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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was withdrawn through cardiac puncture and kidney tissues from sacrificed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