determination of antibodies against angiotensin ii …

DETERMINATION OF ANTIBODIES AGAINST

ANGIOTENSIN II TYPE 1 RECEPTOR (AT1R)

IN THAI KIDNEY TRANSPLANT PATIENTS

BY

MISS SUDARAT VIBOON

A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF

THE REQUIREMENTS FOR THE DEGREE OF THE

MASTER OF SCIENCE (BIOMEDICAL SCIENCES)

GRADUATE PROGRAM IN BIOMEDICAL SCIENCES

FACULTY OF ALLIED HEALTH SCIENCES

THAMMASAT UNIVERSITY

ACADEMIC YEAR 2017

COPYRIGHT OF THAMMASAT UNIVERSITY

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Thesis Title DETERMINATION OF ANTIBODIES

AGAINST ANGIOTENSIN II TYPE 1

RECEPTOR (AT1R) IN THAI KIDNEY

TRANSPLANT PATIENTS

Author Miss Sudarat Viboon

Degree Master of Science (Biomedical Sciences)

Major Field/Faculty/University Molecular Immunology and Molecular Biology

Faculty of Allied Health Sciences

Thammasat University

Thesis Advisor

Thesis Co-Advisor

Prof. Maj. Gen. Oytip Nathalang, Ph.D.

Asst. Prof. Natavudh Townamchai, M.D.

Academic Years 2017

ABSTRACT

Non-HLA antibodies specific to angiotensin II type 1 receptor (AT1R-Ab)

are associated with antibody mediated rejection after kidney transplantation. This study

aimed to determine the prevalence of AT1R-Ab among pretransplant Thai patients and

to compare the association of patient demographics to AT1R-Ab levels. This cohort

study enrolled non-sensitized kidney transplant patients with negative panel reactive

antibodies at the King Chulalongkorn Memorial Hospital, Bangkok, Thailand.

Additionally, 10 samples of unrelated healthy male blood donors at the National Blood

Centre, Thai Red Cross Society, Bangkok, Thailand were also included. AT1R-Ab

level was measured by enzyme-linked immunosorbent assay kit in all pretransplant

serum samples using a cutoff of >17.0 U/mL to distinguish AT1R-Ab (+) and AT1R-

Ab (–) groups. In all, 70 patients enrolled, of which 52 were males with a mean age of

45 (17-68 years). Six patients (8.6%) were positive for AT1R-Ab (>17.0 U/mL). The

mean age of AT1R-Ab (+) was significantly lower than AT1R-Ab (−) groups (p = 0.02,

t-test = 2.395, 95% CI = 2.008 - 22.086). Moreover, the AT1R-Ab (+) group had a

significant increase in those patients <30 years old (50.0% vs. 5.2%, p = 0.03, OR =

0.103, 95% CI = 0.017 - 0.631). No differences were found regarding sex, serum

creatinine level, sensitization history, hypertension, body mass index, ischemic time

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and number of HLA-mismatches between AT1R-Ab positive and negative groups. In

addition, AT1R-Ab (+) was also observed in one healthy blood donor with

hypertension. This study was the first to report the prevalence of AT1R-Ab in Thai

kidney transplant patients. Further studies are suggested for the application of AT1R-

Ab detection for routine testing.

Keywords: AT1R-Ab, Rejection, Transplantation, Thais

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ACKNOWLEDGEMENTS

I would like to express my deepest gratitude and appreciation to my major

advisor, Professor Major General Oytip Nathalang, Ph.D. for her kindness,

encouragement, worthy suggestions and constant support throughout the completion of

this study. I would like to express my deep appreciation to my co-advisor, Assistant

Professor Natavudh Townamchai, M.D., Division of Nephrology, Department of

Medicine, Faculty of Medicine, Chulalongkorn University and King Chulalongkorn

Memorial Hospital for his support of pre-transplant serum samples, patient medical

history, guidance, mentorship, invaluable advice and intellectual discussion.

I would like to acknowledge Associate Professor Sasitorn Bejrachandra,

M.D., National Blood Centre, Thai Red Cross Society for her kind acceptance to be the

chair of my thesis defense committee and Jeeraphong Thanongsaksrikul, Ph.D. Faculty

of Allied Health Sciences, Thammasat University for his kind acceptance to be the

member of my thesis defense committee.

I would like to thank Ms. Pawinee Kupatawintu and Mrs. Sirilak

Phiancharoen, National Blood Centre, Thai Red Cross Society for their support of blood

donor samples and research facilities.

I would like to acknowledge the Organ Donation Centre, Thai Red Cross

Society, which sponsor me for the whole period of my M.Sc.

I gratefully acknowledge financial supports from the Thammasat

University Research Fund for graduate program student.

I would like to extend my cordial and special thanks to seniors, friends and

all my colleagues for their wonderful friendships and invaluable assistance.

Finally, I would like to thank my family for their endless encouragement

and support throughout my time in graduate studies, without them this thesis would not

have been possible.

Miss Sudarat Viboon

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TABLE OF CONTENTS

Page

ABSTRACT (2)

ACKNOWLEDGEMENTS (4)

LIST OF TABLES (8)

LIST OF FIGURES (9)

LIST OF ABBREVIATIONS (10)

CHAPTER 1 INTRODUCTION 1

1.1 Sensitization stage 1

1.2 Effector stage 2

1.3 Hyperacute rejection 2

1.4 Acute rejection 3

1.4.1 Acute cellular rejection (ACR) 3

1.4.2 Acute antibody mediated rejection (AMR) 4

1.5 Chronic rejection 4

CHAPTER 2 OBJECTIVES 6

CHAPTER 3 REVIEW OF LITERATURES 7

3.1 Antibodies against red cell antigens 7

3.2 Antibodies against HLA antigens 8

3.3 Antibodies against non-HLA antigens 9

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TABLE OF CONTENTS (Cont.)

Page

3.3.1 Antibodies against MICA antigens 9

3.3.2 Antibodies against vimentin 10

3.3.3 Antibody against angiotensin II type 1 receptor (AT1R-Ab) 11

3.3.3.1 AT1R genes and structure 11

3.3.3.2 Role of AT1R-Ab in kidney transplantation 12

3.3.3.3 Role of AT1R-Ab in AMR 14

3.4 Current situation in cadaveric kidney transplants among Thai patients 15

CHAPTER 4 MATERIALS AND METHODS 16

4.1 Sample size calculation 16

4.2 Sample population 16

4.3 Sample collection 17

4.4 Study parameters 17

4.4.1 Risk factors for AT1R-Ab development 17

4.4.2 Risk factors for AMR 17

4.5 Determination of HLA antibodies 20

4.6 Determination of red cell antibodies 21

4.7 Determination of AT1R-Ab 21

4.8 Statistical analysis 23

CHAPTER 5 RESULTS 24

5.1 Patient characteristics and AT1R-Ab results 24

5.2 HLA antibodies and red cell antibodies results 28

5.3 Association of AT1R-Ab with kidney allograft rejection 28

5.4 AT1R-Ab and Patients with AMR 28

5.5 Donor Characteristics and AT1R-Ab results 30

CHAPTER 6 DISCUSSION 31

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TABLE OF CONTENTS (Cont.)

