i
ROLE OF INTERLEUKIN-13 ON THE DEVELOPMENT OF MINIMAL
CHANGE NEPHROTIC SYNDROME – A NOVEL ANIMAL MODEL
LAI KIN WAI
(BSc, MMedSc)
A THESIS SUBMITTED
FOR THE DEGREE OF DOCTOR OF PHILOSOPHY
DEPARTMENT OF PAEDIATRICS
NATIONAL UNIVERSITY OF SINGAPORE
2006
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This thesis is dedicated to
Ivy and Joseph
that my life will not be complete without them
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ACKNOWLEDGEMENTS
I would like to express my thankfulness to:
My supervisor, Professor Yap Hui Kim, for her guidance throughout these years.
Professor Gilbert Chiang who has always been supportive in my pursuit of a PhD.

Mr Larry Poh and Dr Liew Lip Nyin for their valuable advices and from whom I
learnt my molecular biology techniques.
Dr Cheung Wai, Dr Gong Wei Kin and Dr Tan Puay Hoon for their helpfulness and
support.
All the friendly and nice people from Department of Paediatrics and other
departments who have helped me in a lot of ways on my experiments.
This work was supported by grants NMRC 0830/2004 from the National Medical
Research Council, Singapore, A*STAR: 04/1/21/19/341 from the Biomedical
Research Council, and R-178-000-081-112 from the University Academic Research
Fund.
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TABLE OF CONTENTS
TITLE PAGE
DEDICATION
ACKNOWLEDGEMENTS i
TABLE OF CONTENTS ii
SUMMARY ix
LIST OF ABBREVIATIONS xi
UNITS OF MEASUREMENT xvi
LIST OF TABLES xix
LIST OF FIGURES xxi
LIST OF APPENDICES xxiv
LIST OF PUBLICATIONS AND CONFERENCE ABSTRACTS xxvi
CHAPTER 1 INTRODUCTION

1
1.1 Interleukin-13 (IL-13)
1
1.1.1 Source of IL-13 1
1.1.2 Gene and protein structure of IL-13 1
1.1.3 IL-13 receptors 2
1.1.4 IL-13 and the immune system 4
1.1.5 Pathological role of IL-13 5
IL-13, asthma and allergic diseases 5
IL-13 and resistance to gastrointestinal nematodes 6
IL-13 and tissue fibrosis 6
IL-13 and tumor immunosurveillance 7
IL-13 and Hodgkin lymphoma 7
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1.1.6 Transgenic model of IL-13 8
1.1.7 In-vivo administration of recombinant IL-13 9
1.1.8 Knockout model of IL-13 9
1.2 Nephrotic syndrome
10
1.2.1 Definition of nephrotic syndrome 10
1.2.2 Classification and etiology of nephrotic syndrome 10
1.3 Pathogenesis of minimal change nephrotic syndrome
16
1.3.1 Minimal change nephrotic syndrome and the immune system 16

1.3.2 Circulating factors 16
Circulating factors and MCNS 17
Circulating factors and FSGS 19
1.3.3 Bias to Th2 cytokines and involvement of IL-13 20
1.4 Genetic aspects of nephrotic syndrome
23
1.4.1 Hereditary diseases manifested with nephrotic syndrome 23
1.4.2 Nephrin and congenital nephrotic syndrome 23
1.4.3 Podocin and steroid-resistant nephrotic syndrome 24
1.4.4
α
-Actinin-4 and familial FSGS
25
1.4.5 Animal models of nephrotic syndrome 25
1.5 Podocyte
27
1.5.1 Podocyte and the glomerular filtration barrier 27
1.5.2 Charge and size selectivity of the glomerular filtration barrier 30
1.5.3 Molecular structure of podocyte foot processes 30
1.5.4 Slit diaphragm 33
1.5.5 The podocyte in idiopathic nephrotic syndrome 34
1.5.6 Mechanism of proteinuria 37
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1.5.7 Podocyte specific/associated protein molecules implicated in

