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Aus der Klinik für Allgemein-, Visceral- und Transplantationschirurgie der
Medizinischen Fakultät Charité – Universitätsmedizin Berlin



DISSERTATION


The urine proteome of kidney transplant patients: A new approach for
the identification of non-invasive rejection markers



zur Erlangung des akademischen Grades
Doctor medicinae (Dr. med.)








vorgelegt der Medizinischen Fakultät
Charité – Universitätsmedizin Berlin





von


Arne Füldner

aus Göttingen


































Gutachter/in: 1. Prof. Dr. med. J. Pratschke
2. Priv.-Doz. Dr. med. I. M. Sauer
3. Prof. Dr. St. Tullius





Datum der Promotion: 04.02.2011
Contents
1 Introduction 1
1.1 Kidney transplantation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1
1.2 Immunosuppression . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4
1.3 Graft rejection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6
1.4 Rejection diagnosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11
1.4.1 Serum creatinine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11
1.4.2 Glomerular filtration rate (GFR) . . . . . . . . . . . . . . . . . . . . 12
1.4.3 Serum albumin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12
1.4.4 Allograft biopsy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14
1.5 New rejection markers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15
1.5.1 Blood-derived markers . . . . . . . . . . . . . . . . . . . . . . . . . . 16
1.5.2 Urine-derived markers . . . . . . . . . . . . . . . . . . . . . . . . . . 16
2 Aims of the study 20
3 Materials and methods 22
3.1 Transplanted patients . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22
3.2 Urine collection and processing . . . . . . . . . . . . . . . . . . . . . . . . . 24
3.3 2-D electrophoresis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 24
3.3.1 Solubilisation and denaturation of the sample proteins . . . . . . . . 26
3.3.2 First dimensional separation by isoelectric focussing . . . . . . . . . . 28
3.3.3 Second dimensional separation by SDS-PAGE . . . . . . . . . . . . . 28
3.3.4 Gel staining . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 28
3.4 Image analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 29
IContents
3.5 Identification of proteins by MS . . . . . . . . . . . . . . . . . . . . . . . . . 30
3.5.1 In-gel digestion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 30
3.5.2 MALDI-TOF MS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 30
3.5.3 MS data analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 32
3.6 ELISA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 32
3.7 Used materials . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 33
4 Results 36
4.1 Establishment of the 2-DE technique: 12.5% vs. 15% vs. 18% gels . . . . . . 36
4.2 The 12% and 18% proteome maps of renal transplant patients . . . . . . . . 38
4.2.1 High-abundance proteins . . . . . . . . . . . . . . . . . . . . . . . . . 40
4.2.2 Low-abundance proteins . . . . . . . . . . . . . . . . . . . . . . . . . 42
4.2.3 Rejection-associated proteins . . . . . . . . . . . . . . . . . . . . . . . 45
4.3 Literature search . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 51
4.4 Profiles of candidate markers . . . . . . . . . . . . . . . . . . . . . . . . . . . 52
5 Discussion 57
5.1 The need for rejection markers . . . . . . . . . . . . . . . . . . . . . . . . . . 57
5.2 Non-invasive sources for markers . . . . . . . . . . . . . . . . . . . . . . . . . 58
5.3 2-DE MALDI-TOF MS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 58
5.4 Transplanted patients selection criteria . . . . . . . . . . . . . . . . . . . . . 60
5.5 The urine proteome map of renal transplant patients . . . . . . . . . . . . . 61
5.6 Rejection-associated proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . 63
5.7 Concluding remarks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 67
6 Summary 69
7 Zusammenfassung 70
A Protein lists 72
IIList of Tables
1 Characterization and terminology of transplantations . . . . . . . . . . . . . 2
2 Major Histocompatibility Complex (MHC) molecules and the HLA system . 3
3 BANFF classification for acute/active rejection of renal allografts . . . . . . 14
4 Clinical information of renal transplant patients analyzed in 12.5% gels . . . 24
5 Clinical information of renal transplant patients analyzed in 18% gels . . . . 25
6 Patient demographics of renal allograft recipients analyzed with ELISA . . . 26
