UNIVERSITI PUTRA MALAYSIA

MERCURY-INDUCED NEPHROTIC SYNDROME: A CORRELATION BETWEEN PATHOLOGICAL CHANGES AND SERUM AND URINE BIOCHEMISTRY PARAMETERS IN RATS

SUHAIDAH IBRAHIM

FPV 1998 9

MERCURY-INDUCED NEPHROTIC SYNDROME: A CORRELATION BETWEEN PATHOLOGICAL CHANGES AND SERUM AND URINE BIOCHEMISTRY PARAMETERS IN RATS

By

SUHAIDAH IBRAHIM

Thesis Submitted in Fulfilment of the Requirement for the Degree of Master of Science in the Faculty of

Veterinary Medicine and Animal Science Universiti Putra Malaysia

March 1998

ACKNOWLEDGEMENTS

All praise to Almighty Allah, the Merciful and the Benevolent. Had it not been

due to His will and favour, the completion of this study would not have been possible.

First of all, I would like to express my sincere gratitude and appreciation to my

supervisor, Associate Professor Dr. Rasedee Abdullah, for his invaluable advice,

guidance, and supervision throughout the duration of the study.

I would like to express my sincere gratitude and appreciation to Dr. Noordin

Mohamed Mustapha for his invaluable advice, guidance, and encouragement towards the

completion of my study. I would also like to express my gratitude and appreciation to

Associate Professor Dr. Noor Azhar Mohd. Shazili for his advice and assistance in trace

element analysis; to Professor Law Ah Tean for his kindness in method of mercury

analysis; to Dr. Zulkifli Idrus and Dr. Nazri Salim for their help in statistical analysis; and

also to Dr. P.G. Joseph and Dr. Abdul Rahim Mutalib for their permission to have the

rats and use the facilities of the experimental animal house for my study.

I also wish to thanks the staff members of Clinical Pathology Laboratory; Dr.

Ragavan Koran, En. Mohd. Halmi Othman, En. Mohd. Jamil Samad, and En. Abu Bakar

Taha, and members of Histology Laboratory; Associate Professor Dr. Mohd. Zamri

11

Saad, En. Abdullah Misron, and En. Mohd. Noh Manaffor their assistance throughout

my study, and also to all other staff members of the Faculty of Veterinary Medicine and

Animal Science, University Putra Malaysia for their invaluable assistance in my study.

Last but not least, I would like to express my sincere appreciation to Associate

Professor Dr. Mohd. Nasir Hassan and his family, Dr. Mohd. Zaki Abdul Rahman, and

Associate Professor Dr. Che Nyonya Abdul Razak and her family of Faculty Science

and Environmental Study, Professor Abdul Rani Bahaman of Institute of Bioscience

Research, Dr. Mohamad Mustafa of MARDI, and also to my friends, Dr. Robert D.

Gaffin of Texas A&M University, Sudesh Kumar of Sa it am a University, and Norhaizan

Mohd. Esa of Surrey University for giving me support and encouragement through the

hard times.

My special thanks goes to my sister Suhawati Ibrahim and my parents for their

understanding of my ambition.

III

TABLE OF CONTENTS

Page ACKNOWLEDGEMENTS ... . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ii LIST OF FIGURES . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . vii LIST OF PLATES . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . viii LIST OF ABBREVIATIONS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . IX ABSTRACT . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . XlI ABSTRAK .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . xv

CHAPTER

1 INTRODUCTION.... . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1

Mercury-Induced Nephrotic Syndrome: A Correlation Between Pathological changes and Serum Biochemical Parameters.. . .. . . . . . . . . . . . . 1

2 LITERATURE REVIEW. .. . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4

Nephrotic Syndrome... . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . .. . . . . .. . . . .. . . . . . . . . . . . . . . . . . . . . 4 Introduction... . . . . . . . . . . . . . . . . . . .. . . .. .. .. ... . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . 4 Classification of Renal Lesion in Nephrotic Syndrome. . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 Etiologies of Nephrotic Syndrome. .. . . . . ...... . . . . . . . . . . . . . . . . . . . . . . . . . . 7 Clinical Signs and Complication of Renal Damage in Nephrotic Syndrome. . . . . . . . . . . . . . . .. . . . . . . .. . . . . . . . . . . . . . . . . . 8 StagesofRenaIFailure . . . . . . . . . . . . . . . . . . .. . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 Laboratory Diagnosis of Renal Damage.. . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . 12