Page

CHAPTER 7 CONCLUSIONS 33

REFERENCES 34

APPENDICES 43

APPENDIX A 44

APPENDIX B 45

APPENDIX C 46

APPENDIX D 47

BIOGRAPHY 48

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LIST OF TABLES

Tables Page

3.1 AT1R-Ab in kidney transplantation 13

5.1 Patient demographics in AT1R-Ab (+) and AT1R-Ab (–) groups. 26

5.2 Characteristics and laboratory results of patients with AMR 29

in AT1R-Ab (+) and AT1R-Ab (-) groups

5.3 Donor characteristics and AT1R-Ab results 30

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LIST OF FIGURES

Figures Page

3.1 Structure of the AT1R 12

3.2 Model for AT1R-Ab related signaling in vascular cells 14

4.1 A conceptual framework for data evaluation between medical history 19

and AT1R-Ab result

4.2 An example of the standard curve for AT1R-Ab determination 22

5.1A A picture of EIA plate, which showed AT1R-Ab results. 25

5.1B The EIA absorbance values for AT1R-Ab determination 25

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LIST OF ABBREVIATIONS

Symbols/Abbreviations Terms

% Percent

× g Gravitational acceleration

°C Degree celsius

α Alpha

Γ Gamma

µl Microlitter (s)

ABOi ABO-incompatibility

ACE Angiotensin converting enzyme

ACEI Angiotensin converting enzyme

inhibitor

ACR Acute cellular rejection

ADCC Antibody-dependent cell-mediated

cytotoxicity

ADPK Autosomal dominant polycystic kidney

disease

AECAs Anti-endothelial cell antibodies

AMR Antibody mediated rejection

AP-1 Activator protein 1

APCs Antigen presenting cells

AR Acute rejection

ARB Angiotensin II receptor blocker

AT1R Angiotensin II type 1 receptor

AT1R-Ab Antibodies against angiotensin II type 1

receptor

AVA Anti-vimentin antibody

BMI Body mass index

CA California

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LIST OF ABBREVIATIONS (Cont.)


CCL-5 C-C motif chemokine ligand-5

CI Confidence interval

CMR Cellular-mediated rejection

CMV Cytomegalovirus

CTL Cytotoxic T lymphocyte

DC Deceased donor

DFPP Double filtration plasmapheresis

DGF Delayed graft function

dl Deciliter (s)

DSA Donor-specific antibody

DTH Delayed type hypersensitivity

DW Distilled water

e.g. Exampli gratis

EIA Enzymed-linked immunosorbent assay

Erks Extracellular signal-regulated kinase

ESRD End-stage renal disease

et al. Et alii

ETAR Endothelin-1 type A receptor

HLA Human leukocyte antigen

HLA-MM Human leukocyte antigen mismatch

HRP The horseradish peroxidase

HSP Heat shock protein

IF/TA Interstitial fibrosis/tubular atrophy

IFN-γ Interferon gamma

IgA Immunoglobulin A

IgG Immunoglobulin G

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LIST OF ABBREVIATIONS(Cont.)


IgM Immunoglobulin M

IL Illinois

IL-2 Interleukin 2

IRB The Institutional Reviewed Boad

ISBT International Society of Blood

Transfusion

IVIG Intravenous immunoglobulin

kDa Kilodalton (s)

LISS Low ionic strength saline

LV Living donor

M Male

MCP-1 Monocyte chemoattractant protein-1

MFI Mean fluorescence intensity

Mg Milligram (s)

MHC Major histocompatibility complex

MICA Major histocompatibility complex class

I related chain A

ml Milliliter (s)

MM Mismatch

mmHg Millimeter of mercury

NA Not applicable

NF-kB Nuclear factor-kB

NK cell Natural killer cell

NKG2D Natural killer group 2D

Nm Nanometer (s)

NT Not test

OR Odds ratio

PBS Phosphate buffer saline

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LIST OF ABBREVIATIONS (Cont.)


PRA Panel reactive antibody

RANTES Regulated upon activation normal T cell

expressed and secreted

RAS Renin-angiotensin system

RBC Red blood cell

RT Room temperature

sCr Serum creatinine

TB Tuberculosis

TNF Tumor necrosis factor

Tpx Transplantation

TX Texas

TxCAD Transplant-associated coronary artery

disease

U Unit

UNOS The United Network for Organ Sharing

USA The United States of America

vs. Versus

VT Vermont

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CHAPTER 1

INTRODUCTION

Currently, kidney transplantation is a successful treatment to decrease mortality rate

among patients with end-stage renal disease (ESRD).1 In Thailand, the numbers of

these patients are increasing but with some limitations due to the lack of living donors

and deceased donors resulting in longer waiting time. According to the regulation of

the Medical Council of Thailand in 2010, living donors should have a blood relationship

with the patients such as father, mother, son, daughter and other family members.

Moreover, only a donor, who is the spouse of a patient with a marriage certificate,

signed at least three years before the kidney transplantation will be accepted.2 Kidney

transplantation from living donors have more advantages compared with deceased

donors because of better matched human leukocyte antigen (HLA) which provides

long-term graft survival. Patients without any living donors need to be registered as

waited-listed patients to obtain a kidney from a deceased donor according to the Allocation

Criteria of the Organ Donation Centre, Thai Red Cross Society. At present, the selection

criteria for kidney transplantation includes ABO compatibility, HLA mismatch (MM),

HLA antibody, waiting time, age and HLA crossmatching. Patients receiving the

highest scores with negative HLA crossmatch will receive kidneys from a deceased

donor.3

However, the recipient’s immune system has developed elaborate and effective

mechanisms to combat foreign agents. These mechanisms are also involved in the

rejection of transplanted kidneys, which are recognized as foreign by the patient’s

immune system. The immune response to a transplanted organ consists of both cellular

(lymphocyte mediated) and humoral (antibody mediated) mechanisms. Although other

cell types are also involved, the T cells are central in the rejection of grafts. The

rejection reaction consists of the sensitization stage and the effector stage.

1.1 Sensitization stage

In this stage, the CD4 or CD8 T cells recognize the alloantigens in the context

of HLA class I or class II protein. There are two possible pathway in which alloantigen

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may be recognized by the immune system including direct and indirect pathways. In

the direct pathway, patient’s T cells recognize intact allogenic HLAs expressed by the

donor APCs. In the indirect pathway, patient’s T cells recognize peptides derived from

the donor HLAs presented by the patient APCs. 4,5

1.2 Effector stage

The most common effector mechanisms participate in allograft rejection are

cell-mediated reactions involving delayed-type hypersensitivity (DTH) and cytotoxic

T lymphocyte (CTL) mediated cytotoxicity; less common mechanisms antibody-

dependent cell-mediated cytotoxicity (ADCC).6 CD8+ CTLs that are generated by

direct pathway recognize graft alloantigens and destroy graft cells that express these

alloantigens. In contrast, any CD8+ CTLs that are generated by the indirect pathway

are self-major histocompatibility complex (MHC) restricted, and they will not be able

to kill the foreign graft cells because these cells do not express self MHC alleles

displaying allogeneic peptides. Therefore, when alloreactive T cells are stimulated by

the indirect pathway, the principal mechanism of rejection is inflammation caused by

the cytokines production including CD8+ or CD4+ effector T cells.7 In addition, CD4+

T cells initiate macrophage-mediated DTH responses and provide help to B cells for

antibody production.8 The antigens most frequently recognized by alloantibodies in

graft rejection are donor HLA molecules, including both HLA class I and class II.9

Depending on the time of onset graft rejection can be classified into 3 categories:

1.3 Hyperacute Rejection

This type of graft rejection is characterized by thrombotic occlusion of the graft

vasculature that begins within minutes to hours after recipient blood vessels are

anastomosed to graft vessels and is mediated by preexisting antibodies in the host

circulation that bind to donor endothelial antigens. In this case the tissue damage occur

due to complement fixation by antibody bound to graft endothelium. Complement

activation leads to endothelial cell injury and exposure of subendothelial basement

membrane proteins that activate platelets. The endothelial cells are stimulated to secrete

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high-molecular-weight forms of von Willebrand factor that cause platelet adhesion and

aggregation. Both endothelial cells and platelets undergo membrane vesiculation

leading to shedding of lipid particles that promote coagulation. Endothelial cells lose

the cell surface heparan sulfate proteoglycans that normally interact with anti-thrombin

III to inhibit coagulation. These processes contribute to thrombosis and vascular

occlusion, and the graft suffers irreversible ischemic damage.10

1.4 Acute rejection

Acute rejection occurs within six months after transplantation. The patterns of

acute rejection are divided into cellular, mediated by T cells, and humoral, mediated by

antibodies, both typically coexist in an acutely rejecting organ.