nephrotic syndrome
41
Nephrin 41
Podocin 42
Dystroglycan 43
α-Actinin-4
44
CD2AP 45
NEPH1 46
FAT 46
1.5.8 Podocyte and IL-13 47
1.5.9 Podocyte and B7-1 48
1.6 Research hypothesis and scope of thesis
49
CHAPTER 2 MATERIALS & METHODS
51
2.1 Cloning of rat IL-13 gene into mammalian expression vector
51
2.1.1 The pCI mammalian expression vector 51
2.1.2 Design of primers 53
2.1.3 RT-PCR using Taq polymerase 53
2.1.4 Restriction enzyme digestion of the XhoI and NotI sites 54
2.1.5 Agarose gel electrophoresis and gel extraction of DNA 55
2.1.6 Ligation of the rat IL-13 gene and the pCI mammalian
expression vector
57
2.1.7 Transformation of ligation product into E. coli 57
2.1.8 Extraction and purification of plasmid DNA 58
2.1.9 Restriction enzyme digestion reaction (for validation) 58
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2.1.10 DNA sequencing (for validation) 59
2.2 Large scale production of cloned plasmid
60
2.3 Animal experiments
61
2.3.1 Experimental protocol 61
Animal used 61
In-vivo electroporation 61
Collection of 24-hour urine sample 61
Collection of blood sample 62
2.3.2 Urine albumin ELISA 67
2.3.3 Serum biochemistry 67
Serum albumin 67
Serum cholesterol 68
Serum creatinine 68
2.3.4 Serum IgE ELISA 68
2.3.5 Serum IL-13 ELISA 69
2.3.6 Sacrificing of animals 70
2.4 Pathological examination
70
2.4.1 Histology 70
2.4.2 Immunofluorescence study 70
2.4.3 Electron microscopy 71
2.5 Glomerular gene expression study

73
2.5.1 Isolation of glomeruli by graded sieving technique 73
2.5.2 Extraction of total RNA from isolated glomeruli by TRIzol
®
reagent
75
2.5.3 Plasmid standards for real-time PCR 75
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2.5.4 Reverse transcription and real-time PCR 76
2.6 Glomerular protein expression study
80
2.7 Flow cytometric analysis of splenic mononuclear cells
83
2.8 Hepatic gene expression study by real-time PCR using
hybridization probes
86
2.9 Gene expression study on hepatocyte cell culture incubated with
recombinant IL-13
89
2.9.1 Incubation of hepatocyte cell line with recombinant IL-13 89
2.9.2 Gene expression study 89
2.10 Statistical analysis
93
CHAPTER 3 OVEREXPRESSION OF INTERLEUKIN-13 AND

DEVELOPMENT OF MINIMAL CHANGE
NEPHROTIC SYNDROME
94
3.1 Introduction
94
3.2 Results
97
3.2.1 Body weight 97
3.2.2 24-hour urine albumin excretion 98
3.2.3 Serum biochemistry 99
3.2.4 Light microscopy & transmission electron microscopy 102
3.2.5 Flow cytometric analysis of splenic mononuclear cells 104
3.3 Discussion
106
3.4 Future directions
112
CHAPTER 4 MOLECULAR MECHANISM OF PROTEINURIA IN
MCNS INDUCED BY INTERLEUKIN-13
113
4.1 Introduction
113
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4.2 Results
116

4.2.1 Quantitation of real-time PCR 116
4.2.2 Glomerular expression of IL-13 receptors and STAT6 118
4.2.3 Glomerular expression of nephrin, podocin and dystroglycan 120
4.3 Discussion
124
CHAPTER 5 ASSOCIATION OF B7-1 WITH PROTEINURIA IN
MCNS INDUCED BY INTERLEUKIN-13
130
5.1 Introduction
130
5.2 Results
132
5.2.1 Glomerular expression of B7-1 132
5.2.2 Correlation between serum IL-13 levels and glomerular gene
expression of B7-1
135
5.3 Discussion
136
CHAPTER 6 EFFECTS OF INTERLEUKIN-13 ON CHOLESTEROL
METABOLIC PATHWAY
140
6.1 Introduction
140
6.1.1 Nephrotic syndrome and hypercholesterolemia 140
6.1.2 Cholesterol metabolism 141
6.1.3 Hepatic enzymes and hypercholesterolemia in nephrotic
syndrome
147
6.1.4 Aim of chapter 148
6.2 Results

