The role and clinical utility of acute kidney injury biomarkers in the setting of renal transplantation

217 Pages·2015·3.48 MB·English

by  Melanie Field

THE ROLE AND CLINICAL UTILITY OF ACUTE KIDNEY
INJURY BIOMARKERS IN THE SETTING OF RENAL
TRANSPLANTATION

by
Melanie Field

A thesis submitted to the University of Birmingham for the degree of Doctorate of
Medicine

Department of Infection and Immunity
School of Medicine
University of Birmingham Medical School
2014
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Abstract
Aim:
To evaluate the role and utility of acute kidney injury biomarkers in renal
transplantation.
Method:
Analysis of levels of a panel of biomarkers in deceased organ donors correlated to
renal graft outcome; ABO incompatible renal transplant recipients correlated to
rejection and HLA incompatible renal transplant correlated to rejection.
Results:
The panel of biomarkers showed comparable ability to creatinine in predicting the
outcome of the renal graft following transplant from deceased donors. Of the panel
of biomarkers tested to predict rejection, notably NGAL and IP-10 had good ability
to predict those HLAi recipients who subsequently developed rejection.
Conclusion:
Biomarkers previously identified in the context of AKI may have a role in the
assessment of deceased organ donor suitability but more promisingly have an
excellent ability to identify those patients at risk of rejection following HLAi
transplant and would now benefit from evaluation in a wider population prior to
adoption in a clinical trial.

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Dedication/acknowledgments
I would very much like to thank the Birmingham QE renal unit and the Queen
Elizabeth Hospital Charities for supporting this work. In particular my supervisors
Nick Inston and Mark Cobbold need thanks for their endless support and
enthusiasm for the project.
I would also like to thank my husband (David) and children (Ben and Darcey) for
their unending support and encouragement throughout this research.

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List of Abbreviations
AMR Antibody mediated rejection
ATN Acute tubular necrosis
CV Co-efficient of variation
DBD Donation after brainstem death
DCD Donation after circulatory death
DGF Delayed graft function
DSA Donor specific antibody
ELISA Enzyme-linked immunosorbant assay
HLA Human Leukocyte Antigen
IFN-γ Interferon-γ
IGF Intermediate graft function
IP-10 Interferon-inducible protein-10
KIM-1 Kidney injury molecule 1
MFI Median fluorescence intensity
MHC Major histocompatibility complex
NGAL Neutrophil Gelatinase Associated Lipocalin
ROC Receiver operator curve
TNF- α Tumour Necrosis Factor α
VEGF Vascular Endothelial Growth Factor

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Contents
Chapter 1: Introduction .............................................................................................. 1

1.1
  Historical Background to Renal Transplantation ........................................... 1

1.1.1
  Barriers to Transplantation ...................................................................... 2

1.1.2
  The Development of Immunosuppression .............................................. 2

1.2
  Success of Renal transplantation .................................................................. 3

1.3
  Classifying Allograft Rejection ....................................................................... 4

1.3.1
  Hyperacute and accelerated rejection .................................................... 4

1.3.2
  Acute rejection ........................................................................................ 5

1.3.3
  Chronic rejection ..................................................................................... 5

1.3.4
  Banff criteria ............................................................................................ 6

1.3.5
  Effect of Rejection on Outcome .............................................................. 7

1.4
  Determinants of outcome in kidney transplantation ...................................... 8

1.4.1
  Early vs. Late loss ................................................................................... 8

1.5
  Donor factors affecting outcome ................................................................. 10

1.5.1
  Sources of Organs for transplantation .................................................. 10

1.6
  The impact of donor type on graft survival .................................................. 14

1.7
  Delayed Graft Function (DGF) .................................................................... 15

1.7.1
  Definition of DGF .................................................................................. 15

1.7.2
  Pathogenesis of Delayed Graft Function (DGF) ................................... 15

1.7.3
  The impact of DGF ................................................................................ 17

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1.7.4
  Delayed Graft Function and rejection ................................................... 18

1.7.5
  Acute Kidney Injury in the donor and Delayed Graft Function .............. 18

1.7.6
  Predictors of DGF ................................................................................. 19

1.7.7
  Interventions for DGF ............................................................................ 20

1.7.8
  Machine perfusion ................................................................................. 21

1.8
  Transplantation across immunological barriers ........................................... 22

1.8.1
  ABO incompatible (ABOi) transplantation ............................................. 23

1.8.2
  HLA incompatible (HLAi) transplantation .............................................. 25

1.9
  Monitoring transplant function and biomarkers in current use ..................... 26

1.9.1
  Biochemical monitoring ......................................................................... 27

1.10
  Biomarkers in Disease Monitoring ............................................................. 30

1.10.1
  The Definition of a Biomarker ............................................................. 30

1.10.2
  Characteristics of Biomarkers ............................................................. 31

1.10.3
  The Evolution of Biomarkers ............................................................... 33

1.11
  Biomarker discovery and application ......................................................... 33

1.12
  Biomarkers in Renal Disease .................................................................... 34

1.12.1
  Kidney Injury Molecule – 1 (KIM-1) ..................................................... 36

1.12.2
  Neutrophil Gelatinase Associated Lipocalin (NGAL) .......................... 43

1.12.3
  Β-2-microglobulin (β2-M) .................................................................... 50

