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Pathogenesis of cholesterol-induced glomerulosclerosis in guinea pigs Al-Shebeb, Taha H. 1987

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Pathogenesis of Cholesterol-Induced Glomerulosclerosis i n Guinea Pigs By Taha H. AL-Shebeb B.V.M.S.. Baghdad University 1975 M.Sc. Baghdad University 1978 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY IN THE FACULTY OF GRADUATE STUDIES (The Department of Pathology) We accept t h i s thesis as conforming to the required standards THE UNIVERSITY OF BRITISH COLUMBIA February, 1987 © Taha H. AL-Shebeb, 1987 In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the head of my department or by his or her representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission. Department of The University of British Columbia 1956 Main Mall Vancouver, Canada V6T 1Y3 Date tfarcA (I?? - i i -Abstract The role of cholesterol-rich diet and of high protein supplement on the development of a glomerular lesion was studied i n male guinea pigs. The possible pathogenesis of lipid-induced glomerulosclerosis was investigated. Four experiments were carried out. Four groups of guinea pigs were used i n experiment I: CONT group was kept on normal guinea pig chow for 70 days; HC group was kept on 2% cholesterol diet for 70 days; HP group was kept on 50% casein diet for 70 days, and HCHP group received 2% cholesterol diet for 30 days and 2% cholesterol/50% casein diet for another 40 days. In experiment II two groups were used: CONT group and acetyl phenylhydrazine (APH)-treated group i n which haemolytic anaemia was induced. In the t h i r d experiment the same dietary regimens as described i n experiment I were used. In experiment IV three groups, namely CONT, HC, and HCHP, were employed. The animals i n experiment IV were s a c r i f i c e d after 5, 10, and 30 days. The f i r s t experiment explored the role of high cholesterol - and high cholesterol/high protein diet i n the development of glomerulosclerosis. The other three experiments were designed to learn about the possible mechanism of lipid-induced glomerulosclerosis. L i p i d analyses of plasma, erythrocytes and kidney tissue as well as complete blood count, erythrocyte osmotic f r a g i l i t y and blood c e l l morphology studies were performed. Kidney histology, histochemistry, immunohistochemistry, electron microscopy, morphometry, and renal and l i v e r function tests were also carried out. De novo cholesterol synthesis was assessed by measuring HMG COA reductase a c t i v i t y and incorporation of t r i t i a t e d water into cholesterol i n the kidneys. - i i i -Cholesterol-fed animals showed decreased weight gain, increased cholesterol concentration i n plasma, erythrocytes, and kidney tissue. Haemolytic anaemia was documented after 70 days on t h i s dietary regimen. Glomerular p r o l i f e r a t i o n lesion was f i r s t noted at day 30 and progressed by day 70. Moderate proteinuria and haematuria were observed at day 70. Addition of protein to the high cholesterol diet led to a further decrease in weight gain. I t also increased the mortality rate to 40% by day 70. The glomerular l e s i o n , proteinuria and haematuria, and possibly haemolysis were more marked i n the HCHP group. No causal relationship was found between l i v e r function, immune complexes, haemolysis and glomerulosclerosis. Serum phosphate levels did not d i f f e r among the groups. The l i p i d found i n the kidney of both HC and HCHP groups was mostly of plasma o r i g i n , since the kidney cholesterol de novo synthesis was suppressed i n these two groups compared to the CONT group. There was a concommitant increase i n the l i p i d content of kidney tissue and the mesangial expansion (MA/GTA) at day 30. No si g n i f i c a n t increase i n the intraglomerular monocyte/macrophage was found at day 30 i n the HCHP group compared to the HC group. However, a s i g n i f i c a n t correlation (r=0.678, p 0.001) was found between the number of these c e l l s and MA/GTA r a t i o among the four experimental groups at day 70. These data indicate that l i p i d deposits i n kidney tissue may induce a glomerulosclerotic lesion i n the absence of monocytes. However, these c e l l s l i k e l y augment the p r o l i f e r a t i o n of mesangial c e l l s . We postulate that high protein diet could worsen the lipid-induced glomerular lesion by increasing delivery of abnormal lipoproteins to the kidney which could trigger mesangial c e l l u l a r p r o l i f e r a t i o n d i r e c t l y and i n d i r e c t l y by a macrophage-mediated process. - i v -Table of Contents Page Abstract i i Table of Contents i v L i s t of Tables i x L i s t of Figures x i Dedication xiv Acknowledgement xv Glossary of abbreviations x v i Introduction I. Preface 1 I I . Plasmas L i p i d i n Renal Disease 3 I I I . Dietary Protein and Renal Disease 5 IV. F a m i l i a l L e c i t h i n : Cholesterol Acyltransferase (LCAT) Deficiency 7 V. Cholesterol-fed Guinea Pig 16 VI. Objectives of This Study 22 Materials and Methods I. Animals, Diets and Materials 23 I I . Experimental Design 26 A. Experiment - I 26 B. Experiment - I I 28 C. Experiment - I I I 30 D. Experiment - IV 33 - V -I I I Methods 35 A. Blood and Urine Collections 35 B. Erythrocyte L i p i d Extraction 35 C. Plasma 35 ( i ) Enzyme assay and l i p i d p r o f i l e : 35 1 - LCAT a c t i v i t y using endogenous substrate 35 2 - LCAT a c t i v i t y using exogenous substrate 37 3 - Total and free cholesterol 38 4 - Phospholipids 38 5 - HDL-Cholesterol 39 6 - Triglycerides 39 ( i i ) Lipoprotein Studies: 39 1 - Lipoprotein i s o l a t i o n 39 2 - Electrophoresis 40 3 - SDS - polyacrylamide gel electrophoresis 40 D. Erythrocyte analyses 41 1 - Complete blood count 41 2 - Erythrocyte Osmotic f r a g i l i t y 41 3 - Erythrocyte Morphology 41 4 - l i p i d analyses 42 E. Kidney: 43 (i ) Renal Function Tests 43 1 - Serum creatinine 43 2 - Blood Urea Nitrogen (BUN) 43 3 - Creatinine clearance 43 4 - Twenty-four hour urinary protein 43 5 - Routine u r i n a l y s i s 44 ( i i ) Renal Histology . 44 1 - Par a f f i n Sections 44 a - Haematoxylin and eosin s t a i n 44 b - Perls Prussian blue stain 44 c - PAS and PASM stains 44 d - Von Kossa stain 44 - v i -2 - Frozen Sections 44 a - O i l red-0 s t a i n 44 b - Immunohistofluorescent 44 c - NSE 45 3 - Electron Microscopy 45 4 - Morphometric Study 45 5 - L i p i d Analysis - Extraction and Measurement 45 a - Tissue free and e s t e r i f i e d cholesterol 46 b - Phospholipids 46 c - Triglycerides 47 6 - Tissue Protein Analysis 47 ( i i i ) E s t i m a t i o n of de novo cholesterol synthesis i n renal tissue 47 1 - Hydroxy methyl g l u t a r y l CoA reductase a c t i v i t y 47 2 - T r i t i a t e d water incorporation into cholesterol 48 F. Liver Function Tests: 49 1 - Serum Total Protein and Albumin 49 2 - Serum BUN (Blood urea nitrogen) 49 3 - Serum aspartate transaminase (SGOT or AST) 49 4 - Serum B i l i r u b i n 50 IV S t a t i s t i c a l Analyses 50 Results I. General Observations 51 I I . Plasma Lipids and Lipoprotein Changes 58 I I I . Plasma LCAT A c t i v i t y 71 IV. Haematological Changes: 76 1. Complete blood count 76 2. Erythrocyte morphology and f r a g i l i t y 76 - v i i -3. Erythrocyte l i p i d p r o f i l e 80 4. Characterization of the vacuolated white blood c e l l s ... 83 V. Changes i n Liver Function 88 VI. Renal Function 91 1. Urinary Findings 91 2. Serum Findings 94 VII Renal Tissue Alterations: 94 1. Histopathology, histochemistry, Histoimmunofluorescence, and electron microscopy 94 2. Morphometry 107 3. Kidney l i p i d analysis 118 VIII Renal de novo Cholesterol Synthesis 122 Discussion I. General Observations 124 I I . Plasma L i p i d Abnormalities 127 1. Effect of the cholesterol-rich diet 127 2. Effect of the cholesterol/protein-rich diet 132 I I I . Haematological Changes 134 1. Effect of the cholesterol-rich diet 134 2. Effect of the cholestrol/protein-rich diet 141 IV. Hepatic Changes 144 V. Renal Structural and Functional Alterations 149 1. Effect of the cholesterol-rich diet 149 a- H i s t o l o g i c a l changes b-,Renal functional 2. Effects of cholesterol/protein-rich diet 152 a- H i s t o l o g i c a l changes b- Renal functional alterations 3. Kidney l i p i d content: o r i g i n and c e l l u l a r interaction .. 