150
6.2.1 Biochemical data 150
6.2.2 Effect of IL-13 on weight of liver 152
6.2.3 Hepatic gene expression of enzymes and proteins associated
with cholesterol metabolism in IL-13 overexpression rat model
153
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6.2.4 Hepatic gene expression of IL-13 receptor subunits and
STAT6 in IL-13 overexpression rat model
155
6.2.5 Correlation studies 157
6.2.6 Gene expression of enzymes and proteins associated with
cholesterol metabolism in mouse hepatocyte cell line
incubated with recombinant IL-13
161
6.2.7 Gene expression of IL-13 receptor subunits and STAT6 in
mouse hepatocyte cell line incubated with recombinant IL-13
163
6.3 Discussion
165
CHAPTER 7 CONCLUSION
171
BIBLIOGRAPHY
174

APPENDICES
217
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SUMMARY
The aim of this thesis is to investigate the role of interleukin-13 (IL-13), a Th2
cytokine, in the induction of minimal change nephrotic syndrome (MCNS) using a
novel IL-13 overexpression rat model. Chapter 1 is a comprehensive literature review
on our current understanding of i) the different aspects of IL-13 including its
physiological functions and pathological roles in different disease conditions, ii) the
characteristics and pathogenesis of MCNS, and iii) recent advances in podocyte
biology and its association with nephrotic syndrome. Chapter 2 is the “Materials &
Methods” section which outlines the principles of all the experimental procedures
used in this study. The complete protocols of all these experimental procedures are
described in detail in the “Appendix” section. Chapter 3 is a study on the effect of IL-
13 on the development of proteinuria and other clinico-pathological features of
MCNS. Rats overexpressing IL-13 developed varying degrees of proteinuria,
hypoalbuminemia and hypercholesterolemia with 17% of the IL-13 transfected rats
developing frank nephrotic syndrome. No significant pathological changes were
found in the glomeruli except for partial effacement of podocyte foot processes. Both
the clinical and pathological features found in the IL-13 transfected rats resembled
human MCNS. In chapter 4, we explored the mechanism of proteinuria in this IL-13
overexpression rat model by studying the effect of elevated level of serum IL-13 on
podocytes. Both the glomerular gene and protein expression of nephrin, podocin and
dystroglycan, which are podocyte-associated proteins important in maintaining the

glomerular filtration barrier, were found to be significantly decreased in the IL-13
transfected rats when compared to the control rats. This was paralleled by an increase
in the glomerular gene and protein expression of IL-4Rα, one of the IL-13 receptor
subunits, suggesting that IL-13 may act directly on the podocytes and cause the
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downregulation of these podocyte-associated proteins. Chapter 5 is an examination on
the association of proteinuria with the glomerular expression of B7-1, a co-
stimulatory signaling molecule on antigen presenting cells which has been implicated
in nephrotic syndrome. Both the gene and protein expression of B7-1 were found to
be significantly increased in the nephrotic rats when compared to the control rats. The
glomerular gene expression of B7-1 also correlated significantly with the serum levels
of IL-13 in the IL-13 transfected rats. Chapter 6 is a follow-up study focusing on the
effect of IL-13 on the cholesterol metabolic pathway. The gene expression of
enzymes and proteins involved in the metabolism of cholesterol were examined in
both in-vivo and in-vitro conditions. The gene expression of HMG-CoA reductase, the
rate limiting enzyme in the cholesterol synthetic pathway, was found to be
upregulated in the liver of the IL-13 transfected rats as well as in hepatocyte cell
culture incubated with recombinant IL-13. Chapter 7 is a summary and conclusion on
the results of this study. The role of IL-13 on the development of MCNS was re-
examined based on our initial hypothesis and results obtained from our IL-13
overexpression rat model. The similarities and differences between human MCNS and
our rat model were discussed.
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LIST OF ABBREVIATIONS
ACAT Acyl-CoA cholesterol acyltransferase
Apo-B Apolipoprotein-B
BSA Bovine serum albumin
CD2AP CD2 associated protein
cDNA Complementary DNA
CERP Cholesterol efflux regulatory protein
CNF Congenital nephrotic syndrome of the Finnish type
CoA Coenzyme A
Cp Crossing point
CTLA-4 Cytotoxic T-lymphocyte-associated antigen-4
CYP7A1 Cytochrome P450, family 7, subfamily a, polypeptide 1
ddH
2
O Double distilled water
DEPC Diethylpyrocarbonate
DMEM Dulbecco’s Modified Eagle’s Medium
DNA Deoxyribonucleic acid
dNTPs Deoxynucleotide triphosphates
EDTA Ethylenediaminetetraacetic acid
ELISA Enzyme linked immunosorbent assay
F-actin Filamentous actin
FBS Fetal bovine serum
FITC Fluorescein isothiocyanate
Frat-1 Frequently rearranged in advanced T cell lymphoma-1

