UNIVERSITI PUTRA MALAYSIA

A CORRELATION BETWEEN PROTEINURIA, ENZYMURIA AND KIDNEY HISTOPATHOLOGICAL CHANGES DURING EARLY RENAL

DAMAGE INDUCED BY GENTAMlClN

SAIRAH BlNTl ABDUL KARlM

FPV 2004 6

A CORRELATION BETWEEN PROTEINURIA, ENZYMURIA AND KIDNEY HISTOPATHOLOGICAL CHANGES DURING EARLY RENAL DAMAGE

INDUCED BY GENTAMlClN

BY

SAIRAH BlNTl ABDUL KARlM

Thesis Submitted to the School of Graduate Studies, Universiti Putra Malaysia, in Fulfilment of the Requirements for the

Degree of Master of Science

May 2004

DEDICATION

To my parents, Mr. Abdul Karim Jabar and Mrs. Noli Othman, my

family members (especially Kak Awi and Abang Shaharul) who encouraged

me to pursue a profession I would enjoy for a lifetime.

To my husband, Mohd. Fadhil and son, Faiz Isqandar, who bring me a

great happiness.

Abstract of thesis presented to the Senate of Universiti Putra Malaysia in fulfilment of the requirement for the degree of Master of Science

A CORRELATION BETWEEN PROTEINURIA AND ENZYMURIA AND KIDNEY HISTOPATHOLOGICAL CHANGES DURING EARLY RENAL

DAMAGE INDUCED BY GENTAMlClN

SAIRAH BlNTl ABDUL KARlM

May 2004

Chairman : Professor Rasedee Abdullah, Ph.D.

Faculty : Veterinary Medicine

This study was carried out to determine the correlation between urine

proteins and urinary enzyme markers, aspartate aminotransferase (AST), y-

glutamyl transferase (y-GT) and alkaline phosphatase (ALP) and tissue

histopathological changes during early renal damage induced by gentamicin.

Fifty five Sprague-Dawley rats, aged 7-8 weeks old were divided into two

groups. One group of ten rats served as controls and forty-five rats were

treated with gentamicin intraperitoneally to induce renal damage. Twenty four

hour urine samples were collected daily for 3 days. On the third day, blood

was withdrawn through cardiac puncture and kidney tissues from sacrificed

rats were taken for histopathological analysis. Urine proteins were separated

by sodium dodecyl sulphate - polyacrylamide gel electrophoresis (SDS-

PAGE). Concentration of urine proteins, enzymes, serum urea nitrogen

(BUN) and creatinine were analysed using a chemistry analyser. The

SDS-PAGE of urine proteins showed that proteinuria in gentamicin-treated

rats consisted primarily of protein with low molecular weight. The separated

urine proteins were mainly located in the molecular weight range of 10 to >

70 kDa. An extra protein band with molecular weight estimated to be

approximately 11 kDa was also detected in the gentamicin-treated group and

suggested to be an indicator of early renal disease. The electrophoretic

patterns of urine proteins also demonstrated that the type of proteins leaking

into urine had also provided some insight on the location and degree of renal

injury. High urine enzymes concentrations specifically ALP had also reflected

that the onset of injury was in the renal proximal tubular cells. This was

confirmed by renal tissue histopathological evidences. Each parameter

showed a high grading in gentamicin-treated group. Grade 3 cytoplasmic

vacuolization (31%), grade 3 tubular dilatation (33.3%) and grade 3 cell

detachment (33.3%) were the most prominent morphological changes in that

group. BUN and serum creatinine did not show any obvious difference

between the two groups. In conclusion, the elevated concentrations of low

molecular weight urine proteins and urine enzymes appeared to be useful

indicators for early renal damage as their elevation occurred before the BUN

and serum creatinine produced any change.