154 - v i i i -VI. General Conclusions and Postulated Mechanisms of Anaemia and Glomerulosclerosis 164 VII. Further Work 166 References 167 G - i x -L i s t of Tables Table Introduction 1. Patients with f a m i l i a l LCAT deficiency 12 2. Frequency of c l i n i c a l findings i n two groups of patients with f a m i l i a l LCAT deficiency 14 3. Methodology of LCAT a c t i v i t y assessment 15 4. S i m i l a r i t i e s i n plasma lipoproteins between f a m i l i a l LCAT deficiency and the cholesterol-fed guinea pig 21 Materials and Methods 5. Composition of the experimental diets 24 Results 6. Weight gain 52 7. Organ weight 57 8. Plasma l i p i d p r o f i l e (experiment I) 61 9. Plasma l i p i d p r o f i l e 64 10. LCAT a c t i v i t y using endogenous substrate 75 11. Complete blood count and erythrocyte osmotic f r a g i l i t y 77 12. Complete blood count (APH-induced anaemia experiment) 86 13. L i p i d p r o f i l e of erythrocyte 87 14. Liver function tests at different periods 89 - X -15. Kidney function tests at day 70 93 16. Blood urea nitrogen and serum creatinine 95 17. Kidney morphometry at different periods 117 18. Kidney l i p i d p r o f i l e and kidney morphometry (APH-induced anaemia experiment) 119 19. Kidney l i p i d p r o f i l e at day 70 120 20. L i p i d p r o f i l e of renal cortex 121 21. De novo cholesterol synthesis i n renal tissue 123 - x i -L i s t of Figures Figures Page Introduction 1. P r i n c i p a l l i p i d reactants i n the LCAT reaction 8 Materials and Methods 2. Flow chart of the design of experiment I 27 3. Flow chart of the design of experiment I I 29 4. Flow chart of the design of experiment I I I 31 5. Flow chart of the experimental design of the assessment of cholesterol de novo synthesis 32 6. Flow chart of the design of experiment IV 34 Results 7. Weight gain 53 8. A severely enlarged spleen from the HCHP group compared to a normal-sized spleen from the CONT group. Affected l i v e r also compared to normal one 56 9. Plasma cholesterol 59 10. Plasma t o t a l cholesterol at different periods 60 11. Plasma free cholesterol 62 12. An electrophoretogram on agarose gel of the CONT, HC, and HCHP groups at day 30 .c 66 - x i i -13. An electrophoretic p r o f i l e on agarose gel of the HC, CONT, and HCHP groups at day 70 68 14. Electrophoretic p r o f i l e of the CONT, HCHP, and HC groups at day 10 on an agarose gel 70 15. Two lipoprotein fractions separated by preparative u l t r a -centrifugation and resolved i n 30% SDS-polyacrylamide 73 16. LCAT a c t i v i t y (MER) - Exogenous substrate 74 17. A peripheral blood smear from an animal kept on a cholesterol-rich diet at day 30 79 18. A peripheral blood smear from a cholesterol-fed guinea pig revealing a target c e l l , few echinocytes, and stomato cyte 79 19. A peripheral blood smear from the HCHP group at day 70 82 20. A peripheral blood smear from the HCHP group at day 70 showing a vacuolated monocyte 82 21. A peripheral blood smear from a cholesterol-fed animal at day 70 depicting two monocytes with oil-Red 0 positive droplets 85 22. A peripheral blood smear from a cholesterol-fed animal at day 70, showing vacuolated NSE-positive c e l l s 85 23. Twenty-four hour urinary protein 92 24. A kidney section from the CONT group at day 70 98 25. A kidney section from the HCHP group at day 70 98 26. A kidney section from the HCHP group at day 70, showing megakaryocytes 100 - x i i i -27. A kidney section from a cholesterol-fed guinea pig at day 70 -Perl's Prussian blue st a i n 100 28. A frozen kidney section from a cholesterol-fed animal at day 70 - 0R0 sta i n 102 29. A frozen kidney section from the HCHP group at day 70 depicts renal tubules with ORO-positive materials 102 30. A glomerulus with NSE-positive c e l l s i n kidney of a cholesterol-fed guinea pig at day 70 104 31. A kidney section from the HCHP group at day 70 stained with PAS sta i n 106 32. Ul t r a t h i n section of a glomerulus of a control animal at day 70 .. 109 33. A glomerular endothelial c e l l with multi-sized l i p i d droplets from a cholesterol-fed animal at day 70 109 34. A glomerulus from a cholesterol-fed animal at day 70 -vacuolated i n t r a c a p i l l a r y monocytes I l l 35. Ul t r a t h i n section from kidney of the HC group at day 70 reveals the expanded mesangium 113 36. An electron micrograph reveals a vacuolated c e l l with some of the c h a r a c t e r i s t i c s of monocyte i n expanded glomerular mesangium i n a HCHP animal at day 70 115 37. Erythrophagocytosis i n the mesangium of an animal i n the HCHP group at day 70 115 - xiv -This thesis i s dedicated To The Glorious IRAQ To The Strong People AFAF KUTAIBA OSAMA - X V -ACKNOWLEDGEMENT My sincere thanks to my supervisor Dr. J. Frohlich for his invaluable guidance and kind support. Also, I would l i k e to thank the members of my supervisory committee, Drs. A. Magil, H. Pritchard, and D. Godin for t h e i r wise suggestions and for allowing me to use the f a c i l i t i e s of t h e i r laboratories. My thanks extend to Mrs. Jasbir G i l l , and Mrs. Caron Fournier for th e i r excellent technical help i n conducting the h i s t o l o g i c a l processing. Also, my thanks to Mrs. L i v i a Srubicki for her assistance i n performing the HMGCOA reductase assay, and to the d i v i s i o n of c l i n i c a l chemistry, Shaughnessy Hospital, for conducting some of the serum analyses. Special greetings and love to my wife Afaf and sons Kutaiba and Osama for tolerating the tough circumstances we have been through. My thanks to my family back i n my beloved country - Iraq, for t h e i r patience and encouragement. My thanks to Anne Bishop, Pat Bernoe, Ruth Hunter, Judith Arnott, Penny Mah and Susan Mah for the excellent typing of t h i s thesis. Special thanks and acknowledgement to the I r a q i government for supporting my scholarship. Also my gratitude i s extended to the Kidney Foundation of Canada for t h e i r f i n a n c i a l support of t h i s project. I t i s the e f f o r t of a l l thanked above and other friends that made t h i s thesis possible. - x v i -Glossary of Abbreviations LCAT Lecithin:cholesterol acyltransferase CONT Control HC High cholesterol HCHP High cholesterol/high protein APH Acetylphenylhydrazine TC Total cholesterol FC Free cholesterol CE Cholesteryl ester TPL Total phospholipids TG Triglycerides HDL-C Cholesterol of high density lipoprotein VLDL Very low density lipoprotein LDL Low density lipoprotein EM Erythrocyte membrane HCC Hypochromic c e l l FER Fractional e s t e r i f i c a t i o n rate MER Molar e s t e r i f i c a t i o n rate Ccr Creatinine clearance BUN Blood urea nitrogen Cr Serum creatinine HMG-COA reductase Hydroxymethylgluteryl coenzyme A reductase LLM Lipid-laden monocyte MA/GTA Mesangial area/glomerular t u f t area r a t i o NSE-positive c e l l s Non-specific esterase positive c e l l s - xviA-INTRODUCTION - 1 -I Preface: This investigation was i n i t i a l l y designed to test a hypothesis that a high protein dietary supplement i s necessary to augment the renal lesion and to induce deterioration of renal function i n patients with f a m i l i a l l e c i t h i n : cholesterol acyltransferase (LCAT) deficiency. This hypothesis formulated on the basis of c l i n i c a l observations i n such patients (1). Typical microscopic findings i n t h i s disorder include accumulation of l i p i d p a r t i c l e s i n different parts of the glomeruli and fusion of foot processes. However, sim i l a r findings were observed i n a biopsy of one patient without proteinuria (3). Flatmark et a l . (1977) reported the same renal lesion i n a normal kidney several months after i t s transplantation into a patient with f a m i l i a l LCAT deficiency, though the renal function was normal (2). Frohlich and Mcleod (1) pointed out that LCAT-deficient patients on high protein diets, e.g. the Scandinavian patients, had a much higher frequency (90%) of renal complications than the Japanese patients on lower protein diets (43%). In addition, the Scandinavians developed uraemia and 60% of them ultimately died of the disease, whereas none of the Japanese were uraemic. (1) A higher r a t i o of BUN to serum creatinine was demonstrated i n those who died of t h e i r kidney disease than the survivors (1). Regarding the fact that urea clearances were i n i t i a l l y normal i n the f i r s t group, the high BUN l e v e l was largely determined by the dietary protein intake. These data suggested that the patients with serious renal involvement were on substantially higher protein diets than the others. The cholesterol-fed guinea pig was chosen as an experimental model, since i t has similar lipoprotein abnormalities, haematological alterations and renal lesions to those described i n LCAT-deficient patients (see - 2 -"Introduction" - Section V). However, although lipoprotein abnormalities and haemolytic anaemia were documented i n t h i s model, no marked reduction i n LCAT a c t i v i t y was demonstrated i n our study. Thus a new set of hypotheses arose: 1- Plasma l i p i d deposition i n glomeruli results i n s i g n i f i c a n t s t r u c t u r a l change. Intraglomerular monocyte i n f i l t r a t i o n may contribute to the str u c t u r a l change. 2- High protein intake augments these changes and may play an important role i n the development of functional impairment. A study that tests the above hypotheses could help us understand the mechanism of renal manifestations found i n some dyslipidaemias, among them LCAT deficiency. Perhaps, more importantly, i t may explain the role of plasma l i p i d s i n the pathogenesis of glomerulosclerosis. In t h i s chapter, the l i t e r a t u r e on renal lesions i n hyperlipidaemias and effect of high protein diet on renal disease w i l l be reviewed. In addition, the cholesterol-fed guinea pig model and i t s relationship to human f a m i l i a l LCAT deficiency w i l l be considered. F i n a l l y , the objectives of t h i s study w i l l be stated. - 3 -I I Plasma Lipids i n Renal Disease The role of l i p i d deposits i n renal glomeruli i n the pathogenesis of renal disease i s unclear. Most studies of the abnormalities (and th e i r possible effects on renal function) have been confined to patients with the nephrotic syndrome. However, hyperlipidaemia i n the nephrotic syndrome i s a secondary one (4) which might be attributed to a renal le s i o n . In t h i s syndrome there i s an increase i n levels of plasma t o t a l cholesterol, t r i g l y c e r i d e s , and phospholipids (5-7) and variable patterns of lipoproteins (6,7). I t has been speculated that the loss of the activator of lipoprotein lipase might lead to persistence of hyperlipaemia due to the disturbance of t r i g l y c e r i d e - r i c h lipoprotein metabolism (8). Al t e r n a t i v e l y , hyperlipidaemia might be due to increased synthesis of lipoproteins by the l i v e r (9). Despite that hyperlipidaemia i s secondary, Moorhead et a l . (1982) suggested