1.12.4
  Fractalkine .......................................................................................... 55

1.12.5
  Cystatin C ........................................................................................... 56

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1.12.6
  Interferon-inducible protein-10 (IP-10) ................................................ 59

1.12.7
  Vascular Endothelial Growth Factor (VEGF) ...................................... 61

1.12.8
  Interleukin-22 ...................................................................................... 62

1.12.9
  Tumour Necrosis Factor-α (TNF-α) ..................................................... 63

1.12.10
  Interferon-γ (IFN-γ) ........................................................................... 64

1.12.11
  Cathepsin L ....................................................................................... 65

1.12.12
  Immunoglobulin Light Chains ............................................................ 66

1.12.13
  Summary ........................................................................................... 66

2
  Chapter 2: Materials and Methods .................................................................... 69

2.1
  Developing an assay to measure the biomarkers ....................................... 69

2.2
  Principles of a Luminex™ assay ................................................................. 69

2.2.1
  Conjugation of proteins to microsphere beads ..................................... 70

2.2.2
  Antibodies used .................................................................................... 73

2.2.3
  Determining the concentration of secondary antibody .......................... 73

2.2.4
  Determining the protocol steps ............................................................. 75

2.2.5
  Altering the duration of the first incubation step .................................... 75

2.2.6
  Altering the duration of the second incubation step .............................. 76

2.2.7
  Determining the effect of one-step assay (removing wash steps) ........ 78

2.2.8
  Standardisation of optimised protocol ................................................... 79

2.2.9
  Exclusion of confounding factors .......................................................... 81

2.2.10
  Assay Variability .................................................................................. 85

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2.2.11
  Adapting the assay to add extra biomarkers ....................................... 86

2.2.12
  Trouble shooting the assay ................................................................. 87

2.2.13
  Assay Quality Control ......................................................................... 88

2.3
  Assessing KIM-1 levels using Lateral Flow device ...................................... 89

2.4
  Statistical analysis methods ........................................................................ 90

2.4.1
  T-test ..................................................................................................... 90

2.4.2
  Mann-Whitney ....................................................................................... 90

2.4.3
  Contingency table analysis ................................................................... 91

2.4.4
  Spearman rank correlation coefficient .................................................. 91

2.4.5
  Receiver operator curve (ROC) analysis .............................................. 91

2.4.6
  Binary logistic regression analysis ........................................................ 91

2.4.7
  Chi squared ........................................................................................... 92

2.5
  Analysis of Deceased Donor Data .............................................................. 92

2.5.1
  Initial Analysis ....................................................................................... 92

2.5.2
  Further Analysis .................................................................................... 92

2.6
  Analysis of Recipient Data .......................................................................... 93

2.7
  Sample collection for studies ....................................................................... 94

2.7.1
  Deceased organ donor samples ........................................................... 94

2.7.2
  Live organ donor samples ..................................................................... 94

2.7.3
  ABOi donor samples ............................................................................. 94

2.7.4
  HLA incompatible transplant samples ................................................... 94

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2.8
  Sample analysis .......................................................................................... 97

2.8.1
  Analysis of deceased samples .............................................................. 97

2.8.2
  Analysis of HLAi samples ..................................................................... 97

2.8.3
  Analysis of Renal Transplant Recipient samples .................................. 98

2.8.4
  Ethical approval .................................................................................... 98

3
  Chapter 3: Results ............................................................................................. 99

3.1
  Biomarkers in the assessment of deceased organ donors ......................... 99

3.1.1
  Study design ....................................................................................... 100

3.1.2
  Method of analysis .............................................................................. 101

3.1.3
  Donor demographics ........................................................................... 102

3.1.4
  Kidney Donation data .......................................................................... 102

3.1.5
  Recipient Demographics ..................................................................... 103

3.1.6
  Group analysis .................................................................................... 104

3.1.7
  Biomarker Panel Analysis ................................................................... 105

3.1.8
  Lateral Flow Assay .............................................................................. 109

3.1.9
  Binary logistic regression analysis ...................................................... 110

3.2
  Kidney biomarker changes in living organ donors ..................................... 114

3.2.1
  Study design ....................................................................................... 114

3.2.2
  Biomarker Panel Analysis from living donors ...................................... 115

3.3
  ABO incompatible transplants ................................................................... 119

3.3.1
  Study design ....................................................................................... 119

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