1.4.1 Acute cellular rejection (ACR)

The principal mechanism of acute cellular rejection is CTL-mediated killing of

cells in the graft. On histologic examination, ACR is characterized by infiltrates of

inflammatory cells inside the tubular walls.11 The destruction of allogeneic cells in a

graft is highly specific, a hallmark of CTL killing. The potential mechanism of CTL

involvement in ACR is mediated by cytotoxic granule-based killing by perforin and

granzyme B- or FasL-induced programmed cell death. In addition, activated CD8+ T

cells also have ability to produce proinflammatory cytokines especially IFN-γ, which

has been shown to enhance alloantigen presentation on target tissues and also serves to

enhance inflammation.12 In kidney transplantation, endothelial cells are major targets

of acute rejection. Endothelialitis or intimal arteritis in medium-sized arteries also

occurs at an early stage of acute rejection and is indicative of severe rejection, which,

if left untreated, will likely result in acute graft failure. Both CD8+ and CD4+ T cells

may contribute to endothelial injury.

1.4.2 Acute antibody mediated rejection (AMR)

AMR has become critical clinically because this form of rejection is usually

unresponsive to conventional anti-rejection therapy, AMR has been recognized as a

major cause of allograft loss. Alloantibodies cause AMR by binding to alloantigens,

mainly HLA molecules, on vascular endothelial cells, causing endothelial injury and

intravascular thrombosis that results in graft destruction. The binding of the

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alloantibodies to the endothelial cell surface triggers local complement activation,

which leads to cell lysis, recruitment and activation of neutrophils, macrophages,

monocytes and thrombus formation.13 Moreover, the biding of alloantibody and

alloantigens on endothelial surface also enhance expression of proinflammatory and

procoagulant molecules.14 The histologic hallmark of this form of acute rejection is

transmural necrosis of graft vessel with acute inflammation.15 Immunohistochemical

identification of the C4d complement fragment in capillaries of renal allografts is used

as an indicator of complement activation via the classical complement pathway and

humoral rejection. However, not only alloantibodies fix complement but the activation

of NK cell in ADCC also causes tissue injury.16

1.5 Chronic rejection

This type of rejection develops six months after transplantation. In kidney

transplantation, chronic rejection results in vascular occlusion and interstitial fibrosis.

A dominant lesion of chronic rejection in vascularized grafts is arterial occlusion as a

result of the intimal smooth muscle cells proliferation and the grafts eventually fail as

a result of ischemic damage. Chronic rejection usually causes by APC of recipient

present the alloantigen of the donor’s vascular smooth muscle cell to recipient CD4+T

cell, which specific to alloantigen thereafter proliferative cytokines are produced such

as IL-2 to induce endothelial and intimal smooth muscle cells proliferation causing the

thickness of blood vessels in transplanted organ. Finally resulting in blood flow in the

graft parenchyma is compromised and is replaced by nonfunctioning fibrous tissue.

This process leads to glomeruli loss function and ischemic graft failure in kidney

transplant patients.17,18

The Organ Donation Centre, Thai Red Cross Society established that waited-listed

patients have to send their current sera to the HLA Laboratory, National Blood Centre

on a monthly basis. After a candidate of a brain-dead donor is selected and the patient

eligibility criteria have been chosen, the patients’ sera of both peak and current sera

are tested with the donor’s T and B cells to determine HLA-A,-B and -DR

compatibility. Only a patient with the highest scores, regarding negative HLA

crossmatch results, will receive the transplant.19 Presently, renal function and survival

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rate of the transplanted kidney depend on HLA class I and II antibody identification in

the patient’s serum before or after transplantation. Highly HLA-sensitized patients may

require treatment with intravenous immunoglobulin (IVIG) and plasmapheresis to

minimize the risk of AMR. Moreover, the extensive use of immunosuppressant has

been established to reduce the occurrence of graft rejection. One report by the Thai

Transplantation Society showed that in kidney transplants, involving Thai patients with

graft loss, acute rejection is the most common cause (15%), followed by death with

functioning graft (15%) and transplant renal artery disease (7%).20 However,

unidentified causes total 10%, suggesting that other non-HLA antigens may play a role

in acute rejection. In 2014, Banasik M. and colleagues reported that 7 of 65 patients

(10%), who had renal allograft injury and graft loss, were due to antibodies against non-

HLA antigens including antibodies against the angiotensin II type 1 receptor (AT1R)

and/or endothelin-1 type A receptor (ETAR).21 Additionally, the Collaborative

Transplant Study reported that antibodies against non-HLA antigens, such as antibodies

against major-histocompatibility-complex class I–related chain A (MICA), AT1R and

vimentin, are involved in AMR and graft loss.22,23 Therefore, determining antibodies

specific to non-HLA antigens in patient’s serum before kidney transplantation may

provide additional useful data to predict graft survival and select the most appropriate

treatment among patients with these antibodies.

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CHAPTER 2

OBJECTIVES

The objective of this study was determined anti-AT1R antibody (AT1R-Ab) in

Thai kidney transplants patients.

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CHAPTER 3

REVIEW OF LITERATURE

According to related studies, antibodies implicated in graft survival and graft

outcome in kidney transplantation comprise three groups.

3.1 Antibodies against red cell antigens

Red cell antigens are expressed on most cells in the body such as red blood cell

(RBC), human tissues, epithelial and endothelial cells.24 These antigens are proteins and

carbohydrates attached to a lipid or protein. Currently, a total of 346 human blood group

antigens are now recognized by the International Society of Blood Transfusion

(ISBT).25, 26 The most important red cell antigens in kidney transplantation are A, B and

H blood group antigens. The immunodominant sugar of the ABH antigens are

carbohydrate epitopes present on different core saccharide chains that are bound to

lipids (glycolipids) or to proteins (glycoproteins).27 The ABH blood group antigens are

characterized by the presence or absence of two carbohydrate alloantigens (A and B)

on RBC membranes and three alloantibodies (anti-A, anti-B, anti-A, B) in plasma.

Antibodies against the ABO blood group system are naturally occurring antibodies,

mainly IgM antibodies, produced in the first year of life by sensitization to

environmental substances, such as food, bacteria, and viruses.28 These antibodies play

an important role in kidney transplantation because antibodies against specific antigens

usually induce hyperacute rejection. Therefore, ABO blood group compatibility

between donor and patient is a necessary prerequisite for successful kidney

transplantation. However, the number of ESRD patients on the waiting-listed is

increasing, while the number of available kidneys is static. An ABO-incompatible

(ABOi) kidney transplant is one strategy to expand the pool of donors, increase kidney

availability and reduce waiting.29 The first successful case of ABOi transplant was

reported by Alexandre GP, et al. in 1987. Pretransplant desensitization protocols were

used. Plasmapheresis and splenectomy were performed to remove anti-A and anti-B

and to prevent additional antibody production.30 Subsequently, most centers have now