a potential role for plasma l i p i d s i n chronic renal disease (10). They postulated that the excessively f i l t e r e d lipoproteins may accumulate i n the mesangium and lead to p r o l i f e r a t i o n of mesangial c e l l s and deposition of matrix tissue. In addition to nephrotic syndrome, glomerular l i p i d deposits have been reported i n Fabry's disease (11), l e c i t h i n : cholesterol ac y l -transferase (LCAT) deficiency (12-15), cholestatic l i v e r disease (16), and arteriohepatic dysplasia ( A l a g i l l e ' s syndrome) (17). The syndrome of p a r t i a l lipo-dystrophy i n humans has also been described with glomerular changes and renal f a i l u r e (9). Edward (1981) noted a s i g n i f i c a n t inverse correlation between fasting serum t r i g l y c e r i d e s and mean glomerular f i l t r a t i o n rate i n patients with analgesic nephropathy (18). Diabetic glomerulosclerosis has also been attributed to fat emboli i n the glomerular c a p i l l a r i e s (19). - 4 -Experimental evidence suggests that l i p i d may have an important role i n the development of some glomerular lesions. Long-term treatment of rats with aminonucleoside and adriamycin resulted i n l i p i d deposition i n the mesangium followed by the development of focal and segmental hyalinosis and scl e r o s i s (20). A si m i l a r finding was reported e a r l i e r by S i l v a et a l . (1979): amino nucleoside-induced focal glomerulosclerosis was enhanced by a l i p i d - r i c h diet while lowering of serum l i p i d s by halofenate protected rats against t h i s effect (21). Kelley and I z u i have demonstrated that an enriched l i p i d diet (51.7% fat) resulted i n increased l i p i d accumulation i n glomeruli and accelerated glomerular injury i n NZBXW mice with lupus nephritis (22). Guinea pigs kept on a 1% cholesterol r i c h diet have developed glomerulosclerosis (23) (see "Introduction" - Section V). Furthermore, i t has been reported that at physiological pH and ionic strength, very low density lipoprotein (VLDL) and low density lipoprotein (LDL) can bind with polyanionic glycosaminoglycans (24). This binding may al t e r the permeability of the glomerular barrier by changing the net charge state of the membrane which normally governs the permeation of the p a r t i c l e s with a surface charge (24). In concert with t h i s report, an increase i n basement membrane l i p i d has been noted (25). In short, a growing body of evidence has been presented which strongly suggests a role for hyperlipidaemia i n the development of renal lesions i n humans and i n experimental animals. Hence, we t r i e d to study the patho-genesis of glomerulosclerosis accompanying hyperlipidaemia, using the cholesterol-fed guinea pig model. - 5 -I I I Dietary Protein and Renal Disease: Examination of the effect of high protein supplementation on l i p i d -induced glomerular lesion i s one of the objectives of t h i s study. This section reviews some of the l i t e r a t u r e dealing with the effect of protein-r i c h diet on renal disease. Renal structural abnormalities i n the experimental animals fed high protein diet were reported early i n t h i s century (26-28). Klahr et a l . (1983) suggested high dietary protein as a r i s k factor i n the progression of chronic renal disease (29). These authors reviewed the evidence of deleterious effect of dietary protein on the course of experimental nephritis. A l l p a r t i a l l y nephrectomized rats (which developed uraemia) were kept on 51% protein-supplemented diet died within 11 days after the surgery. On the other hand, another group of rats subjected to the same surgery but kept on a low-protein dietary regimen l i v e d for 30 days after renal ablation (30). Both renal blood flow and glomerular f i l t r a t i o n rate were increased i n animals kept on the protein r i c h diet (31). Brenner (1983) postulated that the syndrome of progressive azotaemia, proteinuria and glomerulosclerosis i s related to high protein intake (32). He suggested that t h i s dietary supplement to experimental animals with p a r t i a l nephrectomy leads to sustained elevation i n glomerular c a p i l l a r y pressure and flow and ultimately to renal f a i l u r e . In ra t s , renal mass was found to be increased after long-term feeding of protein (33). Conversely, feeding a low protein diet to rats with remnant kidneys has largely prevented the s t r i k i n g increase i n glomerular plasma flow and c a p i l l a r y pressures. Furthermore, the accompanying proteinuria and str u c t u r a l alterations were found to be less - 6 -severe (29, 33). Pennell et a l . (1975) found decreased glomerular f i l t r a t i o n rate (GFR) values i n weaning rats fed a low protein d i e t . Such protein r e s t r i c t i o n also reduced the kidney mass (34). P r o t e i n - r e s t r i c t i o n i n rats accounted for a f a l l i n glomerular c a p i l l a r y plasma flow rate and a reduction i n glomerular cross sectional area (35). The increase i n GFR i n a remnant kidney was associated with remarkable changes i n glomerular structure. There were e p i t h e l i a l c e l l adhesions to Bowman's capsule, detachment of some of the e p i t h e l i a l c e l l s from the underlying basement membrane, and a prominant increase i n mesangial c e l l s and matrix (33). Others described an increase i n glomerular mass, vacuolization of glomerular e p i t h e l i a l c e l l s and effacement of foot processes (36). In humans, r e s t r i c t i o n i n dietary protein may have a sim i l a r effect on renal function to that shown i n experimental animals. Pullman et a l . reported a decrease i n GFR and renal plasma flow i n low-protein diet-consuming healthy individuals (37). Normal subjects also reduced t h e i r GFR on feeding a c a l o r i c - d e f i c i e n t diet (38). Klahr and Alleyne (1981) found decreased values for GFR and renal plasma flow i n ten adults with protein malnutrition; both values returned to normal l e v e l after protein repletion (39). A l l these findings i n both experimental animals and humans support the notion that high dietary protein has a deleterious effect on kidney disease. - 7 -IV F a m i l i a l Lecithin: Cholesterol Acyltransferase Deficiency: The data obtained i n the present study which contribute to an under-standing of lipid-induced glomerulosclerosis are pertinent to the glomerular lesion described i n f a m i l i a l LCAT deficiency. The following review provides a background for t h i s disease and highlights i t s major features; besides, i t f a c i l i t a t e s introducing a comparison between f a m i l i a l LCAT deficiency and the cholesterol-fed guinea pig i n the next section. Lecithin: Cholesterol acyltransferase (EC 2.3.1.A3) catalyzes i n plasma the transfer of a fatt y acyl group from l e c i t h i n to cholesterol (40) (Figure 1). The enzyme i s synthesized and secreted by the l i v e r (41, 42) and activated mainly by apoprotein Al (43) and to a lesser degree by apoproteins CI (44), A IV, E^, and E^ (45). The major portion of LCAT cir c u l a t e s i n plasma with HDL components (46) p a r t i c u l a r l y with the smaller subfraction of HDL (47). F a m i l i a l LCAT deficiency was f i r s t described i n 1968 i n 3 s i s t e r s of a Norwegian family (48). Since then 43 cases have been detected including s i x patients currently being investigated (3) (Table 1). The disease has an autosomal recessive mode of inheritance (12). The LCAT gene i s located between the middle and terminal points of the long arm of chromosome 16 (12). Mclean et a l . (1986) isolat e d the human LCAT cDNA clones (49). The t y p i c a l features of the c l i n i c a l presentation of f a m i l i a l LCAT deficiency are corneal opacities, haemolytic anaemia and various degrees of renal involvement ranging from an increase i n albumin and B 2 microglobulin excretion to uraemia (3) (Table 2). 8 -Fig - 1 C H 2 O - S a t u r a t e d fatty acid CHO — Unsaturated fatty acid l ,p C H 2 0 — P — choline OH Lecithin Cholesterol Lecithin: Cholesterol Acyltransferase i C H 2 O - S a t u r a t e d fatty acid C H O H O II + C H 2 0 - P - choline i OH Lysoleci th in Unsat. fatty acid Cholestery lester Princ ipal lipid reactants in the L C A T reaction - 9 -A l l LCAT deficient patients have a marked decrease i n LCAT a c t i v i t y (Table 3 shows the methodology of LCAT a c t i v i t y assessment), r e l a t i v e l y high concentration of plasma unesterified cholesterol (UC) and phosphatidyl choline (PC), and a l l have low concentrations of cholesteryl ester (CE) and l y s o l e c i t h i n (50). The plasma t r i g l y c e r i d e s (TG) are also increased i n some patients (50). A l l plasma lipoprotein classes i n LCAT deficient patients are abnormal. Upon f i l t r a t i o n through 2% agarose gel, different subtractions of VLDL have been recovered: Large p a r t i c l e s excluded from the gel with more l i p i d r e l a t i v e to protein content and more UC and "PC r e l a t i v e to TG (51); smaller VLDL p a r t i c l e had unchanged r a t i o of CE to TG or UC. Upon electrophoresis of plasma on agarose gel, VLDL demonstrates slow pre-beta migration (51). Low-density lipoprotein p a r t i c l e s of different size and l i p i d composition are also recovered from plasma of LCAT deficient patients (50, 52). Generally, abnormal LDL subfractions have large amounts of FC and PC compared to protein, however, more protein and less l i p i d s are found i n the intermediate-sized subfraction (50). Although the HDL protein i n LCAT deficient patients has been reported to be about one-third normal, the t o t a l concentration