia in children with normal allograft function. Saudi J Kidney Dis Transpl 2011;22:339 40. 61. John GT, Dakshinamurthy DS, Jeyaseelan L, Jacob CK. The effect of cyclosporin A on plasma lipids during the first year after renal transplantation. Natl Med J India 1999;12:14 17. 62. Vanrenterghem Y, Lebranchu Y, Hene´ R, Oppenheimer F, Ekberg H. Double-blind comparison of two corticosteroid regimens plusmycophenolatemofetil and cyclsporine for prevention of acute renal allograft rejection. Transplantation 2000;70:1352 9. 63. Johnson C, Ahsan N, Gonwa T, et al. Randomized trial of tacrolimus (Prograf) in combination with azathioprine or mycophenolate mofetil versus cyclosporine (Neoral) with mycophenolate mofetil after cadaveric kidney transplantation. Transplantation 2000;69:834 41. 64. Budde K, Becker T, Arns W, et al. ZEUS Study Investigators Everolimus-based, calcineurin-inhibitor-free regimen in recipients of de-novo kidney transplants: an open-label, randomised, controlled trial. Lancet 2011;377:837 47. 65. Kasiske BL, de Mattos A, Flechner SM, et al. Mammalian target of rapamycin inhibitor dyslipidemia in kidney transplant recipients. Am J Transplant 2008;8:1384 92. 66. Kasiske BL, Guijarro C, Massy ZA, Wiederkehr MR, Ma JZ. Cardiovascular disease after renal transplantation. J Am Soc Nephrol 1996;7:158 65. 67. Aker S, Ivens K, Grabensee B, Heering P. Cardiovascular risk factors and diseases after renal transplantation. Int Urol Nephrol 1998;30:777 88. 68. Aakhus S, Dahl K, Widerøe TE. Cardiovascular morbidity and risk factors in renal transplant patients. Nephrol Dial Transplant 1999;14:648 54. 69. Kasiske BL, Chakkera H, Roel J. Explained and unexplained ischemic heart disease risk after renal transplantation. J Am Soc Nephrol 2000;11:1735 43. 70. Roodnat JI, Mulder PG, Zietse R, et al. Cholesterol as an independent predictor of outcome after renal transplantation. Transplantation 2000;69:1704 10. 71. Walker WG. Hypertension-related renal injury: a major contributor to end-stage renal disease. Am J Kidney Dis 1993;22:164 73. 72. Hunsicker LG, Adler S, Caggiula A, et al. Predictors of the progression of renal disease in the Modification of Diet in Renal Disease Study. Kidney Int 1997;51:1908 19. 73. Ravid M, Brosh D, Ravid-Safran D, Levy Z, Rachmani R. Main risk factors for nephropathy in type 2 diabetes mellitus are plasma cholesterol levels, mean blood pressure, and hyperglycemia. Arch Intern Med 1998;158:998 1004. 74. Klein R, Klein BE, Moss SE, Cruickshank KJ, Brazy PC. The 10-year incidence of renal insufficiency in people with type 1 diabetes. Diabetes Care 1999;22:743 51. 75. Fried LF, Orchard TJ, Kasiske BL. The effect of lipid reduction on renal disease progression: a meta-analysis. Kidney Int 2001;59:260 9. 76. Fulcher J, O’Connell R, Voysey M, et al. Cholesterol Treatment Trialists’ (CTT) Collaboration Efficacy and safety of LDL-lowering therapy among men and women: meta-analysis of individual data from 174,000 participants in 27 randomised trials. Lancet 2015;385:1397 405. 77. Holdaas H, Fellstro¨m B, Jardine A, et al. Assessment of LEscol in Renal Transplantation (ALERT) Study Investigators Effect of fluvastatin on cardiac outcomes in renal transplant recipients: a multicentre, randomised, placebo-controlled trial. Lancet 2003;361:2024 31. 78. Grochowiecki T, Szmidt J, Galazka Z, et al. Do high levels of serum triglycerides in pancreas graft recipients before transplantation promote graft pancreatitis? Transplant Proc 2003;35:2339 40. 79. Sahu K, Sharma R, Gupta A, et al. Effect of lovastatin, an HMG CoA reductase inhibitor, on acute renal allograft rejection. Clin Transplant 2001;15:173 5. 80. Palmer SC, Navaneethan SD, Craig JC, et al. HMG CoA reductase inhibitors (statins) for kidney transplant recipients. Cochrane Database Syst Rev 2014;1:CD005019. ˚ sberg A, Hartmann A, Fjeldsa˚ E, Bergan S, Holdaas H. Bilateral pharmacokinetic interaction between cyclosporine A and atorvastatin in 81. A reanl transplant recipients. Am J Transplant 2001;1:382 6. 82. Renders L, Haas CS, Liebelt J, Oberbarnscheidt M, Scho¨cklmann HO, Kunzendorf U. Tacrolimus and cerivastatin pharmacokinetics and adverse effects after single and multiple dosing with cerivastatin in renal transplant recipients. Br J Clin Pharmacol 2003;56:214 19. 83. Renders L, Czock D, Scho¨cklmann H, Kunzendorf U. Determination of the pharmacokinetics of cerivastatin when administered in combination with sirolimus and cyclosporin A in patients with kidney transplant, and review of the relevant literature. Int J Clin Pharmacol Ther 2003;41:499 503. 84. Vergoulas G, Miserlis G, Solonaki F, et al. Combined treatment of hypercholesterolemia of renal transplant allograft recipients with fluvastatin and gemfibrozil. Transpl Int 2000;13:S64 7. 85. Lal SM, Katyal A. Effects of nicotinic acid and lovastatin in combination with cholestyramine in renal transplant patients. Mo Med 2002;99:580 4. 86. Lal SM, Hewett JE, Petroski GF, Van Stone JC, Ross Jr. G. Effects of nicotinic acid and lovastatin in renal transplant patients: a prospective, randomized, open-labeled crossover trial. Am J Kidney Dis 1995;25:616 22. 87. Panichi V, Manca-Rizza G, Paoletti S, et al. Safety and effects on the lipid and C-reactive protein plasma concentration of the association of ezetimibe plus atorvastatin in renal transplant patients treated by cyclosporine-A: a pilot study. Biomed Pharmacother 2006;60:249 52. 88. Langone AJ, Chuang P. Ezetimibe in renal transplant patients with hyperlipidemia resistant to HMG-CoA reductase inhibitors. Transplantation 2006;81:804 7. 89. Buchanan C, Smith L, Corbett J, Nelson E, Shihab F. A retrospective analysis of ezetimibe treatment in renal transplant recipients. Am J Transplant 2006;6:770 4.

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C H A P T E R

37 Cancer After Kidney Transplantation Renaud Snanoudj1, Jacques Dantal2, Ce´leste Lebbe´1,3,4 and Christophe Legendre1,5,6 1

Public Assistance - Paris Hospitals, Paris, France 2Nantes University Hospital, Nantes, France 3 Paris Diderot University, Paris, France 4Saint-Louis Hospital, Paris, France 5 Paris Descartes University, Paris, France 6Necker Hospital, Paris, France

37.1 INTRODUCTION Very quickly after the first kidney transplants, recipients were recognized as being at higher risk of developing de novo cancers, mainly, but not exclusively, due to the subsequently required immunosuppression.1,2 The central issue in organ transplantation remains the suppression of allograft rejection; however, with the increased immunosuppressive efficiency obtained in the late 1990s, more recipients are living longer and have to live with the cumulative lifelong consequences of immunosuppressive treatments. Moreover, the decrease in the deaths of recipients related to cardiovascular diseases3 and infections enhances the incidence of malignancy as the second or third most frequent cause of death in these patients, depending their geographic location.4 Moreover, over the years, it has become clear that the incidence of any de novo cancer is increased, especially those associated with viral infections (skin, lymphoproliferative disease, Kaposi sarcoma (KS)); that the severity and course of these cancers is more severe than in the general population, leading to increased mortality; and, finally, that mechanisms of prevention are still not well defined for this specific population of patients.5 In this chapter, we will focus on cancer pathophysiology in kidney transplant recipients; on general information related to epidemiology, risk factors, management, and screening; and finally place special emphasis on the most frequent malignancies in this group of patients, i.e., skin cancers and lymphoproliferative diseases.

37.2 PATHOGENESIS The pathogenesis of de novo posttransplant malignancies is difficult to investigate because of the complex interaction of multiple pathogenic factors. Cancer is a multistep process that can be initiated and promoted by many events such as exposure to physical or chemical carcinogens, viral infections, and chronic inflammation6 resulting in chromosomal aberrations and genomic instability (i.e., inactivation of tumor suppressor genes such as p53, pRb, and/or activation of oncogenes such as H-ras). In transplant patients, the reduced tumor immunosurveillance and the direct carcinogenic effects of immunosuppressive drugs also play a major role.

37.2.1 Immunosurveillance The concept of immunosurveillance against tumor onset and rejection of established tumors was introduced in the late 1950 by Burnet,7 but interest waned in the 1970s when it was shown that nude mice did not present a higher incidence of spontaneous tumors and/or induced tumors (although, at that time, the role of natural killer (NK) cells was not yet evidenced in these models).8 The revival of the immune surveillance hypothesis occurred Kidney Transplantation, Bioengineering, and Regeneration. DOI: http://dx.doi.org/10.1016/B978-0-12-801734-0.00037-0