Mercury . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ..... .. . . . . . . . . . ..... . . . . . . . . . . . . . . . . 1 4 Introduction. .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 4 Uptake and Absorption. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ... . . . . . . . . . . . . . . . 1 5 Distribution. . . . . . . .. . . .. .. . . ... . .. . . . ...... . ... ............ .. .. .. . .... . . .... ......... 17 Metabolism. .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18 Excretion. .. . . . .. . . . . . . .. . . . . . . . . .. . . . .. .. .. ............. ..... .. . . ..... ..... ... ... ..... 20 Toxicity . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . .. . . . . .... . . . . ... . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20

Mercury-Induced Nephrotic Syndrome . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ... . . . . . . . . .. 22

IV

Page 3 MATERIALS AND METHODS . . . . . . . . , . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 24

Animal and Management.. . . . . . . . . . . . . . . . . . . . . . . . .. . . . . ........... . . . . . . . . . . . . . . . . . . . . . . . . 24 Urine and Serum Analyses. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 24 Mercury Analysis. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25

Glasswares and Surgical Instruments. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . 25 Reagents. . . .. . . . ............... .......... . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25 Preparation of Samples. ... . . . . . . ... . .... . . . . . . . ...... . . . . . . . . . . . . . . . . . . . . . . . . . . 26

Equipment. ................. . . . . . . . . . . . . . . .... . . . . . . ... . . . . . . ...... . . . . ... . . . . . . . . '" . . . . . . . . . . . . . 27 Light Microscopy. ..... ..... . . . . . . . . . .. . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 28 Statistical Analysis.. ........ . . .. . . .......... . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . 28

4 EXPERIMENT 1 : THE RESPONSE OF RENAL ASSOCIATED BIOCHEMICAL PARAMETERS TO REPEATED INTRAVENOUS ADMINISTRATION OF MERCURYCIILORIDE .. . . . ... ... .... ... . . . . .. . .. . . ... . . . . .... . . . . . .. . . . . . . . . . . . . . . . . . . 30

Introduction .. .... . . . . . . . ............... .... . . . . . . .. . ..... .... ... .. ... . . ....................... .. 30 Materials and Methods. . . .. . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . .......... . . . . . . . 3 1

Animals and Managements... . . . . . . . . . . . . . . . . . . .. . .................. . . . . . . . . . . 3 1 Urine and Serum Samples. . . . . . . . . . . . . . . . . . . . . . . . . ..... . . . . . . . . . . . . . . . . . . . . . . . 3 1

Results ............................................................................ . .. . . . . . . ........ 3 1 Serum Analysis. .... . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3 1 UrineAnalysis ... ............ . .. .. ....... . ............ ... ... ... . . ...... ........ . . ... . 32

Discussion... . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 40

5 EXPERIMENT 2: TO DETERMINE THE CONCENTRATION OF MERCURY IN RENAL AND LIVER TISSUES AFTER REPEATED INTRAVENOUS ADMINISTRATION OF MERCURY CIILORIDE .. ... . ... . ... ... ... ..... ....... ....... .. .... .. . . . . . . . . . . . . . . . . . . 44

Introduction....... . .. . . . . . . . . . . . . . . ... ..................... ..................................... 44 Materials and Methods ... ... . ... ... ... . . . . . . . . . . . .... . . .. . . ..... . . . . ............ .. . . . . . . . . . 45

Animal and Management. . . . . . . . . . . . . . . . . . . . . .. . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . 45 Tissue Mercury Analysis . .. . . . . . . ... . . . .. . . . . . .. ... . . . .......... . ......... . . . .. 45