Abstrak tesis yang dikemukakan kepada Senat Universiti Putra Malaysia sebagai memenuhi keperluan untuk ljazah Master Sains

PERKAITAN Dl ANTARA PROTElNURlA DAN ENZlMURlA DENGAN PERUBAHAN HISTOPATOLOGI GINJAL SEMASA KEROSAKAN GINJAL

AWAL YANG DIARUH OLEH GENTAMISIN

Oleh

SAIRAH BlNTl ABDUL KARlM

Mei 2004

Pengerusi : Professor Rasedee Abdullah, Ph.D.

Fakulti : Peru batan Veterinar

Kajian ini dijalankan untuk menentukan perkaitan di antara protein urin dan

penanda enzim urin, aspartat aminotransferase (AST), -glutamil transferase

- - .(-GT), dan alkalin fosfatase (ALP) dengan perubahan histopatologi dalam .

kerosakan renal awal yang diaruh oleh gentamisin. Lima puluh lima ekor

tikus Sprague-Dawley, berumur 7-8 minggu, telah dibahagikan kepada dua

kumpulan. Kumpulan pertama dengan sepuluh ekor tikus bertindak sebagai

kawalan, dan empat puluh lima ekor lagi diperlakukan dengan gentamisin

secara suntikan intraperitoneum untuk mengaruhkan kerosakan ginjal.

Sampel dua puluh empat jam urin telah diambil setiap hari selama tiga hari.

Pada hari ketiga darah &paolehi melaluiucdan kardiwrt dantktt ginjat

daripada tikus yang telah dimatikan diperolehi untuk analisis histopatologi.

Protein urin diasingkan melalui elektroforesis agar-agar natrium dodesil sulfat

-poliakrilamida (SDS-PAGE). Kepekatan protein and enzim urin,

nitrogen urea darah (BUN) dan kreatinina serum telah dianalisis

mengguna penganalisis kimia. SDS-PAGE untuk protein urin menunjukkan

proteinuria dalam tikus terperlaku gentamisin terdiri terutama sekali daripada

protein berat molekul rendah. Protein yang terasing itu terletak khususnya

pada julat berat molekul 10 hingga >70 kDa. Satu lagi jalur protein yang

berat molekulnya dianggar pada lebih kurang 11 kDa telah dikesan dalam

kumpulan terperlaku gentamisin dan ini disarankan sebagai petunjuk kepada

penyakit gingal awal. Pola elektroforesis protein urin telah menunjukkan

yang jenis protein yang terkeluar kepada urin telah memberi gambaran

kepada tahap dan tempat berlakunya kecederaan renal. Kepekatan enzim

urin tinggi, khususnya ALP telah mencerminkan yang kecederaan mula

tercetus pada sel tubul proksimal renal. Ini telah terbukti pada histopatologi

tisu renal. Setiap parameter menunjukkan gred yang tinggi bagi kumpulan

yang disuntik gentamisin. Gred 3 vakulisasi sitoplama (31%), gred 3 dilatasi

tubul (33.3%) dan gred 3 penanggalan sel (33.3%) merupakan perubahan-

perubahan morfologi yang ketara ditunjukkan oleh kumpulan tersebut. BUN

dan kreatinina serum tidak menunjukkan sebarang kelainan yang jelas di

antara kumpulan. Adalah disimpulkan yang peningkatan kepekatan protein

berat molekul rendah dan enzim urin merupakan petunjuk berguna dalam

kerosakan renal awal kerana peningkatannya berlaku sebelum ada sebarang

perubahan dalam BUN dan serum kreatinina.

ACKNOWLEDGEMENTS

I would sincerely like to extend my warmest thanks and deepest

appreciation to my main supervisor, Prof. Dr. Rasedee Abdullah for his

guidance, advice and encouragement, which he patiently and unselfishly

gave throughout the course of this study.

I also would like to thank to my co-supervisors, Assoc. Prof. Dr. Abdul

Rahim Mutalib and Assoc. Prof. Dr. Mohd. Hair Bejo for their suggestions

and support.

Sincere gratitude and appreciation are forwarded to Mr. Mohd. Halmi

Othman and Mr. Abdullah Misron, the Hematology Laboratory in Microbiology

and Pathology Department staff for their invaluable technical assistance

during this study.