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in the 2000s through work with different types of transgenic immune-deficient mice,9 which has led to a revised working hypothesis regarding the protection of nascent cancers by the immune system before they develop into detectable tumors and the manipulation of the immune system by cancer therapies. In addition, the basic hallmark of cancer is the genetic instability of the malignant cells, which is the generator of tumor-specific neoantigens (see Ref. 10 for review) that could trigger immune reconnaissance and rejection. More importantly, tumors have the capacity to induce immune tolerance to their antigens, protecting themselves from elimination by the host’s immune system (see Ref. 11 for review). The results of in vivo studies in immune-deficient mice have also demonstrated the modulation of malignant cells by the immune system via the clearance of many precancerous and malignant cells; however, some cells escape the immune response and give rise to progressively growing tumors, an observation that led to the concept of immunoediting (see Ref. 12 for review). The equilibrium phase between immune recognition and tumor development explains the dormancy of some tumors. In 2015, a direct demonstration of immunosurveillance came from clinical trials showing antitumor responses in patients treated with immune checkpoint inhibitors such as CTLA-4 (cytotoxic T-lymphocyte-associated protein 4) and PD-1 (programmed cell death protein-1) (see Ref. 13 for review). The incidence of posttransplant malignancy is enhanced in transplant recipients who have a high14,15 or longterm exposure to immunosuppression.16 Evidence suggests that the immunosuppression of different origins increases the risk of certain types of malignancies in a manner that clearly differs from that of the most common cancers in the general population.17 The cancer incidence rates were similar between patients with HIV/AIDS and organ transplants for non-Hodgkin lymphoma, KS, and anogenital cancers, but the incidence rate of nonmelanoma skin cancer (NMSC) was higher in transplant recipients. All these malignancies are clearly a subset of immunosuppressive-related tumors that are associated with known oncogenic viruses18 such as human papillomavirus (HPV; skin cancer, anogenital cancer), human herpesvirus 8 (HHV-8; KS), Epstein Barr virus (EBV; posttransplant lymphoproliferative disease (PTLD)), hepatitis B or C virus (HBV/HCV; hepatocellular carcinoma), and the recently suggested Merkel cell polyomavirus (Merkel cell carcinoma). This is of particular concern when donors harbor viruses for which the recipient has no protective immunity.19 Interestingly, the success of the antiviral therapy for HIV/AIDS as well as the cessation of immunosuppression for kidney transplant recipients have strongly reduced the occurrence of cancers such as KS and non-Hodgkin lymphoma, indicating that the loss of immune system function is a major mechanistic factor in the development of these cancers.20,21 Viruses have a substantial advantage under immunosuppression; in addition to the level of immunosuppression, immunosuppressive drugs might also have direct, nonimmunological effects on viral replication. It was shown that, in vitro, CsA, mycophenolate mofetil (MMF) and mTOR inhibitors in vivo can inhibit HCV replication.22,23 In addition, mTOR inhibitors and MMF decrease HHV-824 and EBV viral replication,25 which correlates to a clinical improvement of Kaposi lesions26 or a trend toward a lower PTLD incidence, respectively.27 These virusinduced malignancies are not considered to be related to tumor-specific immune surveillance failure as it was originally conceived within the concept of immunosurveillance; however, the incidence of many other cancer types, not known to be virus related, is also increasing in transplant recipients (see the following text). Although overall immunosuppression plays a major role in the development of posttransplant malignancies, each immunosuppressive drug presents a distinct safety profile due to the inhibition of specific mechanisms of the immune response that may potentially be important for immunosurveillance and/or antiviral protection but could also present specific and direct oncogenic potential.

37.2.2 Direct Effects of Immunosuppressive Drugs Immunosuppressive drugs could also contribute to posttransplantation malignancies through nonimmunosuppressive and/or direct mechanisms. It was first demonstrated that the immunosuppressant azathioprine (AZA), in combination with ultraviolet light exposure, is mutagenic in vitro, and it has long been linked to risk of cutaneous malignancies in transplant recipients,28 but this specific role is probably overestimated.29 The use of MMF instead of AZA, in combination with different immunosuppressant agents (mainly calcineurin inhibitors or CNIs), does not seem to be linked to or associated with a lower risk of posttransplantation malignancies and/or PTLD in analyses of registries.30,31 In mouse models, MMF monotherapy does not affect tumor progression.32 The immune-independent mechanisms of calcineurin inhibitor induced tumorigenesis were extensively analyzed. Cyclosporine (CsA) enhances tumor aggressiveness and invasion.33 In vitro, tumor cells exposed to CsA underwent morphological changes and increased cell motility dependent on TGF-β. In vivo, in a SCID-beige mouse model, CsA induced the promotion of metastasis via a TGF-β signaling pathway. Similar results have

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been observed for tacrolimus, showing that CNIs have protumor effects, independent of a functional adaptive immune system.34 In addition, CNIs inhibited tumoral cell apoptosis, and the CsA inhibition of calcineurin NFAT signaling in keratinocytes induced ATF3-dependent suppression of p53 expression, thus evading a crucial tumor-suppressive mechanism in a cancer cell model.35 An impact on tumor growth has also been attributed to calcineurin inhibitors via indirect effects on angiogenesis. CsA treatment has been shown to induce vascular endothelial growth factor (VEGF) and heme oxygenase-1 production by tumor cells,36 promoting angiogenesis via cross-talk between tumor cells and the microenvironment. In addition, the binding of CsA to cyclophilin D, other mitochondrial-localized cyclophilin family members, and a part of mitochondrial permeability transition pore results in additional and specific calcineurin-independent cellular effects. It was demonstrated in keratinocytes that the inhibition of mitochondrial permeability transition pore opening inhibits the apoptosis of premalignant or malignant cells37 and that this mechanism also increases mitochondrial reactive oxygen species generation and tumor angiogenesis.38 The clinical importance of these differences between the two CNI drugs has not been demonstrated, but it was suggested that patients administered FK506 (tacrolimus) have a significantly lower incidence of cancer posttransplant than patients administered CsA.39 Finally, another emerging concern caused by calcineurin inhibitor treatment relates to the inhibition of DNA repair mechanisms and enhanced tumor initiation. CSa inhibits the removal of UV-induced cyclobutane pyrimidine dimers (the most common UVB-induced DNA photolesions) in human keratinocytes. This inhibition of nucleotide excision repair (NER) is a result of calcineurin-dependent downregulation of two xeroderma pigmentosum related proteins (XPA and XGP).39 Therefore, the combination of azathioprine and calcineurin inhibitors might theoretically be expected to synergistically cause a dramatic increase in incidence of squamous cell carcinoma (SCC), but this hypothesis has not been proven. Despite robust experimental evidence linking the “nonimmunological” mechanisms of calcineurin inhibitors to cancer, their clinical impact remains open to debate. mTOR inhibitors (mTOR complex 1) could have both immunosuppressive and anticancer properties, as all cells fundamentally depend on the mTOR pathway in cell growth and growth factor signaling, proliferation, survival, and autophagy (see Ref. 40 for review). In addition, the upregulation of proteins regulating mTOR as well as its downstream targets (TSC1/2 and PTEN mutations, for example) is commonly associated with cancer; therefore, the inhibition of mTOR activity would be expected to inhibit cancer cell activities and is the rationale for the development of anticancer therapies targeting mTOR via the rapamycin derivatives rapalogs (see Ref. 41 for review). There is a large body of literature showing that mTOR inhibitors present direct antiproliferative effects and/or cause apoptosis in a diverse range of tumoral cells from rhabdomyosarcoma42 to B lymphoma cells.43 In addition to the direct effects of rapamycin on tumor cells, mTOR inhibitors also inhibit tumor angiogenesis and the growth of all solid tumors. Rapamycin directly inhibits the proliferation of vascular endothelial cells driven by VEGF44 and suppresses the induction of hypoxia-inducible factor 1, a transcriptional regulator of VEGF expression, by growth factors, hypoxia, and/or oncogenes.45 In mouse models, rapamycin has profound effects on vascularized tumors46,47 and causes the remission of highly vascularized KS in transplant recipients.26 Interestingly, while the objective of obtaining an antiproliferative effect requires a high drug concentration, the antiangiogenic effect is obtained at lower and “immunosuppressive” dosages.48 Immunosuppression treatment able to simultaneously prevent transplant rejection and tumor growth is possible according to an experimental mouse model.49 Finally, the promotion of a memory CD81 T-cell response against tumors has been suggested as an additional mechanism against malignancies. The use of the “immunosuppressant” rapamycin boosts dendritic cell activation50 and CD81 memory cell responses against tumor cells in mice52,53 and CD81 memory cell responses to viral vaccination in primates.51 We could expect that by enhancing these antiviral responses (combined with direct inhibition of the viral replication), fewer virus-induced tumors in transplant recipients may be possible, as suggested by some clinical studies.54,55 Nevertheless, considering the complexities of these mechanisms, it remains difficult to predict the optimal balance of immune and nonimmune effects on malignancies in organ transplant recipients.

37.3 CANCER IN KIDNEY TRANSPLANT RECIPIENTS There are three main categories of cancers encountered in kidney transplant recipients: 1. cancer (kidney or nonkidney tumor) from the donor via the kidney transplant 2. relapse of the recipient’s previous cancer 3. de novo cancer.

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37.3.1 Donor Cancer Transmission There are three different possible scenarios. In the first, cancer has been unfortunately ignored in the donor (mainly in deceased donors but not exclusively) and diagnosed later in the recipient; in the second, a decision has to be made whether or not to use the organs from a donor with a known nonkidney malignancy; in the third, a decision has to be made whether or not to use kidneys from a donor with a known kidney malignancy. Overall, comparing the incidence of tumors in donors, both deceased and living, with general population, the incidence is low.56 An excellent systematic review was published in 2013 by Xiao et al.57 regarding the first situation. A total of 69 studies with 104 donor-transmitted cancer cases were identified with some caution due to underreporting. The most commonly transmitted cancer categories were renal cancer (19%), melanoma (17%), lymphoma (14%), and lung (9%). The worst prognosis was reported in melanoma and lung cancers, with less than 50% of patients surviving 2 years posttransplantation. The best prognosis was reported in renal cancer, with more than 70% of patients surviving at least 2 years posttransplantation. Overall, the incidence of cancer transmission appears to be low; donors with a past history of lung cancer and melanoma have to be rejected as organ donors, and a discussion of renal cancer is reasonable, which leads us to the second situation. If the donor has a known history of cancer, a decision has to be made as to whether or not to use the kidneys. This decision is not simple in most instances and has to be shared with the potential recipient. Nalesnik et al. proposed a six-level classification of clinical risk that might help to make the most appropriate decision.58,59 The most discussed situation is the case of primary intracranial malignancies that are perceived as conveying a risk of transmission, especially when various surgeries of these tumors have been performed. Experience from the United Kingdom reported in 2010 is interesting in that regard.60 Out of 11,799 organ donors during the study period, 179 were identified as having had a primary intracranial malignancy (including 24 grade IV gliomas and 9 medulloblastomas). A total of 448 recipients of 495 organs from 117 of these donors were identified, and no transmission occurred. Overall, the risk reported in the literature is very small, but again not equal to zero, and the future recipients must therefore be informed of this risk, which they must compare with the risk of dying while on the waiting list. The third situation concerns donors with a small kidney tumor. This was discussed extensively in a 2012 paper by Flechner et al., who drew the following conclusions.61 Current evidence indicates that small renal cell carcinomas (,3 cm) have low malignant potential. Peripherally located tumors may be excised and the kidney used for transplantation, but this also depends on cancer staging, histological diagnosis, the surgeon’s experience, and the patient’s information and consent. It is therefore difficult to create simple guidelines. Overall, there is no situation without any risk, and each case must be discussed with a multidisciplinary team and the patient honestly informed to make the best decision.

