Effect of palm oil (Elias guinensis) leaf standardized

extract on progression of renal dysfunction and

arterial stiffness in normal and high fat diet fed

Sprague Dawley rats with induced nephrotoxicity.

By

Zaid O. Ibraheem

Thesis submitted in fulfillment of the requirements for the degree of master of science

2011

ii

ACKNOWLEDGEMENT

I would like to pay thanks to Almighty Allah, who gave me strength to survive in

this research.

I would like to extend my deepest gratitude to my supervisor Professor Munavvar

Zubaid Abdul Sattar and my co-supervisor Dr. Hassaan Rathore for their help and

guidance throughout the period of the project. Their patience and care influenced me to

work hard and demand success and for this, I am very grateful to them for putting at my

disposal every facility that they had which I need during the course of the study.

I wish to extend my thanks to the Dean of the School of Pharmaceutical Sciences

and all the administration staff for their kind support and providing an opportunity to

finish the task in a nice way. I also wish to thank the Dean of the Institute Postgraduate

Studies and his staff who helped me in one way or other. I acknowledge the support

given by non-academic staff of school of Pharmaceutical Science.

I would like to pay my special thanks to Puan Yong and Dr. Gurjeet Kaur for their

exclusive help in pursuing my study. My thanks also go to my lab mates for their

assistance and encouragement throughout the study.

Finally and most importantly, I am indebted to my parents and my friend Tan who

stood by me during the most difficult times.

Zaid

iii

TABLE OF CONTENTS

Acknowledgement ………………………………………………………… ii

Table of contents………………………………………………………… iii

List of figures……………………………………………………………… iv

List of tables……………………………………………………………… xvii

List of equations…………………………………………………………. xx

List of abbreviation………………………………………………………… xxii

Abstrak……………………………………………………………………. xxvi

Abstract………………………………………………………………….. xxx

CHAPTER ONE

INTRODUCTION

1.1 Physiology of kidneys …………………………………………………… 1

1.1.1 General structure ………………………………………………… 1

1.1.1.1 Nephrons ………………………………………………………… 2

1.1.1.2 Renal glomeruli……………………………………………… 3

1.1.1.3 Proximal convoluted tubules (PCT)…………………………… 4

1.1.1.4 Loop of Henle ………………………………………………… 5

1.1.1.5 Distal convoluted tubules ………………………………………… 6

1.1.1.6 Collecting tubules system………………………………………… 6

1.1.2 Renal glomerular filtration and tubular function…………………… 7

1.2 Aminoglycosides induced nephrotoxicity………………………………… 10

iv

1.2.1 Aminoglycosides……………………………………………………. 10

1.2.2 Aminoglycosides induced nephrotoxicity………………………… 11

1.2.2.1 Aminoglycosides induced tubular injury…………………… 12

1.2.2 .2 Gentamicin as an example of nephrotoxic aminoglycoside 15

1.3 Oxidative stress………………………………………………………… 17

1.3.1 Types of free radicals…………………………………………….. 19

1.3.1.1 Reactive oxygen species…………………………………… 19

1.3.1.2 Reactive nitrogen species………………………………… 22

1.3.2 Dietary antioxidants………………………………………………… 24

1.4 High lipid diet………………………………………………………… 25

1.4.1 Lipids…………………………………………………………… 25

1.4.1.1 Fatty acids and their derivitives………………………………… 25

1.4.1.2 Glycerides…………………………………………………….. 28

1.4.1.3 Sterols…………………………………………………..……… 28

1.4.1.3.1 Cholesterol………………………………………………........ 30

1.4.2 Lipoprotein system and lipid circulation………………………… 31

1.4.3 Detrimental role of circulatory fatty acids ……………………… 36

1.4.4 High fat diet and metabolic syndrome …………………………… 38

1.4.4.1 Metabolic syndrome …………………………………………… 38

1.4.4.2 Regulation of fat distribution and food uptake………………… 39

1.4.4.3 Glucose tolerance and high fat diet……………………………… 42

1.5 Arterial blood pressure and pulse wave………………………………… 43

1.6 Pulse wave velocity ……………………………………………………… 52

v

1.7 Factors that affect pulse wave velocity…………………………………… 53

1.8 Objectives…………………………………………………………………. 54

CHAPTER TWO

MATERIALS AND METHOD

2.1 In vitro measurement of the antioxidant activity of the extract …………… 55

2.1.1 Free radical scavenging activity……………………………………… 55

2.1.2 Reducing power assay ……………………………………………… 56

2.1.3 Hydrogen peroxide scavenging activity………………………………. 57

2.1.4 Antilipid peroxidation assay ………………………………………… 58

2.2 LD50 of the extract ……………………………………………………….. 59

2.3 Experimental animals……………………………………………………… 60

2.3.1 Experimental groups ………………………………………………. 61

2.3.2 Animals monitoring ………………………………………………… 64

2.4 Induction of gentamicin induced nephrotoxicity ………………………… 64

2.5 Induction of metabolic syndrome with saturated free fatty acids SAFFAs

rich diet…………………………………………………………………………

65

2.6 Biochemical tests ………………………………………..………………… 66

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2.6.1 Creatinine measurement ……………………………………………… 66

2.6.1.a principle of the method ……………………………………… 67

2.6.1.b Specimen collection and storage …………………………… 67

2.6.1.c Reagents ……………………………………………………… 67

2.6.1.d Procedure …………………………………………………… 68

2.6.1.e Calculation of creatinine concentration ……………………… 68

2.6.1.f Calculation of creatinine clearance ………………………… 69

2.6.2 Urine and plasma sodium and potassium …………………………… 69

2.6.2.a Calculation of both absolute and fractional excretion of sodium and

potassium.....................................................................................................

70

2.6.3 Protein in urine …………………………………………………….. 70

2.6.3.a Principle of the test ………………………………………….. 71

2.6.3.b Reagents ……………………………………………………… 72

2.6.3.c Procedure …………………………………………………… 73

2.6.4 Malonyldialdehyde (MDA) in renal homogenate …………………… 73

2.6.4.a Principle of the test …………………………………………… 74

2.6.4.b Reagents …………………………………………………… 74

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2.6.4.c Procedure …………………………………………………… 74

2.6.5 Oral glucose tolerance test (OGTT)………………………………… 76

2.6.5.a Procedure …………………………………………………… 76

2.6.6 Lipid profile ………………………………………………………… 77

2.6.6.a Triglyceride ………………………………….......................... 77

2.6.6.a.1 Principle of the test …………………………………… 77

2.6.6.a.2 Reagents …………………………………………… 77

2.6.6.a.3 Method ……………………………………………… 78

2.6.6.b Cholesterol …………………………. …………………………… 79

2.6.6.b.1 Principle of the test ……………………………………… 79

2.6.6.b.2 Reagents ………………………………………………… 79

2.6.6.b.3 Method …………………………………………………… 79

2.7 Acute study ………………………………………………………………… 80

2.7.1 Surgical preparation of the animal …………………………………… 81

2.7.2 Pulse wave velocity measurement …………………………………… 82

2.7.2.a Propagation distance ……………………………………… 82

2.7.2.b Propagation time ……………………………………………. 83

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2.8 Pulse wave analysis…………………………………………………………. 84

2.9 Histology of kidney tissue ………………………………………………… 86

2.9.1 Tissue fixation ………………………………………………………. 87

2.9.2 Tissue procession …………………………………………………… 87

2.9.3 Microtom sectioning and fixed slides preparation………………….. 89

2.9.4 Staining procedure ………………………………………………….. 89

2.9.5 Slide mounting ………………………………………………………. 91

2.10 Materials……………………………………………………………........... 92

2.10.1 List of equipment ………………………………………………… 92

2.10.2 List of chemicals …………………………………………………… 94

CHAPTER THREE

RESULTS

3.1 Results of the preliminary study for nephrotoxicity induction............................96

3.2 Body weight …………………………………………………….......................96

3.3 Food uptake ………………………………………………………………….97

3.4 Urine flow rate ………………………………………………………………..97

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3.5 Absolute excretion of sodium …………………………………………………99

3.6 Fractional excretion of sodium ……………………………………………….101

3.7 Absolute excretion of potassium ……………………………………………..102

3.8 Fractional excretion of potassium ……………………………………………

104

3.9 Serum creatinine ………………………………………………………………106

3.10 Creatinine clearance …………………………………………………………108

3.101 Absolute creatinine clearance …………………………………………108

3.10.2 Creatinine clearance / 100 g (body weight)……………………………110

3.10.3 Creatinine clearance / kidney tissue ……………………………………113

3.11 Kidney index………………….……………………………………………..114

3.12 Urinary excretion of protein….……………………………………………….114

3.13 Serum electrolytes……………………………………………………………115

3.14 Kidney tissue content of malonyldialdehyde (MDA). ……………………….115

3.15 adiposity indices ……………………………………………………………..116

3.15.1 Mesentric fat index…………………………………………………116

3.15.2 Retroperitoneal fat index ……………………………………………116

3.15.3 Epididymal fat index……………………………………………….117

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3.15.4 Obesity index ……………………………………………………….119