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stopped performing splenectomy for ABOi kidney transplant due to its association with

bleeding complications.31,32 Presently, selective methods of antibody removal such as

immunoadsorption, double filtration plasmapheresis (DFPP) and standard

immunosuppression are performed.33 In Thailand, the first successful case of ABOi

kidney transplantation was reported in 2009. To reduce anti-ABO titers, patients

undergo DFPP and receive intravenous anti-CD20 monoclonal antibodies. The short

term outcome of ABOi kidney transplant patient is good. Hence, ABOi kidney

transplantation is an alternative therapy for ESRD patients, especially to expand the

pool of donors and reduce waiting time for transplantation.34

3.2 Antibodies against HLA antigens

HLA comprise the antigens that concentrate on the surface of white blood cells and

most other body tissues. HLA genes are located on the short arm of chromosome 6 and

are characterized in three groups. The first group is MHC class I encoded for HLA-A,

HLA-B and HLA-C antigens. These antigens can be found on the surface of all

nucleated cells and platelets and are responsible for endogenous peptide presentation to

CD8 + T lymphocyte. The second group is MHC class II, encoded for HLA-DR, HLA-

DQ and HLA-DP antigens. These antigens can be found on the surface of

macrophages, dendritic cells, B lymphocytes and monocytes involved in the

presentation of exogenous peptides to CD4+ T lymphocyte. The last group is MHC

class III, encoded for several components of the complement system, e.g., C2, C4a,

C4b, Bf. They are responsible for the levels of components of the complement system.

Moreover, these genes are encoded for inflammatory cytokines, tumor necrosis factor

(TNF) and heat shock proteins (HSP). Because they are not membrane proteins, they

have no role in antigen presentation but are important in the immune response 3 5 . For

kidney transplantation, HLA-B and -DR mismatch between patient and donor affects

graft survival.36 Concerning a report of The United Network for Organ Sharing (UNOS)

in 2002, three-year graft survival rates for zero HLA-DR mismatched normal kidney

grafts were high, similar to recipients of zero HLA-BDR mismatched grafts. On the

other hand, those with one or two DR antigen-mismatched had significantly poorer graft

outcomes (p < 0.001).37

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Because HLA have highly polymorphism, immunization after transplantation, blood

transfusion or pregnancy can lead to the development of HLA antibodies. These

antibodies may be directed against HLA class I or class II molecules and vary with

respect to the titer, affinity, immunoglobulin isotype and specificity.38 In the past

decade, HLA antibodies play an importance role in transplantation since they are

postulated as the cause of graft rejection. The screening HLA antibody before

transplantation were used to define a state of pre-sensitization, with a higher risk of

early kidney graft failure. Hence, Allocation Criteria of the Organ Donation Centre,

Thai Red Cross Society for cadaveric kidney transplantation include ABO

compatibility, HLA-A, -B and -DR mismatch between donors and patients, panel

reactive antibody (PRA) of the patients, waiting time and patients’ age. Patients with

the highest scores and negative HLA crossmatch would be first accepted for kidney

transplantation. 19

3.3 Antibodies against non-HLA antigens

Antibodies against non-HLA antigens located on endothelial cells or called anti-

endothelial cell antibodies (AECAs)39 and their properties may be either autoantibody

and alloantibody40 These antibodies can induce acute rejection leading to vascular

endothelial cell injury.41 Antibodies in this group consist of major histocompatibility

complex class I related chain A antigens (MICA), Vimentin and Angiotensin II type 1

receptor (AT1R).

3.3.1 Antibodies against MICA antigens

MICA antigens constitute a glycoprotein expressed on the surface of

endothelial cells, dendritic cells, fibroblasts, epithelial cells, monocytes and tumor cells

but not peripheral blood lymphocytes.42 These molecules share limited homologies

with classical HLA-class I antigens including extracellular domain (α1, α2 and α3),

transmembrane domain and cytoplasmic tail but are not associated with ß 2

microglobulin domain. MICA is a ligand for natural killer group 2D (NKG2D)

activation receptor, which is expressed on NK cells and of T cell subsets.43 It is

upregulated in stressed conditions, such as ischemia reperfusion injury and viral and

bacterial infections. Engagement of the donor’s MICA and NKG2D receptors on the

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recipient’s NK cells, results in not only induced cytotoxicity but also elicit cytokine

production.44 MICA genes appear highly polymorphic, consisting of 84 alleles encoded

for 71 proteins.45 The routes for MICA alloimmunization are mainly involved in HLA

alloimmunization. Nevertheless, the presence of anti-MICA antibodies might represent

cross-reactivity against epitopes shared between microbial agents and MICA antigens. 46 A

previous study reported that before transplantation, preformed anti-MICA antibodies

were demonstrated (11.4%) in 1,910 kidney transplant patients. One year graft survival

in these patients was significantly lower than patients without antibodies (p = 0.01).47

In addition, the presence of de novo MICA antibodies following transplantation also

reduced long-term graft survival or chronic rejection.48 In contrast, the association

between anti-MICA antibodies and acute AMR is uncertain because no correlation of

these antibodies with C4d+ rejection has been determined.49 Accordingly, screening

tests for anti-MICA antibodies among kidney transplant patients is used to follow up

and evaluate risk of graft survival in chronic rejection.

3.3.2 Antibodies against vimentin

Vimentin is a cytoskeleton intermediate filament protein, expressed on the

surface of apoptotic T cells, endothelial cells and smooth muscle cells.50 The vimentin

function is to support and strengthen the nuclear envelope and also regulates leukocyte

migration. Anti-vimentin antibodies (AVA) can be produced in patients who recognize

different antigen epitopes of vimentin.51 AVA play a key role in cardiac transplantation

as previously demonstrated in non-human primate models confirming that cardiac

allograft injury is caused by AVA.52,53 Additionally, Jurcevic S. and colleagues reported

that AVA showed significant correlation with the development of transplant-associated

coronary artery disease (TxCAD) (p < 0.0001). Therefore, AVA is a predictive marker

to identify patients at high risk of cardiac rejection.54 For kidney transplantation,

patients with graft failure were more likely to form AVA when patients had HLA-DQ2

positive (p < 0.001).55

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3.3.3 Antibody against angiotensin II type 1 receptor (AT1R-Ab)

3.3.3.1 AT1R genes and structure

AT1R comprises seven transmembrane-spanning G-protein-coupled

receptors, consisting of extracellular, glycosylated region connecting to the seven

transmembrane α-helices linked by three intracellular and three extracellular loops

(Figure 3.1).56 AT1R is responsible for physiologic and pathophysiologic actions that

can be mediated by angiotensin II. Angiotensin II is a bioactive peptide of the renin-

angiotensin system (RAS), which is considered to play an important role in regulating

blood pressure, electrolyte balance, aldosterone release and sodium reabsorption.57

When plasma sodium concentration is lower than normal or renal blood flow is reduced,

juxtaglomerular cells in the kidney convert prorenin (an intracellular protein) into renin

and then secrete it directly in the blood circulation.58 Thereafter, renin converts

angiotensinogen that is mainly secreted by the liver to angiotensin I, which is a

physiologically inactive substance. Angiotensin I is rapidly converted to angiotensin II

by angiotensin converting enzyme (ACE) which present mainly in lung capillaries.

