University of Ghana http://ugspace.ug.edu.gh UNIVERSITY OF GHANA COLLEGE OF HEALTH SCIENCES RENAL HISTOLOGY AND FUNCTION IN GENTAMICIN- INDUCED ACUTE KIDNEY INJURY (AKI) IN RATS GIVEN NATURAL COCOA BY MARIAN NAA ANYEMA ASAH-OPOKU (ID NUMBER – 10186448) THIS THESIS IS SUBMITTED TO THE UNIVERSITY OF GHANA, LEGON IN PARTIAL FULFILLMENT OF THE REQUIREMENT FOR THE AWARD OF MPHIL. ANATOMY DEGREE DEPARTMENT OF ANATOMY JULY, 2015 i University of Ghana http://ugspace.ug.edu.gh DECLARATION BY CANDIDATE This is a study based on my own research which I carried out at the Animal Experimentation Unit, Korle-Bu Campus and under supervision in accordance with regulations of the School of Research and Graduate Studies, University of Ghana. I hereby declare that having duly acknowledged the references to works of other researchers, this study is original to me and has not been submitted either in whole or part for the award of any other degree in this or another University. Signature: …………………………… Date: ………………………….. Marian Naa Anyema Asah-Opoku (Mrs.) DECLARATION BY SUPERVISORS We declare that the practical work and presentation of this thesis were supervised by us in accordance with guidelines on supervision of thesis laid down by the University of Ghana. Principal Supervisor: …………………………… Date: ………....................... Prof. Frederick K. Addai Co-Supervisors: …………………................ Date: ……………………… Rev’d Prof. Seth A. Ayettey …………………………… Date: ……………………… Dr. John Ahenkorah i University of Ghana http://ugspace.ug.edu.gh DEDICATION This thesis is dedicated to my lovely husband, Dr. Kwaku Asah-Opoku and my very supportive mum, Mrs. Comfort Okine. ii University of Ghana http://ugspace.ug.edu.gh ACKNOWLEDGEMENTS I thank God Almighty for His grace and mercies bestowed on me throughout the period of study. I am very grateful to the Head of Department, Prof. Frederick K. Addai and the entire Anatomy Department for the immense financial support as well as monthly allowances granted me to be able to complete this study. My sincerest thanks go to my Academic Supervisors, Prof. Frederick K. Addai, Rev’d Prof. Seth Ayettey and Dr. John Ahenkorah for their directions and inputs to ensure a good job was done. I am greatly indebted to Drs. Vincent Boima, a Consultant Nephrologist at the Renal Dialysis Unit, Korle-Bu Teaching Hospital and Benjamin Ako- Boham for their immense contributions from the time of developing my proposal to the completion of this thesis. I am also very grateful to Dr. George Asare (Major Rtd) of the Chemical Pathology Department, University of Ghana, for his tremendous help throughout my preliminary study and teaching me how to perform cardiac puncture. To my colleague, Mr. Ernest Asiamah, I say a very big thank you for the sleepless nights we spent together in the laboratory to complete this study. God bless you. I wish also to express my profound gratitude to the technologists at the Anatomy Department, Oral Pathology Unit, School of Medicine and Dentistry and Animal Experimentation Unit: Mr. Armah, Mr. Samuel Mensah, Mr. Paul Atiah, Mrs. Sethina Adjetey, Mr. Samuel deGraft Mensah and Miss Linda Azuyie for giving me all the assistance I needed to carry out the laboratory works. Finally, to all my friends and loved ones whose prayers and support saw me through, I say God richly bless you. iii University of Ghana http://ugspace.ug.edu.gh TABLE OF CONTENTS DECLARATION BY CANDIDATE i DEDICATION ii ACKNOWLEDGEMENTS iii TABLE OF CONTENTS iv LIST OF TABLES viii LIST OF ABBREVIATION x ABSTRACT 1 CHAPTER ONE 3 1.0 INTRODUCTION 3 1.1 Background to study 3 1.2 Problem Statement 11 1.3 Justification 13 1.4 Working Hypothesis 13 1.5 Aim 13 CHAPTER TWO 15 2.0 LITERATURE REVIEW 15 2.1 Gross structure of the human kidney 15 2.2 Gross structure of rat kidney 16 2.3 Histomorphometry of the nephron 17 2.4 Functions of the kidney 19 2.5 Risk of kidney injury 20 2.6 Types of kidney injury 21 2.6.1 Acute kidney injury - definition 21 2.6.2 Incidence of AKI 22 2.6.3 Causes of AKI 22 2.6.3.1 Pre-renal 22 2.6.3.2 Intrinsic or intrarenal 23 2.6.3.3 Post-renal 23 2.7 Clinical presentation of AKI 24 iv University of Ghana http://ugspace.ug.edu.gh 2.8 Management of AKI 24 2.9 Nephrotoxicity as a cause of AKI 25 2.10 Drugs as cause of nephrotoxicity 25 2.11 Aminoglycosides 26 2.11.1 Gentamicin as a common cause of nephrotoxicity 27 2.11.2 GM-induced nephrotoxicity as a model of AKI 28 2.12 Reactive oxygen species (ROS), free radicals and oxidative stress as a cause 2 8 of tissue damage 2.13 GM induces production of ROS 29 2.14 Gentamicin (GM) induces free radical production 29 2.15 Mechanism of GM uptake and renal damage 30 2.16 Effects of nephrotoxic injury on renal