3.16 Oral glucose tolerance test (OGTT)………………………………………….120

3.17 Serum triglycerides…………………………………………………………..120

3.18 Serum Cholesterol……………………………………………………………122

3.19 Surgical results ………………………………………………………………122

3.20.1 Hemodynamic parameters ……………………………………………122

3.20.1. 1 Diastolic blood pressure (DBP)……………………………122

3.20.1.2 Systolic blood pressure (SBP)………………………………122

3.20.1.3 Mean arterial blood pressure (MABP)…………………….123

3.20.1.4 Pulse pressure (PP)…………………………………………123

3.20.1.5 Dicrotic notch(DN)…………………………………………124

3.20.1.6 Heart rate …………………………………………………124

3.21.2 Arterial stiffness parameters ………………………………………………125

3.21.2.1 Augmentation index ………………………………………………125

3.21.2.2 Pulse wave analysis………………………………………………125

3.21.2.3 β index……………………………………………………………126

3.22 Histology results ………………………………………………………126

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3.23 In vitro tests for antioxidant assessment………………………………126

3.23.1 DPPH test ………………………………………………………126

3.23.2 Reducing power assay…………………………………………127

3.23.2 Hydrogen peroxide scavenging activity ………………………127

3.23.3 Antilipid peroxidation assay........................................................127

3.24 Tables ……………………………………………………………………….128

3.25 Figures………………………………………………………………………..148

Discussion ……………………………………………………………………. … 230

Conclusion ……………….. ……………………………………………… ……. 252

References ………………………………………………………………………… 254

LIST OF FIGURES

Figure 1.1 Kidney and nephron structure………………………………… 3

Figure 1.2 The 3 ring structure of kanamycin as an aminoglycosed basic structure 14

Figure 1.3 Chemical structure of gentamicin ……………………………………. 16

Figure 1.4 Lipid peroxidation pathway ………………………………………….. 18

Figure 1.5 Formation of reactive oxygen species (ROS)………………………… 21

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Figure 1.6 Detoxification of hydrogen peroxide by glutathione …………………. 22

Figure 1.7 Fate of nitric oxide……………………………………………………. 23

Figure 1.8 Ferulic acid structure………………………………………………….. 25

Figure 1.9 Fatty acid synthesis……………………………………………………. 26

Figure 1.10 Sterol nucleus structure …………………………………………….. 29

Figure 1.11 Cholesterol synthesis………………………………………………….. 30

Figure 1.12 Pressure wave in aorta in absence of reflection wave…………………… 48

Figure 1.13 Progression of pressure wave by ventricular spurt ……………………… 49

Figure 1.14 Effect of reflected wave on propagation wave………………………….. 49

Figure 1.15 Travel of reflection wave in stiffer artery ………………………………. 50

Figure 1.16 Inflection point with earlier reflection wave …………………………….. 50

Figure 1.17 Effect of earlier arrival of reflection wave on the central systolic pressure

and pulse pressure……………………………………………………….

51

Figure 1.18 Effect of the earlier arrival of the reflection wave on ventricular load ….. 51

Figure 2.1 Experimental protocol of treatment……………………………………. 63

Figure 2.2 Macro-Bradford assay standard curve …………………………………. 73

Figure 2.3 Principle of triglyceride determination test ……………………………. 78

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Figure 2.4 Principle of cholesterol determination test ……………………………. 80

Figure 2.5 Daistolic phase center measurement ………………………………… 83

Figure 2.6 Measurement of pulse wave velocity from both carotid and iliac arteries

wavefronts…………………………………………………………….

84

Figure 2.7 Pulse wave analysis…………………………………………………….. 86

Figure 3.1.1 Change in serum creatinine during the preliminary study……………….. 149

Figure 3.1.2 Change in absolute creatinine clearance during the preliminary study. … 149

Figure 3.2.1 Percent of weekly body weight change for C, H, HG and G groups…….. 150

Figure 3.2.2 Percent of weekly body weight change for C, H, HP0.5, HP1 and HP2

groups …………………………………………………………………………………. 151

Figure 3.2.3 Percent of weekly body weight change for C,H, HG, HG0.5, HG1 and

HG2 groups. ………………………………………………………………………….. 152

Figure 3.2.4 Percent of weekly body weight change for C, G, G0.5, G1 and G2 groups. 153

Figure 3.3.1 Daily Calories uptake for C, H, HG and G groups………………….. 154

Figure 3.3.2 Daily Calories uptake for C, H, HP0.5, HP1 and HP2 groups……….. 155

Figure 3.3.3 Daily Calories uptake for C,H, HG, HG0.5, HG1 and HG2 groups. … 156

Figure 3.3.4 Daily Calories uptake for C, G, G0.5, G1 and G2 groups…………….. 157

Figure 3.4.1 Urine flow rate for C, H, HG and G groups………………………… 158

Figure 3.4.2 Urine flow rate for C, H, HP0.5, HP1 and HP2 groups……………. 159

Figure 3.4.3 Urine flow rate for C,H, HG, HG0.5, HG1 and HG2 groups. ……… 160

Figure 3.4.4 Urine flow rate for C, G, G0.5, G1 and G2 groups…………………. 161

Figure 3.5.1 Absolute excretion of sodium for C, H, HG and G groups………… 162

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Figure 3.5.2 Absolute excretion of sodium for C, H, HP0.5, HP1 and HP2

groups……………………………………………………………………………….. 163

.Figure 3.5.3 Absolute excretion of sodium for C,H, HG, HG0.5, HG1 and HG2

groups. ………………………………………………………………………………. 164

Figure 3.5.4 Absolute excretion of sodium for C, G, G0.5, G1 and G2 groups. … 165

Figure 3.6.1 Absolute excretion of potassium for C, H, HG and G groups. . ……… 166

Figure 3.6.2 Absolute excretion of potassium for C, H, HP0.5, HP1 and HP2 groups.

……………………………………………………………………………….. 167

Figure 3.6.3 Absolute excretion of potassium for C,H, HG, HG0.5, HG1 and HG2

groups. ……………………………………………………………………………… 168

Figure 3.6.4 Absolute excretion of potassium for C, G, G0.5, G1 and G2 groups. 169

Figure 3.7.1 Fractional excretion of sodium for C, H, HG and G groups. ……….. 170

Figure 3.7.2 Fractional excretion of sodium for C, H, HP0.5, HP1 and HP2 groups. 172

Figure 3.7.3 Fractional ecretion of sodium C,H, HG, HG0.5, HG1 and HG2 groups. 173

Figure 3.7.4 Fractional excretion of sodium for C, G, G0.5, G1 and G2 groups…… 174

Figure 3.8.1 Fractional excretion of potassium for C, H, HG and G groups………… 175

Figure 3.8.2 Fractional excretion of potassium for C, H, HP0.5, HP1 and HP2 groups 1176

Figure 3.8.3 Fractional excretion of potassium for C,H, HG, HG0.5, HG1 and HG2

groups. …………………………………………………………………………………. 1177

Figure 3.8.4 Fractional excretion of potassium for C, G, G0.5, G1 and G2 groups…... 1178

Figure 3.9.1 Serum Creatinine for C, H, HG and G groups. ………………………… 179

Figure 3.9.2 Serum Creatinine in µmol/l for C, H, HP0.5, HP1 and HP2 groups. …… 180

Figure 3.9.3 Serum Creatinine in µmol/l for C,H, HG, HG0.5, HG1 and HG2 groups 181

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Figure 3.9.4 Serum Creatinine in µmol/l for C, G, G0.5, G1 and G2 groups……….. 182

Figure 3.10.1 Absolute Creatinine clearance for C, H, HG and G groups…………. 183

Figure 3.10.2 Absolute Creatinine clearance for C, H, HP0.5, HP1 and HP2 group…. 184

Figure 3.10.3 Absolute Creatinine for C,H, HG, HG0.5, HG1 and HG2 groups. …… 185

Figure 3.11.1 Creatinine clearance / 100 g B.W for C, H, HG and G groups. …….. 186

Figure 3.11.2 Creatinine clearance / 100 g B.W for C, H, HP0.5, HP1 and HP2

groups………………………………………………………………………………… 187

Figure 3.11.3 Creatinine clearance / 100 g B.W. for C,H, HG, HG0.5, HG1 and HG2

groups. ………………………………………………………………………………… 188

Figure 3.11.4 Creatinine clearance / 100 g B.W for C, G, G0.5, G1 and G2 groups. 189

Figure 3.12.1 Creatinine clearance/Kidney tissue for C, H, HG and G groups…… 190

Figure 3.12.2 Creatinine clearance/Kidney tissue For C, G, G0.5, G1 and G2 groups. 190

Figure 3.12.3 Creatinine clearance/Kidney tissue for C,H, HG, HG0.5, HG1 and HG2

groups…………………………………………………………………………… 191

Figure 3.12.4 Creatinine clearance/Kidney tissue for for C, H, HG and G

groups………………………………………………………………………………… 191

Figure 3.13.1 Kidney index of control, HFD, HFDG and G groups throughout their

treatment period. ……………………………………………………………………….. 192

Figure 3.13.2 Kidney index for C, H, HP0.5, HP1 and HP2 groups…………......... 192

Figure 3.13.3 Kidney index for C,H, HG, HG0.5, HG1 and HG2 groups. …………... 193

Figure 3.13.4 Kidney index for C, G, G0.5, G1 and G2 groups. ……………............... 193

Figure 3.14.1 Urinary protein excretion for C, H, HG and G groups………….. ….. 194

Figure 3.14.2 Urinary protein excretion in mg/hr. for C, H, HP0.5, HP1 and HP2 194

xvi

group…………………………………………………………………………………..