of HDL UC was i n the normal range due to the high proportion of UC i n the HDL subfractions (53). Upon f i l t r a t i o n through Sephadex G200, the HDL of LCAT deficient patient has been subfractionated i n t o : large, normal and small-sized components (50). The large HDL subfraction forms stacked discs (54) and contains apoprotein E (55). In short, the plasma l i p i d and lipoprotein abnormalities might d i r e c t l y or i n d i r e c t l y be implicated i n the development of renal disease i n f a m i l i a l LCAT deficiency syndrome. Employing an experimental animal model - 10 -which has a substantial number of these abnormalities could help i n v e s t i -gating the pathogenesis of renal disease i n hyperlipidemic patients. M *• LCAT - deficient patients have moderate t a r g e t - c e l l haemolytic anaemia with reduced compensatory erythropoiesis (56), and normochromic erythrocytes. Several investigators (57-59) have reported an increase i n erythrocyte free cholesterol, PC and a decrease i n phosphatidylethanolamine. Moreover, the erythrocyte l i p i d abnormalities can be reverted when patients' erythrocytes are incubated i n normal plasma (12). Different features of these abnormalities have been reported i n cholesterol fed guinea pigs (see next section). Those patients have sea-blue histocytes i n spleen and bone marrow (60). I t has been found that the lamellar-arranged membranes detected inside these c e l l s consisted of PC and UC (13, 61) which might be due to phagocytosis of abnormal lipoproteins by reticuloendothelial c e l l s (62). However, no foam c a l l s have been reported i n the blood c i r c u l a t i o n of LCAT deficient patients. On the other hand, foam c e l l s have been described i n the renal lesion (12). Thus, the interrelationships between plasma l i p i d a l terations, the li p i d - l a d e n phagocytes and renal lesion are considered, too, i n t h i s study. Also, the possible role of cholesterol-rich erythrocytes i n the development of a renal le s i o n was investigated i n our study. Renal disease i s the most serious outcome of f a m i l i a l LCAT deficiency syndrome. Moderate proteinuria has been reported early i n l i f e i n most patients (12, 62). Beside protein, urine of those patients contains erythrocytes and hyaline casts; albumin, alpha-1, and alpha-2 globulins are detected on urine electrophoresis (12, 62). Nevertheless, renal function i n LCAT-deficient patients indicated by serum creatinine and urea concentration i n addition to the creatinine, i n u l i n , and PAH clearance i s normal before - l i -the end-stage disease (62). Grossly, the kidneys of LCAT deficient patients are pale and s l i g h t l y enlarged while microscopically, the glomeruli show expanded mesangial areas and Bowman's capsule i s thickened (12). The glomerular t u f t c a p i l l a r i e s usually have thickened walls and lumens containing amorphous materials (12, 62). Ul t r a s t r u c t u r a l studies of the lumen material have shown that the c a p i l l a r y lumens were partly f i l l e d with a membraneous meshwork (13, 14). Frequent loss of c a p i l l a r y endothelial c e l l s and fusion of e p i t h e l i a l foot processes are frequently noted (12). Some of these findings have been described e a r l i e r i n cholesterol-fed guinea pigs (see next section). In addition, the subendothelial l i p i d deposits and the presence of foam c e l l s i n the tunica media of renal a r t e r i o l e s of LCAT deficient patients (12) have been reported i n cholesterol-fed guinea pigs (23). Ah' additional feature of the renal manifestations i n f a m i l i a l LCAT deficiency i s the increase i n the l i p i d content i n kidney tissue (63). Implication of large LDL as causally related to renal injury i n f a m i l i a l LCAT deficiency i s s t i l l equivocal (64, 1). However, the presence of plasma l i p i d and lipoprotein abnormalities besides the l i p i d deposition i n kidney tissue may implicate a causal relationship with renal l e s i o n . The mechanism of such a r e l a t i o n could be studied i n an animal model which exhibits a simil a r plasma l i p i d and kidney s t r u c t u r a l a l t e r a t i o n . Hence, we used cholesterol-fed guinea pigs for t h i s purpose (see next section). - 12 -Table 1: Patients with F a m i l i a l LCAT Deficiency RENAL FAMILY PATIENT SEX DOB ETHNIC ANAEMIA INVOLVEMENT ORIGIN I 1 F 1936 Norwegian + + 2 F 1934 Norwegian + + 3 F 1946 Norwegian + + I I 4 F 1921 Swedish + + 5 M 1935 Swedish + + I I I 6 F 1926 Norwegian + + 7 M 1932 Norwegian + + IV 8 M 1918 Norwegian + + 9 F 1914 Norwegian + — V 10 M 1942 I t a l i a n + + 11 M 1944 I t a l i a n + + VI 12 M 1955 Indian - -VII 13 M 1945 Eng-Cdn + 14 M 1940 Eng-Cdn + + VIII 15 F 1934 French + + 16 F 1937 French + IX 17 M 1959 Ital-Dutch + + 18 F 1954 Ital-Dutch + -X 19 F 1946 Japanese + MIN 20 M 1948 Japanese + + 21 M 1950 Japanese + + XI 22 M 1950 Eng-Irish + + 23 F 1955 Eng-Irish + — - 13 -Table 1 Cont'd RENAL FAMILY PATIENT SEX DOB ETHNIC ANAEMIA INVOLVEMENT ORIGIN XII 24 F 1932 I r i s h + + 25 F 1925 I r i s h — — 26 M 1932 I r i s h + + 27 F 1931 I r i s h + — XIII 28 F 1954 Norwegian + -XIV 29 F 1949 Germ-Irish + MIN 30 M 1958 Germ-Irish - + 31 M 1955 Germ-Irish — + XV 32 M 1933 English MIN* MIN XVI .33 M 1965 I t a l i a n - + XVII 34 F 1937 Japanese + 35 M 1933 Japanese + — XVIII 36 M 1934 Japanese + + 37 F 1924 Japanese + -* Min - Minimum - 14 -Table 2: Frequency of C l i n i c a l Findings i n Two Groups of Patients with F a m i l i a l LCAT Deficiency Scandinavian A l l Other Number (10 patients) (27 patients) Affected Corneal opacities 100% 100% 37 Anaemia 100% 82% 32 Proteinuria 80% 74% 22 Uraemia 70% 14% 10 Death 60% 4% 7 - 15 -Table 3 Methodology of LCAT A c t i v i t y Assessment Method Substrate for LCAT Reaction" Remarks I. Glomset and Wright Pooled human plasma (heat denatured substrate) I I . Stokke and Norum Patient's own plasma (endogenous substrate) I I I . Enzyme Immunoassay — IV. Common-Substrate Method Apo Al-containing liposome (exogenous substrate) -Requires long incubation time -Low r e p r o d u c i b i l i t y -Time consuming -Influenced by the quality of substrate -Quantitative measure-ment of the enzyme mass -Low s e n s i t i v i t y may be due to complete Ag-Ab reaction (antigenic determinants of the enzyme are obscured by HDL/LCAT complex) -Preferred method -Overcomes the l i m i t a -tions imposed by the v a r i a b i l i t y of the endogenous substrate -Rapid, reproducible, and sensitive -Correlates well with enzyme mass measurement - 16 -V-Cholesterol-fed Guinea Pig:  H i s t o r i c a l Background The investigation of the b i o l o g i c a l effect of a cholesterol-rich diet on guinea pigs goes back to 1939 when Okey and Greaves (64) studied the metabolic a c t i v i t i e s of cholesterol. They chose guinea pig over rat because the f i r s t , l i k e human, has a gallbladder and requires ascorbic acid i n i t s diet. In that study they reported a severe anaemia with splenic enlargement and fatty l i v e r i n guinea pigs fed a 1% cholesterol-rich diet. Severe anaemia appeared after f i v e weeks of the experiment and the enormous splenomegaly was evident after 7-9 weeks. In 1944 Okey demonstrated that haemolytic anaemia and splenic hyperplasia were correlated with the excess e s t e r i f i e d cholesterol i n l i v e r and free cholesterol i n plasma (65). Also, they showed that phagocytic c e l l s play a role i n removing cholesteryl ester from the l i v e r . Most of these data were l a t e r confirmed and studied i n d e t a i l by Ostwald and Shanon (1964), as discussed below (66). In 1967 French et a l . reported a glomerulosclerotic lesion i n cholesterol-fed guinea pigs (23). ( i ) Plasma Lipids and Lipoprotein Abnormalities: Guinea pigs kept on a 1% cholesterol diet for 10-14 weeks have an increased l e v e l of both UC and CE i n plasma (66, 67); UC was increased 11-fold while EC showed' a 2.5-fold increase. Total phospholipid and phosphatidylcholine (PC) concentrations i n plasma were also elevated; the plasma CE had a decreased proportion of l i n o l e i c acid (66). The most s t r i k i n g abnormalities i n cholesterol-fed guinea pig which resemble those i n f a m i l i a l LCAT deficiency have been seen i n the plasma lipoproteins (Table 4) (68, 69). On agarose gel electrophoresis, B-or slow - 17 -pre-B VLDL was demonstrated. Compared to the protein content, VLDL of guinea pigs fed a 1% cholesterol diet has increased UC (50). The concen-t r a t i o n of UC and phospholipid was also increased i n the LDL of the same animals. The subfractions of LDL which have been described i n LCAT deficient patients after f i l t r a t i o n through 2% agarose gel have the same charac t e r i s t i c s as those i n the cholesterol-fed guinea pig. Glomset and Norum (1973) pointed out that besides the s i m i l a r i t y i n size d i s t r i b u t i o n of the LDL subfractions to those i n patients of f a m i l i a l LCAT deficiency, the large and intermediate LDL p a r t i c l e s unlike the smaller ones were r i c h i n UC and phospholipid (50). Sardet et a l . (1972) demonstrated LDL heterogeneity on electron microscopy (69). Large and intermediate molecular weight subfractions appeared as f l a t discs of 80-100 nm i n diameter while the small subfraction was indistinguishable from LDL of the control animals. The HDL of cholesterol-fed guinea pigs has been shown to be r i c h i n UC and phospholipid and large enough to be excluded from Sephadex -G200 (68). They formed stacked discs upon electron microscopy (69). F i n a l l y , plasma of cholesterol-fed guinea pigs contains the