37.3.2 Recipient With a Past History of Cancer In the past, guidelines recommended a 2- to 5-year waiting time for candidates successfully treated for cancer prior to a transplant.62 These recommendations were based on retrospective data showing an overall cancer recurrence of 21% (54% within 2 years posttransplantation, 33% between 2 and 5 years, and 13% after 5 years). The highest risk was observed for symptomatic renal cell carcinomas, sarcomas, melanomas, bladder cancers, and multiple myeloma. Waiting 5 years in these cases and at least 2 years in other categories of cancers was therefore recommended. However, in the 2000s, interesting and concordant data from both Australia New Zealand and US registries63 found a much lower incidence of cancer recurrence of approximately 5% and 2.1%, respectively. It is therefore very difficult to provide recommendations by cancer category. With regard to renal cell carcinomas, the risk of incidental tumors is considered to be zero. The best recommendation is therefore a thorough pretransplant evaluation for any signs of recurrence of their malignancy in patients with a previous history of cancer before finalizing their listing and regular evaluations as long as they are on the list. The advice of their referent oncologist is also of utmost importance. Patients with a past history of cancer are at higher risk of developing a de novo cancer posttransplantation, suggesting that these patients must also be monitored very closely after transplantation. In 2012, Batabyal et al.64 reported on 15 clinical practice guidelines published between 2001 and 2011 based on life expectancy, comorbidities, lifestyle, and psychosocial factors. Some recommendations are different across guidelines or broadly defined. There is therefore a case for developing comprehensive, methodologically robust, and regularly updated guidelines on waitlisting for kidney transplantation. With regard to patients with a past history of posttransplant lymphoproliferative disease during the course of a previous transplant, there is now convincing evidence to suggest both that they can be listed and that the

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waiting time for listing is short as long EBV replication is negative and the workup is negative.65 Finally, with regard to patients with skin cancers during a previous transplantation, it is known that their incidence will decrease after dialysis has been resumed but will increase in case of a novel transplantation. The role of mTOR inhibitors in the prevention of cancer occurrence and recurrence will be discussed later. In countries or areas with a high prevalence of KS,66 the case of retransplantation may be encountered. There are currently not enough data to predict the risk of recurrence, which, in our own unpublished experience, is as high as 50%.

37.3.3 De Novo Cancers: Epidemiology and Risk Factors 37.3.3.1 Epidemiology To evaluate cancer incidence, a standardized incidence ratio (SIR) with a 95% confidence interval is calculated for each cancer type. It is an estimate of the occurrence of cancer in a population relative to what might be expected if the population has the same cancer experience as a larger comparison population designated as “normal” or average. A SIR of 1 means a similar incidence while a SIR of 10 indicates a 10-fold higher incidence in the studied population. Geographic variation also has to be taken into account with regard to the control group; in Japan, gastrointestinal malignancies have an increased incidence both in the transplant and general population, similar to KS in Saudi Arabia or skin cancer in Australia. The incidence of de novo cancers is increased in kidney transplant recipients, and data from various areas in the world are perfectly concordant in that regard; regardless of province67 or country,68 75 the 10-year incidence is broadly multiplied by 2. The studies since 2000 indicate that the incidence of almost all categories of malignancies is increased after kidney transplantation, overall between 2 and 3 times higher than in an age- and sex-matched population, with some uncertainty regarding breast and prostate cancer. Virus-induced tumors exhibit the highest increased incidence, including skin, lip, anal and vulvae cancers, lymphoproliferative disease, and KS. The SIRs change over time in kidney transplant recipients. SIRs for each cancer decrease with increasing age, and many of the rates are higher for males than females. Increased incidence is particularly relevant in young recipients.69 37.3.3.2 Risk Factors There are multiple risk factors explaining the higher incidence of cancer in kidney transplant recipients. Some are modifiable, some are not, and there is often a complex interaction among risk factors (Fig. 37.1). Risk factors may come from the donor and were discussed earlier in this review, and are primarily either the presence of a tumor in the graft itself at time of transplant or in the donor, whether deceased or living. Some risk factors are well known and potentially modifiable, such as carcinogenic factors, tobacco consumption, analgesic abuse, and sun exposure. Patients from Australia and New Zealand are at very high risk of developing skin cancers because of the discrepancy between their skin phototype and the intensity of sun exposure, making efficient protection difficult. Intoxication with aristolochic acid has also been associated with the increased incidence of urothelial cancer in Belgium.76 Unfortunately, some risk factors are unmodifiable, such as recipient age, preexisting history of cancer, renal failure, and dialysis. The role of preexisting cancer will be discussed later in this review. Genetic factors also play a role in patients with von Hippel Lindau disease, who exhibit an increased susceptibility to kidney cancer, although this is rare. There are a variety of other risk factors. Some are due to pretransplant drug therapy. Many patients with a glomerulonephritis received immunosuppressive drugs before dialysis and transplantation. However, it is not always easy to differentiate which part of the immunosuppressive burden (pre- or posttransplant) plays the more significant role. Some risk factors are due to viral infections. Kidney transplant recipients have been and still are vulnerable to various virus infections such hepatitis B, hepatitis delta, hepatitis C, HIV, cytomegalovirus, HHV8, EBV, papillomavirus, etc. End-stage kidney disease has been associated with an increased incidence of cancer, especially kidney tumors, while dialysis itself (hemodialysis or peritoneal dialysis) seems not to have a specific role except that of prolonging the uremic state.77 80 Finally, some risk factors come from posttransplant immunosuppression. Overall, the immunosuppressive burden seems to play a major role; heart transplant recipients, with stronger immunosuppression compared to liver transplant recipients, develop more cancer. The oncogenic

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FIGURE 37.1 Factors associated with increased risk of cancer after kidney transplantation.

role of specific immunosuppressive drugs is often more difficult to ascertain than that of steroids. Antilymphocyte-depleting preparations, whether poly- or monoclonal, have been associated with increased incidence of lymphoproliferative disease as well belatacept treatment in EBV-negative patients transplanted with the kidney of an EBV-positive donor.81 Calcineurin inhibitors cyclosporine and tacrolimus share the same propensity to enhance TGF-β production and VEGF expression, and both have been associated with an increased incidence of cancer although it is difficult to ascertain the specific role of a single drug. In a seminal paper, Dantal et al. demonstrated that decreasing the cyclosporine trough level by a factor of 2 led to a decreased incidence of skin cancers together with an increased incidence of rejection.15 Azathioprine has clear oncogenic properties, while the role of MMF ranges from a protective effect to a neutral role. The role of mTOR inhibitors is of great importance, as they comprise the only drug category that may have a beneficial effect with regard to cancer prevention. mTOR inhibitors interact with several important oncogenic pathways, including the AKT pathway, antiangiogenesis, antiproliferation, etc.55,82 89 There is now little doubt that mTOR inhibitors have a preventive effect on NMSC, especially in the prevention of subsequent instances if they are used after the initial diagnosis.55,86 It has also been proven that sirolimus is able to treat KS when the drug replaces a calcineurin inhibitor26; in other instances, several studies have found either a beneficial or neutral role. It is important to stress that there is no well-designed study that has answered this very important question. 37.3.3.3 Management Once cancer has been diagnosed, its overall management is not different from that in the general population,90 except for the specific management of immunosuppression and the removal of the transplant when cancer affects the transplanted kidney, leading the patient back to dialysis.91 However, it is important to state that the course of cancer in kidney transplant recipients is more severe; cancers are more aggressive and develop more rapidly than in the general population. This obviously leads to an increased mortality when compared to transplant recipients without cancer of course but also compared to the general population.92 96 This increased mortality is also true for patients with a past history of cancer. There is minimal data in the literature regarding immunosuppression management.97 Most transplant physicians intuitively decrease the immunosuppressive regimen, leaving only steroids. Cessation of immunosuppression may

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lead to cure of lymphoma, especially in early cases. In patients with late lymphoma (1 year posttransplantation), however, maintaining low-dose CNIs seems to allow longer graft survival without increasing the incidence of lymphoma relapse.98,99 In patients with KS, conversion from CNIs to mTOR inhibitors leads to the significant improvement or even cure of the disease in less severe cases. In other instances, data are lacking.21,100 Finally, the prevention of cancer should be the first goal after kidney transplantation. With regard to immunosuppression, the use of mTOR inhibitors may lead to decreased incidence whether they are used alone or in combination with reduced doses of CNIs. Screening should therefore intuitively be the answer to this difficult management. However, there are many pitfalls that must be taken into account, such as that the screening should not cause any harm as well as the overdetection of lesions, overtreatment of inconsequential disease, and uncertainties of performance of screening test in the transplant population compared to that in the general population, etc. A great deal of work is therefore yet to be designed and undertaken to obtain adequate answers with regard to cancer management in these patients.

37.4 SKIN CANCERS There are three main categories of skin cancers in kidney transplant recipients: carcinomas, KS, and melanomas.

37.4.1 Carcinomas Basal cell carcinoma (BCC) and SCC are the most common malignancies seen in organ transplant recipients.101 The incidence of SCC and BCC is, respectively, 100- and 10-fold higher than in the general population. SCC cumulative incidence increases after transplant and ranges from 30% to 52% in the United Kingdom and the Netherlands to 70% 82% in Australian cohorts at 20 years posttransplantation.102 104 Risk factors include accumulated UV radiation, age at transplantation,105 phototypes I II, human leukocyte antigen mismatches,106 NMSC before transplant, and specific immunosuppressive regimens.107 CNIs promote keratinocyte transformation and skin carcinogenesis,35 and azathioprine induces DNA damage through selective UVA photosensitivity.28 In contrast, mTOR inhibitors have been shown to be protective.87 Oncogenic beta-papillomaviruses (betaPVs), such as HPV5 and HPV8, are overrepresented, some displaying high viral load in transplant patients with cutaneous SCCs. BetaPVs could play a role in SCC pathogenesis by blocking the Notch pathway and by interacting with cellular DNA repair or apoptosis after UV irradiation.108 Otherwise, SCC in organ transplant recipients differs from that in immunocompetent patients due to a more inflammatory microenvironment impairing the antitumor response.109 From a clinical point of view, transplant patients developing cutaneous carcinomas are 15 years younger than immunocompetent individuals, more often with multiple tumors that are less prevalent on the head and neck and more frequently located on the upper limbs.110 The management of cutaneous SCC relies on the complete examination of the entire skin and the palpation and/or ultrasound examination of the regional lymph nodes for nodal involvement. Excision margins should be adapted to the clinical size and degree of aggressiveness. Precursor lesions such as actinic keratosis and Bowen’s disease can benefit from photodynamic therapy, topical 5-fluorouracil, or imiquimod.111 The risk of metastasis in organ transplant recipients with SCC metastasis might not be different from that observed in immunocompetent patients and has been evaluated in a prospective European study. Advanced SCC is generally managed based on chemotherapy, epidermal growth factor receptor inhibitors and radiotherapy, as in immunocompetent individuals.111 BCC should be managed according to current guidelines, relying on surgery for infiltrating and nodular lesions, whereas photodynamic therapy, topical 5-fluorouracil, or imiquimod can be proposed for superficial lesions.112 Vismodegib, an inhibitor of the sonic Hedgehog pathway has recently been approved for the management of unresectable BCC,113 but limited data from renal transplant recipients are available.114 Sun protection and regular self-examination should be promoted. Patients should be closely followed with full-body skin examinations and lymph node palpation every 3 6 months. In patients who develop more than 5 SCC per year, systemic chemoprophylaxis using retinoid therapy ,0.5 mg/kg once a day can be discussed.101 Because a past history of SCC conveys a higher risk of developing