Figure 3.14.3 Urinary protein excretion for C,H, HG, HG0.5, HG1 and HG2 groups. 195

Figure 3.14.4 Urinary protein excretion for C, G, G0.5, G1 and G2 groups. 195

Figure 3.15.1 Malonyldialdehyde (MDA) in kidney homogenate for C, H, HG and G

groups. ……………………………………………………………………………..… 196

Figure 3.15.2 Malonyldialdehyde (MDA) in kidney for C, H, HP0.5, HP1 and HP2

groups……………………………………………………........................................... 196

Figure 3.15.3 Malonyldialdehyde (MDA) in kidney for C,H, HG, HG0.5, HG1 and

HG2 groups. ………………………………………………………………………… 197

Figure 3.15.4 Malonyldialdehyde (MDA) in kidney homogenate for C, G, G0.5, G1 and

G2 groups. ……………………………………………………………………….. 197

Figure 3.16.1 Mesenteric fat index for C, H, HG and G groups…………………… 198

Figure 3.16.2 Mesenteric fat index for C, H, HP0.5, HP1 and HP2 groups………. 198

Figure 3.16.3 Mesentric fat index for C,H, HG, HG0.5, HG1 and HG2 groups. ….. 199

Figure 3.16.4 Mesentric fat index for C, G, G0.5, G1 and G2 groups. ……………. 199

Figure 3.17.1 Retroperitoneal fat index for C, H, HG and G groups………………… 200

Figure 3.17.2 Retroperitoneal fat index for C, H, HP0.5, HP1 and HP2 groups……. 200

Figure 3.17.3 Retroperitoneal fat index for C,H, HG, HG0.5, HG1 and HG2 groups. 201

Figure 3.17.4 Retroperitoneal fat index for C, G, G0.5, G1 and G2 groups. ………… 201

Figure 3.18.1 Epididymal fat index for C, H, HG and G groups…………………… 202

Figure 3.18.2 Epididymal fat index for C, H, HP0.5, HP1 and HP2 groups…………. 202

Figure 3.18.3 Epididymal fat index for C,H, HG, HG0.5, HG1 and HG2 groups. …... 203

Figure 3.18.4 Epididymal fat index for C, G, G0.5, G1 and G2 groups. ……………. 203

xvii

Figure 3.19.1 Obesity index for C, H, HG and G groups……………………………. 204

Figure 3.19.2 Obesity index for C, H, HP0.5, HP1 and HP2 groups………………… 204

Figure 3.19.3 Obesity index for C,H, HG, HG0.5, HG1 and HG2 groups…………… 205

Figure 3.19.4 Obesity index for C, G, G0.5, G1 and G2 groups. …………………….. 205

Figure 3.20.1 Area under the curve of the oral glucose tolerance for C, H, HG and G

groups. ………………………………………………………………………………… 206

Figure 3.20.2 Area under the curve of the oral glucose tolerance test for C, H, HP0.5,

HP1 and HP2 roups…………………………………………………………………….. 206

Figure 3.20.3 Area under the curve for C,H, HG, HG0.5, HG1 and HG2 groups… 207

Figure 3.20.4 Area under the curve for C, G, G0.5, G1 and G2 groups…………… 207

Figure 3.20.5 Oral glucose tolerance curve for control …………………………… 208

Figure 3.20.6 Oral glucose tolerance curve for H group. …………………………… 208

Figure 3.20.7 Oral glucose tolerance curve for HG group…………. …………… 209

Figure 3.20.8 Oral glucose tolerance curve for G group. …………………………… 209

Figure 3.20.9 Oral glucose tolerance curve for C, H HG and G groups at the end of the

treatment period. ………………………………………………………………… 210

Figure 3.21.1 Serum triglyceride concentration for C, H, HG and G groups................ 211

Figure 3.21.2 Serum triglyceride concentration for C, H, HP0.5, HP1 and HP2. …… 212

Figure 3.21.3 Serum triglyceride for C, H, HG, HG0.5, HG1 and HG2 groups. …… 213

Figure 3.21.4 Serum T.G concentration for C, G, G0.5, G1 and G2 groups. ……… 214

Figure 3.22.1 Serum cholesterol concentration for C, H, HG and G groups. …… 215

Figure 3.22.2 Serum cholesterol concentration for C, H, HP0.5, HP1 and HP2 groups 216

Figure 3.22.3 Serum cholesterol for C,H, HG, HG0.5, HG1 and HG2 groups…… 217

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Figure 3.22.4 Serum cholesterol concentration for C, G, G0.5, G1 and G2 groups. 218

Figure 3.23.1 Diastolic blood pressure for C, H, HG and G groups. ………………. 219

Figure 3.23.2 Diastolic blood pressure (DBP) in mmHg for C, H, HP0.5, HP1 and HP2

groups. ……………………………………………………………………………. 219

Figure 3.23.3 Diastolic blood pressure in mmHg for C,H, HG, HG0.5, HG1 and HG2

groups. ………………………………………………………………………………….. 220

Figure 3.23.4 Diastolic blood pressure for C, G, G0.5, G1 and G2 groups………… 220

Figure 3.24.1 Systolic blood pressure for C, H, HG and G groups. ………………….. 221

Figure 3.24.2 Systolic blood pressure for C, H, HP0.5, HP1 and HP2 groups……….. 221

Figure 3.24.3 Systolic blood pressure for C,H, HG, HG0.5, HG1 and HG2 groups…. 222

Figure 3.24.4 Systolic blood pressure for C, G, G0.5, G1 and G2 groups……………. 222

Figure 3.25.1 Mean blood pressure for C, H, HG and G groups. …………………….. 223

Figure 3.25.2 Mean blood pressure for C, H, HP0.5, HP1 and HP2 groups………….. 223

Figure 3.25.3 Mean blood pressure for C,H, HG, HG0.5, HG1 and HG2 groups. … 224

Figure 3.25.4 Mean blood pressure for C, G, G0.5, G1 and G2 groups……………… 224

Figure 3.26.1 Augmentation index for C, H, HG and G groups……………………… 225

Figure 3.26.2 Augmentation index for C, H, HP0.5, HP1 and HP2 groups………….. 225

Figure 3.26.3 Augmentation index for C,H, HG, HG0.5, HG1 and HG2 groups……. 226

Figure 3.26.4 Augmentation index for C, G, G0.5, G1 and G2 groups………………. 226

Figure 3.27.1 Pulse wave velocity for C, H, HG and G groups...................................... 227

Figure 3.27.2 Pulse wave velocity for C, H, HP0.5, HP1 and HP2 ………………….. 227

Figure 3.27.3 Pulse wave velocity for C,H, HG, HG0.5, HG1 and HG2 groups…….. 228

Figure 3.27.4 Pulse wave velocity for C, G, G0.5, G1 and G2 groups………………. 228

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Figure 3.28 Kidney histology slides for control (C), gentamicin treated group (G) and

gentamicin treated group along with high fat diet (HG). …………………………. 229

Figure 4.1 Factors affect arterial stiffness…………………………………………….. 240

LIST OF TABLES

Table 2.1 Division of the groups used in the study………………………… 61

Table 2.2 Nutritional constituents of the experimental high fat diet as

compared to the standard rodents chow…………………………

66

Table 2.3 Preparation of simple for MDA estimation……………………… 75

Table 2.4 Steps of tissue procession steps for histological slides preparation.. 88

Table 2.5 Steps for histological slide staining using E&H………………… 90

Table 2.6 List of equipments used in the study………………………………. 92

Table 2.7 List of chemicals used in the study…………………………… 94

Table 3.1 Percent of weekly body weight change for all the treated groups ... 128

Table 3.2 Food uptake in Kcal/100 g B.W. day for all the treated groups …..129

Table 3.3 Urine flow rate (µl/min/100 g B.W) for all the treated groups……130

Table 3.4 Absolute excretion of sodium (mmol/hr) for all the treated groups.131

Table 3.5 Absolute excretion of potassium (mmol/he) for all the treated 132

xx

groups ……………………………………………………………

Table 3.6 Fractional excretion of sodium for all the treated groups. ……133

Table 3.7 Fractional excretion of potassium for all the treated groups …… 134

Table 3.8 Serum creatinine in µmol/l for all the treated groups ……………135

Table 3.9 Creatinine clearance/100 g B.W for all the treated groups ………136

Table 3.10 Absolute creatinine clearance (ml/min) for all the treated groups 137

Table 3.11 Kidney index and creatinine clearance/ kidney tissue for all the

treated groups ……………………………………………………138

Table 3.12 Urinary protein excretion I mg/hr for all the treated groups……… 139

Table 3.13 Serum concentration of sodium in mmol/l for all the treated groups 140

Table 3.14 Serum concentration of potassium in mmol/l for all the treated

groups …………………………………………………………… 141

Table 3.15 MDA concentration in renal homogenate for all the treated groups.142

Table 3.16 Obesity indices for all the treated groups…………………………..143

Table 3.17 Oral glucose tolerance test for all the treated groups………………144

Table 3.18 Serum triglyceride in mmol/l for all the treated groups……………145

Table 3.19 Serum cholesterol in mmol/l for all the treated groups…………….146

xxi

Table 3.20 Heamodynamic parameters for all the treated groups……………...147

Table 3.21 Arterial stiffness parameters for all the treated groups…………….148

LIST OF EQUATIONS

Equation 1.1 Factors that affect pulse wave velocity…………………………… 53

Equation 2.1 Calculation of creatinine concentration in plasma ……………… 68

Equation2. 2 Calculation of creatinine concentration in urine ……………….. 68

Equation 2.3 Calculation of absolute creatinine clearance…………………….. 69

Equation 2.4 Calculation of creatinine clearance/100 g (B.W) …………….. 69

Equation 2.5 Calculation of absolute excretion of sodium…………………….. 70

Equation 2.6 Calculation of absolute excretion of potassium………………….. 70

Equation 2.7 Calculation of fractional excretion of sodium………………….. 70

Equation 2.8 Calculation of fractional excretion of potassium………………….. 70

Equation 2.9 Calculation of MDA (Malonyldialdehyde) concentration in a test

sample………………………………………………………..