abnormal lipoprotein LP-X. This p a r t i c l e has the composition of the abnormal intermediate-sized LDL reportedly si m i l a r to the LP-X described by Seidel et a l . (1969) (70). Upon electron microscopy they are disc-shaped with a major axis of 40-60 nm (71, 72). ( i i ) Haematologic Abnormalities: Haemolytic anaemia has been found consistently after 7-10 weeks i n guinea pigs fed 1% cholesterol-rich diet (66, 73, 74). The h a l f - l i f e of erythrocyte i s decreased (66) and the f a l l i n the erythrocyte count i s accompanied by an increase i n the erythrocyte mean corpuscular volume (MCV). - 18 -Sardet et a l . (1972)demonstrated a 10-35% increase i n reticulocytes i n anaemic cholesterol-fed guinea pigs (75). Numerous studies have shown that cholesterol content of erythrocyte i s increased i n t h i s animal model (67, 68, 73, 75). In an i n v i t r o study, cholesterol-loaded RBC from cholesterol-fed guinea pig l o s t i t s excess cholesterol when incubated with normal guinea pig's plasma; i n the same way, unesterified cholesterol mostly transferred from a hypercholesterolaemic plasma to normal RBC (75) (also, see "Discussion"). Erythrocyte cholesterol has been shown to increase within 1-3 days after supplementation of the guinea pig diet with 1% cholesterol, simultaneously with the increase i n plasma cholesterol content (76). Sardet et a l . (1972) suggested that UC transfers from the abnormal HDL to erythrocyte membrane i n guinea pigs fed a 1% cholesterol-rich diet (69). They found that worsening of haemolytic anaemia was concomittant with the appearance of new species of HDL and abnormal LDL p a r t i c l e s . Cholesterol-r i c h erythrocytes could be a possible contributor to the lipid-induced glomerulosclerosis; hence, we investigated t h i s p o s s i b i l i t y i n a separate experiment. In conclusion, cholesterol-fed guinea pig has s t r i k i n g s i m i l a r i t i e s i n plasma l i p i d and lipoprotein abnormalities to those i n the f a m i l i a l LCAT deficiency syndrome. On the other hand, although t h i s animal model presents haemolytic anaemia after feeding cholesterol-rich diet, i t has some differences i n the haematological findings: f i r s t , haemolytic anaemia i s severe i n the animal model (23, 66, 69); second, the erythrocytes of cholesterol-fed guinea pigs are mainly spur c e l l s (69), while i n LCAT deficient patient they are mainly of target c e l l type (as described i n a previous section). (Also see next section for further conclusions.) - 19 -( i i i ) Renal Findings: The renal findings i n cholesterol-fed guinea pigs are sim i l a r to those i n human LCAT deficiency (see section IV). French et a l . (1967) demonstrated the following i n the kidneys of anaemic guinea pigs on 1% cholesterol-rich diet: (1), a focal or diffuse thickening of the glomerular stalk with an increase i n the number of stalk c e l l and the connective tissue elements; (2) , hyaline material i n glomeruli together with Sudan IV positive droplets; (3) , fat and hyaline i n the media and the subintima of the renal a r t e r i o l e s (23). E p i t h e l i a l c e l l s of the proximal convoluted tubules loaded with haemosiderin were noted i n another study (77). This finding indicates the massive f i l t r a t i o n of haemoglobin due to cholesterol-induced haemolytic anaemia. There are several differences i n the renal lesions between LCAT-deficient patients and cholesterol-fed guinea pigs. Extramedullar haemato- poiesis and erythrophagocytosis were detected i n the affected glomeruli of the guinea pigs (23) but not i n LCAT deficient patients. Mesangial hype r c e l l u l a r i t y was demonstrated i n the animal model, while i n f a m i l i a l LCAT deficiency the glomeruli are normocellular despite the increase i n mesangial matrix (15). As well, i n LCAT deficiency the lumens of the glomerular c a p i l l a r i e s have a mottled structure with a meshwork of membranes and p a r t i c l e s , a finding which has not been described i n cholesterol-fed guinea pigs, which show intraluminal l i p i d - l a d e n macrophages (78). Unlike i n LCAT deficiency, no glomerular basement membrane thickening was demonstrated i n cholesterol-fed guinea pigs. - 20 -Since the cholesterol-fed guinea pig i s intended, i n our study, to be a model for the investigation of the pathogenesis of the renal lesion i n hyperlipidaemic diseases, we review here the features of renal lesions i n two of these diseases. In Fabry's disease (11) the kidney lesion i s characterized by the foamy vacuolation of the c e l l s of the glomerular t u f t , (endothelial, mesangial, and e p i t h e l i a l ) . The foamy c e l l s contain O i l Red. 0-positive material which i s c l e a r l y recognizable by electron microscopy. Furthermore, irreg u l a r thickening of the glomerular basement membrane and subendothelial l i p i d deposition are described, as well. In hepatic disease, Hovig et a l . (1978) reported several cases of hyperlipidaemia with renal involvement (79). Kidney biopsy revealed widening of mesangial region, i r r e g u l a r thickening of basement membranes, and occasional pronounced glomerulosclerotic alterations. Again, l i p i d deposition i n different parts of the glomeruli were detected by electron microscopy. In Fabry's disease, no mesangial p r o l i f e r a t i o n was reported, while i n cases of hepatic disease moderate glomerulo- s c l e r o s i s was present. In the hyperlipidemic diseases described above including f a m i l i a l LCAT deficiency and i n the cholesterol-fed guinea pig model, a consistent r e l a t i o n i s found between hyperlipidaemia, l i p i d deposition i n renal tissue, and the glomerular le s i o n . This observation constitutes the basis for the most current hypothesis to explain the glomerular lesion i n hyperlipidaemic diseases (10). Moreover, since the cholesterol-fed guinea pig has most of the histopathological features of the renal lesion presented i n human hyperlipidaemic disease, i t may serve as a suitable model to study the mechanism of development of the renal pathology reported i n these diseases. - 21 -Table 4 S i m i l a r i t i e s i n plasma lipoproteins between f a m i l i a l LCAT deficiency and cholesterol-fed guinea pig 1. Very low density lipoprotein: a-Increase UC b-Slow pre-beta mobility 2. Low density li p o p r o t e i n : a-Increase UC b-Yield of three subfractions upon 2% agarose gel f i l t r a t i o n c-Large and intermediate molecular weight subfractions of (b) are r i c h i n UC and phospholipids d-Upon electron microscopy, large and intermediate molecular weight subfractions are f l a t discs e-On a n a l y t i c a l centrifugation, presence of fast f l o a t i n g components of S f7 to 12 and S f 12 ft. 3. High density lipoprotein : (large HDL) a-Excluded from sephadex G200 b-Increased UC and phospholipid c-Yield stacked discs upon electron microscopy 4. Presence of LP-X HDL i s v i r t u a l l y absent i n normal guinea pig - 22 -VI Objectives of This Study: This investigation aimed to: A. Study the effect of cholesterol feeding on the kidney i n guinea pigs, and to elucidate the mechanism underlying renal alterations. The role of the following i n the development of renal lesion was studied: 1- L i p i d deposition per se and kidney cholesterol de novo synthesis 2- Haemolysis 3- Liver changes 4- Immune complex mediation 5- Monocyte/macrophage mediation B. Study the role of high protein intake i n augmenting the renal changes. Again, the above-suggested mechanisms were examined. -22A-MATERIALS AND METHODS - 23 -I. Animals, diets and materials Animals and diets Hartley male guinea pigs were supplied by the animal unit at the University of B r i t i s h Columbia; t h e i r i n i t i a l weights ranged from 330 and 370 gm. Normal guinea pig chow based on Reid and Brigg's formula and modified high protein diet (4% corn o i l , 50% protein (casein), 37.5% carbohydrate, 8.5% vitamins and minerals, plus 7 gms. fiber/lOOgm diet) (Table 5) were supplied by ICN ( N u t r i t i o n a l Biochemical, Cleveland, Ohio). To prepare the 2% cholesterol-enriched diet and 2% cholesterol-enriched high protein d i e t , ether-dissolved cholesterol was added to both normal and modified high protein diets. - 24 -Table 5 [ Composition of the experimental diets ] Type of diet Cholesterol gm/lOOgm diet Fat Protein (casein) Carbohydrate (dextrose) Vitamins & minerals Fibres gm/lOOgm diet Normal Guinea Pig Chow - 4% (8) 18.5% (20) 69% (72) 8.5% 7 Cholesterol-Rich Diet 2 4% (8) 18.5% (20) 69% (72) 8.5% 7 Protein-rich Diet - 4% (8) 50% (52) 37.5% (40) 8.5% 7 Cholesterol/ Protein-Rich Diet 2 4% (8) 50% (52) 37.5% (40) 8.5% 7 The numbers between brackets represent the percentage of t o t a l c a l o r i e s . - 25 -Materials 1. For composing an a r t i f i c i a l substrate for the measurement of LCAT a c t i v i t y ; egg yolk phosphatidylcholine was purchased from Sigma (type III-E 5mg/ml i n absolute ethanol). Free cholesterol was Sigma CH-S (lmg/ml i n absolute ethanol). Apoprotein Al was p u r i f i e d by PBE 94 chromatofocussing by Roger Mcleod - Shaughnessy Hospital. Bovine serum albumin (BSA) was Sigma and was e s s e n t i a l l y f a t t y acid free. 2. Reagents and quality control materials used i n the determination of plasma t o t a l and free cholesterol and t r i g l y c e r i d e s were supplied by Abbott and Eastman Kodak Ektachem. 3. Plasma lipoprotein electrophoresis was done on universal electrophoresis f i l m agarose which was supplied by Corning Universal. 4. The radioactive substances used were purchased from New England Nuclear (Boston, Massachusetts, USA). 