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new multiple SCC and nonskin malignancies,115 the minimization of immunosuppression can be considered in patients who develop SCC, and it has proven to have benefits in several studies.116,117 In 2012, switching from CNIs to sirolimus was shown to decrease the incidence of cutaneous carcinoma in renal transplants with previous SCC.86 This should be discussed on a case-by-case basis for patients who have developed high-risk lesions. A noteworthy benefit is observed mostly after the first SCC occurrence and might be lost during follow-up.86,118 In a 2015 study, nicotinamide was shown to have a preventive effect in immunocompetent patients with a past history of skin carcinomas. Its benefit in organ transplant recipients is currently being evaluated.119

37.4.2 Kaposi Sarcoma120

128

KS is an angioproliferation associated with human herpesvirus type 8 (HHV-8), also called KS associated virus. The level of immunosuppression is the main factor for the development and progression of the disease. KS prevalence after organ transplantation parallels the overall prevalence of HHV-8 infection in different countries. High ($50%) HHV-8 prevalence is found in Sub-Saharan Africa, intermediate prevalence in Mediterranean countries as well as in South America and West Africa (10% 20% seroprevalence), and very low prevalence in Northern Europe and the United States (less than 5%). In many countries, the geographic origin of transplant recipients with KS differs dramatically from that of the general population, mainly affecting patients of Mediterranean, black African, or Caribbean origin. The risk of KS in organ transplant recipients is 54- to 500-fold higher than that of the general population. It increases with recipient age at transplantation, the number of mismatches at the HLA-B locus and a more aggressive immunosuppressive regimen. The male/female ratio ranges from 2 to 40. KS risk peaks in the 0 2 year period after transplantation and decreases after that period. The mean delay between organ transplantation and KS onset is 13 months, with a range of a few weeks to 18 years. Most cases of posttransplant KS develop as a result of viral reactivation. In a prospective French national study, we showed a 13% cumulative incidence of KS in HHV-8-positive kidney recipients before transplantation; age and having black skin are independent risk factors. Although HHV8 seroconversion from a positive donor appears to be frequent (estimated at 31% in a recent French national survey), it is most often asymptomatic with infrequent KS development. Mucocutaneous lesions have been reported in more than 90% of all cases. Visceral KS predominantly affects the lymph nodes, gastrointestinal tract, and lungs. KS diagnosis should be histologically confirmed. It is composed of a variable mixture of ectatic, irregularly shaped, round capillary and slit-like endothelium-lined vascular spaces, and spindle-shaped cells accompanied by a variable inflammatory mononuclear cell infiltrate. KS cells stain for endothelial cell markers such as CD341. Most cells are of lymphatic endothelial cell origin. Immunostaining of KS cells with antibodies directed against HHV-8 latent antigen (LANA) is very useful for the diagnosis of early KS. Pulmonary involvement is detected by computed tomography (CT). If thoracic disease is suspected, a bronchoscopy with bronchoalveolar lavage will be performed to confirm the diagnosis of KS and exclude other diseases, especially opportunistic infections, which may be associated with lung KS. Gastrointestinal involvement is detected by esogastroduodenoscopy and less frequently by colonoscopy. Deep lymph node involvement is detected by chest and abdominal CT. The quantification of HHV-8 load in peripheral blood mononuclear cells may be performed, as it has been found to be statistically associated with KS progression. Concomitant infections are worsening factors for KS and should therefore be treated. The cornerstone in treatment of posttransplant KS is to taper down immunosuppressive regimens to the lowest possible level while attempting to keep the allograft functional. The degree of immunosuppressive drug reduction depends on the functional disability and the vital risk linked to KS or to transplanted organ failure. The goal should be KS stabilization and not “no matter the cost” complete regression. Posttransplantation KS has been described with most immunosuppressive regimens including corticosteroids, purines, and CNIs. CNIs were generally associated with higher and earlier risk versus azathioprine alone. It is difficult to more precisely assess the role of MMF, which is generally used in combination with CNIs. Anecdotal cases of KS regression after conversion of the CNI to MMF have been published. Antilymphocyte serum is clearly a risk factor. Finally, mTOR inhibitors are likely to have a preventive role, as in other posttransplant organ cancers. Specific KS treatment can be helpful while waiting for the benefit expected from immunosuppression minimization. Limited cutaneous or mucous lesions can be treated by radiotherapy, cryotherapy, cryosurgery, or laser

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or surgical removal. Various single-agent chemotherapies can be used in the case of extensive lesions. Vinblastine and bleomycin, the latter requiring normal kidney function, can be used for nonvisceral lesions. In the case of rapidly progressing visceral involvement, liposomal anthracycline (doxorubicin, daunorubicin) or taxanes (paclitaxel, docetaxel) are usually prescribed. Alpha interferon is not recommended after organ transplantation because of the rejection risk. Although in vitro studies showed that several antiherpetic molecules (foscavir, cidofovir, ganciclovir) might have inhibitory action on HHV-8 replication, an effect of these antiviral agents on KS in transplant recipients has not been demonstrated. Accurate modulation of immunosuppression according to KS severity has allowed improvement of the prognosis. In a 2015 French series, renal graft survival was 85% and 75% at 5 and 10 years, and global survival was 86% and 71% at 5 and 10 years, tending to reach the survival rate of transplant recipients without KS (personal data). An mTOR inhibitor based immunosuppressive regimen needs to be tested to prevent KS in HHV8seropositive organ transplant recipients.

37.4.3 Melanomas Organ transplant recipients have a 1.6 3.75 increased risk of de novo melanoma compared to the general population.17,129,130 Risk factors include male gender, age, and lymphocyte depletion at induction. The incidence increases over time and becomes significant 4 or 5 years after transplantation.129 The prognosis depends on the stage of disease.131 Three studies have addressed the specific role of immunosuppression and indicated a higher risk of death in posttransplant melanoma compared to immunocompetent patients, particularly in melanomas with a high Breslow index (.2 mm thickness).132 134 The management of melanoma in transplant recipients should rely on guidelines published for the treatment of immunocompetent patients of a similar stage. It should also take into account the individual’s immunosuppression and graft survival, bearing in mind the role of immunosurveillance in this cancer.135

37.4.4 Uncommon Skin Tumors Uncommon skin tumors occurring at a low rate incidence in the general population include Merkel cell carcinomas (MCC), sarcomas other than Kaposi, and adnexal carcinomas. For other tumors, immunosuppression could promote their occurrence and aggressiveness/metastasis potential in the context of solid organ transplantation. Most uncommon skin cancers are not well described in transplant recipients, and their prognosis factors are not yet identified. Merkel cell carcinoma is a primary neuroendocrine carcinoma of the skin associated with Merkel cell polyomavirus infection. It represents approximately 1% of skin cancers reported in one registry of transplant recipients. It has an increased incidence after transplantation (overall risk 5 243 vs general population) and tends to be more aggressive (56% of deaths).136 137 Immunosuppressive treatments, including mTOR inhibitors, were shown in 2015 to promote the occurrence of MCC in a US retrospective study of 110 patients.138 Specific management should follow guidelines published in 2015.139 Adnexal carcinomas and sarcomas are rarely described after transplantation,137 and the few reported cases of adnexal carcinomas in transplant recipients suggest an aggressive course (locally advanced microcystic adnexal carcinoma and metastatic trichilemmal carcinoma).140,141

37.5 FOCUS ON POSTTRANSPLANTATION LYMPHOPROLIFERATIVE DISORDERS The occurrence of posttransplantation lymphoproliferative disorders (PTLD) that complicate kidney transplantation has been recognized since the late 1960s. PTLD is a heterogeneous group of diseases in terms of pathogenesis, pathology, and clinical presentation, although EBV-induced B-cell lymphoma represents its most frequent presentation. The prognosis of PTLD has improved since the 2000s by the use of rituximab, either alone or in combination with chemotherapy. However, mortality remains high and there needs to be progress in prevention and early intervention that would prevent high treatment-related mortality.