75

Equation 2.10 Calculation of MDA content in kidney tissue……………….…… 76

Equation 2.11 Calculation of augmentation index (AI%)……………………….. 85

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Equation 2.12 Calculation of β index……………………………………………... 86

LIST OF ABBREVIATIONS

Ad libitum To be taken as wanted

ADH Anit-diuretic hormone

AGRP Agouti related peptide

AI% Augmentation index

ANP Anti -natruitic peptide

AT1 Angiotensin receptor

ATP Adenosin triphosphate

B.W Body weight

BAT Brown adipose tissue

BHT Butylated hydroxyl toleun

CD Conjugated diens

CETP Cholesterol ester transfer protein

cGMP Cyclic guanosine mono phosphate

CHO. Cholesterol

Cr. Cl. Creatinine clearance

CSF Colony stimulating factor

CT Collecting tubules

DBP Diastolic blood pressure

DCT Distal convoluted tubules

xxiii

DN Dicrotic notch

DNA Deoxyribo nucleic acid

DPC Dextrapropoxyphene

DPPH 1-1-Diphenyl-2-Picryl-Hyrazil

ECF Extracellular luid

ECV Extracellular volume

ETC Electron transport chain

FE Fractional excretion

Eq. Equation

FeCl3 Ferric chloride

FFA Free fatty acids

FRSA Free radical scavenging activity

g Gram

g.m.wt Gram molecular weight

GFR Glomerular filtration rate

GIT Gastro-intestinal tract

GN Glomerulonephritis

GPx. Glutathione peroxidase enzyme

Gr. Gluthaion reductase

GSH Reduced form of glutathion

GSSG Oxidized frm of glutathione

H & E Heamatoxylin and eosine

HBSS Hangs balance salt solution

xxiv

HDL High density lipoprotein

HMG-Co-A reductase 3-hydroxy-4-glutaryl-CoA-reductase enzyme

I.P Intraperitoneal

I.R Insulin resistance

I.U International unite

ICAM Intracellular adhesion molecule

IDL Intermediate density lipoprotein

IL Interleukin

IRS-1 Insulin resistance substrate -1

Kcal Kilo calorie

kg Kilogram

KI Kidney index

LCAT Lecithin cholesterol acyl transferase enzyme

LDL Low density lipoprotein

MAP Mean arterial pressure

MDA Malonyldialdehyde

MDAR Monohydroascorbate reductase

MIC Minimum inhibitory concentration

ml Milliliter

MMps Metaloproteinase enzyme

MPO Myeloperoxidase

MSH-α Melanocyte stimulating hormone

MUSFFAs Mono-unsaturated free fatty acids

xxv

N/S Normal saline

NADP Ncotonomide adenine dinucleotide

NF-КB Natural factor- КB

NO. Nitric oxide

NO2- Nitrite ion

NO3- Nitrate ion

NOS Nitric oxide synthase enzyme

O.D Optical density

OGTT Oral glucose tolerance test

ONOO. Peroxynitrate

PBC Hydrostatic pressure in Bowman capsule

PCT Proximal convoluted tubules

PDGF Platelet derived growth factor

PGC Glomerular capillary hydrostatic pressure

Pi Inflection point

PKC-Ǿ Protein kinase C-theta

PLPT Phospholipids transfer protein

P.O Per oral.

POLE Palm oil leaf extract

PP Pulse pressure

PPAR Peroxosome proliferation activating factor

Puf Ultrafiltartion pressure

PUFFAs Polyunsaturated free fatty acids

xxvi

PUSAFFA Poly unsaturated free fatty acids

PWV Pulse wave velocity

RAAS Renin Angiotensine aldosteron system

RBCs Red blood corpuscles

RNS Reactive nitrogen species

ROS Reactive oxygen species

RPM Revolution per minute

S.E.M Standard error of Mean

SAFFAs Saturated free fatty acids

SBP Systolic blood pressure

SOD Super oxide dismutase

SREBP Sterol regulatory element binding protein

STD Standard

T.G Triglyceride

TBA Thiobarbituric acid

TCA Tri-chloro acetic cid

TNF Tumor necrosis factor

UCP Uncoupling protein

Ucr Concentration of creatinine in urine

UFFAs Un-esterified free fatty acids

UFR Urine flow rate

UK+ Concentration of potassium in urine

UNa+ Concentration of sodium in urine

xxvii

USAFFAs Unsaturated free fatty acids

Uvol. Urine volume

VCAM Vascular cell adhesion molecule

VD Volume of distribution

VLDL Very low density lipoprotein

WAT White adipose tissue

∏BC Oncotic pressure in Bowman capsule

∏GC Oncotic pressure in glomerular capillary

µg Microgram

4-HHE 4- Hydroxyhexenal

4-HNE 4-Hydroxynonenal

xxviii

Kesan ekstrak daun sawit (pechan etanol

terstandard) terhadap kemajuan kegagalan ginjal

dan kekakuan arteri dalam tikus Sprague-Dawley

yang normal dan yang diberi diet lemak tinggi

melalui kenofrotolsikan

ABSTRAK

Asid lemak bebas tepu (saturated free fatty acids, SAFFA) mengaruh kesan mudarat

mereka melalui aruhan laluan inflamatori intrasel yang berkaitan dengan rintangan

insulin. Dalam bidang bioperubatan, keadaan ini dikenali sebagai sindrom

metabolisme. Gentamicin merupakan suatu antibiotik aminoglikosid yang digunakan

dengan meluas. Tindakan nefrotoksiknya dicirikan oleh tubul berlingkar proksimal dan

kerosakan pada membran glomerular. Mekanisme kenefrotoksikan berkaitan rapat

dengan stres oksidatif dan penjanaan radikal bebas.

Dewasa ini ditemui bahawa ekstrak daun sawit mempunyai beberapa kepentingan

bioperubatan kerana polifenol yang terkandung di dalamnya boleh bertindak balas bagi

mengurangkan stres oksidatif.

Kajian kami bertujuan menentukan impak daripada menggantikan elemen diet

penting dalam makanan yang diperkaya dengan SAFFA terhadap kemajuan kerosakan

ginjal dan kaitannya dengan perubahan hemodinamik dan metabolisme dalam model

tikus yang kenefrotoksikan diaruh dengan gentamicin. Kajian ini juga menilai kesan

xxix

profilaktik dalam ekstrak daun sawit (palm oil leaf extract POLE) terhadap kemajuan

gangguan ini.

Ketidakfungsian ginjal dinilai melalui ukuran serum kreatinin, klearans kreatinin,

pecahan dan kumuhan / eksresi mutlak daripada natrium dan kalium. Malonildialdehid

(suatu biopenanda bagi stres oksidatif) dalam homogen ginjal dan kumuhan protein

urin. Keputusan menunjukkan bahawa terdapat insidens kenefrotoksikan yang amat

tinggi dalam tikus yang diberi diet yang diperkaya dengan SAFFA dibandingkan

dengan tikus yang diberi diet yang standard. Kenefrotoksikan digred sebagai sedikit

bagi kumpulan gagal-ginjal yang diberi diet yang standard. Sebaliknya, digred sebagai

sederhana hingga teruk, bagi yang diberi diet yang diperkaya dengan SAFFA. Dos

POLE yang tinggi boleh membataskan kemajuan kenefrotoksikan dan perkembangan

stres oksidatif dalam tisu ginjal. Dari sudut lain, sindrom metabolisme dinilai melalui

ujian tolerans oral-glukos (oral-glucose tolerance test, OGTT), ukuran profil lipid dan

indeks obesiti / kegemukan. Kajian menunjukkan kemerosotan dalam sindrom

metabolisme pada tikus yang diberi diet yang diperkaya dengan SAFFA. Pemberian

gentamicin bersama diet ini menyebabkan kemerosotan yang amat sedikit dalam

tolerans glukosa tanpa impak yang ketara terhadap profil lipid. Sindrom metabolisme

yang diaruh dengan SAFFA dikaitkan dengan perubahan kardiovaskular yang dicirikan

oleh hipertensi dan kekakuan yang amat tinggi pada arteri. Selepas pemberian

gentamicin bersama diet SAFFA, didapati tekanan semakin berkurangan dan kekakuan

kekal. Sebaliknya, injeksi gentamicin pada tikus yang diberi diet standard, tidak

menunjukkan sebarang perubahan kardiovaskular. Kemajuan sindrom metabolisme dan

perubahan kardivaskular terbatas sedikit selepas pemberian POLE.

xxx

Secara keseluruhan, sindrom metabolisme yang diaruh SAFFAs mempercepat

kemajuan kenefrotoksikan dalam mekanisme yang berkaitan untuk penjanaan radikal

bebas yang tinggi. POLE sebagai suatu produk makanan kesihatan menghasilkan kesan

propilaktik yang amat sedikit terhadap impak ini. .

xxxi

Effect of palm oil (Elias guinensis) leaf

standardized extract on progression of renal

dysfunction and arterial stiffness in normal

and high fat diet fed Sprague Dawley rats

with induced nephrotoxicity.