FSGS Focal segmental glomerulosclerosis
FXR Farnesoid X receptor
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GBM Glomerular basement membrane
GM-CSF Granulocyte macrophage – colony stimulating factor
GN Glomerulonephritis
HBSS Hank’s balanced salt solution
HDL High-density lipoprotein
HDLR High-density lipoprotein receptor
HMG-CoA 3-hydroxy-3-methylglutaryl-CoA
HMG-CoAR 3-hydroxy-3-met hylgluta ry l-CoA reductas e
HRP Horseradish peroxidase
IFN Interferon
Ig Immunoglobulin
IL Interleukin
IL-4Rα
Interleukin-4 receptor alpha
IL-13Rα1
Interleukin-13 receptor alpha 1
IL-13R
α
2
Interleukin-13 receptor alpha 2
JAK Janus tyrosine kinase

LB Luria-Bertani
LCAT Lecithin cholesterol acyltransferase
LDL Low-density lipoprotein
LDLR Low-density lipoprotein receptor
LM Light microscopy
LPS Lipopolysaccharides
LXR Liver X receptor
mAB Monoclonal antibody
MCNS Minimal change nephrotic syndrome
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MHC Major histocompatibility complex
M-MLV Moloney murine leukemia virus
mRNA Messenger RNA
Ms Mouse
NEPH1 Nephrin homolog 1
NHERF2 Na
+
/H
+
exchanger regulatory factor 2
NK Natural killer
OD Optical density
OPD o-Diphenylenediamine
p p-value

PAGE Polyacrylamide gel electrophoresis
PAN Puromycin aminonucleoside
PAS Periodic acid-Schiff
PBMC Peripheral blood mononuclear cell
PBS Phosphate buffered saline
PCR Polymerase chain reaction
P-cadherin Proto-cadherin
PE Phycoerythrin
PI3K Phosphoinositide 3-OH kinase
PMA Phorbol myristate acetate
PMSF Phenylmethylsulfonyl fluoride
r Coefficient of correlation
RBC Red blood cell
RNA Ribonucleic acid
RS Reed-Sternberg
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RT Reverse transcription
RT-PCR Reverse transcription – Polymerase chain reaction
SD Standard deviation
SDS Sodium dodecylsulphate
SEM Standard error mean
SH2 Src homology 2
SNP Single nucleotide polymorphism
SRB-1 Scavenger receptor B-1

SREBP Sterol regulatory element binding protein
SRNS Steroid-resistant nephrotic syndrome
SSNS Steroid-sensitive nephrotic syndrome
STAT Signal transducer and activator of transcription
Taq Thermus aquaticus
TBE Tris borate EDTA
TBS Tris buffered saline
TCR T cell receptor
TE Tris-EDTA
TEM Transmission electron microscopy
TGF-β
Transforming growth factor - beta
Th T helper
TLR Toll-like receptor
T
m
Melting temperature
TNF-α
Tumor necrosis factor-alpha
Tyk Tyrosine kinase
VEGF Vascular endothelial growth factor
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VLDL Very low-density lipoprotein
VLDLR Very low-density lipoprotein receptor

VPF Vascular permeability factor
WT-1 Wilms’ tumor - 1
ZO-1 Zonula occludens-1
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UNITS OF MEASUREMENT
bp Base pair
bp/s Base pair per second
cm Centimeter
cm
2
Centimeter square
o
C Degree Celsius
EU/mg Enzyme units per milligram
g Gram
g Gravity
g/24hr Gram per 24 hour
g/100g Gram per 100 gram
g/L Gram per litre
hr Hour
I.U. International units
kb Kilobase
kDa KiloDalton
L Litre