Last but not least, I would like to extend my gratitude to my loving

husband, Mr. Mohd. Fadhil Ahmad, my son, Faiz Isqandar, my parents and

family members for the constant support, love and encouragement.

vii

I certify that an Examination Committee met on 18 '~ May 2004 to conduct the final examination of Sairah Bte Abdul Karim on her Master of Science thesis entitled "A Correlation Between Proteinuria, Enzymuria and Kidney Histopathological Changes During Early Renal Damage Induced by Gentamicin" in accordance with Universiti Pertanian Malaysia (Higher Degree) Act 1980 and Universiti Pertanian Malaysia (Higher Degree) Regulations 1981. The Committee recommends that the candidate be awarded the relevant degree. Members of the Examination Committee are as follows:

Mohamed Ali Rajion, Ph.D. Professor Faculty of Veterinary Medicine Universiti Putra Malaysia (Chairman)

Abdul Rahman Omar, Ph.D. Associate Professor Faculty of Veterinary Medicine Universiti Putra Malaysia (Member)

Jasni Sabri, Ph.D. Associate Professor Faculty of Veterinary Medicine Universiti Putra Malaysia (Member)

Nabishah Mohamad, Ph.D. Professor Faculty of Medicine Universiti Kebangsaan Malaysia (Independent Examiner)

GULAM RU ~ r o f e s s o r / ~ e ~ c ) t ~ dean . .

School of Graduate Studies Universiti Putra Malaysia

Date: 1 7 JUN 2004

The thesis submitted to the Senate of Universiti Putra Malaysia and has been accepted as fulfilment of the requirement for the degree of Master of Science. The members of the Supervisory Committee are as follows:

Rasedee Abdullah, Ph.D. Professor Faculty of Veterinary Medicine Universiti Putra Malaysia (Chairman)

Mohd. Hair Mohd. Bejo, Ph.D. Associate Professor Faculty of Veterinary Medicine Universiti Putra Malaysia (Member)

Abdul Rahim Mutalib, Ph.D. Associate Professor Faculty of Veterinary Medicine Universiti Putra Malaysia (Member)

AlNl IDERIS, Ph.D. Professor/Dean School of Graduate Studies Universiti Putra Malaysia

DECLARATION

I hereby declare that the thesis is based on my original work except for quotations and citations which have been duly acknowledged. I also declare that it has not been previously or concurrently submitted for any other degree at UPM or other institutions.

TABLE OF CONTENTS

Page

DEDICATION ABSTRACT ABSTRAK ACKNOWLEDGMENTS APPROVAL DECLARATION LlST OF FIGURES LlST OF TABLES LlST OF PLATES LlST OF ABBREVIATION

CHAPTER I INTRODUCTION

I I LITERATURE REVIEW Progressive Renal Disease

lntroduction Mechanisms Treatment for Kidney Diseases New Therapeutic Strategies

Current Diagnostic Approach of Renal Diseases Urine Osmolality Urine Protein Serum Creatinine Blood Urea Nitrogen Other Parameters of Renal Damage Renal Biopsy

Development of Diagnostic Assays of Renal Damage Urine Assays Urine Chemical lndicators Urine Enzymes lndicators Serum lndicators of Renal Damage

Gentamicin Introduction Pharmacokinetics Mechanisms of Nephrotoxicity Gentamicin-Induced Renal Damage Gentamicin-Induced Proteinuria Gentamicin-Induced Enzymuria Gentamicin-Induced Renal Lesions

Conclusion

.. I I iii v vii viii X

xiv xv xvi xvii

MATERIALS AND METHODS Animals and Management Experimental Designs Blood Collections Urine Collections Kidney Tissues Collections Statistical Analysis

EXPERIMENT 1: DETERMINATION OF THE URINARY PROTEIN PATTERNS IN SPRAGUE-DAWLEY RATS AFTER ADMINISTRATION OF A SINGLE DOSE OF GENTAMICIN. lntroduction Materials and Methods

Animals and Management Urine and Serum Samples Protein Analysis Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis (SDS-PAGE)