Angiotensin II regulates blood pressure and mediates its vasoconstrictor effects by

stimulating AT1R in vascular smooth muscle cells. Moreover, angiotensin II stimulates

the release of aldosterone from the zona glomerulosa of the adrenal gland resulting in

a further rise in blood pressure related to sodium and water retention.59,60 AT1R is

localized in various tissues including the liver, adrenal, brain, lung, kidney, heart and

vasculature.61 The AT1R coding gene is located on chromosome 3 comprising 359

amino acids, with a molecular weight of 41 kDa.62 This gene has at least five exons; the

last exon containing the coding sequence.63 The AT1R gene has been found to be highly

polymorphic, but only the A1166 polymorphism is associated with increased response

to angiotensin II, cardiovascular abnormalities and renal pathologies. 64-66

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Figure 3.1 Structure of the AT1R

3.3.3.2 Role of AT1R-Ab in kidney transplantation

AT1R-Ab has been shown to be directly involved in the

pathophysiology of immune-mediated vascular diseases such as preeclampsia and

malignant hypertension.67,68 In addition, previous studies have confirmed that AT1R-

Ab has been associated with acute AMR in kidney transplantation, as shown in Table

3.1.69

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Table 3.1 AT1R-Ab in kidney transplantation

Studies

Cohort

Time of Ab

detection

Key findings

Reinsmoen NL, et al.70 • 63 patients with no HLA-DSA or

MICA-DSA, including 16 patients

with AR (7 AMR, 9 ACR)

Pre & post

transplantation

• AT1R-Ab associated with increased

AMR but not ACR

• 6 of 7 patients with AMR did not

have C4d deposition

Taniguchi M, et al.71 • 134 patients with abnormal

biopsies

• 217 control patients

Pre & post

transplantation

• AT1R-Ab associated with abnormal

biopsy samples

• De novo AT1R-Ab associated with

increased graft failure

Giral M, et al.72 • 283 AT1R-Ab (+) patients

• 316 AT1R-Ab (-) patients

Pretransplantation • Pre-formed AT1R-Ab associated

with increased AR within

4 months and increased graft failure

>3 years post transplantation

In JW, et al.73 • 7 AT1R-Ab (+) patients

• 72 AT1R-Ab (-) patients


with increased AMR but not ACR

Lee J, et al.74 • 12 patients with AMR

and no HLA-DSA

Pre & post

transplantation

• 9 of 10 patients had AT1R-Ab (+)

sera before transplantation

• 10 of 12 patients were AT1R(+) at

the time of biopsy

Lee J, et al.75 • 98 AT1R-Ab (+)

• 64 AT1R-Ab (-)


with increased AMR

Abbreviation: AT1R-Ab, antibody against angiotensin II type 1 receptor; ACR, acute cellular rejection; AMR, antibody-mediated

rejection; CMR, cellular-mediated rejection; C4d, complement degradation product C4d; DSA, donor-specific antibody; Tpx, transplant.

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3.3.3.3 Role of AT1R-Ab in AMR

AT1R-Ab belong to complement fixing antibodies (IgG1 and IgG3)

but the mechanism can operate through a complement independent mechanism.76

AT1R-Ab bind to the second extracellular loop of AT1R and initiates signal

transduction cascades by inducing extracellular signal-regulated kinase (Erks) 1/2

phosphorylation in endothelial cells and vascular smooth muscle cells. This results in

the increased activity of transcription factors and activates the activator protein 1 (AP-

1) and nuclear factor-kB (NF-kB), subsequently increasing the expression of

inflammation and coagulation related genes. The expression of pro-inflammatory

markers include monocyte chemoattractant protein-1 (MCP-1), regulated on activation,

normal T cell expressed and secreted (RANTES) or C-C motif chemokine ligand-5

(CCL-5) and pro-coagulator genes (tissue factor). This might account for intravascular

inflammatory cell infiltration and thrombotic angiopathy (Figure 3.2).77 Graft biopsies

of patients, who had vascular rejection with positive anti-AT1R antibodies, did not

show any evidence of complement deposition (C4d: negative).78

Figure 3.2 Model for AT1R-Ab related signaling in vascular cells

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3.4 Current situation in cadaveric kidney transplants among Thai patients

At present, for cadaveric kidney transplantation, substantial development in

immunosuppressive regimens, HLA typing and HLA antibody detection, have led to

continuous improvements in patient allograft survival. In Thailand, the 2014 Annual

Report of Organ Transplantation revealed that 88.8% of kidney transplant patients had

negative PRA. The major cause of graft loss within one-year after transplantation was

graft rejection (39.1%), followed by vascular/urologic (18.6%), death with functioning

graft (13.8%), other causes (13.8%), interstitial fibrosis/tubular atrophy (IF/TA)

(10.0%) and malignancy/noncompliance/glomerular diseases (2.4%), respectively.79

Additionally, a previous report from Siriraj Hospital demonstrated that among 444 Thai

patients undergoing kidney transplantation from January 2005 to December 2012, acute

AMR was diagnosed in 25 patients (5.36%). Of these, 17 highly sensitized patients had

positive PRA more than 20%. DSA was detected during a rejection episode in seven

patients, four patients had HLA class I DSA, two patients had HLA class II DSA and

one patient had both class I and II. The other eight patients with negative PRA might

have had antibodies specific to non-HLA antigens, which could not be determined.80

Therefore, determining AT1R-Ab in patients before kidney transplantation would be

beneficial to predict graft survival and to diagnose acute AMR to select the most

appropriate treatment.

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CHAPTER 4

MATERIALS AND METHODS

4.1 Sample size calculation

The sample size was calculated using a single proportion equation.

N = Z2PQ/D2

N = required sample size

Z = confidence level as at 95% (standard value of 1.96)

P = estimated prevalence of AT1R-Ab positive in Polish kidney transplant

patients is 10%21

Q = 1-P (0.99)

D = desired precision (0.07)

For calculation

N = 1.962 x 0.10 x (1-0.10)/0.072

N = 70

Therefore, 70 samples of kidney transplant patients were used in this study.

4.2 Sample population

Altogether, 80 serum samples were included in this study. Seventy pretransplant

serum samples of consecutive kidney transplant patients were obtained from the

Division of Nephrology, Department of Medicine, Faculty of Medicine, Chulalongkorn

University and King Chulalongkorn Memorial Hospital, Bangkok, Thailand. All

patients were transplanted with ABO-identical kidneys from January 1, 2010 to

December 27, 2016. HLA antibodies and crossmatch results of all patients were

negative before kidney transplantation. The clinical data and kidney allograft biopsy

after transplantation were retrieved from medical history. Criteria for AMR diagnosis

was defined by the presence of C4d on frozen sections using the immunofluorescence

technique, according to the basis of the Banff 2013 classification.81 Among 70 patients,

52 and 18 were males and females, respectively. Their ages ranged from 17 to 68 years,

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with a mean age of 45 years and a standard deviation (SD) of 12.2 years. Of these, 48,

19 and 3 patients received kidneys from deceased donors, living related and living

unrelated (spouse) donors, respectively.

Ten samples of unrelated healthy male blood donors at the National Blood Centre,

Thai Red Cross Society, Bangkok, Thailand were also tested for AT1R-Ab. All

donated blood between January 12 and February 15, 2017. Their ages ranged from 19

to 52 years, with a mean of 29 years (SD 7.1) years.

This study was approved by the Institutional Review Board, Faculty of Medicine,

Chulalongkorn University (IRB No. 633/59), the Research Ethics Committee, National

Blood Centre, Thai Red Cross Society, Bangkok (COA No. NBC 13/2016) and the

Committee on Human Rights Related to Research Involving Human Subjects,

Thammasat University, Pathumtani, Thailand (COE No. 051/2560).

4.3 Sample collection

All serum samples of patients and donors were kept at -80°C until the day of

measurement for HLA antibodies, red cell antibodies and AT1R-Ab.

4.4 Study parameters

A conceptual framework for data evaluation between medical history and AT1R-Ab

results is illustrated in Figure 4.1. The study parameters were divided in two groups.

4.4.1 Risk factors of AT1R-Ab development

Risk factors for AT1R-Ab development before kidney transplantation were

determined. Parameters were analyzed including serum creatinine (sCr) level, history

of sensitization, hypertension, body mass index (BMI) and the use of anti-hypertensive

drugs (angiotensin-converting enzyme inhibitors, ACEI and angiotensin II receptor

blocker, ARB).