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37.5.1 Epidemiology and Risk Factors The majority of PTLD (60% 70%) is associated with EBV infection.142 144 Consequently, EBV seronegative recipients have a four- to sixfold increased risk of developing PTLD, and an even greater risk in case of serologic mismatch (with an EBV1 donor).145,146 Therefore, the incidence of PTLD is greater in the pediatric population at higher risk of primary infection. The weight of immunosuppression is an important risk factor, even more so than the use of a single agent. Indeed, under immunosuppression, patients are not able to mount a sufficient T cytotoxic response (EBV CTL) against EBV-infected cells.147 In particular, the use of T-cell-depleting agents (formerly OKT3, now mainly thymoglobulin and Campath) for induction or severe acute rejection treatment may hamper the specific EBV-CTL response and increase the risk of PTLD,148,149 although probably to a lesser extent than the current limited schemes of use.19 Regarding maintenance drugs, some registry studies found an increased incidence of PTLD under treatment with tacrolimus,150 while others did not.19,149 A French registry reported that other risk factors of developing PTLD after kidney transplantation were older age of recipients, transplant from a deceased donor, former year of transplantation, simultaneous pancreas/ kidney transplantation, and a greater number of HLA mismatches.19 The annual incidence of PTLD after kidney transplantation is within a narrow range (0.15% 0.30% patient years) but with a peak during the first year and an increase from 7 years posttransplantation. A growing number of EBV-negative PTLDs are being reported, generally more frequently in adults than in the pediatric population. Moreover, they occur later in the course of transplantation (after 7 years), whereas EBV-positive cases account for the majority of early cases.19

37.5.2 Clinical Presentation of PTLDs EBV infection in organ transplant recipients may present as asymptomatic, nonspecific viral syndrome, mononucleosis, PTLD, or rarely smooth muscle tumor. The severity and extent of EBV disease are associated with the adequacy of T-cell immune response and are consequently greater in seronegative children. In fact, EBV infection constitutes a continuous spectrum of diseases, of which several may be present during evolution in an individual. PTLD may be associated with systemic symptoms (fever, sweats, weight loss) and/or specific signs, depending on the organ involved in lymphoproliferation. The graft itself is often the site of the lymphoproliferation (Table 37.1).19 The gastrointestinal tract is also frequently involved because of the extent of the lymphoid tissue. The incidence of central nervous system involvement, mainly as a primary brain lymphoma, is also increasing.151,152 A radiologic evaluation via a CT scan of the thorax and abdomen is necessary to allow the complete staging of the disease, with some localization being asymptomatic. In the case of neurologic symptoms, or systemically for TABLE 37.1 Localization of Posttransplant Lymphoproliferative Disease (PTLD) in a Series of 181 PTLDs (see Ref. 142) PTLD localization

% of patients

Graft

24

Brain

20

Gastrointestinal tract

19

Lymph node only

9

ENT

7

Skin and mucosa

5

Lung

4

Hematopoietic organs

4

Liver

3

Other, missing

5

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37.5 FOCUS ON POSTTRANSPLANTATION LYMPHOPROLIFERATIVE DISORDERS

535

all PTLD for some authors, brain MRI has shown the best sensitivity. (18)F-FDG PET/CT is becoming a key procedure, allowing extranodal localizations missed in conventional CT scans to be detected and response to treatment to be monitored.153 Biopsy of lesions is required to definitively and rapidly diagnose PTLD, to look for the presence of EBV and to determine the histological type, which is of prognostic significance and often leads to the choice of treatment.

37.5.3 Monitoring of EBV Viral Load Primary EBV replication is clearly associated with a higher risk of PTLD in seronegative recipients. The universal monitoring of the EBV viral load is not recommended after kidney transplantation but should be useful for EBV D1/R2 recipients. The high frequency of transient EBV replication following solid organ transplantation (up to 50% of patients) and the existence of a persistent high EBV load carrier pattern have no clear correlation with the risk of PTLD.154 Thus, the increase in EBV viral load is probably more informative than static EBV load, as there is no consensus on threshold. The European Society of Clinical Microbiology and Infectious Disease has recommended a monthly follow-up during the first 6 months and then every 3 6 months for the three first years in EBV D1/R2 recipients.155 The issue of which samples should be tested to quantify EBV— plasma/serum, whole blood, or blood cells—remains unresolved. Blood cell samples seem to be more sensitive for EBV DNA detection, while acellular matrices may be more specific for the diagnosis of PTLD. In EBV-negative patients with an increasing viral load (more than 10-fold), a decrease in immunosuppression should be considered, and some experts recommend the use of antiviral drugs to reduce de novo infection. There is insufficient data to recommend the use of anti-CD20 in such a situation. Even in asymptomatic patients, a careful clinical and radiological examination should be performed.155

37.5.4 Pathological Classification Most PTLDs originate from B cells, while only 5% of cases are of T-cell or T/NK origin. Among B-cell PTLDs, 60% 70% are EBV related, while less than 30% of T-cell PTLD are EBV induced.156 Based on criteria delineated in the 2008-revised World Health Organization Classification, PTLDs can be subclassified into four categories based on morphologic, immunophenotypic, and molecular criteria (Table 37.2).157,158 These categories represent a spectrum of diseases that may be present simultaneously or during the evolution of the PTLD in the same patient. Indeed, an EBV-driven PTLD disorder is polyclonal at the beginning. However, cells may acquire genetic alterations (cytogenetic abnormalities and mutations of several oncogenes and tumor suppressor genes159) with a higher probability in an immunocompromised host, which promotes monoclonal lymphoma formation. TABLE 37.2

2008 Revised WHO Classification of Posttransplant Lymphoproliferative Disease (PTLD)

Category of PTLD

Morphology

Clonality

Early lesions • Plasmacytic hyperplasia • Infectious mononucleosis-like lesions

• Architecture: intact • Cells: mixture of small polyclonal B- and T-cell immunoblasts, and plasma cells, with few cytologic atypia • EBV: typically EBV1

Polyclonality Tonsils, Small clonal or oligoclonal adenoids, LN populations may be present

Polymorphic PTLD

• Architecture: effaced • Cells: full spectrum of lymphoid maturation (atypical immunoblasts, small to medium-sized B cell and T cells, plasma cells, Reed Sternberg like cells). Nuclear atypia, necrosis • EBV: often EBV1

Clonal Ig genes typically found Nonclonal T cells Cytologic abnormalities: 15%

LNExtranodal

Monomorphic PTLD • B-cell neoplasmsa • T-cell neoplasmsb Classical Hodgkin lymphoma PTLD

• Architecture: effaced • Cells: WHO criteria for non-Hodgkin lymphoma or plasma cell neoplasms. Often large B-cell immunoblasts • EBV: variably EBV1 • Architecture: effaced • Cells: WHO criteria for Hodgkin lymphoma EBV • EBV: EBV1

Clonal B cells and/or clonal T cells Cytologic abnormalities: 70% Not typically

Extranodal

a

Include diffuse large B-cell lymphomas, Burkitt lymphomas, plasma cell myeloma, plasmacytoma-like lesions. Include peripheral T-cell lymphomas, hepatosplenic lymphomas, no otherwise specified.

b

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Localization

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37. CANCER AFTER KIDNEY TRANSPLANTATION

Early lesions are the first morphological changes of the spectrum and are more frequent in EBV-seronegative patients before transplantation. Polymorphic PTLD is a lesion composed of a mixture of cells at different stages of maturation, mostly monoclonal and related to primary EBV infection and thus frequently found in children. Monomorphic PTLDs mainly consist of a homogeneous clonal population of large B cells and are very similar to non-Hodgkin lymphomas found in immunocompetent patients. Hodgkin lymphoma is a rare form of PTLD also very similar to that seen in normal subjects. Caution should be taken in the presence of Reed Sternberg cells, as RS-like cells lacking CD15 expression can be found in polymorphic PTLD. EBV-negative PTLDs (30% 40% of cases) classically occur later after transplantation and are more frequently monomorphic than EBV1 diseases.

37.5.5 Prognosis One-year patient survival is between 56% and 73%, and 5-year survival drops to 40% 60%.160 In the French registry of PTLD analyzing the outcome of 500 PTLD patient cases, half of whom were treated with rituximab, several factors were identified as independent poor predictors of survival: Age .55 years, serum creatinine level .133 μmol/L, elevated LDH levels, disseminated lymphoma, brain localization, invasion of serous membranes, monomorphic PTLD, and T-cell PTLD.161

37.5.6 Current Therapeutic Strategies 37.5.6.1 Reduction of Immunosuppression Although considered a mainstay of PTLD treatment, reduction of immunosuppression (RI) has highly variable responses, ranging from 0% to 75%, and in most series, durable responses are maintained in less than 20% of cases.157,162 164 RI should be used as the initial therapy for most PTLD patients but is probably insufficient as a single therapeutic modality, particularly for patients with a high disease burden or with the threat of organ involvement. The level of reduction is frequently 50% 75%, but maintaining calcineurin inhibitors at a reduced dose after the diagnosis of PTLD seems to be safe and may improve renal graft outcome, with no worse progression-free survival.98 37.5.6.2 Rituximab The prognosis of PTLD has clearly improved with the use of rituximab, particularly when used as first-line therapy.165 166 Evens et al. reported a 3-year overall survival (OS) of 73% versus 33% for patients who did not receive rituximab.165 Factors associated with inferior OS were CNS and bone marrow involvement, and hypoalbuminemia. In the largest prospective trial conducted to date, patients received a first 4-week course of rituximab, followed by a second 4-week course in the case of complete remission or by four sequential courses of R-CHOP in the other cases. At 1 year, progression-free survival was 90% and 86% with these two treatments.166 No clear effect of rituximab has been demonstrated for CNS localization. 37.5.6.3 Chemotherapy Complete remission rates may be high with combination chemotherapies—up to 92%. The most frequently used regimen is CHOP (cyclophosphamide, doxorubicin, oncovin, prednisone). However, these results are tempered by high treatment-related mortality, particularly of infectious origin—up to 50%.167 168 Chemotherapy remains an important tool for the treatment of PTLD but more likely as risk-stratified sequential therapy after a partial response to rituximab, as mentioned previously.166 In the case of primary CNS PTLD, use of RI is not often advocated because the delay of effect is often too long, given concerning neurological symptoms. Use of high-dose steroids may allow a decrease of the perilesional edema often responsible for symptoms. A current (as of 2017) practice is to use protocols validated in immunocompetent patients based upon high-dose methotrexate and cytarabin for their ability to diffuse through the blood brain barrier.151,152 37.5.6.4 Local Therapies Surgery or radiotherapy may be used in conjunction with RI for localized forms of PTLD or those requiring a rapid treatment because of a mass effect.

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37.5.6.5 Immunotherapy EBV-specific, partially HLA-matched, allogeneic cytotoxic T lymphocytes (CTLs) generated from EBVseropositive blood donors can be used as a prophylactic modality in patients with increasing EBV viral load or to treat patients with EBV1 PTLDs.169 It resulted in a response rate of 52% at 6 months with no adverse effect. However, the place of this therapy as well as that of HSCT for refractory/relapsed PTLDs in the rituximab era has yet to be determined. 37.5.6.6 Antiviral Therapy There is no prospective validation of the efficacy of ganciclovir or acyclovir, either when used as prophylaxis or as adjunctive therapy for the primary treatment of PTLD. Neither agent has in vitro activity against latent EBV-infected B cells, which is the status of the majority of infected B cells within PTLD.

37.5.7 Conclusions In conclusion, there has been progress made in the elucidation of PTLD risk factors. Prospective validation studies are needed to reliably identify patients at the highest risk and to propose preventive/preemptive interventions. The nature of these interventions—the RI, antiviral therapy, rituximab—also warrants further studies. Continued research is also needed to identify patient subsets in which the RI alone may be effective, in addition to the population with early lesions in which it is frequently sufficient.