ABSTRACT

Saturated free fatty acids (SAFFAs) trigger their deleterious effects through

inducing the intracellular inflammatory pathway related to insulin resistance. This

results in a constellation of biomedical disorders known as metabolic syndrome.

Gentamicin is a widely used aminoglycoside antibiotic. Its nephrotoxic action is

characterized by both proximal convoluted tubules and glomerular membrane damage.

The mechanism of nephrotoxicity is closely related to oxidative stress and free radicals

generation.

It is found nowadays that palm oil leaf extract has got some biomedical importance

due to its content of polyphenols which act to counteract the oxidative stress.

Our study aimed to find the impact of replacing the essential dietary elements in

food by SAFFAs enriched fats on progression of renal damage and its associated

hemodynamic and metabolic changes in rat's model of gentamicin induced

xxxii

nephrotoxicity. The study also evaluated the prophylactic effect of palm oil leaf extract

(POLE) on progression of these disorders.

Renal dysfunction was assessed through measuring serum creatinine, creatinine

clearance, fractional and absolute excretion of both sodium and potassium,

malonyldialdehyde (a biomarker of oxidative stress) in renal homogenate and the

urinary protein excretion. The results showed a higher incidence of nephrotoxicity in

the rats fed with the diet enriched with SAFFAs as compared to those fed with the

standard diet. The nephrotoxicity was graded as mild for the renal failure group fed with

the standard diet and moderate to severe for those fed with the SAFFAs enriched diet.

High dose of POLE could have limited the progression of nephrotoxicity and oxidative

stress development in the renal tissue. Metabolic syndrome was assessed through

performing oral-glucose tolerance test (OGTT), measuring the lipid profile and obesity

indices. The study showed deterioration in metabolic syndrome after feeding the rats

with the diet rich in SAFFAs. Gentamicin co-administration along with this diet

deteriorated the glucose tolerance without producing a noticeable impact on lipid

profile. The metabolic syndrome induced by the SAFFAs was associated with

cardiovascular changes characterized by hypertension and higher arterial stiffness. The

blood pressure was reduced after gentamicin co-administration along with the SAFFAs

enriched diet while the stiffness remained comparable. Moreover, gentamicin injection

to the rats fed with the standard diet did not produce any cardiovascular change.

Progression of metabolic syndrome was limited after POLE co-administration.

xxxiii

Overall, metabolic syndrome induced by SAFFAs hastens the progression of

nephrotoxicity in a mechanism related to higher free radicals generation. POLE as a

health product produced a prophylactic effect against these impacts.

1

Chapter One

Introduction

1.1 Physiology of kidneys

Kidneys are bean shaped organs, located in the abdominal cavity. They have a

contact with the diaphragm and the posterior abdominal muscles and are surrounded by

both the 11th and 12th ribs. Both kidneys are surrounded by the epirenal adipose

connective tissue and some visceral organs. (Kinne, 1989).

1.1.1 General structure

Kidneys are oval shaped organs. They have two parallel borders; the concave border

which is directed toward the body’s midline and the convex border which is directed

laterally. There is a slit in the middle of the concave border called helium (the site

where the kidney receives the renal artery and both the renal vein and ureter emerge

(Lote, 2000).

Renal tissue is made up of two layers; cortex and medulla. It is surrounded by a

layer of loose connective tissues, called renal capsule. The cortex appears as a red

colored spotted area due to the presence of numerous capillary bundles that represent

Malpighian corpuscles. The saggital section of the kidney shows the presence of 8-18

pyramidal triangular areas whose bases are on the cortex and their apices are inside the

2

medulla. These areas are known as renal pyramids, they are marked by fine converging

lines known as medullary rays which represent the collecting ducts. Renal pyramid

apices are known as renal papillae where the urine that is formed in conducting tubules

leave the pyramids through some small orifices into a space called the renal sinus. Then

it drains to the renal pelvis and ureter (Kinne, 1989; Lot, 2000).

1.1.1.1 Nephrons

Nephrons are the basic functional units of renal tissue. There are about one million

nephrons in each kidney. They are composed of the filtration unit that is known as the

glomerulus and a tubular system; responsible for re-absorption and secretion of

biochemical entities. Each renal glomerulus (Malpighian corpuscle) is made of a

globular bundle of capillaries surrounded by a crescent shaped chamber (Bowman’s

capsule). The glomerular capillary is made of separate loops of capillaries but it appears

as a tangled mass of vessels. Bowman’s capsule is made up of a single layer of flat cells

known as podocytes (Kinne, 1989; Sherwood, 2008).

The tubular system of nephron is made up of four distinct zones; proximal

convoluted tubule (PCT), loop of Henle, distal convoluted tubule (DCT) and collecting

tubule (CT) that deliver urine into the orifices present on renal papillae. Both PCT and

DCT are the twisted and coiled portions located in the renal cortex. Loop of Henle &

CT constitute the converging lines which mark the medullary renal pyramid (Sherwood,

2008; Kinne, 1989).

3

Figure 1.1 a) Saggital structure of kidney, b) General structure of nephron c) Detailed

outline of renal tubules. (Adapted from http://www.google.com.my/images

?hl=en&q=kidney+structure+image).

1.1.1.2 Renal glomeruli

Renal glomeruli are made up of a capillary bundle surrounded by a crescent shaped

chamber, known as Bowman’s capsule. Blood enters the glomerular capillary tuft

through the afferent arteriole and leaves it through the efferent arterioles. Along its

passage through the capillary tuft, some of the plasma passes into the lumen of

Bowman’s capsule through the glomerular membrane (Kinne, 1989; Sherwood, 2008).

4

The glomerular membrane is made up of the wall of glomerular capillary loop and

the alignment of epithelial cells called podocytes which constitute the wall of Bowman

capsule. The glomerular membrane sieve is made of three filtration layers; the first is

represented by the fenestrae distributed among the capillary endothelial cells. It is a

coarse filter layer which retains high gram molecular weight (g.m.wt) proteins, fat

globules and blood cells. Underneath the capillary endothelium, there is another

filtration unit. It is represented by an alignment of negatively charged glycoprotein and

glycolipid molecules. It hinders penetration of the negatively charged proteins across

the glomerular membrane and permits penetration of molecules with radii exceeding 60

°A. The third unit is represented by podocytes. Podocytes are irregularly shaped and

possess a number of intermingled primary and secondary processions leaving slits of

rectangular pores measuring 140 x 40 °A, providing the most physically restrictive filter

(Ota, et al, 1980).

1.1.1.3 Proximal convoluted tubules PCT

PCT is a joining segment between Bowman’s capsule and loop of Henle. It is lined

by simple cuboidal epithelial cells which are uniform in all parts of the tubule with

some ultra-structural differences. Their plasma membrane plays an important role in

their function. The site of the membrane that faces the tubular lumen is called the apical

membrane while the one which faces the interstitial fluid space is called the basolateral

membrane. The apical membrane is covered by a border made of densely packed

microvilli, called brush border which increases the surface area upon which re-

5

absorption takes place while the basolateral one contains proteins related Na+-K+ pump

(Kinne, 1989). Their cytoplasm is densely packed, acidophilic in nature due to plenitude

of mitochondria. Under the light microscope there is no obvious discrete margin among

cells except for lateral interdigitations near the apical membrane creating a tight

junction between the neighboring cells and leaving a space (called the intercellular

space) between them at the site of basolateral membrane (Moffat, 1975). Most of the

biochemical entities cross the PCT wall into the interstitial space either by trans-cellular

pathway (crossing both the apical and basolateral membranes) or by crossing the tight

intercellular junction into the intercellular space and then to interstitial fluid (para-

cellular pathway) ( Neumann and Rector, 1979).

According to the gross appearance, PCT is divided into; pars convoluta & pars

recta. Pars convoluta is the initial convoluted portion while recta are the straight

descending second portion. PCT is subdivided also according to the functional

difference into S1 and S2 segments or according to ultra structural difference into S1,

S2 and S3 (Tisher and Osborn, 1969 ; Barrett and Heidger, 1975).