M Molar
mg Milligram
mg/24hr Milligram per 24 hour
mg/100g Milligram per 100 gram
mg/dL Milligram per decilitre
mg/ml Milligram per millilitre
ml Millilitre
ml/24hr Millilitre per 24 hour
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mm Millimeter
mM Millimolar
mmol Millimole
mmol/L Millimole per litre
mol/L Mole per litre
M
r
Molecular mass
ms Millisecond
N Normal
ng Nanogram
ng/ml Nanogram per millilitre
nm Nanometer
OD Optical density
pg Picogram

pg/ml Picogram per millilitre
pmole Picomole
rpm Revolutions per minute
s Second
U Unit
U/ml Unit per milliliter
U/µl
Unit per microlitre
µ
g
Microgram
µg/ml
Microgram per millilitre
µg/µl
Microgram per microlitre
µ
l
Microlitre
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µM
Micromolar
µ
mol/L
Micromole per litre

V Volt
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LIST OF TABLES
Table 1.1 Interspecies IL-13 sequence identity 2
Table 1.2 Histopathological patterns of primary glomerulonephritis in
Singapore children
13
Table 1.3 Distribution of patients by histopathology in children
primary nephrotic syndrome
14
Table 2.1 List of primary antibodies used for immunofluorescence
study
72
Table 2.2 Nucleotide sequence of primers and probes used in real-time
PCR for rat glomerular gene expression study
78
Table 2.3A List of primary antibodies used for glomerular protein
expression study
81
Table 2.3B List of secondary antibodies used for glomerular protein
expression study
82
Table 2.4 List of primary antibodies used for flow cytometry 85
Table 2.5 Nucleotide sequence of primers and probes used in real-time

PCR for rat hepatic gene expression study
87
Table 2.6 Nucleotide sequence of primers and probes used in real-time
PCR for mouse hepatocyte gene expression study
91
Table 3.1 Mean serum levels of IL-13, albumin, cholesterol, creatinine
and IgE when the rats were sacrificed (day 70)
99
Table 3.2 Mean 24-hour urine albumin excretion and mean serum IL-
13, albumin and cholesterol levels of control rats, proteinuric
rats (Group I) and nephrotic rats (Group 2) when they were
sacrificed (day 70)
101
Table 4.1 Glomerular gene expression of nephrin, podocin and
dystroglycan in control rats, proteinuric IL-13 transfected
rats (Group I) and nephrotic IL-13 transfected rats (Group II)
121
Table 6.1 Summary of biochemical data of control rats and IL-13
transfected rats (when sacrificed) included in cholesterol
metabolism study
150
Table 6.2 Hepatic gene expression of SREBP-2, HMG-CoA reductase,
CYP7A1 and LDL receptor in the control rats and the IL-13
transfected rats
153
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Table 6.3
Hepatic gene expression of IL-4Rα, IL-13Rα1, IL-13Rα2
and STAT6 in the control rats and the IL-13 transfected rats
155
Table 6.4 Gene expression of SREBP-2, HMG-CoA reductase and
LDL receptor, standardized against β-actin, in mouse
hepatocyte cell line incubated with 50 ng/ml of recombinant
mouse IL-13 for 8 hours
161
Table 6.5
Gene expression of IL-4Rα, IL-13Rα1, IL-13Rα2 and
STAT6, standardized against β-actin, in mouse hepatocyte
cell line incubated with 50 ng/ml of recombinant mouse IL-
13 for 8 hours
163
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LIST OF FIGURES
Figure 1.1 Cross section of the glomerular capillary wall 28
Figure 1.2 High power view of the podocyte tertiary foot processes 29
Figure 1.3 Schematic drawing of the molecular equipment in podocyte
foot processes
32

Figure 1.4 Effacement of podocyte tertiary foot processes 36
Figure 2.1 pCI mammalian expression vector and multiple cloning
region maps
52
Figure 2.2 DNA products of restriction enzyme digestion at the XhoI
and NotI sites separated by agarose gel electrophoresis
56
Figure 2.3 Experimental protocol of animal experiments 62
Figure 2.4 In-vivo electroporation 63
Figure 2.5 Metabolic cage 64
Figure 2.6 Collection of blood from the tail 65
Figure 2.7 Collection of blood by heart puncture technique 66
Figure 2.8 Graded sieving technique 74
Figure 3.1 Mean body weight of control rats and IL-13 transfected rats
measured at 10-day intervals
97
Figure 3.2 Mean 24-hour urine albumin excretion of control rats and IL-
13 transfected rats measured at 14-day intervals
98
Figure 3.3 Correlation between serum IL-13 and serum cholesterol
levels in IL-13 transfected rats
100
Figure 3.4 LM and TEM study of the glomeruli 103
Figure 3.5 Comparison on the weight of spleen expressed as net weight
and g/100g body weight between the control group and the
IL-13 transfected group
104
Figure 3.6 Relative proportions of T cells, B cells and macrophages in
the spleen between the control group and the IL-13
transfected group