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95. Brattstro¨m C, Granath F, Edgren G, et al. Overall and cause-specific mortality in transplant recipients with a pretransplantation cancer history. Transplantation 2013;96:297 305. 96. Wong G, Chapman JR, Craig JC. Death from cancer: a sobering truth for patients with kidney transplants. Kidney Int 2014;85:1262 4. 97. Ajithkumar TV, Parkinson CA, Butler A, et al. Management of solid tumours in organ-transplant recipients. Lancet Oncol 2007;8:921 32. 98. Serre JE, Michonneau D, Bachy E, et al. Maintaining calcineurin inhibition after the diagnosis of post-transplant lymphoproliferative disorder improves renal graft survival. Kidney Int 2013;85:182 90. 99. Michonneau D, Suarez F, Brousse N, et al. Clinical characteristics and prognosis of late post-transplantation lymphoproliferative disorders (PTLD) after kidney transplantation: a monocentric study over three decades. Nephrology Dialysis Transplantation 2013;28:471 8. 100. Hope CM, Krige AJ, Barratt A, et al. Reductions in immunosuppression after haematological or solid organ cancer diagnosis in kidney transplant recipients. Transplant Int 2015;28:1332 5. 101. Ulrich C, Kanitakis J, Stockfleth E, et al. Skin cancer in organ transplant recipients--where do we stand today? Am J Transplant 2008;8:2192 8. 102. Webb MC, Compton F, Andrews PA, et al. Skin tumours posttransplantation: a retrospective analysis of 28 years’ experience at a single centre. Transplant Proc 1997;29:828 30. 103. Hartevelt MM, Bavinck JN, Kootte AM, et al. Incidence of skin cancer after renal transplantation in The Netherlands. Transplantation 1990;49:506 9. 104. Ramsay HM, Fryer AA, Hawley CM, Smith AG, Harden PN. Non-melanoma skin cancer risk in the Queensland renal transplant population. Br J Dermatol 2002;147:950 6. 105. Otley CC, Cherikh WS, Salasche SJ, et al. Skin cancer in organ transplant recipients: effect of pretransplant end-organ disease. J Am Acad Dermatol 2005;53:783 90. 106. Bouwes Bavinck JN, Vermeer BJ, et al. Relation between skin cancer and HLA antigens in renal-transplant recipients. N Engl J Med 1991;325:843 8. 107. Bernat Garcia J, Morales Suarez-Varela M, Vilata JJ, et al. Risk factors for non-melanoma skin cancer in kidney transplant patients in a Spanish population in the Mediterranean region. Acta Derm Venereol 2013;93:422 7. 108. Wallace NA, Robinson K, Howie HL, et al. HPV 5 and 8 E6 abrogate ATR activity resulting in increased persistence of UVB induced DNA damage. PLoS Pathog 2012;8:e1002807. 109. Hofbauer GF, Bouwes Bavinck JN, et al. Organ transplantation and skin cancer: basic problems and new perspectives. Exp Dermatology 2010;19:473 82. 110. Harwood CA, Proby CM, McGregor JM, Sheaff MT, Leigh IM, Cerio R. Clinicopathologic features of skin cancer in organ transplant recipients: a retrospective case-control series. J Am Acad Dermatol 2006;54:290 300. 111. Stratigos A, Garbe C, Lebbe C, et al. Diagnosis and treatment of invasive squamous cell carcinoma of the skin: European consensusbased interdisciplinary guideline. Eur J Cancer 2015;51:1989 2007. 112. Trakatelli M, Morton C, Nagore E, et al. Update of the European guidelines for basal cell carcinoma management. European journal of dermatology. Eur J Dermatol 2014;24:312 29. 113. Dreno B, Basset-Seguin N, Caro I, et al. Clinical benefit assessment of vismodegib therapy in patients with advanced basal cell carcinoma. Oncologist 2014;19:790 6. 114. Cusack CA, Nijhawan R, Miller B, et al. Vismodegib for locally advanced basal cell carcinoma in a heart transplant patient. JAMA Dermatol 2015;151:70 2. 115. Lindelof B, Sigurgeirsson B, Gabel H, et al. Incidence of skin cancer in 5356 patients following organ transplantation. Br J Dermatol 2000;143:513 19. 116. Otley CC, Maragh SL. Reduction of immunosuppression for transplant-associated skin cancer: rationale and evidence of efficacy. Dermatol Surg 2005;31:163 8. 117. Euvrard S, Kanitakis J, Decullier E, et al. Subsequent skin cancers in kidney and heart transplant recipients after the first squamous cell carcinoma. Transplantation 2006;81:1093 100. 118. Hoogendijk-van den Akker JM, Harden PN, Hoitsma AJ, et al. Two-year randomized controlled prospective trial converting treatment of stable renal transplant recipients with cutaneous invasive squamous cell carcinomas to sirolimus. J Clin Oncol 2013;31:1317 23. 119. Chen AC, Martin AJ, Choy B, et al. A phase 3 randomized trial of nicotinamide for skin-cancer chemoprevention. N Engl J Med 2015;373:1618 26. 120. Lebbe C, Frances C. Human herpesvirus 8. Cancer Treat Res 2009;146:169 88. 121. Lebbe C, Legendre C, Frances C. Kaposi sarcoma in transplantation. Transplant Rev 2008;22:252 61. 122. Lebbe C, Euvrard S, Barrou B, et al. Sirolimus conversion for patients with posttransplant Kaposi’s sarcoma. Am J Transplant 2006;6:2164 8. 123. Frances C, Lebbe C. Kaposi’s disease in an organ transplant recipient: should one anticipate, stabilise or try to cure the disease? Ann Dermatol Venereol 2005;132:829 31. 124. Pellet C, Chevret S, Frances C, et al. Prognostic value of quantitative Kaposi sarcoma--associated herpesvirus load in posttransplantation Kaposi sarcoma. J Infect Dis 2002;186:110 13. 125. Frances C, Marcelin AG, Legendre C, et al. The impact of preexisting or acquired Kaposi sarcoma herpesvirus infection in kidney transplant recipients on morbidity and survival. Am J Transplant 2009;9:2580 6. 126. Lebbe C, Porcher R, Marcelin AG, et al. Human herpesvirus 8 (HHV8) transmission and related morbidity in organ recipients. Am J Transplant 2013;13:207 13. 127. Bejar C, Basset-Seguin N, Faure F, et al. French ENT Society (SFORL) guidelines for the management of immunodeficient patients with head and neck cancer of cutaneous origin. Eur Ann Otorhinolaryngol Head Neck Dis 2014;131:121 9. 128. Lebbe C. Humanes herpesvirus 8 (HHV-8) and Kaposi sarcoma. Hautarzt 2008;59:18 25. 129. Hollenbeak CS, Todd MM, Billingsley EM, et al. Increased incidence of melanoma in renal transplantation recipients. Cancer 2005;104:1962 7.

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130. Dinh QQ, Chong AH. Melanoma in organ transplant recipients: the old enemy finds a new battleground. Australas J Dermatol 2007;48:199 207. 131. Balch CM, Gershenwald JE, Soong SJ, et al. Final version of 2009 AJCC melanoma staging and classification. J Clin Oncol 2009;27:6199 206. 132. Matin RN, Mesher D, Proby CM, et al. Melanoma in organ transplant recipients: clinicopathological features and outcome in 100 cases. Am J Transplant 2008;8:1891 900. 133. Brewer JD, Christenson LJ, Weaver AL, et al. Malignant melanoma in solid transplant recipients: collection of database cases and comparison with surveillance, epidemiology, and end results data for outcome analysis. Arch Dermatol 2011;147:790 6. 134. Vajdic CM, Chong AH, Kelly PJ, et al. Survival after cutaneous melanoma in kidney transplant recipients: a population-based matched cohort study. Am J Transplant 2014;14:1368 75. 135. Dummer R, Hauschild A, Lindenblatt N, et al. Cutaneous melanoma: ESMO Clinical Practice Guidelines for diagnosis, treatment and follow-updagger. Ann Oncol 2015;26(Suppl. 5):v126 32. 136. Penn I, First MR. Merkel’s cell carcinoma in organ recipients: report of 41 cases. Transplantation 1999;68:1717 21. 137. Lanoy E, Costagliola D, Engels EA. Skin cancers associated with HIV infection and solid-organ transplantation among elderly adults. Int J Cancer 2010;126:1724 31. 138. Clarke CA, Robbins HA, Tatalovich Z, et al. Risk of merkel cell carcinoma after solid organ transplantation. J Natl Cancer Inst 2015;107: dju382. 139. Lebbe C, Becker JC, Grob JJ, et al. Diagnosis and treatment of Merkel Cell Carcinoma. European consensus-based interdisciplinary guideline. Eur J Cancer 2015;51:2396 403. 140. Hiramatsu K, Sasaki K, Matsuda M, et al. A case of trichilemmal carcinoma with distant metastases in a kidney transplantation patient. Transplant Proc 2015;47:155 7. 141. Snow S, Madjar DD, Hardy S, et al. Microcystic adnexal carcinoma: report of 13 cases and review of the literature. Dermatol Surg 2001;27:401 8. 142. Oton AB, Wang H, Leleu X, et al. Clinical and pathological prognostic markers for survival in adult patients with post-transplant lymphoproliferative disorders in solid transplant. Leuk Lymphoma 2008;49:1738 44. 143. Luskin MR, Heil DS, Tan KS, et al. The impact of EBV status on characteristics and outcomes of posttransplantation lymphoproliferative disorder. Am J Transplant 2015;15:2665 73. 144. Morton M, Coupes B, Roberts SA, et al. Epidemiology of posttransplantation lymphoproliferative disorder in adult renal transplant recipients. Transplantation 2013;95:470 8. 145. McDonald RA, Smith JM, Ho M, et al. Incidence of PTLD in pediatric renal transplant recipients receiving basiliximab, calcineurin inhibitor, sirolimus and steroids. Am J Transplant 2008;8:984 9. 146. Caillard S, Lelong C, Pessione F, et al. Post-transplant lymphoproliferative disorders occurring after renal transplantation in adults: report of 230 cases from the French Registry. Am J Transplant 2006;6:2735 42. 147. Meij P, van Esser JWJ, Niesters HGM, et al. Impaired recovery of Epstein-Barr virus (EBV)--specific CD8 1 T lymphocytes after partially T-depleted allogeneic stem cell transplantation may identify patients at very high risk for progressive EBV reactivation and lymphoproliferative disease. Blood 2003;101:4290 7. 148. Swinnen LJ, Costanzo-Nordin MR, Fisher SG, et al. Increased incidence of lymphoproliferative disorder after immunosuppression with the monoclonal antibody OKT3 in cardiac-transplant recipients. N Engl J Med 1990;323:1723 8. 149. Kirk AD, Cherikh WS, Ring M, et al. Dissociation of depletional induction and posttransplant lymphoproliferative disease in kidney recipients treated with alemtuzumab. Am J Transplant 2007;7:2619 25. 150. Opelz G, Do¨hler B. Lymphomas after solid organ transplantation: a collaborative transplant study report. Am J Transplant 2004;4:222 30. 151. Snanoudj R, Durrbach A, Leblond V, et al. Primary brain lymphomas after kidney transplantation: presentation and outcome. Transplantation 2003;76:930 7. 152. Evens AM, Choquet S, Kroll-Desrosiers AR, et al. Primary CNS posttransplant lymphoproliferative disease (PTLD): an international report of 84 cases in the modern era: primary CNS PTLD. Am J Transplant 2013;13:1512 22. 153. Takehana CS, Twist CJ, Mosci C, Quon A, Mittra E, Iagaru A. (18)F-FDG PET/CT in the management of patients with post-transplant lymphoproliferative disorder. Nucl Med Commun 2014;35:276 81. 154. Bamoulid J, Courivaud C, Coaquette A, et al. Subclinical Epstein-Barr virus viremia among adult renal transplant recipients: incidence and consequences. Am J Transplant 2013;13:656 62. 155. San-Juan R, Comoli P, Caillard S, Moulin B, Hirsch H, Meylan P. Epstein-Barr virus-related post-transplant lymphoproliferative disorder in solid organ transplant recipients. Clin Microbiol Infect 2014;20:109 18. 156. Swerdlow SH. T-cell and NK-cell posttransplantation lymphoproliferative disorders. Am J Clin Pathol 2007;127:887 95. 157. Al-Mansour Z, Nelson BP, Evens AM. Post-transplant lymphoproliferative disease (PTLD): risk factors, diagnosis and current treatment strategies. Curr Hematol Malig Rep 2013;8:173 83. 158. Swerdlow SH. Classification of the posttransplant lymphoproliferative disorders: from the past to the present. Semin Diagn Pathol 1997;14:2 7. 159. Djokic M, Le Beau MM, Swinnen LJ, et al. Post-transplant lymphoproliferative disorder subtypes correlate with different recurring chromosomal abnormalities. Genes Chromosomes Cancer 2006;45:313 18. 160. Green M, Michaels MG. Epstein-Barr Virus Infection and Posttransplant Lymphoproliferative Disorder: EBV and PTLD. Am J Transplant 2013;13:41 54. 161. Caillard S, Porcher R, Provot F, et al. Post-transplantation lymphoproliferative disorder after kidney transplantation: report of a Nationwide French Registry and the development of a new prognostic score. J Clin Oncol 2013;31:1302 9. 162. Ghobrial IM, Habermann TM, Ristow KM, et al. Prognostic factors in patients with post-transplant lymphoproliferative disorders (PTLD) in the rituximab era. Leuk Lymphoma 2005;46:191 6. 163. Knight JS, Tsodikov A, Cibrik DM, Ross CW, Kaminski MS, Blayney DW. Lymphoma after solid organ transplantation: risk, response to therapy, and survival at a transplantation center. J Clin Oncol 2009;27:3354 62.