compartments 31 2.17 Recovery of renal function in AKI 31 2.18 Induction of AKI in rats with gentamicin 32 2.19 Antioxidants role in GM nephrotoxicity 32 2.19.1 Sources of antioxidants 33 2.19.2 Theobroma cacao – Novel source of powerful antioxidants 34 2.19.3 Bioavailability of cocoa 35 2.19.4 Health benefits of natural cocoa 36 3.0 MATERIALS AND METHODS 38 3.1 PILOT STUDY 38 3.2 EXPERIMENTAL PROTOCOL FOR MAIN STUDY 40 3.2.1 Study design 40 3.2.2 Study population 40 3.2.3 Inclusion and exclusion criteria 40 3.2.4 Acquisition and acclimatization of animals 40 3.3 Blood sampling for kidney function test 41 3.4 Preparation of 2% (w/v) natural cocoa suspension (NCS) 42 3.5 Gentamicin (GM) dosage for acute kidney injury (AKI) 43 3.6 Grouping and Treatment of animals 44 3.7 Harvesting of rat kidneys 46 v University of Ghana http://ugspace.ug.edu.gh 3.8 Determination of weight and volume of kidneys 47 3.9 Sampling of kidney tissues 47 3.10 Histological preparation 48 3.11 Stereological assessment of rat kidney 49 3.12 STATISTICAL ANALYSIS 51 CHAPTER FOUR 55 4.0 RESULTS 55 4.1 Behavioural changes 55 4.2 Mortality 55 4.3 Body weight 56 4.4 GM and weight 56 4.5 Cocoa and weight 60 4.6 Fluid intake 61 4.8 Selected biochemical indicators of kidney function 64 4.8.1 Blood urea nitrogen (BUN) level 64 4.8.2 Serum creatinine (SCr) level 64 4.9 Stereological assessments 68 4.9.1 Glomerular volume 68 4.9.2 number-weighted volume fraction of proximal tubular epithelial cells 69 4.10 Histological features of kidneys sections of rats of various experimental 70 groups CHAPTER FIVE 78 5.0 DISCUSSIONS 78 5.1 General 78 5.1.1 Mortality 81 5.1.2 Effect of cocoa on fluid consumption 81 5.1.3 Body weight 81 5.1.4 Effect of GM on selected biochemical markers 82 5.2 Histopathologic changes 83 5.3 Effects of regular consumption of cocoa as opposed to discontinuous intake 85 5.4 Summary of key findings 87 vi University of Ghana http://ugspace.ug.edu.gh 5.5 Conclusion 88 5.6 Limitations of the study 88 5.7 Recommendations 89 REFERENCES 90 APPENDIX I 108 APPENDIX II 109 APPENDIX III 112 vii University of Ghana http://ugspace.ug.edu.gh LIST OF TABLES Table 1: Experimental Interventions 45 Table 2: Summary on mortality for experimental period 56 Table 3: Summary of statistics on mean body weight (g) at various treatment 58 points Table 4: Summary of weight analysis of cocoa and non-cocoa (NNCS) treated 60 groups (6 weeks post NCS treated) Table 5: Summary of Kruskal Wallis analysis of daily fluid intake (mL) per rat in 61 each group Table 6: Summary of post HOC test for median fluid intake 62 Table 7: Summary of statistics on biochemical analysis 65 Table 8: Summary of absolute glomerular volume among treatment groups 68 Table 9: Summary of post HOC analysis for glomerular volume 68 Table 10: Summary of ANOVA test of PCT volume. 69 Table 11: Post HOC analysis of PCT volume 69 viii University of Ghana http://ugspace.ug.edu.gh LIST OF FIGURES Figure 1 : A diagram showing gross appearance of the kidney in longitudinal 16 section Figure 2: Micrographs of H & E (Haematoxylin and Eosin) stained kidney section 19 showing cortical tissue. Figure 3: Picture showing a rat in a dessicator being anaesthetized by inhalation of 52 chloroform. A – dessicator (without dessicant), B - rat Figure 4: Picture of a rat being perfused. 53 Figure 5: A picture of stereological grid (0.5cm × 0.5 cm) superimposed on kidney 54 section to count PCT epithelial cells Figure 6: A picture of stereological grid (1 cm × 1 cm) superimposed on kidney 54 section to estimate glomerular volume Figure 7: Bar chart of rat weight at different times of experiment (weeks 0, 6 and 7) 59 Figure 8: Line graph showing daily median fluid intake (mL) of experimental 63 groups Figure 9: Line graph showing mean blood urea nitrogen (BUN) levels of 66 experimental groups at weeks 0, 6 and 7 Figure 10: Line graph showing serum creatinine (SCr) levels at weeks 0, 6 and 7 67 Figure 11(G1 – G5): Micrographs of PAS stained kidney sections showing proximal 70 tubular necrosis. (See legend to Figure 13 on next page). Figure 12(G1 – G5): Micrographs of PAS stained kidney sections showing 72 mononuclear cell infiltration in GM treated groups. (See legend on next page). Figure 13 (G1 – G4): Micrographs of PAS stained kidney sections showing colloidal 74 material in renal tubules of GM treated rats (G1, G2 and G4). Figure 14(G3 – G4): Micrograph of PAS stained kidney sections of GM only (G4) 75 and control group (G3) showing glomerular shrinkage (g) in G4 and normal glomerulus (G) in G3. Figure 15(G1 – G5): Micrographs of PAS stained kidney sections showing loss of 76 brush border in GM treated groups (See legend on next page). ix