Results .. . . . . . . . . . . . . . ........ . . ... . . . ......... ... .. ...... . .... . . ..... . ... . . . . ... ... . . . . . . . . . . . . ... . 45 Discussion... . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 48

v

6 EXPERIMENT 3 : THE STAGES OF CHRONIC RENAL FAILURE AND REPARATIVE PHASE OF THE KIDNEYS FOLLOWING REPEATED MERCURY CIll.,ORIDEEXPOSURE . . . . . . . . . . . . .. . . . . . . . . . . . ... . . . . . . . . . . . . . . . . . . . . . . . . . .. .... . . . . . 51

Introduction. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ... . . . . . . . . .. . . . . . . . . . . ... . . . . . . . . 5 1 Materials and Methods. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 52

Animals and Managements. . . .. . . . . . . . . .. . . . . . . . . . . . . . . . . ... . . . . . . . . . ... . . . . . . 52 Light Microscopy. . . . . . . . . .... . . . . . . . . . . . . . . . . . . . ..... . . . . . . . . . . . . . . . . . . . . . . . . . . .. . 53

Results . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . ... . . . . . . . . . . . . . .. . . . . . . . . . . . . . . .... . . . . . . 53 Discussion. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 60

7 GENERALDISCUSSION . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . 62

REFERENCES . .. . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 67

APPENDIX ........ . . . . . . . . . . . . ................... . . . . .. . . ........ . . . . ........... . . . . . . . . . . . . .......... . . . . ...... . . . . 74

BIOGRAPHY OF THEAUTHOR. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. .. . . . . . . . . . . . . . . . .. . . . . . . ... 84

VI

LIST OF FIGURES

Figures Page

3 . 1 Diagrammatic set-up of the cold vapour atomic absorption spectrophotometer. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 29

4. 1 The concentration of blood urea nitrogen following repeated exposure to mercury chloride... . . . . . . . . . . . . . . . . . . . . . . . . . . . 34

4 .2 The concentration of serum creatinine following repeated exposure to mercury chloride.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3 5

4.3 The 'concentration of serum total protein following repeated exposure to mercury chloride. . . . . .. . . . . . . . . . . . . . . . . . . . . . . . 36

4.4 The concentration of serum albumin following repeated exposure to mercury chloride. . . . . . . .. . . . . . .. . . . . . . . . . . . . . . 37

4.5 The concentration of urine total protein following repeated exposure to mercury chloride. . . ... . . . . . . . . . . . . . . . . . . . . . . . . 38

4 .6 The concentration of urine albumin following repeated exposure to mercury chloride.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . 39

5 . 1 The concentration of renal mercury following repeated exposure to mercury chloride. . . . . . . . . . . . . ... . . . . . . . . . . . . . . 46

5 .2 The concentration of hepatic mercury following repeated exposure to mercury chloride. .. . . . . ... . . . . . . . . . . . . . . . . . . . . 47

6. 1 Abnormal kidney cells following repeated exposure to mercury chloride... . . . . . . . . . . . . . . . . . . . . . . . . . . . 55

6.2 Epithelisation in the kidney following repeated exposure to mercury chloride. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 56

VII

Plates

6. 1

6.2

6.3

6.4

6.5

LIST OF PLATES

Photograph, kidney, treated group. The kidneys were enlarged

Page

(shownby arrow head) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 57

Photomicrograph, kidney, control group. Damage limited to mild evidence of karyolysis and vacoulated cytoplasm only (100Xmagnification) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 57

Photomicrograph, kidney, treated group. An early damage (day 1 8) with karyolysis (Cy), vacoulated cytoplasm (V c), swollen cells (Sc),

pycnotic (P), and sloughing-off cells in lumen (S) (200Xmagnification) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 58

Photomicrograph, kidney, treated group. Late stage damage (day 3 8) showing loss of renal architecture (200Xmagnification) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 58

Photomicrograph, kidney, treated group. An epithelisation process taking place on day 14. The young cells were bluish in colour (Ep), (200Xmagnification) . . . . . . . . ... . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 59