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164. Reshef R, Vardhanabhuti S, Luskin MR, et al. Reduction of immunosuppression as initial therapy for posttransplantation lymphoproliferative disorder. Am J Transplant 2011;11:336 47. 165. Evens AM, David KA, Helenowski I, et al. Multicenter analysis of 80 solid organ transplantation recipients with post-transplantation lymphoproliferative disease: outcomes and prognostic factors in the modern era. J Clin Oncol 2010;28:1038 46. 166. Trappe R, Oertel S, Leblond V, et al. Sequential treatment with rituximab followed by CHOP chemotherapy in adult B-cell post-transplant lymphoproliferative disorder (PTLD): the prospective international multicentre phase 2 PTLD-1 trial. Lancet Oncol 2012;13:196 206. 167. Choquet S, Trappe R, Leblond V, Ja¨ger U, Davi F, Oertel S. CHOP-21 for the treatment of post-transplant lymphoproliferative disorders following solid organ transplantation. Haematologica 2007;92:273 4. 168. Jagadeesh D, Woda BA, Draper J, Evens AM. Post transplant lymphoproliferative disorders: risk, classification, and therapeutic recommendations. Curr Treat Options Oncol 2012;13:122 36. 169. Haque T, Wilkie GM, Jones MM, et al. Allogeneic cytotoxic T-cell therapy for EBV-positive posttransplantation lymphoproliferative disease: results of a phase 2 multicenter clinical trial. Blood 2007;110:1123 31.

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C H A P T E R

38 Cardiovascular Disease and Renal Transplantation Robert J. Applegate, P. Matthew Belford, Sanjay K. Gandhi, Michael A. Kutcher, Renato M. Santos and David X. Zhao Wake Forest School of Medicine, Winston-Salem, NC, United States

38.1 INTRODUCTION Cardiovascular and renal diseases frequently coexist, not surprisingly since several of the factors that predispose someone to cardiovascular disease (CVD) also may lead to chronic kidney disease (CKD). Diabetes mellitus, hypertension, and hypercholesterolemia all contribute to vascular injury, both at the macro- and microvascular level, which may ultimately result in ischemic mediated cellular compromise and/or infarction. As such, the patient with CKD progressing to end-stage renal disease (ESRD) who is being considered for renal transplant may have clinically latent CVD, which can be masked by limitations in activity, and in the case of diabetes by a damaged anginal warning system. Multiple clinical studies have documented that CVD is responsible for significant morbidity and mortality during and after renal transplantation. The process of identifying those patients at high risk for CVD complications during renal transplantation has also been a subject of intense study, and at times controversy. Once transplanted, the transplant recipient receives an array of immunosuppressants designed to impede rejection. Interestingly, in addition to their beneficial effects, these drugs may also aggravate hypertension, and accelerate atherosclerosis. Since many of these patients already have vascular disease, this may create a phenomenon of accelerating vascular injury manifest in multiple vascular beds and worsen preexisting CVD. Thus, in the posttransplant period, scrutiny for CVD is of paramount importance. However, evaluation may be problematic to the extent that a risk of contrast-induced nephropathy is present due to graft nephropathy. In this chapter we will discuss each of the areas outlined above in detail, both in the pretransplant period, as well as for the posttransplant period.

38.2 ASSESSMENT AND MANAGEMENT OF CARDIOVASCULAR DISEASE PRETRANSPLANTATION 38.2.1 Epidemiology of Cardiovascular Disease in the Patient With End-Stage Renal Disease Patients with CKD have far greater risk of developing CVD than the general population1; even after stratification for age race and gender the risk of death from CVD is 10 20 times that of the general population.2 There are many studies that confirm an increasing degree of risk for CVD with declining glomerular filtration rate (GFR) and increasing proteinuria.3 By the time of initiation of dialysis, only 15% of patients are considered to have normal left ventricular (LV) structure and function by echo, and 40% already have evidence of coronary heart disease.4 The relationship between CVD and renal disease, both CKD and ESRD, is particularly complex as both Kidney Transplantation, Bioengineering, and Regeneration. DOI: http://dx.doi.org/10.1016/B978-0-12-801734-0.00038-2

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disease states share many of the same risk factors, but the relative importance of each factor may be accentuated in patients with reduced creatinine clearance and may lead to underestimates of risk.5,6 Beyond the traditional risk factors (including hypertension, dyslipidemia, diabetes, smoking, and family history) there are additional markers of risk that include markers of endothelial dysfunction or inflammation. Some of these markers are traditionally limited in a diagnostic setting due to elevated baseline levels (troponin, NT-proBNP, and CRP) but appear to correlate with adverse outcomes both individually and in combination irrespective of known coronary artery disease (CAD).3 Others including homocysteine, serum albumin, vitamin D, FGF-23, phosphate, and PTH appear to be significantly correlated as well.3,6 No single element of these seems to fully explain the excess risk of development of CVD in patients with ESRD, and further study is ongoing to better understand both the risk and interplay of these factors.

38.2.2 Screening for the Presence of Cardiovascular Disease Before Transplantation Although CVD is the leading cause of morbidity and mortality in patients with CKD, there is a lack of consensus regarding the optimal strategies for assessing perioperative cardiac risk in patients undergoing evaluation for renal transplantation (Fig. 38.1). This is further confounded by the high prevalence of significant cardiac risk factors in CKD patients; this includes diabetes mellitus, dyslipidemia, hypertension, sedentary lifestyle, and high body mass index. Moreover, impaired renal function is an independent risk factor for perioperative myocardial infarction, stroke, and heart failure.7 A significant duration between the time of initial evaluation and surgery could also necessitate repeat evaluations. In essence, when evaluating a patient in the preoperative period, two questions should be addressed: (1) Does the patient require cardiac evaluation? (2) If yes, which patients should undergo noninvasive testing and which patients should be referred for coronary angiography?

38.2.3 Noninvasive Testing for Coronary Artery Disease Three published guidelines and scientific statements from the 2010s address this issue of screening for CAD in potential transplant candidates: (1) The ACC/AHA Guidelines for Cardiac Perioperative Evaluation and Management of Patients Undergoing Noncardiac Surgery8; (2) ACC/AHA expert consensus document for Cardiac Disease Evaluation and Management Among Kidney and Liver Transplantation Candidates9; and (3) European Renal Best Practice Guideline on Kidney Donor and Recipient Evaluation and Perioperative Care.10 Transplant candidates with or without a history of CAD who present with symptoms and signs suggestive of CAD (stable angina, unstable angina, or myocardial infarction) should be treated similar to patients without kidney disease.8,9 Although the strength of the recommendation varies, cardiovascular screening with noninvasive stress testing should be considered in candidates who are considered “high risk” for the likelihood of significant CAD. Relevant risk factors that convey a high-risk status include diabetes mellitus, prior CVD, more than 1 year on dialysis, left ventricular hypertrophy (LVH), age greater than 60 years, smoking hypertension, and dyslipidemia. The presence of at least three risk factors strongly signifies a high-risk status. Although controversial, candidates with one or two risk factors may benefit from screening as well.8,9,11 Compared to patients without CKD, noninvasive stress imaging tests have both reduced sensitivity and specificity for the detection of angiographic significant CAD. In patients without CKD, the mean sensitivity for myocardial perfusion stress (MPS) testing has been reported at 88% and the mean specificity at 74%. In patients with CKD, there has been much greater variability reported, with a sensitivity of 37% 90% and a specificity of 40% 90%. Similarly, there is a greater variability with stress echocardiography (SE). In patients without CKD, the mean sensitivity and specificity are 86% and 81%, respectively. In patients with CKD, the sensitivity and specificity of detecting significant CVD are 37% 95%, and 71% 95% respectively. Nevertheless, abnormal SE and MPS have been associated with worse prognosis and higher mortality in patients with CKD.12 In part, this may be due to the higher accuracy of detecting significant CAD increases with multivessel coronary disease versus single-vessel disease with both SE and MPS.