Results Discussion

EXPERIMENT 2: DETERMINATION OF THE URINARY ENZYME CHANGES AND SERUM PARAMETERS IN SPRAGUE-DAWLEY RATS AFTER ADMINISTRATION OF A SINGLE DOSE OF GENTAMICIN Introduction 39 Materials and Methods 40

Animals and Management 40 Urine and Serum Samples 40 Urinary Enzymes and Serum Biochemistry Analysis 40

Results 4 1 Discussion 47

EXPERIMENT 3: DETERMINATION OF THE EXTENT OF RENAL DAMAGE IN SPRAGUE-DAWLEY RATS AFTER ADMINISTRATION OF A SINGLE DOSE OF GENTAMICIN lntroduction Materials and Methods

Animals and Management Histopathological Examination

Results Discussion

xii

GENERAL DISCUSSION AND CONCLUSIONS

BIBLIOGRAPHY APPENDICES BIODATA OF THE AUTHOR

... X l l l

LIST OF FIGURES

Figure

1

Page

Urinary proteins excretion after a single dose of gentamicin administration (1 00 mglkg body weight)

SDS-PAGE of urinary proteins pattern on days post- gentamicin administration (1 00 mglkg body weight)

Urine aspartate aminotransferase (AST) excretion after single dose gentamicin administration (100 mgfkg)

Urine gamma glutamyl transferase (GGT) excretion after single dose gentamicin administration (1 00 mgfkg)

Urine alkaline phosphatase (ALP) excretion after single dose gentamicin administration (1 00 mglkg)

Percentage of tubular necrosis in the kidney sections of control and gentamicin-treated group

Percentage of tubular dilatation in the kidney sections of control and gentamicin-treated group

Percentage of cell detachment in the kidney sections of control and gentamicin-treated group

Percentage of denuded basement membrane in the kidney sections of control and gentamicin-treated group

Percentage of apical cytoplasmic vacoulisation in the kidney sections of control and gentamicin-treated group

Calibration curve of standard protein separated by SDS-PAGE

xiv

LIST OF TABLES

Table

1 Serum urea (BUN) and creatinine concentration on day one and day three post-gentamicin administration

Serum electrolytes concentration on day one and day three post-gentamicin administration

Urinary protein concentrations at different days from gentamicin treated rat and control.

Urine aspartate transferase activities at different days from gentamicin treated rat and control

Urine gamma glutamyl transferase activities at at different days from gentamicin treated rat and control

Urine alkaline phosphatase activities at different days from gentamicin treated rat and control

Page

LIST OF PLATES

Plate Page

Kidney section from gentamicin-treated rat. Severe vacuolization of tubular cells (arrow) was noted in injured tubular epithelium (HE, x200)

Kidney section from gentamicin-treated rat. The tubules devoid of epithelial cells (arrow), leaving denuded basement membrane (M), filled with amorphous, eosinophilic debris and intraluminal collection of necrotic cells (HE, x200)

Kidney section from gentamicin-treated rat. Tubules were filled with eosinophilic debris (E) and necrotic cells (N) (HE, x200)

Kidney section from gentamicin-treated rat. Congestions was noted in glomerulus and tubular cells(C). The tubules re were filled with eosinophilic debris (E) and intra-luminal collections of necrotic cells (N) (HE, x200)

Kidney section from gentamicin - treated rat . Tubules were filled with necrotic cells (N) and eosinophilic debris (E). Congestion (C ) also was noted (HE, x200)

Kidney section from gentamicin treated rat. The tubules devoid of epithelial cells, leaving denuded basement membrane (M), filled with amorphous, eosinophilic debris (E) and intraluminal collection of necrotic cells (HE, x200)

Kidney section from gentamicin-treated rat. Tubular dilatation (D) and congestion (C) were noted (HE, x200)