4.4.2 Risk factors of AMR

Risk factors of AMR were analyzed. Factors included patient’s sex, history of

sensitization, donor’s age, type of donor, ischemic time, delayed graft function (DGF),

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HLA-A, -B, -DR incompatibility, tuberculosis (TB) infection, cytomegalovirus (CMV)

infection and de novo HLA-DSA.

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Figure 4.1 A conceptual framework for data evaluation between medical history and AT1R-Ab result

Abbreviations: ACEI, angiotensin converting enzyme inhibitor; AMR: antibody mediated rejection; ARB, angiotensin receptor blocker;

BMI, body mass index; CMV, cytomegalovirus; DGF, delayed graft function; DSA, donor-specific antibody; HLA, human leukocyte

antigen; sCr: serum creatinine; TB, tuberculosis.

Kidney

transplantation

Graft rejection (AMR)

Transplant

-Ischemic time

-DGF

-HLA-A, -B, -DR

mismatch

-TB infection

-CMV infection

- de novo HLA-DSA

Donor

-Age

-Type of donor

Patient

-Sex

-Sensitization

AT1R-Ab

ACEI ARB

BMI

Hypertension sCr level

Sensitization

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4.5 Determination of HLA antibodies

HLA antibodies were evaluated in serum samples of patients and donors using

fluorescence-based solid phase immunoassay (LABScreen PRA; One Lambda, Inc.,

Canoga Park, CA, USA). This test was performed on a Luminex platform (Luminex,

Austin, TX, USA), according to the manufacturer instructions. First, 20 μl of serum

samples were incubated with 5 μl of LABScreen PRA class I and LABScreen PRA

class II beads, coated with purified Class I or Class II HLA antigens, in each well of a

96-well plates for 30 minutes in a dark at room temperature (RT) with gentle shaking.

Second, 10X wash buffer was diluted with distilled water (DW) to make a 1X wash

solution. Third, 150 μl of 1X wash buffer was added to each well of the plate after

incubation. Then the plate was covered with tray seal, mixed and centrifuged at 1,300

× g for 5 minutes. Wash buffer from the wells of the plate was removed by flicking.

Fourth, 200 μl of 1X wash buffer was added to each well of the plate. The plate was

covered with tray seal, mixed and centrifuged at 1,300 × g for 5 minutes. The

supernatant from the wells of the plate was removed by flicking and this step was

repeated once. Fifth, 1 μl per test of 100X PE-conjugated anti-human IgG was diluted

with 99 μl of 1X wash buffer to make a 1X solution. Sixth, 100 μL of 1X PE-conjugated

anti-human IgG were added to each well. The plate was covered with tray seal, mixed

and incubated in the dark for 30 minutes at RT with gentle shaking. Seventh, the plate

was centrifuged at 1,300 × g for 5 minutes. The supernatant from the wells of the plate

was removed by flicking and the washing step was repeated twice. Thereafter, 80 μl of

1X PBS was added to each well. The plate was covered with tray seal and mixed.

Finally, samples were read on the LABScan3D™ flow analyzer (One Lambda, Inc.,

Canoga Park, CA, USA). The cut-off for reactive sample was based on calculations

performed by HLA Fusion Software, Version 3.4 Service Pack (One Lambda, Inc.,

Canoga Park, CA, USA). The results were expressed as mean fluorescence intensity

(MFI) value for each HLA antibody. MFI value equal to or more than 1,000 was defined

as a positive result for the presence of HLA antibodies.

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4.6 Determination of red cell antibodies

Red cell antibody detection among patients and donors was determined using the

column agglutination test (Bio-Rad, Cressier sur Morat,, Switzerland). Fifty microliters

of 1% screening cells O1 and O2 (National Blood Centre, Thai Red Cross Society,

Bangkok, Thailand), suspended in Diluent-II (Modified-LISS), were added in

microtubes of the ID-Card (Bio-Rad, Cressier sur Morat,, Switzerland). Thereafter, 25

μl of serum samples were added to each microtube. The ID-Card was incubated for 15

minutes at 37°C and centrifuged at 85 × g for 10 minutes in the ID-centrifuge (DiaMed

AG, Cressier sur Morat, Switzerland), and the results were read and recorded. A

positive result was indicated when the agglutinated cells formed a red cell line on the

surface of the gel or agglutinates dispensed in the gel. The positive reaction of 1+ to 4+

indicated the presence of the red cell antibodies according to the manufacturer

instructions. On the other hand, a negative result was expressed by a compact button of

cells on the bottom of the microtube indicating the absence of the red cell antibodies.

4.7 Determination of AT1R-Ab

Detection of AT1R-Ab was performed using an enzyme-linked immunosorbent

assay (EIA) kit (One Lambda, Inc., Canoga Park, CA, USA) according to the

manufacturer instructions. First, 96 wells of microtiter strips were labeled for each

sample. Second, the serum samples were diluted with sample diluent 1:100. Third, 100

μl of duplicate serum samples were added to each test well and incubated at 2-8°C for

2 hours. Fourth, the fluid was removed from wells and washed three times with 300 μl

wash buffer. After the last washing step, the microtiter strips were inverted and placed

on a clean paper towel to remove the fluid. Fifth, horseradish peroxidase (HRP)

conjugate was diluted with conjugate diluent 1:100 and 100 μl of diluted HRP conjugate

was dispensed to each well and incubated for 1 hour at RT with gentle shaking. Sixth,

the washing step was repeated and 100 μl of 3, 3′, 5, 5′-tetramethylbenzidine substrate

solution was added to each well and incubated for 20 minutes at RT in the dark.

Thereafter, 100 μl of stop solution was added to each well. The absorbance was

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measured at 450 nm with the BioTek Microplate Reader (BioTek Instruments, Inc.

Winooski, VT, USA). The presence of antibodies bound to AT1R was detected using

a colorimetric assay change. A standard curve was generated to allow the quantitation

of AT1R-Ab, using five standard sera samples (2.5, 5, 10, 20 and ≥40 U/ml) and

analyzed by AT1R Software, Version 1.0.0 (One Lambda, Inc., Canoga Park, CA,

USA.). X-axis: linear, AT1R-Ab standard concentrations. Y-axis: linear, absorbance.

Sample concentrations could be calculated from this standard curve (Figure 4.2).

Positive and negative controls were run in parallel. The level of >17, 10-17 and <10

U/ml were considered as AT1R-Ab positive, at risk and negative, respectively

according to the manufacturer instructions.

Figure 4.2 An example of the standard curve for AT1R-Ab determination

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4.8 Statistical analysis

The independent sample t-test was used to determine the differences of the means of

continuous variables between the AT1R-Ab (+) and AT1R-Ab (−) groups. The Chi-

square test and Fisher's exact test were used to compare categorical variables. The

analysis was performed using SPSS Software, Version 16.0 (SPSS Inc., Chicago, IL,

USA), and p-values less than 0.05 were considered statistically significant.

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CHAPTER 5

RESULTS

A total of 80 serum samples were included in this study. Seventy pretransplant

patient samples from the Division of Nephrology, Department of Medicine, Faculty of

Medicine, Chulalongkorn University, King Chulalongkorn Memorial Hospital and 10

samples of healthy blood donors from the National Blood Centre, Thai Red Cross

Society, Bangkok, Thailand were tested for HLA antibodies, red cell antibodies and

AT1R-Ab.