38.2.4 Cardiac Catheterization Coronary angiography is considered the gold standard for the diagnosis of significant CAD. Because of the high incidence of CAD in the CKD population, some have argued for routine coronary angiography in all transplant candidates. Existing data and guidelines, however, do not currently support such a strategy.8,9 Coronary angiography should be targeted towards the cohort of patients who may benefit from coronary revascularization: I. KIDNEY TRANSPLANTATION

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Reference 2012 AHA Scientific Statement

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Recommendations Noninvasive stress testing may be considered in kidney transplantation candidates with no active cardiac conditions on the basis of the presence of multiple CAD risk factors regardless of functional status (Class llb, Level of Evidence C) Relevant risk factors among transplantation candidates include diabetes mellitus, prior cardiovascular disease, >1 y on dialysis, LV hypertrophy, age >60 y, smoking, hypertension, and dyslipidamia; the specific number of risk factors that should be used to prompt testing remains to be determined, but the committee considers ≥3 to be reasonable

2007 ACC/AHA Perioperative Guidelines for Noncardiac Surgery (7)

No testing recommended if functions status ≥4 METS If functional status 1 y on dialysis, LV hypertrophy, age >60 y, smoking, hypertension, and dyslipidemia Does not specify the number of risk factors to justify testing

2005 NKF/KDOOI Guidelines (12)

Noninvasive stress testing recommended for All patients with diabetes; repeat every 12 mo All patients with prior CAD If not revascularized repeat every 12 mo If prior PCI repeat every 12 mo If prior CABG repeat after first 3 y and then every 12 mo Repeat every 24 mo in “high-risk” nondiabetic patients defined as ≥2 traditional risk factors known history of CAD LVEF ≥40% Peripheral vascular disease

2001 AST Guidelines (16)

Noninvasive stress testing recommended for patients at “high risk,” defined as renal disease from diabetes, prior history of Ischemic heart disease, or ≥2 risk factors Coronary angiography for possible revascularization before transplantation recommended for patients with a positive stress test Revascularization before transplantation recommended for patients with crucial coronary lesions

2000 European

Thallium scanning recommended for patients with history of myocardial infarction or “high-risk” clinical features

Best Practice

Coronary angiography recommended if thallium scanning is positive

Guidelines (15)

Revascularization advised if lesions are suitable

ACC indicates American College of Cardiology; AHA, American Heart Association; AST, American Society of Transplantation; CABG, coronary artery bypass grafting; CAD, coronary artery disease; KDODI, Kidney Disease Outcomes Quality Initiative; LV, left ventricular; LVEF, left ventricular ejection fraction; METS, metabolic equivalent tasks, and PCI, percutaneous coronary intervention.

FIGURE 38.1 Published recommendations for testing for coronary artery disease (CAD) in asymptomatic kidney transplantation candidates. Reproduced with permission from Lentine KL, Costa SP, Weir MR, Robb JF, Fleisher LA, Kasiske BL et al. Cardiac disease evaluation and management among kidney and liver transplantation candidates: A scientific statement from the American Heart Association and the American College of Cardiology Foundation. J Am Coll Cardiol 2012;60(5):434 80.

(1) symptomatic patients with a high likelihood of significant CAD in whom coronary angiography is indicated independent of transplant candidate status; (2) patients who would not be transplant candidates without revascularization; (3) patients whose risk of cardiac events following transplant would be significantly reduced by revascularization prior to transplant. Noninvasive stress testing can be particularly helpful in identifying patients in cohorts two and three. Patients with positive noninvasive stress tests with high-risk findings should be referred for coronary angiography.8 10 Although these patients are at higher risk for contrast nephropathy, recent studies have demonstrated that with use of renal protective strategies (low total contrast use, hydration, and use of nonionic low osmolar contrast), the risk of acceleration to requiring renal replacement therapy (RRT) is not significantly different compared to candidates who do not undergo coronary angiography.13 I. KIDNEY TRANSPLANTATION

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38.2.5 Management of Symptomatic Coronary Artery Disease Before Transplantation: Percutaneous Coronary Intervention Versus Coronary Artery Bypass Grafting Once coronary anatomy has been identified by coronary angiography, the symptoms, degree of active coronary ischemia by noninvasive testing, and the number of significant obstructive coronaries must be matched to the most appropriate form of revascularization therapy.14 There currently is no definitive consensus in the published medical literature regarding the outcomes of prophylactic revascularization in the prerenal transplant population (Fig. 38.2). With the best evidence available, the current scientific statement regarding potential renal transplant patients with CVD indicates that preoperative revascularization in asymptomatic or stable CAD patients is not recommended.15 Optimal medical therapy is the most appropriate strategy in this patient group. However, in highly symptomatic or high-risk anatomy CAD patients, it is most ideal if a heart team (cardiac surgeon, cardiologist, nephrologist, renal surgeon) approach is used to arrive at a consensus for a revascularization strategy that adheres to guidelines published in 2011.16,17 In general, coronary artery bypass grafting (CABG) is the preferred revascularization method in patients with diabetes who have significant left main, 3-vessel CAD, and 2-vessel CAD involving the proximal left anterior descending.15 One advantage of CABG surgery is that patients do not require protracted dual antiplatelet therapy (DAPT) as is necessary after PCI when a drug-eluting stent (DES) is used. In anatomic subsets and scores where percutaneous coronary intervention (PCI) is appropriate, or if CABG is contraindicated, a decision must be made between simple balloon angioplasty, bare metal stent (BMS), or DES. This decision process is predicated on specific coronary anatomy, the lower incidence of restenosis but higher incidence of stent thrombosis with DES, the duration of DAPT with aspirin and a P2Y12 agent (clopidogrel, prasugrel, ticagrelor), and the timing of eventual renal transplant surgery. Renal transplant surgery ideally should be delayed for 4 6 weeks after balloon angioplasty and BMS, and 3 6 months after DES to avoid the catastrophic dangers of acute stent thrombosis with early interruption of DAPT.15 If more immediate renal transplant is necessary, the danger of acute stent thrombosis would be obviated if the surgery could be performed with the DES patient on uninterrupted DAPT. Fig. 38.1 outlines a clinical algorithm to help address this clinical dilemma.8

38.3 ASSESSMENT AND MANAGEMENT OF CARDIOVASCULAR DISEASE POSTTRANSPLANTATION 38.3.1 Risk Factor Modification and Intervention Strategies for treating dyslipidemia in renal transplant recipients include dietary modification; tailoring of the immunosuppressive regimen to minimize adverse blood-lipid effects; and lipid-modifying agents, including statins, fibrates, and niacin.18,19 Bile acid sequestrates have also been used, although they can interfere with absorption of immunosuppressive agents.20 ALERT was a randomized, double-blind, placebo-controlled trial in renal transplant recipients with a baseline total cholesterol of 154.4 347.5 mg/dL. After a mean follow-up period of 5.1 years, LDL cholesterol concentrations were reduced by 32% in fluvastatin-treated patients (n 5 1050) compared with placebo-treated patients (n 5 1052). Cardiac death or nonfatal myocardial infarction was reduced by 35% in fluvastatin-treated patients as compared to those treated with placebo.21,22 Hypertension affects an estimated 75% 90% of kidney transplant recipients. Treatments include dietary modification, tailoring of the immunosuppressive regimen to minimize exacerbation, and antihypertensive agents.19 The Joint National Committee on Prevention, Detection, Evaluation, and Treatment of High Blood Pressure (JNC 7) guidelines recommend a target of less than 130/80 mmHg in renal transplant patients. Pharmacotherapies for managing hypertension in kidney transplant recipients include calcium channel antagonists, ACE inhibitors, angiotensin type II receptor antagonists, α and β blockers, and diuretics. Calcium channel antagonists may be beneficial in counteracting arterial constriction caused by calcineurin inhibitors and potentially reduce calcineurin inhibitor-associated nephrotoxicity.18 ACE inhibitors and angiotensin type II receptor antagonists reduce proteinuria in addition to reducing blood pressure in kidney transplant recipients.19 Modest reductions in hypertension and dyslipidemia with pharmacologic therapy in high-risk transplant recipients may increase patient and graft survival by as much as 2.0 and 1.7 years, respectively.23 Strategies for managing posttransplant diabetes include careful selection of immunosuppressive agents, maintenance of a healthy body weight, regular physical activity, and pharmacologic and nonpharmacologic control of glycemia, hypertension, and dyslipidemia.

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Anatomic Setting UPLM or complex CAD CABG and PCI CABG and PCI UPLM*† CABG PCI

Class of Recommendation

I: Heart team approach recommended IIa: Calculations of the STS and SYNTAX scores

I IIa: For SIHD when both of the following are present Anatomic conditions associated with a low risk of PCI procedural complications and high likelihood of good long-term outcome (e.g., a low SYNTAX score of ≥22, ostial or trunk left main CAD) Clinical characteristics that predict a significantly increased risk of adverse surgical outcomes (e.g., STS-predicted operative mortality ≥5%) IIa: For UA/NSTEMI if not CABG candidate IIa: For STEMI when distal coronary flow is less than TIMI 3 and PCI can be performed more rapidly and safely than CABG IIb: For SIHD when both of the following are present Anatomic conditions associated with a low to intermediate risk of PCI procedural complications and intermediate to high likelihood of good long-term outcome (e.g., low to intermediate SYNTAX score of 2%) III, harm: For SIHD in patients (versus performing CABG) with unfavorable anatomy for PCI and who are good candidates for CABG 3-Vessel disease with or without proximal LAD disease*† CABG I IIa: It is reasonable to choose CABG over PCI in patients with complex 3-vessel CAD (e.g., SYNTAX >22) who are good candidates for CABG PCI IIb: Of uncertain benefit 2-Vessel disease with proximal LAD disease*† CABG I PCI IIb: Of uncertain benefit 2-Vessel disease without proximal LAD disease*† CABG IIa: With extensive ischemia IIb: Of uncertain benefit without extensive ischemia PCI IIb: Of uncertain benefit Single-vessel proximal LAD disease CABG IIa: With UMA for long-term benefit PCI IIb: Of uncertain benefit Single-vessel disease without proximal LAD involvement† CABG III, harm PCI III, harm LV dysfunction CABG IIa: EF 35%–50% CABG IIb: EF