Kidney section from control rat received normal saline. Renal tubules (R) were normal (HE, x200)

xvi

LIST OF ABBREVIATIONS

AAP

ACE

ALP

ARF

AST

BUN

CRF

ESRD

GFR

GGT

LDH

NAG

NEP

PFD

RAS

RBP

RRT

SDS-PAGE

alanine aminopeptidase

angiotensin converting enzyme

alkaline phosphatase

acute renal failure

aspartate transferase

blood urea nitrogen

chronic renal failure

end stage renal disease

glomerular filtration rate

gamma glutamyl transferase

lactate dehydrogenase

neutral endopeptidase

Pirfenidone

rennin angiotensin system

retinal-binding protein

renal replacement therapy

Sodium dodecyl sulfate polyacrylamide gel electrophoresis

xvii

CHAPTER l

INTRODUCTION

Among human diseases, chronic renal failure (CRF) remains a serious health

problem and the prevalence is increasing worldwide. Chronic renal failure is

the most significant result of chronic kidney disease, characterized by a

progressive loss of renal function. Damage to the kidneys over a period of

ten years or more, may result in terminal kidney failure or end-stage renal

disease (ESRD), which is the most feared consequence of kidney disease.

According to Pierro et a/., (2001) the prevalence of ESRD is a good indicator

of the burden of renal disease in a country. Therefore, ideally, effective

programs should be directed at preventing the development of chronic renal

insufficiency which could subsequent progress to ESRD. The early detection

of kidney disease is very important.

Prevention through early detection of kidney diseases may serve as a

solution to reduce the prevalence of renal disease. Treatment at the earlier

stages of kidney disease will arrest the progression of the established kidney

disease or even l ead to the regression of the renal p arenchymal d amage.

One epidemiological study suggested patients with kidney disease detected

earlier had almost twice greater survival rate than those evaluated at a late

stage (Mat Edelson, 2003). Unfortunately, many kidney diseases tended to

be latent at onset and progress slowly making detection difficult. This is

because kidneys have very high compensatory abilities that can compensate

up to 70% loss of functional mass and this unique feature had contributed to

the difficulty of detection of renal insufficiency at very early stages.

At present, identification of patients with early renal disease relies heavily on

blood and urine analysis in patients at increased risk for renal disease

before the presence of significant symptoms. Analysis of urinary proteins

and enzymes are accepted as reliable biomarkers and is very useful for the

early diagnosis of tubule damage.

Even though there are numerous studies on enzymuria and proteinuria, it

seems there is lack of information on their correlation with renal disease

especially at the early stages of renal damage. Hypothesis of this study is

that there are correlation between proteinuria, enzymuria and renal

histopathological changes during early renal damage. Thus, this study was

undertaken to determine their correlation and consequently ascertain the

earliest indicator of renal damage. Gentamicin, an aminoglycoside antibiotic

was used as the inducer for renal damage in this study and its effects were

assessed to determined:

a. the relevant biochemical parameters that may be used as

indicators of renal insufficiency during very early stage.

b. the rate of excretion of selected serum and urine biochemical

parameters (blood urea nitrogen, serum creatinine, electrolytes,

protein, alkaline phosphatase, aspartate arninotransferase, gamma

glutamyl transferase) during early renal damage.

c. the correlation between the extent of renal damage and urinary

enzyme and protein excretions.

CHAPTER ll

LITERATURE REVIEW

Progressive Renal Disease

Introduction

The progressive loss of renal function to end-stage renal disease (ESRD) is

a common p henomenon i n renal p atients i rrespective of the i nitial d isease

(Mai et a/., 2000). The most important causes of ESRD, at least in the United

States, were hypertension and diabetic nephropathy (Raine, 1994), which

are reliable predictors of risk for kidney disease. Patients who recover from

an episode of acute renal failure may be at risk of developing chronic renal

disease (Salem, 1999).

Nowadays, patients with various diabetic and non-diabetic glomerulopathies

form the m ain contingent for renal replacement therapy (RRT) (Mai et a/.,

2000). The number of patients receiving dialysis and transplantation

continues to increase (Carl, 1995). These therapies are expensive, so that

prevention is of prime importance. Arresting the rate of the deterioration of

kidney failure will have a great impact and this may be achieved through

accurate detection of early abnormal renal changes (Mai et a/., 2000).