5.1 Patient characteristics and AT1R-Ab results

The AT1R-Ab results and the absorbance values are shown in Figure 5.1A + B The

patients were divided in 2 groups according to the level of AT1R-Ab: >17.0 U/ml,

AT1R-Ab (+) and ≤17.0 U/ml, AT1R-Ab (–). When the AT1R-Ab concentrations were

analyzed by AT1R Software, it was found that AT1R-Ab (+) were detected in 6 of 70

(8.6%) patients. The mean age in the AT1R-Ab (+) groups was significantly lower than

in the AT1R-Ab (−) groups (34.0 ± 14.6 vs. 46.0 ± 11.5, p = 0.02, t-test = 2.395, 95%

CI = 2.008 - 22.086). Moreover, patients were divided in two age groups of <30 and

≥30 years, the AT1R-Ab (+) group had a significant increase in those patients <30 years

old (50.0% vs. 5.2%, p = 0.03, OR = 0.103, 95% CI = 0.017 - 0.631). On the other hand,

no statistical differences were observed between AT1R-Ab (+) groups and AT1R-Ab

(−) groups in terms of sex, sCr level, sensitization history, hypertension, body mass

index, ischemic time and number of HLA-MM (Table 5.1).

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Figure 5.1A A picture of the EIA plate, showing AT1R-Ab results.

Wells 1A-2A: negative control serum, 1B-2B: positive control serum, 1C-2C, 1D-2D,

1E-2E, 1F-2F and 1G-2G: standard sera 2.5, 5, 10, 20 and ≥40 U/mL, respectively.

Wells 1H-2H: sample blank. The AT1R-Ab level >17.0 U/mL was considered as

AT1R-Ab (+). Patient samples with AT1R-Ab (+) presented in wells 3B-4B, 3E-4E,

3F-4F, 5B-6B and 11A-12A boxed, respectively.

Figure 5.1B The EIA absorbance values for AT1R-Ab determination

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Table 5.1 Patient demographics in AT1R-Ab (+) and AT1R-Ab (–) groups.

AT1R-Ab (+) AT1R-Ab (−) p-value

Total number of patients 6 64 NA

Patient characteristics

Male 5 47 1.00

Female 1 17

Mean patient age 34.0 ± 14.6 46.0 ± 11.5 0.02

Patient age <30 years 3 6 0.03

Patient age ≥30 years 3 58

sCr level (mg/dL) 10.4 ± 3.3 9.4 ± 2.5 0.45

BMI 19.5 ± 4.1 21.6 ± 3.7 0.19

Retransplant 0 3 0.76

Blood transfusion 1 25 0.27

Pregnancy 0 6 0.57

Hypertension 6 53 0.34

ACEI 1 8 0.58

ARB 2 26 0.54

Transplant characteristics

Donor’s age 32.5 ± 4.3 37.1 ± 11.8 NA

Ischemic time (hours) 6.3 ± 8.6 12.9 ± 8.7 0.08

DGF 0 21 NA

Living related donors 4 15 NA

Living unrelated donors 0 3 NA

Deceased donors 2 46 NA

No. of HLA mismatches 3 (2-3) 3 (0-6) 0.45

Time from transplant to rejection (years) 1.7 2.4 NA

TB infection 0 2 NA

CMV infection 0 2 NA

de novo HLA-DSA 0 2 NA

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Table 5.1 Patient demographics in AT1R-Ab (+) and AT1R-Ab (–) groups (Cont.)

AT1R-Ab (+) AT1R-Ab (−) p-value

Causes of ESRD

Chronic glomerulonephritis 1 6 NA

Diabetic nephropathy 1 9 NA

Obstructive uropathy 1 1 NA

ADPK 0 4 NA

Congenital cystic disease 0 1 NA

Hypertensive nephropathy 0 5 NA

IgA nephropathy 0 5 NA

Lupus nephritis 0 1 NA

Membrane proliferative 0 1 NA

Unknown 3 31 NA

Abbreviations: ACEI, angiotensin converting enzyme inhibitor; ADPK, autosomal

dominant polycystic kidney disease; ARB, angiotensin II receptor blocker; BMI: body

mass index; CMV, cytomegalovirus; DGF, delayed graft function; DSA, donor-specific

antibody; ESRD, end-stage renal disease; HLA, human leukocyte antigen; NA, not

applicable; sCr, serum creatinine; TB, tuberculosis.

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5.2 HLA antibodies and red cell antibody results

As noted, 80 serum samples including 70 pretransplant serum samples of

consecutive kidney transplant patients and 10 samples of male blood donors underwent

HLA and red cell antibody detection. In this study all samples were negative for both

HLA and red cell antibodies.

5.3 Association of AT1R-Ab with kidney allograft rejection

Among 6 patients who had AT1R-Ab (+), the mean AT1R-Ab level was 18.5 ± 2.9

U/mL. Kidney biopsies were performed 1.5 ± 1.4 years after transplantation.

Concerning kidney biopsy result, 2 patients had AMR, 2 patients presented T cell-

mediated rejection, 1 patient had acute tubular necrosis and 1 patient presented BK

nephropathy

5.4 AT1R-Ab and patients with AMR

Regarding 7 kidney transplant patients, with a diagnosis of AMR, C4d staining was

positive in transplant kidney biopsies. Two patients were AT1R-Ab (+) and five

patients were AT1R-Ab (−). When the HLA-MM were compared in both groups, 3

HLA-MM and 3 to 6 HLA-MM were found in AT1R-Ab (+) and AT1R-Ab (−) groups,

respectively. Moreover, time of AMR diagnosis tended to decrease among 4 patients of

AT1R-Ab (−) ranging from 3 to 11 days after transplantation. Only 1 patient with 6

HLA-MM was diagnosed at day 221 and de novo HLA-DSA was demonstrated. On the

contrary, time of AMR diagnosis in 2 patients of AT1R-Ab (+) was 523 and 555 days

after transplantation, respectively (Table 5.2).

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Table 5.2 Characteristics and laboratory results of patients with AMR in AT1R-Ab (+) and AT1R-Ab (-) groups

Abbreviations: AMR, antibody mediated rejection; DC, deceased donor; DSA, donor-specific antibody; HLA, human leukocyte antigen;

LV, living donor; M, male; MM, mismatch; NT, not test

Pre-transplant Post-transplant

No. Sex Age Ischemic time

(hours)

Type of

transplant

AT1R-Ab

level (U/ml)

HLA-MM Biopsy

result

Time

(days) C4d result

de novo

HLA-DSA A B DR

AT1R-Ab (+) group (n = 2)

005 M 57 0:52 LV 17.48 1 1 1 AMR 555 Positive Negative

010 M 17 17:25 DC 17.00 1 1 1 AMR 523 Positive Negative

AT1R-Ab (-) group (n = 5)

018 M 27 9:29 DC 11.59 1 2 2 AMR 7 Positive NT



055 M 54 1:20 LV 13.50 2 2 2 AMR 221 Positive Positive

070 M 34 1:58 LV 8.18 1 2 2 AMR 3 Positive Negative

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5.5 Donor characteristics and AT1R-Ab results

This study performed AT1R-Ab in 10 samples of male blood donors. The mean of

AT1R-Ab level was 8.2 ± 4.5 U/ml. The positive results of AT1R-Ab were

demonstrated in 1 of 10 donors (10.0%). Interestingly, a positive AT1R-Ab result

(≥40.0 U/ml) was found in a 23-year-old male donor. The sample was obtained at the

second donation. Blood pressure levels recorded at the first and second donations were

132/80 and 144/90 mmHg, respectively (Table 5.3).