Vlll

AAS

AT III

ATPase

BOD

BUN

Cd

CGN

Cu

EDTA-Naz

EP

Factor V

Factor VII

Factor VIII

Factor IX

Factor X

Factor XII

g

J-lg

LIST OF ABBREVIATIONS

Atomic absorption spectrophotometer

Antithrombin III

Adenosine triphosphatase

Biological oxygen demand

Blood urea nitrogen

Calcium ion

Cadmium

Chronic glomerulonephritis

Cooper

Ethylenediamine tetraacetic acid disodium salt

Extraction procedure

Cofactor proaccelerin

Proconvertin (enzyme)

Antihaemophilic factor

Christmas factor / Plasma thromboplastin component

Stuart-Prower factor (enzyme)

Hageman factor

Gram

Microgram

IX

GBM

GFR

HCI

H&E

Hg

HgCl2

RN03

H2S04

IgG

IgA

IgM

IgE

K+

K2Cr207

kg

KMn04

mg

ml

MPGN

MT

NaCI

NS

Glomerular basement membrane

Glomerular filtration rate

Hydrochloride acid

Haematoxylin and Eosin

Mercury

Mercury chloride

Nitric acid

Sulphuric acid

Immunoglobulin G

Immunoglobulin A

Immunoglobulin M

Immunoglobulin E

Potassium ion

Potassium dichromate

Kilogram

Potassium permanganate

Milligram

Millilitre

Mesangial proliferative glomerulonephritis

Metallothionein

Sodium ion

Sodium chloride

Nephrotic syndrome

x

pbb Part per billion

Se Selenium

SG Specific gravity

SLE Systemic lupus erythromatosus

Snel2 Stannous chloride

Zn Zinc

Xl

Abstract of thesis submitted to the Senate ofUniversiti Putra Malaysia in fulfilment of the requirements for the degree of Master of Science

MERCURY-INDUCED NEPHROTIC SYNDROME: A

CORRELATION BETWEEN PATHOLOGICAL CHANGES AND

SERUM AND URINE BIOCHEMISTRY PARAMETERS IN RATS

by

SUHAIDAH mRAHIM

March 1998

Chairman: Associate Professor Rasedee Abdullah, Ph.D

Faculty: Veterinary Medicine and Animal Science

Nephrotic syndrome (NS) is a renal disease featured mainly by proteinuria,

hypoalbuminemia, oedema, and ascites. The etiologies could be diverse while the signs

and symptoms are detected only at late stages ofthe disease. This study was conducted

to assess the response of serum and urine biochemical indicators of renal failure and the

compensatory mechanisrnls that may be involved in maintaining optimum renal function

following repeated exposure to mercury chloride (HgCI2). A total of forty-five

Sprague-Dawley rats aged between eight to ten weeks were injected intravenously

through tail vein with 0.5 mg ofHgCli kg body weight every alternate days for ten days.

The same number of rats were injected with 1 ml of normal saline/ kg body weight and

Xl1

served as controls. Five rats from each group were killed every four days commencing

from the fourth day of the last injection.

There were significant changes observed in the concentration of blood urea

nitrogen (BUN), serum creatinine, serum total protein, serum albumin, urine total

protein, and urine albumin during the 42-day experimental period. The concentration

of BUN begun to increase significantly (P<0.05) by day 22, but returned to normal

values after the initial increase on day 30 . While serum creatinine concentration

fluctuated with two peak values on days 34 and 42. Loss of albumin from plasma was

observed to be intermittent and urine total protein showed a late increase on day 34.

Urine albumin showed a significant earlier increase (p<O.05) on day 18, but decreased

toward control values for the next 8 days before increasing back to a peak value on day

42.

The deposition of mercury (Hg) following chronic exposure was high in the

kidneys and the liver. The concentration of renal Hg was at peak values from day 14 to

day 22 and gradually decreased thereafter. The renal tubular damage was observed to

begin on day 1 8 and increased in intensity 26 days into the experiment reaching peak on

day 42. There was also epithelisation of renal tubular epithelium. This response was

greater on day '14 and quickly decreased thereon to disappear completely by day 28.

The extensive damage of renal tubules which began on day 18 onwards could be due to

an excessive loading of the metal beyond tissue elemental saturation and to the long

retention ofHg in the tissues.