Mechanisms

The mechanism underlying progression of renal disease is likely to be

multifactorial. The nature of the progressive renal damage with various

etiologies is governed by such factors as hemodynamics, participation of the

renin-ang iotensin system (RAS) and progressive proteinuria (Mai et a/. ,

2000). All histological manifestations from patients with impaired kidney

function show a rather similar histological picture of glomerulosclerosis,

interstitial fibrosis, tubular atrophy and an interstitial i nfiltration (Vleming et

a/., 1999). These seem to suggest that the progressive loss of kidney

function results from a pathologic process that serves as a final cascade of

events before renal failure. These events may be independent of the original

etiology of the disease (Vleming et a/., 1999).

Disturbances in lipid metabolism, participation of cellular and molecular

factors and subsequent accumulation of extra-cellular matrix component with -

. the development of kidney fibrosis are closely related to the renal disease

progression (Mai et a/., 2000). Recently much attention has been paid to the

importance of persistence proteinuria, as an independent risk factor of renal

disease progression (Giuseppe, 2000). There are now evidences which

suggest a strong correlation between the degree of proteinuria and the rate

of progression of renal failure in both diabetic (Breyer et a/., 1996) and non-

diabetic (Ruggenenti et a/., 1998) renal disease patients. For

also, the National Kidney Foundation (NFK) has recommended that high-risk

individuals undergo routine screening for proteinuria (Verna, 1999).

Treatment for Kidney Diseases

Treatment of kidney diseases is a complex issue and depends on the type of

disease, the underlying cause, and the duration of the diseases. Clinically it

is important to differentiate acute renal failure (ARF) from chronic renal

failure (CRF) because generally acute disease is potentially reversible while

the chronic form is not. Unfortunately, ARF may eventually go on to develop

into CRF or end-stage-renal-failure if no treatment is instituted. Up to 10% of

patients who experience ARF will suffer irreversible renal failure (Paula,

2000).

Treatment usually starts with addressing the original causes. In the case of

acute kidney failure, treating the underlying cause may return the kidneys to

normal function. Normally, initial treatments are focused o n correcting fluid

and electrolyte balances and uremia (Malay and Richard, 2000). Sometimes

dietary restrictions (less salt and protein) are required until the kidneys are

able to handle these substances. Diuretic drugs may help the body to

excrete more water and salts. However, in CRF, drugs are used to stop the

progression of the disease to ESRD.

When kidney disease does not respond to treatment with dietary restrictions

and drugs, dialysis or kidney transplantation are the next treatments to

consider. At least 500 000 people throughout the world are being routinely

maintained by renal dialysis (Pierro et a/., 2001). Dialysis is a technique used

to remove waste products from the blood and excess fluids from the body of

renal failure patients. Kidney transplantation on the other hand is a surgical

procedure in which the diseased kidney is removed and replaced with a

healthy kidney from a donor. This is the most effective form of renal disease

therapy, with more than 12 000 procedures performed per year in the United

State (David and Christopher, 1999). However, renal replacement therapy is

expensive.

New Therapeutic Strategies

Renoprotection is a strategy, which has been developed to interrupt or

reverse the progression of renal disease. Various modalities are now being

used includes low-protein diet, anti-hypertensive therapy, and anti-fibrotic

therapy (Mai et a/., 2000). However, it seems that the benefit of a low-protein

diet in slowing the progression of renal failure is negligible (Pierro et a/.,

2001). Instead an adequate early anti-proteinuric therapy may be a better

approach. Anti-proteinuric therapy may arrest the progression of the renal

disease or even lead to regression of the disease (Mai eta/., 2002).

Hypertension is recognized to be a strong independent risk factor for ESRD

(Klag et a/., 1996). Renoprotection through lowering the blood pressure

uniformly has shown to slow the disease progression and reduces renal

damage (Anderson et a/., 1986). Among the anti-hypertensive drugs, the

angiotensin-converting-enzyme (ACE) inhibitors are most effective in