5. The purity of cholesterol used i n preparing d i f f e r e n t dietary regimens was equivalent to USP s p e c i f i c a t i o n . 6. A l l reagents and quality control materials used i n the analyses of the l i v e r function tests were supplied by (Eastman Kodak EKTA Ghem). 7. A l l other reagents used i n t h i s study were of a n a l y t i c a l grade. 8. Plasma ultracentrifugation for lipoproteins i s o l a t i o n was done on (Beckman L8-70). (Details of preparation for ultracentrifugation i s described i n Section I I I ) . 9. Acetylphenylhydrazine was purchased from BDH ( B r i t i s h Drug House). - 26 -I I Experimental Design A l l the animals were fed normal guinea pig chow for one week as an adaptation period, and kept i n couples per (60 x 45 x 45cm) cage. Throughout the experimental period the animals had free access to food and water. Water was supplemented with ascorbic acid (lgm/L) to meet the animal's daily requirement. Blood and urine samples were obtained and weights were recorded at the beginning of each experiment and at different experimental periods (see de t a i l s about blood and urine c o l l e c t i o n i n Section I I I ) . A. Experiment I This experiment was done to study the effect of cholesterol-rich diet i n inducing glomerulosclerosis i n guinea pigs and to determine the eff e c t of a cholesterol/protein-rich diet i n aggravating t h i s l e s i o n . Twenty-nine male guinea pigs were used. The animals were randomly allocated to four groups: Control group (CONT) was kept on normal guinea pig chow for seventy days; high cholesterol diet (HC) group received 2% cholesterol-rich diet for seventy days; high protein (HP) diet group was kept on 50% casein-enriched diet for seventy days, and high-cholesterol high-protein diet (HCHP) group started on 2% cholesterol-rich diet for 30 days then was shifted to a cholesterol/ protein-rich diet (2% cholesterol/50% casein-rich diet) for another 40 days (Figure 2). - 27-Fig - 2 Flow chart of the experimental design of experiment -1 time course 29 guinea pigs 330 - 370 gm Adaptation period (7 days) CONT 6 • 30 days 70 days kept on normal guinea pig chow HC 7 kept on 2% cholesterol-rich diet HP 6 kept on 50% casein-rich diet HC HP » (animals/ I U group) kept on 2% cholesterol ' rich diet kept on 2% cholesterol/50% casein-rich diet CONT = Control group HC = High cholesterol group HP = High protein group HCHP = High Cholesterol/High Protein Group - 28 -B. Experiment I I This experiment was conducted to examine the effect of haemolytic anaemia i n inducing a glomerulosclerotic lesion i n guinea pigs. Six male guinea pigs were used with a s t a r t i n g weight of 500 gm. They were injected with acetyl phenylhydrazine (APH) (30mg/kg body weight/every other day). The experiment continued for forty days and the res u l t s were compared to those of the control group i n Experiment I. Animals i n t h i s experiment were kept on normal guinea pig chow. Blood samples were obtained at day 0 and day 40, i n addition to random sampling along the experimental periods. Urine samples were collected at day 40 (Figure 3). - 29 -Fig - 3 Flow chart of the experimental design of the experiment - II t ime course 40 days 12 animals 500 gm adaptation period (7 days) group 1 6 kept on normal guinea pig chow and received APH 30mg /Kg /eve ry other day group 2 o (animals/ D group) kept on normal guinea pig chow (from experiment I) APH = Acetylphenyl hydrazine - 30 -C. Experiment I I I This experiment aimed to study the possible pathogenesis of cholesterol-induced glomerulosclerosis and the role of the high-protein supplement i n aggravating such a lesion (see "Objectives" i n "Introduction", too). Twenty-six male guinea pigs were used. They were assigned randomly to the following groups: HCHP group had ten animals; HC group had seven animals; HP group had five animals and CONT group had four animals. A l l the groups ran through a si m i l a r time course and received the same diets described i n Experiment I (Figure 4). In t h i s experiment, sixteen additional animals were used i n the assessment assay of HMG-COA reductase a c t i v i t y . Five animals served as the HC group, f i v e animals as the HCHP group, three animals as the HP group and three animals as the CONT group. To measure cholesterol de novo synthesis by another method, namely, measuring incorporation of t r i t i a t e d water into cholesterol, another ten animals were used i n the following groups: HC group (three animals), HCHP group (three animals), HP group (two animals), and CONT group (two animals) (Figure 5). - -31-Fig - 4 Flow chart of the experimental design of experiment - III t ime c o u r s e 26 guinea pigs 330 - 370 gm adaptation period (7 days) CONT HC 4 7 30 days 70 days kept on normal guinea pig chow HP 5 kept on 2% cholesterol-rich diet kept on 50% casein-rich diet HC HP 10 (animals/ group) kept on 2% cholesterol-rich diet kept on 2% cholesterol/50% casein-rich diet CONT = Control group HC = High cholesterol group HP = High protein group HCHP = High Cholesterol/High Protein Group - 32 -Fig - 5 Flow chart of the experimental design of the assessment assays of cholesterol de novo synthesis t ime couse 26 guinea pigs 330 - 370 gm adaptation period (7 days) 30 days 70 days the day 70 Cont 5 HC 8 HP 5 kept on normal kept on 2% guinea pig chow cholesterol-rich diet kept on 50% casein-rich diet HC HP O (animals ° /group) kept on 2% cholesterol-rich diet kept on 2% cholesterol/50% casein-rich di< measurement of H M G - C O A reductase activity measurement of incorporation of tritiated water into cholesterol - 33 -D. Experiment IV This experiment was performed to sequentially follow up the changes i n plasma and kidney tissue l i p i d s versus the kidney histopathological alterations i n an e f f o r t to elucidate the pathogenetic role of plasma l i p i d and lipoproteins i n inducing glomeruloclerosis. A t o t a l of thirty-nine male guinea pigs were used. Groups of animals were s a c r i f i c e d at day 5, 10, and 30 (Figure 6). In the r e s u l t analysis, the data for day 70 were derived from the Experiment I I I . The following dietary regimens were i n s t i t u t e d : fi v e animals on the cholesterol-rich d i e t , f i v e animals on the cholesterol/ protein-rich d i e t , and three animals on the normal control d i e t . _ 34 _ Fig - 6 Flow chart of the experimental design of experiment - IV time course 39 guinea pigs 330 - 370 gm adaptation period (7days) 5 days 10 days 30 days 70 days CONT 1 1 1 ! 3 HC mi 5 on normal diet on 2% cholesterol-rich diet i n i n 3 5 on normal diet on 2% cholesterol-rich diet HC HP i m f- (animals/ O group) on 2% cholesterol/ 50% casein-rich diet Hi on 2% cholesterol/ 50% casein-rich diet J J i l on normal diet on 2% cholesterol- on 2% cholesterol/ rich diet 50% casein-rich diet I I data obtained from experiment III - 35 -I I I Methods A. Blood and Urine Collections: Blood was collected into EDTA containing tubes from ether anaesthetized animals from a 1mm long i n c i s i o n i n the l a t e r a l metatarsal vein. After separation of plasma by centrifugation at lOOOxg for 15 minutes, whole RBC and plasma l i p i d extractions were performed. 24-hour urine co l l e c t i o n s were done at day 70 i n a l l of the animals i n Experiment I I I . The animals spent one day i n metabolic cages as an adaptation period, then two separate 24-hour urine specimens were obtained. B. Erythrocyte l i p i d extraction: The solvent system used to extract erythrocyte l i p i d was chloroform: isopropanol (2:1 v/v) with solvent-to-plasma sample r a t i o (10:lv:v). Samples were re-extracted to increase l i p i d recovery, and a l l extractions were performed at 0°C i n an ice bath (80). The l i p i d extracts were dried under a nitrogen stream and reconstituted with 200ul of chloroform:methanol (2:1 v/v). Butylated hydroxy toluene (BHT) 0.05% (w/v) was added to the solvents to prevent l i p i d peroxidation. C. Plasma: ( i ) Enzyme assay and l i p i d P r o f i l e : 1. Lecithin:cholesterol acyltransferase a c t i v i t y using endogenous substrate: Plasma LCAT a c t i v i t y was measured according to Stokke and Norum (1971) (81). Plasma sample (0.25ml) was incubated with 50ul of dithionitrobenzoic acid 4.2 mM (DTNB) for 30 minutes at 37°C i n a shaking water bath. 3H-cholesterol/50% bovine serum albumin emulsion (75ul) was added and the - 36 -mixture was incubated for 4 hours at 37°C water bath. After t h i s incubation period, 50ul of 2-mercaptoethanol was added and a 150ul aliquot of the mixture was removed to represent a c t i v i t y at time 0 (t=0). After one hour incubation another 150ul aliquot was removed into a separate tube to represent a c t i v i t y after one hour ( t = l ) . Each aliquot was mixed with 2ml of chloroform:methanol (2:1 v/v) to allow l i p i d extraction. The extract was recovered from the chloroform layer after mixing with saline and centrifugation. The l i p i d extract was dried down under a nitrogen stream at 60°C; i t was reconstituted by chloroform and streaked onto 10 x 20cm s i l i c a gel G p l a s t i c plates. Separation of free cholesterol and cholesteryl ester was obtained by using petroleum ether:ether:acetic acid solvent (70:10:1 v/v/v) and the spots were visu a l i z e d by iodine vapour. Free cholesterol and cholesteryl ester spots were mixed with 5mls Omniflour i n toluene and l i q u i d s c i n t i l l a t i o n counting was done i n window (A15) for 5 minutes. Fractional e s t e r i f i c a t i o n rate (FER) was calculated as follows: FER = CPM - EC x 100 CPM - EC + CPM-FC 1 hr where CPM = Count per minute EC = E s t e r i f i e d cholesterol FC = Free cholesterol - 37 -To calculate molar e s t e r i f i c a t i o n rate (MER): MER = FER/hr x FC (nmol) = nmol FC/hr/ml plasma 2. Lecithin:cholesterol acyltransferase a c t i v i t y - using a r t i f i c i a l (exogenous) ethanolosome substrate: Liposomes were prepared by mixing 0.26mls egg yolk phosphatidylcholine (PC) (5mg/ml i n absolute ethanol), 0.15mls free cholesterol (FC) (lmg/ml i n absolute ethanol), and 12ul 3H-FC (lmCi/ml), giving a molar r a t i o of PC:FC of 4:1. The mixture was dried down and taken up i n 125ul of absolute ethanol and injected rapidly into lOmls of lOmM Tris-HCl pH 7.4/5mM EDTA/0.15M NaCl. The f i n a l volume of 2.5ml was obtained by concentration with an Amicon Ym-30 membrane. Thirty m i c r o l i t r e s of liposomes were pre-incubated at 37°c for 30 minutes with apo A l (20ul of 0.5mg/ml) and Tris-HCL buffer. 