105
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Figure 4.1 Example of a standard curve created by real-time PCR with
hybridization probes using serial dilutions of plasmid
standards
116
Figure 4.2 Example of real-time PCR results showing the Cp value and
the copy number of target gene in each sample
117
Figure 4.3
Glomerular gene expression of IL-4Rα, IL-13Rα1, IL-
13Rα2 and STAT6 in control rats and IL-13 transfected rats
118
Figure 4.4
Immunofluorescence study on the expression of IL-4Rα in
the glomeruli
119
Figure 4.5 Glomerular gene expression of nephrin, podocin and
dystroglycan in control rats, proteinuric IL-13
transfected
rats (Group I) and nephrotic IL-13 transfected rats (Group II)
120
Figure 4.6 Immunofluorescence study on the expression of nephrin,
podocin and dystroglycan in the glomeruli

122
Figure 5.1 Glomerular gene expression of B7-1 as shown by agarose gel
electrophoresis
132
Figure 5.2 Glomerular gene expression of B7-1 in the control rats,
proteinuric IL-13 transfected rats (Group I) and nephrotic IL-
13 transfected rats (Group II)
133
Figure 5.3 Protein expression of B7-1 in the glomeruli by
immunofluorescence technique
134
Figure 5.4 Correlation between serum IL-13 levels and glomerular gene
expression of B7-1 in IL-13 transfected rats
135
Figure 6.1 Cholesterol uptake, metabolism, and secretion in the
hepatocyte
143
Figure 6.2 The role of SREBP-1 in regulation of cholesterol
biosynthesis
145
Figure 6.3
Induction and repression of cholesterol 7
α
-hydroxylase
activities
146
Figure 6.4 Time course study of the mean serum levels of albumin and
cholesterol and mean urine albumin excretion in IL-13
transfected rats with the highest levels of serum cholesterol
151

Figure 6.5 Comparison of the net weight and the relative weight of the
liver of control rats and IL-13 transfected rats
152
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Figure 6.6 Hepatic gene expression of SREBP-2, HMG-CoA reductase,
CYP7A1 and LDL receptor in the control rats and the IL-13
transfected rats
154
Figure 6.7
Hepatic gene expression of IL-4Rα, IL-13Rα1, IL-13Rα2
and STAT6 in the control rats and the IL-13 transfected rats
156
Figure 6.8 Correlation between serum albumin levels and serum
cholesterol levels in IL-13 transfected rats with the highest
levels of serum cholesterol
157
Figure 6.9 Correlation between urine albumin levels and serum
cholesterol levels in IL-13 transfected rats with the highest
levels of serum cholesterol
157
Figure 6.10 Correlation between serum albumin levels and hepatic gene
expression of HMG-CoA reductase in IL-13 transfected rats
with the highest levels of serum cholesterol
158

Figure 6.11 Correlation between serum albumin levels and hepatic gene
expression of CYP7A1 in IL-13 transfected rats with the
highest levels of serum cholesterol
158
Figure 6.12 Correlation between the hepatic gene expression of HMG-
CoA reductase and CYP7A1 in IL-13 transfected rats with
the highest levels of serum cholesterol
159
Figure 6.13 Correlation between the hepatic gene expression of HMG-
CoA reductase and serum cholesterol levels in IL-13
transfected rats with the highest levels of serum cholesterol
160
Figure 6.14 Gene expression of SREBP-2, HMG-CoA reductase and
LDL receptor, standardized against β-actin, in mouse
hepatocyte cell line incubated with 50 ng/ml of recombinant
mouse IL-13 for 8 hours
162
Figure 6.15
Gene expression of IL-4Rα, IL-13Rα1, IL-13Rα2 and
STAT6, standardized against
β
-actin, in mouse hepatocyte
cell line incubated with 50 ng/ml of recombinant mouse IL-
13 for 8 hours
164
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