X1ll

The study suggests Hg accumulated predominantly in the kidneys and produced

a biphasic response of renal-associated biochemical parameters in which urine albumin

is the possible early indicator to renal damage. Tubular epithelisation could be one of the

mechanisms involved in maintaining the optimum renal function.

XlV

Abstrak tesis yang dikemukakan kepada Senat Universiti Putra Malaysia bagi memenuhi syarat-syarat untuk ijazah saIjana sains

SINDROM NEPROSIS TERARUH MERKURI: SUATU PERKAITAN

DI ANTARAPERUBAHANPATOLOGI DANPARAMETER

BIOKIMIA

Oleh

SUHAIDAH IBRAIDM

Mac 1998

Pengerusi: Professor Madya Rasedee Abdullah, Ph.D

Fakulti: Kedoktoran Veterinar dan Sains Peternakan

Sindrom nefrosis (NS) ialah satu penyakit renal yang dinyatakan oleh proteinuria,

hipoalbuminemia, edema, dan asitis. Etiologinya mungkin pelbagai, sambil petanda dan

simptom pula hanya dapat dikesan pada peringkat lewat penyakit ini. Kajian ini

dikendalikan untuk menilai gerak balas petunjuk biokimia serum terhadap kegagalan

renal dan mekanisme pampasan yang terlibat dalam penyenggaraan fungsi renal optimum

berikutan pendedahan berulang kepada merkuri klorida (HgC12). Empat puluh lima ekor

tikus Sprague-Dawley berumur lapan hingga sepuluh minggu disuntik secara intravena

menerusi vena ekor dengan 0.5 mg HgCI/kg berat badan setiap selang satu hari selama

sepuluh hari. Sejumlah sarna tikus disuntik dengan 1 ml 0.85% natrium klorida

xv

(NaCI2)/kg berat badan bertindak sebagai kawalan. Lima ekor tikus daripada setiap

kumpulan dimatikan setiap empat hari bermula empat hari selepas suntikan terakhir.

Perubahan tererti (P<O. 05) telah dicerapkan dalam kepekatan nitrogen urea darah

(BUN), kreatinin serum, protein sepenuh dan albumin serum, protein sepenuh dan

albumin urin sepanjang tempoh 42 hari ujikaji. Kepekatan BUN mula meningkat secara

tererti (P<O. 05) pada hari 22 dan kembali kepada normal selepas peningkatan awal, pada

hari 30. Sambi! itu kepekatan kreatinin serum beralun dengan dua nilai kemuncak pada

hari 34 dan 42. Kehilangan albumin daripada plasma dicerapkan tidak selanjar dan

protein sepenuh urin menunjukkan peningkatan lewat pada hari 34. Albumin urin

menunjukkan peningkat tererti (P<0.05) lebih awal pada hari 1 8 tetapi menurun semula

ke arah nilai kawalan selama 8 hari berikutannya sebelum ia meningkat semula kepada

nilai kemuncak pada hari 42.

Pengenapan merkuri (Hg) berikutan pendedahan kronik adalah tinggi dalam

ginj al dan hati. Kepekatan Hg renal berada pada nilai kemuncak daripada hari 14 hingga

22 dan beransur kurangan sejurus selepas itu. Kerosakan tubul renal dicerap bermula

pada hari 1 8 dan meningkat keamatannya selepas 26 hari ke dalam tempoh ujikaji dengan

mencapai kemuncak pada hari 42. Keepiteliuman tubul renal juga berlaku. Gerak balas

ini lebih tinggi pada hari 14 dan cepat mengurang selepas itu untuk hilang terus pada hari

28 . Kerosakan teruk tubul renal yang bermula pada hari 1 8 mungkin disebabkan oleh

pembebanan berlebihan logam ini hingga melebihi ketepuan unsur tisu dan oleh

penahanan Hg dalam tisu.

XVi

Kajian ini menyarankan Hg terkumpul secara keutamaan dalam ginjal dan

menghasilkan gerak balas dwifasa parameter biokimia berkaitan renal, yang mana

albumin urin mungkin merupakan petunjuk awal kepada kerosakan ginjal. Keepiteliuman

tubul mungkin merupakan satu daripada mekanisme yang terlibat dalam menyengarakan

fungsi renal optimum.