50ul bovine serum albumin (BSA), and lOul of 2-mercaptoethanol were added and the reaction was started by adding 15ul of plasma. After 30 minutes incubation, the reaction was stopped by adding 4mls chloroform:methanol (2:1). The extracted l i p i d was dried under nitrogen, and 20ul of FC/esterified cholesterol (EC) were added together with 70ul chloroform and the mixture was streaked on s i l i c a gel TLC plates. FC and EC spots were separated and ra d i o a c t i v i t y was determined. Calculation of the a c t i v i t y : MER = FER x 6.21 (nmol FC esterified/hr/ml). - 38 -3. Total and Free Cholesterol. Cholesterol was measured enzymatically (82) using an "ABBOTT (ABA-100) Bichromatic Analyzer". The p r i n c i p l e of t h i s assay i s converting cholesterol to cholest-4-ene-3-one and H 20 2 by cholesterol oxidase; ^2®2 r e a c t s with 4-aminoantipyrine i n the presence of phenol and hydrogen peroxidase to produce quinoneimine dye that absorbs at 500nm. 4. Phospholipids. Plasma phospholipids were determined by one dimensional thi n layer chromatography (TLC) with subsequent assay of the phosphorus content of indi v i d u a l resolved phospholipids according to the B a r t l e t t method (83). An aliquot of 200ul of a chloroform:methanol (2:1 v/v) reconstituted l i p i d extract was applied to pre-coated s i l i c a gel F-254 (0.2mm thickness, Brinkman) which was activated by heating at 110°C for 30 minutes. The solvent mixture contained chloroform:methanol:ammonia (14:6:1 v/v/v). V i s u a l i z a t i o n of phospholipid spots was obtained by iodine vapor; aminophospholipids were visualized after ninhydrin spraying. Individual phospholipids were hydrolyzed by 70% perchloric acid at 230°C for 1/2 hr; after cooling down and mixing with water and centrifuging the mixture, an aliquot from the supernatant was reacted with 5% ammonium molybdate and ANS (l-amino-2-napthol-4-sulfonic acid) i n a b o i l i n g water bath for 7 minutes. The cooled samples were read at 830nm. Plasma t o t a l phospholipids were estimated according to Anderson and Davis (1982) (also, see kidney l i p i d analysis for more d e t a i l ) . - 39 -5. HDL-Cholesterol (HDL-C). Plasma HDL-C was measured after heparin-manganese pr e c i p i t a t i o n of the lipoproteins containing apo-B. A mixture of manganese chloride l.Omol/L and heparin 4000u/mL was used to precipitate chylomicrons, VLOL, and LDL. The HDL-C found i n the supernatant was determined as described above (82). 6. Triglycerides. Triglycerides were determined enzymatically by "ABBOTT (ABA-100) Biochromatic Analyzer" based on the method of Bucolo and David (1973) (84). The t r i g l y c e r i d e s are hydrolyzed to glycerol and fatty acids by l i p a s e . The produced glycerol i s phosphorylated by ATP which i s catalyzed by glycerol kinase producing ADP. ADP i s rephosphorylated by phosphoenol pyruvate to produce pyruvate. In the presence of NADH + H + and lactate dehydrogenase, pyruvate i s converted to l a c t a t e . The decrease i n absorbance at 340nm i s proportional to the concentration of g l y c e r o l . ( i i ) Lipoprotein studies. 1. Lipoprotein i s o l a t i o n . Lipoproteins were isolated according to t h e i r hydrated densities. The lipoprotein with hydrated density 1.006 was isolated by putting one volume of plasma i n a centrifuge tube and the l e v e l was marked. One ha l f volume of d=1.006g/ml saline was layered over the sample and centrifuged i n a 75 T i rotor for 18 hr at 40,000 rpm, 15°C. The upper two mis were removed by a tube s l i c i n g technique and placed i n a clean tube, whereas the infranatant under the intermediate clear zone proceeded for further lipoprotein - 40 -i s o l a t i o n . To i s o l a t e the lipoprotein fraction at the hydrated density range of d=1.006 - 1.063, 0.5 volume of 1.182g/ml saline solution was mixed with 1 volume of the infranatant recovered from the f i r s t i s o l a t i o n . The tube was sealed and centrifuged under the same conditions for 20 hrs. The upper two mis were kept as the lipoprotein f r a c t i o n with hydrated density of d=l.006-1.063. After removing the middle clear zone, one volume of the infranatant was mixed with 0.5 volume of d=1.478g/ml saline solution. The sealed centrifuge tubes were spun at 40,000 rpm, 15°C for 24 hrs. The top two mis contained the lipoprotein f r a c t i o n with hydrated density of (d=l.063-1.210). The isolated lipoprotein fractions were dialysed prior to SDS-polyacrylamide gel electrophoresis. 1. Electrophoresis. One u l plasma was applied to 1% agarose gel (1% agarose/barbital buffer, pH 8.6). After 35 minutes electrophoresis gels were dried and overlayered with fat s t a i n . The stained gels were dried under hot a i r . 2. SDS Polyacrylamide gel electrophoresis. Acrylamide 30% (73gm acrylamide and 2gm Bis, made up into 250ml) was used after f i l t r a t i o n . To prepare gel, lOmls acrylamide 30% was added to 7.5ml lower buffer (1.5M Tris-HCl pH 8.8/0.4% SDS) and 12.6ml water. To the mixture lOul TEMED and 150uL ammonium persulfate were added. To the casted gel, stacking gel [3ml acrylamide 30%, 5ml upper buffer (0.5M Tris-HCl pH 6.8/0.4% SDS), 12ml_ d H20, 20uL TEMED, and 60 uL ammonium persulfate] was added. After insertion of an appropriate comb, the stacking gel was allowed to polymerize for one hour. One volume of sample buffer pH 6.8 (20gm - 41 -g l y c e r o l , 4.6gm SDS, 0.76gm Tr i s base/in 50mls d h^O; plus 1 part mercaptoethanol and 1/2 part 0.5% bromophenol blue) was added to 9 parts (2 x concentration) of the sample buffer. This mixture was used i n the electrophoresis after mixing 1 volume sample buffer (dilu t e ) with 1 volume dialyzed sample and heating i n a b o i l i n g water bath for 3-5 minutes and cooling down to room temperature. Electrophoresis was carried out at a constant 200 volts with tapwater cooling of the electrophoresis chamber. The gel was stained with Coomassie blue R250 overnight at room temperature. Destaining of the background was achieved by using destaining solution (lOOmls acetic acid, 450mls methanol, and 450 mis dH 20). Low molecular weight standards were included i n the separation. D. Erythrocyte Analyses: 1. Complete blood count: I t was performed using an ELT-8 (Ortho instruments) automatic counter. 2. Erythrocyte osmotic f r a g i l i t y : An aliquot of packed RBC was used to measure osmotic f r a g i l i t y as described by Godin et a l . (1978) (58). Erythrocytes were incubated i n different concentrations of NaCl, and the degree of hemolysis was monitored by spectrophotometry at 540nm. 3. Erythrocyte morphology: Peripheral blood films were stained with: - 42 -* Wright-Giemsa stain (Giemsa st a i n 5ml/L mixed with Wright s t a i n 3g.L i n absolute methanol). * Oil-Red 0 s t a i n : Blood films were fixed by exposure to formalin fume. A saturated solution of O i l Red 0 (0.25-0.5%) i n isopropyl alcohol was mixed with water 6:4 (v/v) and the s l i d e s were stained i n a closed container for 10-15 minutes. * Nonspecific esterase (NSE) s t a i n : Monocytes i n peripheral blood and macrophages i n renal tissue were detected by the alpha-naphthyl acetate for NSE s t a i n based on the method described by Yam et a l . (1971 (85). The non-specific esterase of these c e l l s l i b r a t e s alpha-napthol from the substrate alpha-napthyl acetate. The alpha-napthol i s coupled to the dye hexazonium pararosaniline to form dark red granules i n the cytoplasm. Estimation of vacuolated monocytes i n the peripheral blood: The NSE-positive white blood c e l l s are mainly monocytes (85); neutrophils are s l i g h t l y p o s i t i v e l y stained. D i f f e r e n t i a t i o n between the two c e l l types was achieved by applying other morphological c r i t e r i a . One hundred monocytes were counted i n each blood f i l m and the percentage of the vacuolated monocytes was derived. Scanning the periphery of each smear by a zig-zag pattern was the method applied to a l l samples. 4. L i p i d analyses were carried out as mentioned previously (see section I I I ( i ) ) . - 43 -E. Kidney: ( i ) Renal Function Tests. 1. Serum creatinine: Serum creatinine was determined on EKTACHEM automatic analyzer. In t h i s method, serum creatinine i s hydrolyzed by creatinine iminohydrolase into N-methylhydantoin and ammonia. The produced ammonia reacted with bromophenol blue (ammonia indicator) and produced a blue dye. The r e f l e c t i o n was measured at 600nm. 2. Blood urea nitrogen (BUN): BUN was measured by EKTACHEM automatic analyzer. The p r i n c i p l e i s based on releasing ammonia from urea by the action of urease; the rest of the measurement followed the same reaction described under serum creatinine. 