Table 5.3 Donor characteristics and AT1R-Ab results

No. Age AT1R-Ab level

(U/ml)

Blood pressure (mmHg)

1st time donation 2nd time donation

AT 071 26 3.37 130/90 124/84

AT 072 51 3.98 120/80 127/89

AT 073 52 13.31 125/76 130/90

AT 074 28 13.38 134/62 130/70

AT 075 42 3.47 120/87 129/80

AT 076 38 4.62 130/70 130/80

AT 077 48 10.03 129/80 130/84

AT 078

AT 079

AT 080

19

23

42

13.79

≥ 40

8.15

121/66

144/90

133/77

120/80

132/82

120/80

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CHAPTER 6

DISCUSSION

The presence of pretransplant antibodies against HLA and non-HLA has been

proven to be an important factor of allograft rejection and decreased graft survival in

kidney transplantation. In this study, serum samples of kidney transplant patients and

healthy donors were tested for HLA and red cell antibodies and AT1R-Ab. All samples

were negative for HLA and red cell antibodies. According to these results the AT1R-

Ab may be involved in graft dysfunction of Thai kidney transplant patients.

As previously mentioned, other factors could affect AT1R-Ab development. First,

sCr level and BMI were analyzed and no association was found among AT1R-Ab (+)

and AT1R-Ab (–) groups, similar to a previous study of Norwegian patients.82 Second,

AT1R-Abs can be developed after blood transfusion, pregnancy or transplantation;

however, in this study no association was found between AT1R-Ab and history of

sensitization, consistent with related studies among American and French patients.83,84

Third, hypertension and the use of antihypertensive drugs including ACEI and ARB

were analyzed. Even though related studies revealed that AT1R-Ab developed in

patients with malignant hypertension, no association was found in our patient

groups.76,85 Additionally, ACEI and ARB are used to reduce kidney failure and

cardiovascular events, in our patient groups no association was found with AT1R-Ab

development, similar to a related study.86

Concerning the development of non-HLA antibodies, especially AT1R-Ab, it has

been well known to be associated with AMR contributing allograft rejection and

decreased long-term graft survival in kidney transplantation.69 In this study, the AT1R-

Ab was evaluated in serum samples of kidney transplant patients and healthy blood

donors. The AT1R-Ab (+) and AT1R-Ab (–) groups were classified based on a cut-off

value >17.0 U/mL and the prevalence of AT1R-Ab (+) among patients’ pretransplant

samples was 8.6%, similar to related reports regarding American and Korean

patients.70,73 Comparing AT1R-Ab and patient characteristics, the frequency of AT1R-

Ab (+) significantly increased among patients at younger age (<30 years). Our data

differed from a report among Caucasian American patients in that AT1R-Ab (+)

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significantly differed among older patients aged >45 years.71 On the contrary, no

significant difference was found in age between patients in the AT1R-Ab (+) and

AT1R-Ab (−) groups among French and Korean patients.72-75 This may be due to

different ethnic groups and patient selection in each study.

In this study, 7 patients with an episode of AMR did not have pre-existing HLA

antibodies before kidney transplant. Notably, 4 patients in the AT1R-Ab (−) group had

early signs of acute AMR rejection. They received kidneys from deceased donors with

3 to 5 HLA-MM, which may have caused this outcome.37,87,88 Unfortunately, de novo

HLA-DSA were negative in 1 patient and HLA-DSA detection could not be performed

in 3 patients. Over time, HLA-DSA could not be confirmed as the cause of acute

rejection without explanation. In addition, 1 patient, who received a kidney from living-

related donor, could be used to conclude that AMR was associated with HLA-DSA.

Regarding the time of AMR diagnosis, the correlation between AT1R-Ab level in

pretransplant subjects and consequent graft loss from 3 years post transplantation was

reported among French patients.72 Meanwhile, other reports among Korean and Polish

patients showed that high levels of pretransplant AT1R-Ab were associated with

developing acute rejection during the first year of transplantation. 74,75,89 In this study,

2 patients with AT1R-Ab (+) in pretransplant samples exhibited early signs of AMR

after 1 year of transplantation, consistent with related reports. Based on the data of

French patients, graft loss after 3 years post-transplantation may have occured among

our patients. An effective therapeutic reduction of AT1R-Ab level following early signs

of AMR would benefit graft outcome. To determine this, well-designed cohort studies

are suggested. Moreover, the remaining 4 AT1R-Ab (+) patients were diagnosed

involving as T cell-mediated rejection (n=2), acute tubular necrosis (n=1) and BK

nephropathy (n=1). Interestingly, AT1R-Ab (+) was also observed in 1 healthy blood

donor, similar to a related finding.71 The high level of AT1R-Ab may be associated

with hypertension70 and to conclude this finding, long term follow up and a larger

sample size will be needed.

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CHAPTER 7

CONCLUSIONS

HLA antibodies found among kidney transplant patients are associated with

hyperacute, acute and chronic AMR. Among patients with negative PRA receiving

compatible HLA crossmatch kidney donors and presenting signs of AMR, non-HLA

antibodies should be determined. This study was the first to report the prevalence of

AT1R-Ab in pretransplant samples of Thai kidney transplant patients. The presence of

high AT1R-Ab levels before kidney transplantation was significantly associated with

patients in younger ages (<30 years). Moreover, 2 patients presenting AT1R-Ab (+) in

pretransplant samples exhibited early signs of AMR after 1 year of transplantation.

Further studies among patients using larger sample sizes are suggested to confirm our

findings.

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APPENDICES

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APPENDIX A

HUMAN ETHIC APPROVAL OF THE COMMITTEE ON

HUMAN RIGHTS RELATED TO RESEARCH INVOLVING

HUMAN SUBJECTS, THAMMASAT UNIVERSITY

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APPENDIX B

HUMAN ETHIC APPROVAL OF THE RESEARCH ETHICS

COMMITTEE, NATIONAL BLOOD CENTRE,

THAI RED CROSS SOCIETY, BANGKOK

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APPENDIX C

HUMAN ETHIC APPROVAL OF THE INSTITUTIONAL

REVIEW BOARD, FACULTY OF MEDICINE,

CHULALONGKORN UNIVERSITY

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APPENDIX D

MANUSCRIPTS ASSIGMENT LETTER FROM

TRANSPLATATION PROCEEDING JOURNAL

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BIOGRAPHY

Name: Miss Sudarat Viboon

Date of Birth: December 24 , 1984

Educational records:

2015-present

M.Sc. student in Master Program (Biomedical Sciences),

Graduate Program in Biomedical Sciences,

Faculty of Allied Health Sciences,

Thammasat University, Thailand

2003-2006 B.Sc. in Bachelor’s Degree of Science of Medical Technology,

Faculty of Medical Technology,

Huachiew Chalermprakiet University, Thailand

Work Experience:

2007- 2009

2009- Present

Medical Technologist,

Prapokklao Hospital, Chanthaburi, Thailand

Medical Technologist,

The Organ Donation Centre,

Thai Red Cross Society, Bangkok, Thailand

Official address: 1871 Terdprakiat Building, Henry Dunant Road,

Patumwan, Bangkok 10330, Thailand

Home office: 324/488 LPN park Rattanathibet-Ngamwongwan,

Rattanathibet Road, Bangkrasor,

Nonthaburi 11000, Thailand

E-mail address: [email protected]

Publications:

1. Kupatawintu P, Tatawatorn A, Phiencharoen S, Viboon S, Choketaweesak N,

Nathalang O, et al. Allocation criteria to increase chances of kidney transplantation

for highly HLA-sensitized patients. J Hematol Transfus Med. 2014;24:103-9.

2. Viboon S, Ounjai S , Khanoonthong S, Phiancharoen S , Kupatawintu P, Nathalang

O, et al. Changing of panel reactive antibody levels in kidney transplant waited-

listed patients. J Hematol Transfus Med. 2015;25:193-200.

Ref. code: 25605812040011ZLR

determination of antibodies against angiotensin ii …

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