1.1.1.4 Loop of Henle

It is the joining segment between PCT and DCT. It is made up of two parallel tubes

penetrate deep into medulla where they connect in a hair pin like manner. It is

surrounded by an anastomosis of capillary system called vasa recti renis. Both loop of

Henle and vasa recta renis play an important role in countercurrent multiplication

6

mechanism that is responsible for water re-absorption from the collecting tubules

(Moffat, 1975; Kinne, 1989).

1.1.1.5 Distal convoluted tubules

It is the joining segment between loop of Henle and the collecting tubular system. It

is lined by simple cuboidal epithelial cells, similar to that in PCT with some minor

differences as paucity of brush border, making their lumen wider than that of PCT.

Their cytoplasm is less acidic and less densely packed than that of PCT due to their

lower content of mitochondria. DCT is responsible for re-absorption of some ions as

chloride, sodium, potassium and calcium and fine adjustment of tubular fluid and PH. A

part of DCT gets near the afferent arteriole; its epithelial cells are more tightly packed

and specialized. It is called macula densa that is responsible for blood osmolality

monitoring (Moffat, 1975; Kinne, 1989).

1.1.1.6 Collecting tubules system

A series of tubules drain the nephronal tubular fluid from DCT into renal papilla.

The lining epithelium of the CT system participates in electrolyte homeostasis through

potassium ion secretion and sodium re-absorption, pH adjustment and the anti-diuretic

hormone (ADH) mediated water re-absorption (Haas, et al., 1979).

7

1.1.2 Renal glomerular filtration and tubular function

Plasma free fluid transverses the three abovementioned sieving layers of the

glomerulus into Bowman capsule. Rate of transfer is determined by Starling’s

hypothesis which states that the filtration pressure of any capillary system is the

algebraic sum of the opposing hydrostatic and oncotic pressures (Renkin and Robinson,

1974).

PUF= (PGC+∏ BC)-(PBC+∏ GC)

PUF, PGC, PBC, ∏ BC, ∏ GC are the ultra-filtration pressure, glomerular capillary

hydrostatic pressure, hydrostatic pressure in Bowman’s capsule, oncotic pressure in

Bowman’s capsule and oncotic pressure in glomerular capillary, respectively. ∏ BC is

mostly set to be zero due to virtual exclusion of proteins in the glomerular filtrate

(Sherwood, 2006).

The ultra-filtration pressure is higher at the afferent end of glomerular capillary; it is

about 12 mmHg and drops to zero at the efferent end of glomerular capillary as the

blood transverses the length of glomerular capillary. This drop is attributed to the slight

decrease in hydrostatic pressure due to resistance to flow caused by glomerular capillary

and to the increase of intra-glomerular capillary oncotic pressure that result from the

filtration of the protein free plasma, a factor that offsets the hydrostatic pressure and

drops the ultra-filtration pressure fall to zero (Renkin and Robinson, 1974).

Kidney has a system of glomerular filtration auto-regulation which relies on

regulation of the caliber and resistance of both the afferent and efferent arterioles, in

such a way that the intra-glomerular capillary hydrostatic pressure is balanced

Eq. 1.1

8

according to the requirement (Christensen, et al., 2003). Normally, the caliber of

afferent arteriole is bigger than that of efferent, a disproportionality that elevates intra-

glomerular hydrostatic pressure and increases vascularity of the glomerular bed

(Sherwood, 2006).

Auto regulation mechanism is controlled by renal sympathetic nerve fiber and a

series of systemic and local autacoids as angiotensin II, catecholamines, prostaglandins,

thromboxans, adenosine, dopamine, nitric oxide (NO.), endotheline and ANP (Anti-

natriutic peptide). Angiotensin II, catecholamines, thromboxans, adenosine and

endotheline act as afferent and efferent arterioles vasonstrictors leading to reduction of

glomerular filtration rate while the rest have apposite function afferent and efferent

arterioles. Auto regulation of glomerular filtration is accomplished by one of the two

mechanisms; the myogenic where vasoconstriction incurs as a response to any stretch or

the tubulo-glomerular feedback mechanism, in which, the increase in sodium delivered

to DCT triggers macula densa to activate the juxta-glomerular apparatus to release

some autacoids as angiotensin II and adenosine which induce afferent arterioles

vasoconstriction (Salman, et al., 1999; Vallon, 2003; Christensen, et al., 2003).

Renal tubular function is associated with the re-absorptive and secretory job of the

ductile system that starts from the beginning of PCT and ends up in ducti of Billini

(Thies, 1995).

PCT absorbs about 67% of electrolytes and water and almost all filtered glucose and

amino acids. Sodium ion plays an important role in PCT’s function, since most of the

sym-porters and anti-porters require sodium for proper functioning. Sodium re-

9

absorption creates the required osmotic gradient for water re-absorption. Mechanism of

sodium re-absorption in the early segment is different from that in the late segment. In

the early segment, it is coupled with bicarbonate, phosphate and a number of organic

molecules, such as; glucose, amino acids and lactate while it is coupled with chloride in

the late segment. It is so avid that these solutes are almost completely cleared from

tubular fluids in this segment leading to a great change in tubular fluid composition

between the first and second halves. Most of the water soluble electrolytes can follow

para-cellular route down their electrochemical gradient potential by a process called

solvent drag. PCT cells can excrete both organic cations and anions through specific

symporters and antiporters present on both the apical and basolateral membrane (Beck,

et al., 1973; Neuman and Rector, 1976).

Tubular fluid leaves PCT towards loop of Henle where the counter current

multiplication mechanism takes place in cooperation with vasa recta renis. This

mechanism aims to increase osmolality of the renal interstitium at the deep inner

medullary portion; an action that facilitates reabsorption of water from the medullary

CT toward renal interstitium by the aid of ADH (anti diuretic hormone). Unlike the

descending limb, thick ascending limb of loop of Henle is impermeable to water and

permeable to ions. Its apical membrane is endowed with lots of frusemide sensitive

Na+-K+-Cl- symporters which can actively extrude these ions from the tubular fluid.

Like other tubular epithelia, basolateral membrane is endowed with lots of Na+-K+

ATPase pump mechanism which extrude Na+ toward the interstitium. The reabsorbed

K+ can be recycled again to tubular fluid and translocated into the DCT.

10

Accumulation of sodium in the interstitium elevates the interstitial osmolality and

makes water to transfer from descending limb of the loop of Henle into the interstitium

according to the osmolar gradient. Repetition of this process leads to a high osmotic

pressure at the tip of the loop. Vasa recta renis acts to wash the reabsorbed fluid into the

deep intra-medullary interstitium towards the cortical region (Layton and Weinstein,

2002).

DCT is the site where Na+ is reabsorbed by the aid of Na+-Cl- co-transporters in the

apical membrane and Na+-K+ ATPase pump in the basolateral membrane of the lining

epithelium. Accordingly other electrolytes are re-absorbed by solvent drag mechanism

along with reabsorbed water through the para-cellular shunt. Inside the CT ducti system

urine volume and electrolyte composition are regulated. The system is rich in two types

of cells; principle and intercalated cells. Principle cells are responsible for sodium re-

absorption in replacement of potassium ion. It is facilitated by basolateral Na+-K+

ATPase pump, apical membrane Na+ and K+ channels and the electrochemical gradient

that push potassium to CT lumen. Intercalated cells are found mainly in the medullary

region. They transfer some K+ and reabsorbed water in response to ADH from the

medullary CT toward renal interstitium (Thies, 1995).

1.2 Aminoglycosides induced nephrotoxicity

1.2.1 Aminoglycosides

Aminoglycosides are a group of structurally complicated antimicrobial compounds,

composed of modified amino-sugar moieties linked by O-glycosidic linkage. They are

11

extracted from some species of Streptomyces bacteria or Micromonospora fungi and

chemically modified for a stronger antimicrobial activity or to enervate its toxicity

(Mitsuhashi, 1975). Their antimicrobial action is attributed to their ability to indispose

bacterial protein synthesis and dismount the cell wall integrity (Lutwyche, et al., 1998).

Toxicity of aminoglycosides is demarcated by being an oculo-, vestibulo-, cochlear and

nephrotoxicity due to the specific cellular uptake mechanisms that epithelia of these

organs possess for molecules having a cationic nature like aminoglycosides (Arya,

2007; Mingut-Leclerq, et al., 1999). Leading to some notorious clinical signs and

symptoms, such as oscillopsia (bouncing vision), blurred vision, tinnitus, vertigo,

difficulty in balance, mental fogginess, short term memory lapse, tiredness and change

in urine volume (Kelly, 2006). Hairy cells of vestibulocochlear apparatus in the middle

ear, the oculo-retinal cells, the lining epithelium of renal proximal convoluted tubules,

the neuromuscular junctions and the renal glomerular basement membrane of renal

glomeruli are the main targets of aminoglycosides (Arya, 2007; Mingut-Leclerq, et al.,

1999).

1. 2.2 Aminoglycosides induced nephrotoxicity

Aminoglycosides induced nephrotoxicity is a multi-step process, characterized by

prominent changes in renal glomeruli and PCT (Rougier, et al., 2003).

