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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).
1
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