XVll

CHAPTER 1

INTRODUCTION

Mercury-Induced Nephrotic Syndrome: A Correlation Between

Pathological Changes and Serum Biochemical Parameters

In recent years, there has been growing concern over the extent of environmental

contamination with toxic metals due to industrial development. Among the fourteen

environmental contaminants currently specified under the US Extraction Procedure (EP)

Toxicity Test, Hg is rather high on the list.

The concentration of Hg in the environment is, in part, the result of waste

products from manufacturing processes which utilise Hg or the disposal of products

containing Hg. On a global basis, it is estimated about 1 0 metric tons mercuric waste

per year is released into fresh water and about 480 metric tons per year into oceans

(Von Burg and Greenwood, 1991 ). These mercuric wastes may sediment at the bottom

of the lakes, rivers, and seas. There, the bacteria and fungi methylate the inorganic Hg

to organic form and introduces the threat into the food chain (Hansen et aI., 1989).

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2

Accumulated Hg in the food chain, with fish and shellfish being the dominant

reservoir, responsibles for poisoning in some communities, or undergoes

biotransformation and released into th e atmosphere as elemental Hg.

The most common forms of Hg exposed to humans and animals can be

categorised into three classes; metallic Hg, inorganic Hg, and organic Hg (Clarkson,

1972). Each class ofHg has different pharmacokinetic properties with regard to their

uptake and absorption, distribution in the body , metabolism, and toxic effects.

Based on present evidence, Hg is not considered essential for living organisms

(Underwood, 1977). In fact, Hg is extremely toxic even in relatively small amounts and

is bioaccumulative. Mercury is known particularly to be a potential nephrotoxic agent

(Bariety et al., 1971). Methy lmercury is by far recognised as th e most toxic form ofHg

and it represents great risks of irreversible functional damage to both humans and

animals.

The two main routes of Hg entry into the body leading to toxicosis are ingestion

( the food chain) and inhalation ( atmostphere). Regardless of the chemical form and

route of entry, following exposure, the kidney h as been recognised to accumulate the

highest concentration ofHg ( Greenwood et a!., 1990). Th e association ofHg toxicosis

and neph rotic sy ndrome h as long been established (Mandema et al., 1963).

3 Studies so far have shown that chronic exposure to HgCl2 could lead to an

induction of biphasic membranous glomerulonephritis in Brown Norway rats ( Sapin et

al., 1 977; Druet et al., 1 978). A similar immunological process was also observed in

human.

The kidneys, although vulnerable, have a very high compensatory ability. It

could compensate up to 70% loss of functional mass. However, this unique feature has

contributed to the difficulty of detecting renal insufficiency at very early stages. Both

biochemical and morphological techniques are still incapable of detecting early damage.

Thus, this study was undertaken to determine the effects ofHg as an inducer of

NS with the following objectives:

a. to determine relevant biochemical parameters that may be used as early

indicators of renal damage.

b. to determine the correlation between pathological changes and selected

serum and urine biochemical parameters.

c. to determine the Hg concentration in kidney and liver tissues in

conjunction with the development of nephrosis.

CHAPTER 2

LITERA TURE REVIEW

Nephrotic Syndrome

Introduction

Nephrotic syndrome (NS) is a renal disease of varied etiologies characterised

by hypoalbuminemia, proteinuria, ascites, and oedema (Coggins and Maffiy, 1985). The

hallmark of this condition is attributed to an increase in the glomerular permeability due

to the loss of fixed negative charges on the glomerular membrane. This in tum allows

negatively charged polyanions, particularly albumin, to enter renal tubules and to be

excreted into urine (Coggins and Maft1y, 1985).

Massive proteinuria is the most prominent feature ofNS. A nephrotic patient

can excrete as much as 3. 5 g of protein per day through the urine (Coggins and Maft1y,

1985; Wilson, 1986a). The loss of protein into the urine directly decreases the plasma

concentration of albumin and forces the plasma fluid out of blood vessels into tissue

interstitial spaces and causing ascites and oedema. In addition, the phenomenon of

elevated plasma renin and aldosterone activity due to hypovolemia, as a result of plasma

protein loss, causes the kidney to retain more sodium and water (Wilson, 1986a). The

4

5

retention of sodium, water and other electroly tes further contributes to the increase in

extracellular fluid accumulation, and this explains the contraindications of removing

excess fluid which could further aggravate the edematous condition.