12

1.2.2.1 Aminoglycosides induced tubular injury

It commences with the uptake of aminoglycosides by the simple cuboidal epithelial

cells of pars recti segment of PCT. Due to their cationic nature, aminoglycosides can

easily bind to acid phosphates that incrust the brush border as a precursory step for their

subsequent intracellular internalization. Then they bind to megalin (a trans-membrane

protein that binds to polybasic compounds like aminoglycoside and internalize them

into the cytosol. The selectivity of aminoglycosides induced toxicity on PCT

epithelium, hairy cells of vestibulocochlear apparatus and retinal epithelia is attributed

to the megaline on their surface) (Nagai, et al., 2001; Pruiska, et al., 1997; Kanig et al.,

2008). As soon as they access the cytosol, they are engulfed by endosomes which

provide an endocytic membrane transport shunt from plasma membrane to lysosomes

(Sandoval, et al., 1998) where the environment is more acidic (PH=5), rendering

aminoglycosides to be more cationic and increase their potency to bind to lysosomal

membrane acid phosphates (that constitute about 5-20% of the membrane), leading to a

cellular change known as lysosomal phospholipidosis (Schank, et al., 1992). In this

case, lysosomal phospholipase and sphingomylinase enzymes lose their ability to break

down phospholipids molecules. This in turn leads to a conspicuous accumulation of

myeloid bodies in the form of intracellular aggregations of phospholipids, cholesterol

and proteins (Kacew, 1987). When the critical threshold of intralysosomal concentration

of aminoglycoside is reached, It bursts out to the cytosol inducing destructive changes

in mitochondria and both the apical and basolateral membranes (Guirgea-Marion, et al.,

1986; Giuliano, et al., 1987). Inside the mitochondria, aminoglycosides bind with iron

to form a complex with Fe++ which initiates lipid peroxidation and affects membrane

13

integrity that in turn leads to damping of mitochondrial internal respiration and decrease

in ATP synthesis (Msastrasinh, et al., 1982; Zorov, 2010). The passive effect of

gentamicin on apical and basolateral membranes integrity is attributed to lipid

peroxidation and inhibition of ATP dependent transport mechanisms (Blias, et al.,

1993).

Hydropic degeneration may inflict tubular cells due to a decrease in ATP that is

required for the active Na+-K+ pump, leading to Na+ retention and accumulation of an

equimolar amount of water inside the cytoplasm as cytoplasmic vacuoles (Solez, 1986).

If the destructive action pursues, necrosis may inflict injury to the cells. It is

characterized by loss of cellular outlines and nuclear changes (karyopyknosis,

karyorrehix and karyolysis) (Turton and Hooson, 1998). Progression of these events is

controlled by chemical structure of the aminoglycoside which affects the ability of the

drug to bind into the negatively charged phospholipids that incrust the brush border

(Malis, 1984) and mode of the drug administration, as dosing and time interval

(nephrotoxicity pattern differs if the drug is given in low or high dose and if the drug is

given once a day, multiple doses or by continuous infusion). The single dosing system

is less nephrotoxic as compared with the multiple dosing systems, as it creates just one

peak of the drug plasma level while the multiple dosing systems create more (Reiner,

1987). This is due to the nature of the cellular uptake of aminoglycosides which is a

saturable process such that a finite quantity of drug transposes into the intracellular

compartment after surpassing a certain critical threshold (Giuliano, et al., 1986).

There are many approaches that have been proposed to halt aminoglycosides induced

nephrotoxicity. One of the approaches proposed using agents that make a complex with

14

aminoglycosides extracellularly or compete with the aminoglycosides at the binding

sites. (For example; the series of polyaspartic acid derived compounds, like

daptomycin) (Williams, 1985). Changing the mode of drug administration is another

way to ameliorate the nephrotoxic effect of gentamicin (by switching the patient into

the single dosing system, as mentioned above) (Reiner, et al., 1978).

Recently a series of aminoglycosides with a modified structure have been evolved.

They have lower ability to bind to brush border or lysosomal phospholipids, e.g.

amikacin, arbekacin and isepamicin (Carlier, et al., 1983). Another approach proposed

is by using antioxidants and iron chelators. They intervene with the cascade sequential

processes associated with intracellular aminoglycosides induced lipid peroxidation

(Walker , et al., 1987).

.

Figure 1.2:- The structure of kanamycin (The standard chemical structure of aminoglycoside molecule. IUPAC name (2-(aminomethyl)- 6-[4,6-diamino-3- [4-amino-3,5-dihydroxy-6-(hydroxymethyl) tetrahydropyran-2-yl]oxy- 2-hydroxy- cyclohexoxy]- tetrahydropyran- 3,4,5-triol). Adapted from http://chemistry.about.com/od/factsstruc -tures/ig/Chemical-Structures K/Kanamycin.htm).

O

OO

O

NH2

OH

OH

H2N

NH2

OH

H2N

OH

HO

H2N

HO

IIII

II

15

1.2.2 Gentamicin as an example of nephrotoxic aminoglycoside

Gentamicin is an aminoglycoside with methyl-N-substitution. Its chemical formula

is C21H43N5O7 and its g.mwt is 488 gm/mole. It is a water soluble crystalline white to

yellow colored powder. It is obtained from Micromonospora species; a genus of gram

positive bacteria, disseminated widely in nature. It has a broad spectrum of bactericidal

action against an array of gram negative bacteria, such as pseudomonas, niesseria,

legionella and various coliform bacteria (Mitsuhashi, 1975). Gentamicin is more toxic

as compared to other aminoglycosides due to its stronger ability to bind into acid

phosphates (the targets of aminoglycosides-induced toxicity). This deterrent

characteristic makes physicians more cautious while prescribing it (Carlier, 1980).

Bioavailability of gentamicin after oral administration is very poor while it is higher

if it is given parenterally. Its hydrophilicity and molecular size allows it to permeate

easily through the capillary endothelium (Segal, et al., 1988). Its Vd (volume of

distribution) is affected by changes in total volume of extra-cellular fluids. In edema,

the concentration decreases due to the increase in VD while in dehydration, the

concentration goes up (Kelman, et al., 1984). Its binding to plasma proteins is weak and

reaches 10-30%. Gentamicin is eliminated primarily by kidneys. Gentamicin has a very

short half life which is higher in infancy as compared to higher ages. This difference

may be due to the difference of GFR and renal function with different age groups.

(Vozeh, et al., 1979).

16

Figure 1.3:- Chemical structure of gentamicin. IUPAC name (diamino-3 3-amino-6-1-(methylamino)ethyl]oxan-2-yl]oxy}-2-hydroxycyclohexyl]oxy}-5-methyl-4-(methylamino)oxane-3,5-diol) (Mitsuhashi, 1975).

Therapeutic plasma concentration of gentamicin ranges from 2 to 12 µgm/ml. The

minimum value represents the MIC (minimum inhibitory concentration) for bacteria

while the highest one represents the critical threshold concentration. So as an antibiotic,

it should be given in a dose that keeps the concentration between the two thresholds. In

human, the therapeutic dose that maintains it within the abovementioned range is about

4-7 mg/kg(B.W)/ day. Higher doses make the concentration higher than this range. In

rats, the dose that maintains the concentration within the therapeutic level is about 10-

20 mg/Kg (B.W)/day. It is documented that nephrotoxicity in rats is induced at a dose

exceeding 40 mg/ Kg (B.W)/day. In one study, after a single injection of 100 mg/kg

gentamicin, the plasma level rose to168 µgm/ml (Stahlmann, et al., 1988).

17

1.3 Oxidative stress

Oxidative stress is evolved due to constant interaction of the body with oxygen

during intracellular respiration resulting in unleashing of highly reactive molecules,

known as free radicals. Cells are endowed with a redox system, responsible for

mopping free radicals out by scavenging them (Sies, 1991). Free radical molecules

hareve an atom with an unpaired electron in its outer shell, making them highly reactive

species with a tendency to attack various biomolecules. It is formed due to heterophilic

breakdown of covalent bonds during cellular metabolism (Nonhebel and Walton, 1974).

When the free radical is formed, a series of chain propagation reactions are triggered.

They are called electron transport chain (ETC). In these reactions, each newly formed

radical looks to return into its original state by stealing another electron with antiparallel

spin from the surrounding environment. This in turns evolves more new radicals. ETC

mostly happens inside the mitochondria where oxygen is used to generate energy and

acts as an electron acceptor (Gropper, et al, 2008).

Free radicals target double bonds in poly unsaturated free fatty acids (PUFFAs)

resulting in a heterophilic breakdown of C-H bonds attached to the double bonds.

PUFFAs contain one or more methelene groups between the double bonds. They are

highly reactive to oxidizing agents and can easily lose their hydrogen after forming

carbon centered radicals (Gropper, et al, 2008). In an effort to stabilize it, molecular

rearrangement occurs which converts the molecule into more stable form called

conjugated diene (CD). CDs tend to react with other oxygen molecule to form

18

peroxyradicals which look to attack other PUFFAs' double bonds, leading to continuous

repetition of this process (radical chain propagation reaction) (Gropper, et al, 2008).