8 Differentially expressed spots in the 12% proteome map . . . . . . . . . . . . 45
9 Differentially expressed spots in the 18% proteome map . . . . . . . . . . . . 49
10 Spot identifications in the proteome maps . . . . . . . . . . . . . . . . . . . 72
11 Keratin types identified and taken out of the list . . . . . . . . . . . . . . . . 85
IIIList of Figures
1 Most important co-stimulatory molecules on APC and their ligands on T cells 8
2 Intercellular adhesion molecules . . . . . . . . . . . . . . . . . . . . . . . . . 10
3 Two-dimensional electrophoresis gel from a renal transplant patient . . . . . 27
4 Schematic illustration of MALDI-TOF-MS . . . . . . . . . . . . . . . . . . . 31
5 Optimisation of 2-D electrophoretic gels . . . . . . . . . . . . . . . . . . . . 37
6 The 12% urine proteome map of renal transplant patients . . . . . . . . . . . 38
7 The 18% urine proteome map of renal transplant patients . . . . . . . . . . . 39
8 The composition of urine proteins . . . . . . . . . . . . . . . . . . . . . . . . 43
9 Individual spot expression kinetics of high-abundance proteins in a control
and a rejecting patient. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 53
10 Individual spot expression kinetics of rejection-associated proteins in a control
and a rejecting patient. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 54
11 Urinary concentration of osteopontin . . . . . . . . . . . . . . . . . . . . . . 55
12 Individual kinetics of urinary osteopontin concentration . . . . . . . . . . . . 56
IVList of abbreviations
2-DE ............ Two-dimensional Electrophoresis
ALG ............. Anti-Lymphocyte Globulin
AMBP ........... Alpha-1-Microglobulin/Bikunin Precursor
APC ............. Antigen-Presenting Cell
aRx .............. Acute Rejection
ATG ............. Anti-Thymocyte Globulin
BL ............... Borderline Rejection
CAN ............. Chronic Allograft Nephropathy
CE .............. Capillary Electrophoresis
CE-MS .......... Capillary Mass Spectrometry
CHAPS .......... 3-[(3-Cholamidopropyl)-Dimethylammonio]-1-Propane Sulfonate
CIT .............. Cold Ischemia Time
CMV ............ Cytomegalovirus
CsA ............. Cyclosporine A
DC .............. Dendritic Cells
DNA ............ Deoxyribonucleic Acid
DTT ............. Dithiothreitol
ELISA ........... Enzyme-linked Immunosorbent Assay
ESRD ........... End Stage Renal Disease
Eta-1 ............ Early T lymphocyte activation 1
FasL ............. Fas Ligand
FOXP3 .......... Forkhead Box P3
GFR ............. Glomerular Filtration Rate
HLA ............. Human Leucocyte Antigen
IAA ............. Iodoacetamide
ICAM-1 ......... Intercellular Adhesion Molecule 1
IEF .............. Isoelectric Focussing
IFN ............ Interferon gamma
IL-2 ............. Interleukin-2
kDa .............. kiloDalton
LFA-1 ........... Lymphocyte Function-associated Antigen 1
LOX-1 ........... Lectin-like Oxidized Low-Density Lipoprotein Receptor-1
MALDI-TOF MS Matrix-associatedLaserDesorptionIonizationTime-of-flightMassSpec-
trometry
MHC ............ Major Histocompatibility Complex
VList of abbreviations
MIG ............. Monokine Induced By IFN-gamma
mRNA ........... Messenger Ribonucleic Acid
MS .............. Mass Spectrometry
MW ............. Molecular Weight
NF-B ........... Nuclear Factor B
NGAL ........... Neutrophil-Gelatinase-Associated Lipocalin
OPN ............. Osteopontin
PBMC ........... Peripheral Blood Mononuclear Cells
PI-9 ............. Serine Proteinase Inhibitor-9
PMF ............ Peptide Mass Fingerprinting
RANTES ........ Regulated On Activation Normal T-cell Expressed And Secreted
RBP ............. Retinol-binding Protein
RNA ............. Ribonucleic Acid
RT-PCR ......... Reverse Transcriptase Polymerase Chain Reaction
SDS ............. Sodium Dodecyl Sulphate
SELDI-TOF-MS . Surface-enhanced Laser Desorption Ionization Time-of-flight Mass Spec-
trometry
SIBLING ........ Small Integrin-binding Ligand N-linked Glycoprotein
sICAM-1 ......... Soluble Intercellular Adhesion Molecule-1
sIL-2R ........... Soluble Interleukin-2 Receptor
TBM ............ Tubular Basement Membrane
TCR ............. T Cell Receptor
TEMED ......... Tetramethylethylendiamin
TLR ............. Toll-like Receptor
TNF and ..... Tumour Necrosis Factors and
Tris .............. Tris(hydroxymethyl)aminomethane
UTI ............. Urinary Tract Infection
VCAM-1 ......... Vascular Adhesion Molecule 1
VLA-4 ........... Very Late Antigen-4
VI

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