Classification of Renal Lesion in Nephrotic Syndrome

Glomerulonephritis, either primary or secondary , is a bilateral inflammatory

disease of the kidney which begins in the glomerulus (Wilson, 1986b). Approximately

50% of chronic glomerulonephritis (CGN) will eventually give rise to NS (Coggins and

Maffiy , 1985). Primary glomerulonephritis representing 75% of the incidence (Coggins

and Maffly , 1985) and can further be subclassified into four histologic entities, namely ;

membranous nephropathy ; minimal lesion glomerulonephritis; focal glomerular sclerosis;

and mesangial proliferative glomerulonephritis (MPGN) (Wilson, 1986b).

Membranous Nephropathy

Almost one-third of NS cases originated from lesions of membranous

nephropathy (Coggins and Maffiy , 1985). In this case, microscopic and ultrastructural

studies revealed thickened glomerular capillary wall due to deposits of immune

complexes (Row et aI., 1975; Wilson, 1986b). The involvement of the immune

mechanism can be demonstrated by immunoflourescence staining to show the presence

ofIgG and complement deposits (Wilson, 1986b). In the United States, nearly half of

6

the total number of adult patients with idiopathy showed this form ofNS at biopsy

(Glassock et aI. , 1981).

The response of membranous nephropathy to corticosteroid and

immunosuppressive therapy is poor with variable prognosis which either show complete

remission, improvement but still having proteinuria, persistent nephritis, or slowly

progressing to chronic renal failure (Row et aI. , 1975; Noel et aI. , 1979).

Minimal Lesion Glomerulonephritis

Minimal lesion glomerulonephritis, also known as lipoid nephrosis or foot

process disease (Wilson, 1986b), represents 15% of the incidence of NS (Cameron et

al., 1974). This is the only form of NS which does not involve immunopathology

(Cameron et al., 1974; Wilson, 1986b). The glomeruli may appear normal or nearly

normal under light microscope but lipid accumulation is seen in the epithelial cells of the

tubules whilst electron microscope studies revealed fusion of the foot processes (Wilson,

1986b). The contributing factors associated with this ty pe of lesion are diverse. It

could be food allergy, foreign agents, or Hodgkin's disease (Moorthy et aI., 1976). This

ty pe of NS accounted for 80 - 90% of cases affecting children of primary

glomerulonephritis aged between one to five y ears old (Coggins and Maffiy , 1985). The

prognosis is very good since the disease responds readily to corticosteroid treatment and

rarely progress to renal failure (!<ida et aI. , 1977).

7

Focal Glomerular Sclerosis

The third typical form of glomerulonephritis is focal glomerulosclerosis. A

sclerotic process which takes place within the glomeruli with ensuing renal insufficiency.

The pathogenetic mechanism initially involves deeper juxtamedullary nephrons and it

may or may not be detected at early renal biopsy (Wilson, 1986b). It is usually

associated with tubular defects which lead to glycosuria, aminoaciduria, renal tubular

acidosis, and phosphaturia. It has also been reported as a complication of AIDS (Rao

et aI., 1984).

Mesangial Proliferative Glomerulonephritis

Mesangial proliferative glomerulonephritis (MPGN) involves the proliferation

of mesangial cells (Wilson, 1986b). Immunoflourescent studies showed deposition of

IgA or IgM in the mesangium. The thickened glomerular basement membrane gave rise

a lobular or "wire-loop" appearance under light microscope. This type of lesion is

closely associated with lupus nephritis. A common manifestation of MPGN is

hypertension and microscopic haematuria. Normally the prognosis is poor and may

slowly progress to chronic renal failure (Wilson, 1986b).

Etiologies of Nephrotic Syndrome

The causes of NS could be diverse, encompassing infectious organisms of the

urinary tract, secondary to certain systemic diseases, or toxic agents. In most cases, the