After the formation of peroxy radicals, either they are reduced to hydroperoxides

which are relatively more stable or they suffer sequence of reactions associated with

intramolecular cyclization, leading to generation of a broad range of products (Mittler,

2002) (Figure 1.4).

Figure 1.4:-Lipid peroxides fate pathways (adapted from Mittler, 2002).

Final products of lipid peroxidation are either aldehydes, like formaldehyde,

acetyladehyde, acrolien, malonyldialdehyde (MDA),4-hydroxyhexenal (4-HHE) and 4-

hydroxnonenal (4-HNE), oxaloaldehydes such as glyoxal and methyl glyoxal, ketones

19

such as; acetone and butanone and some alkanes such as; hexane, cyclohexane and

heptanes (Mittler, 2002). MDA is the most convenient biomarker of lipid peroxidation

among them because of its plain reaction with thiobarbituric acid (TBA) to form TBA-

MDA adduct that is easy to be detected calorimetrically (Akhgari, et al., 2003).

Recently, F2- isoprostane is used as a marker as well (Tianying, et al., 2004). Aldehyde

products of lipid peroxidation are relatively less harmful than the free radicals, although

they have a tendency to form Schiff bases with lysine, histidine and cysteine residues of

proteins and nitrogen bases of DNA backbone leading to conformational changes of the

proteins along with loss of their function and some genotoxic and mutagenic effects due

to DNA binding (Cederbaum, 2001; Cohen , et al., 1984; Kharbanda , et al., 2002).

1.3.1 Types of free radicals

Free radicals are either reactive oxygen species (ROS) or reactive nitrogen species

(RNS) depending on whether oxygen or nitrogen is centered in (Nonhebel and Waton,

1974).

1.3.1.1 Reactive oxygen species

Reactive oxygen species (ROS) include; superoxide anion (•O2-), hydrogen peroxide

(H2O2), hydroxyl radical (•OH), alkoxy radicals (RO•), peroxy radicals (ROO•) and

hypochlorous acid (HOCl). Oxygen has a tendency to be reduced in sequential

univalent processes releasing intermediates such as •O2-, H2O2 and •OH (Uday, 1999).

20

Leakage of electrons during ETC reaction in mitochondria results in oxygen

reduction. ETC reaction accompanies steps associated with oxidation of hydroxyacids

icas those in citric acid cycle, amino acids or fatty acids. Nicotinamide adenine

dinucleotide (NADPH) acts as a source of reducing equivalents in these reactions. It

donates electrons to cytochrom P450 via flavoproteins. Cytochrom P450 reduces

oxygen to water without the formation of •O2-. Any disturbance in mitochondrial

function reaction leads to leakage of electrons and release of •O2-.

As soon as •O2- is released, it is dismutated spontaneously or by the aid of super

oxide dismutase enzyme (SOD) forming H2O2. •O2- is rather inactive and has very poor

penetration through plasma membrane while hydrogen peroxide is more active and

especially ferrous and cuprous ions induce release of •OH by a reaction called Fenton

reaction ( Uday, et al., 1999). These metal ions are not found freely and mostly are

bound to a tissue protein called metalloprotein. They are released during cellular

degeneration and tigger Fenton reaction (Aruoma, 1989). •OH is extremely active and

due to its low diffusion capacity, it damages any molecule in its vicinity (Uday, et al.,

1999).

Inside the body, there are two defensive mechanisms to mop ROS out; the primary

one which is represented by antioxidant enzymes and the secondary one which is

represented by the antioxidants which exist in dietary supplements. SOD, catalase and

glutathione system represent the enzymatic system that mop out ROS. SOD is a

metalloprotein. It contains copper and zinc as cofactor ions. It dismutates •O2- to H2O2.

Catalase enzyme is present in peroxosomes. It is a hem-protein that catalyzes

conversion of H2O2 to water and oxygen.

Figure 1.5: - Cascadradical generation. Odismutase enzyme, Hmyeloperoxidase enzy

Glutathione system i

glutathione reductase

from NADPH to hydr

(Thorp, et al., 2004; M

Furthermore, immu

Neutrophiles produce

through action of mey

Hypochloro

O2.

O2

XO or mitochondrial

21

e sequential reaction of ROS form2

.:- singlet oxygen or superoxide 2O2:- hydrogen peroxide, OH:- hyme (Thorp, et al., 2004).

s made up of glutathione, glutathio

enzyme. It works along with ascor

ogen peroxide or other hydroperoxid

ay, et al., 1996).

ne system lays an important role in

H2O through NADPH oxidase enz

loperoxidase enzyme on H2O2 (Gropp

respiration

us acid MPOx

ROOH Peroxy radical

. OH

Fenton

H

SOD

H2O + O2

Catalase

ation after superoxide free radical, SOD:- superoxide

droxyl radical and MPOx:-

ne peroxidase enzyme and

bic acid to shuttle electrons

es as illustrated in Fgure1.6

triggering oxidative stress.

yme and hypochlorous acid

er, et al., 2008).

reaction with iron

2O2

22

Figure 1.6:- Glutathion-ascorbate cycle to detoxify H2O2. Apx:-Ascorbate peroxidase enzyme, MDA: - Monohydroascorbate, MDAR: - Monohydroascorbate reductase, GPx:- glutathione peroxidase enzyme, Gr:- glutathione reductase enzyme, GSH:-reduced form of glutathione, GSSG:-Oxidized form of glutathione (May, et al., 1996).

1.3.1.2 Reactive nitrogen species RNS

Nitric oxide (NO.) and peroxynitrate (ONOO) are the RNS forms of free radicals.

NO. is produced extensively by endothelial cells as a short lived paracrine physiological

transmitter that mediates vascular smooth muscles dilation and inhibition of platelets

aggregation through activation of cGMP and decreasing intracytosolic calcium

concentration. Furthermore, it inhibits endothelial function by acting as a gene

modulator preventing the expression of surface adhesion molecules as VCAM-1 and

ICAM-1which are required for leukocyte adhesion and development of atherosclerotic

events (Van der, et al., 1999; Walker, et al., 2001).

During oxidative stress, NO. reacts with superoxide radical generating peroxynitrate

(ONOO.). Peroxynitrate is a strong free radical. It induces free radical chain propagation

reaction and nitrosation of tyrosine residues of proteins and nitrogen bases of DNA

H2O2

H2O + O2

Ascorbate

MDA

MDAR

GSSG

GSH

APx

NADPH +H+

NADP

GPx

NADPH oxidase

Gr

leading to cellular dysfunction and mutation (Van der, et al., 1999; Walker, et al.,

2001). The human body fends this pathway through triggering nitrate-nitrite-nitric oxide

pathway. When the amount of NO. exceeds the threshold, NO. is oxidized to nitrite

(NO2-) and then to nitrate (NO3

-) by aid of multicopper oxidase system, cerruloplasmin

and oxyhaemoglobin which converts to methaemoglobin (Van der, et al., 1999; Walker,

et al., 2001).

Conversion of NO. to NO2- and NO3

- is a bidirectional process which occurs either

spontaneously after disturbance of the chemical balance or triggered by enzymes. On

the other hand, NO. could be evolved from NO2- and NO3

- by aid of nitrate reductase

enzyme produced by gastrointestinal commensal bacteria (Lundberg, et al., 2008).

Regulation of protein function through nitrosylating its thiol groups by NO. is another

mechanism that the body is endowed to discard excessive NO. radical (Walker, et al.,

2001).

Figure 1.7:-Fate of nitr

O-

Nitrous oxide

Nitroso Hb

Arginine NO2 and NO3

NO. Synthase enzyme

nitrotyrosine

Superoxide radical ONO

23

ic oxide inside the body (Walker, et al., 2001).

24

1.3.2 Dietary antioxidants

Diet provides plenty of biochemical compounds able to counteract oxidative stress

and enforce the defensive processes against free radicals generation. These compounds

are either vitamin products , as vitamin A, vitamin E and vitamin C which are found in

animal and plants products such as some carotenoid derivatives (zeaxanthine, lycopene

and lutein) and polyphenols (Shahidi, 1997).

Polyphenols are water soluble chemical compounds that bear more than one phenolic

groups. Phytochemical polyphenols are classified into:-tannins, flavonoids and

phenylpropanoids (Shahidi, 1997).

Palm oil leaf extract used in our study, contains polyphenols with catechin and

ferrulic acid constituting the majority. Catechin is a tricyclic flavonoid derivative. It

possesses an antioxidant activity due to the presence of phenolic groups attached to

benzene ring (Chumbalov, et al., 1995). Ferrulic acid is a phenylpropanoid derivative,

found in plant cell wall as a covalent side chain attached to arabinoxylan and cellulose

of the cell wall. Inside the plant, it serves to crosslink lignine to polysaccharides adding

some rigidity to the cell wall. Previous studies reveal that after ingestion, ferrulic acid is

absorbed in gastrointestinal tract after cleavage of its linkage with lignines by pancreatic

acids (Pan, et al., 1999). It has better bioavailability in plasma as compared to other

polyphenols (Scheliner, 1968). Ferrulic acid has a unique antioxidant power due to the

presence of carboxyl group in its structure which acts as a free fatty acid anchor in

membranous structures resulting in higher anti lipid peroxidation effect (figure 1.8)

(Kanski, et al., 2002).