3. Creatinine clearance: Serum and urine creatinine were measured by EKTACHEM automatic analyzer as described above. Creatinine clearance was obtained by applying the equation: Creatinine clearance (mL/min) = urinary creat. cone, x volume of urine/24hrs. serum creat cone 4. Twenty-four hour urinary protein: Urinary protein was measured by ABBOTT ABA-100 automatic analyzer (86). Coomassie b r i l l i a n t blue G250 was used i n the presence of phosphoric acid and 95% ethanol. The diluted dye reagent i s bound to the protein i n urine samples and absorbance i s measured spectrophotometrically at 595nm. - 44 -5. Routine u r i n a l y s i s (chemical and microscopic): A semiquantitative assessment of white blood c e l l s , b i l i r u b i n , and erythrocyte i n urine was achieved by using a dipstick method. ( i i ) Renal histology. 1. Blocks of lcm"5 size were taken from kidneys and fixed i n Karnovsky's f i x a t i v e (2 mis of 70% gluteraldehyde, 14mls of 38-40% formalin, and 126mls phosphate buffer of pH 7.2). Thin sections of 3 microns were obtained from paraffin blocks and stained with different s t a i n s . a. Routine h i s t o l o g i c a l s t a i n (hematoxylin and eosin s t a i n ) . b. Perls Prussian blue s t a i n : Iron containing pigments are soluble i n acids, and insoluble i n a l k a l i s and fat solvents. The specimens were exposed to a fresh solution of equal parts of 2% aqueous potassium ferrocyanide and 2% hydrochloric acid for 30 minutes. After a wash with d i s t i l l e d water, a counter s t a i n (1% neutral red) was applied for 10-15 seconds. The ferric-iron-containing pigments appeared blue. c. Periodic acid-Schiff (PAS) and PASM stains were used to elucidate the connective tissue elements (collagenous and e l a s t i c f i b r e s ) , (for PASM, 1-micron thick sections were used after embedding of tissues i n polyglycol methacrylate). d. Von Kossa st a i n for calcium. 2. Renal frozen sections. a. Oil-Red 0 Stain (as described above). b. Immunohistofluorescent study: - 45 -Fluorescein conjugated goat anti-guinea pig IgG (heavy and l i g h t chain s p e c i f i c ) (Cappell, Cockranvile, PA) was used with an antibody protein concentration of 4.2mg//ml. Frozen sections were examined, after incubation, for immunofluorescent complexes. c. Non s p e c i f i c estrase (NSE) stain (as described above). 3. Electron microscopy: Blocks of 1mm3 were taken from kidney and fixed i n ice cold 3% glutaraldyehyde i n 0.1M sodium phosphate, pH 7.3 (Sorensen's solution). After washing i n 0.1M phosphate buffer for approximately 30 minutes, the tissue was post fixed i n 1% osmium tetroxide i n 0.1M phosphate buffer for one hour at 4°C. After washing i n phosphate buffer, the tissues were dehydrated i n a graded ethanol series and embedded i n an epon-araldite (Polyscience In. Co., PA). Ult r a t h i n sections were cut from appropriate blocks with a diamond knife. The sections were stained with uranyl acetate followed by lead c i t r a t e and examined by a Zeiss EM 109 electron microscope. 4. Morphometric study: Glomerular mesangial expansion was measured by examining h i s t o l o g i c a l sections of kidney stained by PASM sta i n at high magnification (lOOOx) using an eyepiece net micrometer (20 divisions/cm). The r e l a t i v e mesangial area (mesangial area/glomerular t u f t area) was determined by a standard point-counting method (87). Mesangial c e l l s were also determined. 5. L i p i d analysis - extraction and measurement: Kidney tissues were homogenized and l i p i d was extracted with - 46 -chloroform:methanol 2:1 (v/v). The extract was dried down under nitrogen and reconstituted with chloroform to be streaked on the TLC plates. The TLC solution solvent system used was petroleum ether:ethyl ether:acetic acid (85:15:3, v/v/). The phospholipid spots were visualized under u l t r a v i o l e t l i g h t , while the spots of the different l i p i d s were vi s u a l i z e d by b r i e f exposure to iodine vapour. Phospholipids were eluted by chloroform:methanol: acetic acid:water (25:15:4:2, v/v/v/v) then by methanol alone followed by methanol:acetic acid:water (95:1:5, v/v/v) (88). Free cholesterol, cholesteryl esters and t r i g l y c e r i d e s were eluted from the s i l i c a powder by a mixture of chloroform:methanol (89). a. Tissue free and e s t e r i f i e d cholesterol. Cholesterol was determined by a modified Liebermann-Burchard reaction. This reaction involves strong acid medium-sulfuric acid, and g l a c i a l acetic acid. F e r r i c chloride i s added to y i e l d t e t r a e n y l i c cation which absorbs at 560nm. b. Phospholipids. Phospholipids were measured according to Anderson and Davis (1982) (90). Phospholipids were digested with concentrated sulphuric acid at 155°C. To accomplish oxidation of the organic compounds hydrogen peroxide was used subsequently. Colorimetric reaction was achieved when a mixture of lO.lnmol/L ammonium molybdate and 0.28mol/L ascorbic acid (1:1, v/v) was added; the absorbance was read at 797nm. - 47 -c. Triglycerides. Triglycerides i n renal tissue extract were determined according to Fossati and Lorenzo (1982) (91). Triglyceride was converted by lipase into glycerol and free fatty acids. By virtue of glycerol kinase and i n the presence of ATP, glycerol-l-phosphate was formed. The l a t t e r i s oxidized by glycerol phosphate oxidase i n the presence of 0 2 to produce H 20 2 which gives a quinoneimine dye after reacting with 4-amino antipyrine. The produced dye absorbed at 515nm. 6. Assessment of tissue protein was done by Lowry et a l . method (92). The value of protein served i n expressing the l i p i d values i n kidney t i s s e ; the unit was ug/mg protein. ( i i i ) Estimation of cholesterol de novo synthesis i n renal tissue: 1. Measurement of hydroxy methyl glutaryl-COA (HMG-COA) reductase a c t i v i t y . Measurement of HMG-COA reductase a c t i v i t y was performed according to Goodwin and Marglis (93). Kidneys were homogenized and the microsomal fra c t i o n was prepared as follows: The homogenate (1 part tissue:5 parts of 225mM sucrose/25mM T r i s HC1 buffer pH 7.8) was spun at 2500 rpm for 10 minutes at 4°C. The supernatant was spun again at 8000 rpm for 20 minutes at 4°C. A t h i r d spin of the supernatant was performed at 25000 rpm (105000g) for 60 minutes at 4°C. The p e l l e t was resuspended i n 1ml (lOOmM sucrose/50mM KCl/40mM KH2P04/30mM EDTA buffer, pH 7.2). Fresh 20mM d i t h i o t h r e i t o l (30.86mg/10ml) was added to the resuspension before storing at -20°C. The assay i s conducted according to the following p r i n c i p l e : - 48 -3H-HMG-C0A (known dpm and (added by known protein from sample) HMG-COA reductase Mevalonic acid (measure) known moles of HMGCOA) NADPH NADP (excess i n the reaction) gluconolactone glucose-6P dehydrogenase ( i n excess) glucose-6-phosphate (excess i n the reaction) The reaction was stopped at every 15 sec. by adding 12N HC1, and incubated for 30 minutes at 37°C. Sodium s u l f i t e was added to raise the pH of the reaction mixture to 6.5 which enhanced the retention of HMG COA i n the aqueous phase. The produced mevalonate was extracted by toluene. S c i n t i l l a t i o n f l u i d was added to 10ml of the extract and r a d i o a c t i v i t y was counted. 2. Estimation of incorporation of t r i t i a t e d water into cholesterol: Guinea pigs were injected intravenously with 200mCi/kg t r i t i a t e d water between 9:00 and 11:00 a.m. (94). One hour l a t e r , the animals were s a c r i f i c e d and blood samples were taken for the measurement of the s p e c i f i c a c t i v i t y . Kidneys were removed; thin s l i c e s were prepared and washed with cold isotonic s a l i n e . The tissues were weighed and homogenized i n 0.9% NaCl/3mM EDTA. Tissue l i p i d s were extracted with chloroform:methanol (2:1, v/v). After adding s a l i n e , the mixture was centrifuged at 2500 rpm for 5 min. and the bottom phase was recovered for further l i p i d separation. Free cholesterol and cholesteryl esters were separated by TLC chromatography (as described above). Radioactivity detected i n the cholesteryl esters was neg l i g i b l e . Thus, the t r i t i a t e d water was incorporated into the free cholesterol fraction only. The calculations were performed according to Turley et a l . (95). - 49 -F. Liver Function Tests 1. Serum t o t a l protein and albumin: A l l analyses were done on an EKTACHEM automatic analyzer by the chemistry unit i n Shaughnessy Hospital. The method for determining t o t a l protein was based on the biuret reaction (96) i n which a violet-colored complex i s generated when protein i s treated with cupric ion (Cu^ +) i n an alkaline medium. The density of the resu l t i n g complex i s related to the concentration of t o t a l protein i n the sample, and a spectrophorometric measurement can be achieved at 540nM. Serum albumin was measured using the same automatic analyzer. The method was based on the binding of bromcresol green dye to albumin, re s u l t i n g i n a substantial s h i f t i n the wavelength of l i g h t absorbed by the free dye. The density of the albumin-bound dye was related to the concentration of albumin i n the sample. I t was measured spectrophoto-metrically at 630nm. 2. Blood urea nitrogen, (see above). 3. Serum aspartate transaminase (SGOT or AST). In t h i s enzymatic assay, the amino group of L-aspartate i s transferred to alpha-keto glutarate i n the presence of sodium pyridoxal-5-phosphate to produce glutamate and oxalo-acetate. Oxalo-acetate i s converted to malate by malate dehydrogenase i n the presence of NADH which i s oxidized at 37°C to NAD+. The oxidation of NADH i s monitored by reflectance spectrometry at 340nm. - 50 -4. Serum b i l i r u b i n . Determination of both conjugated and unconjugated b i l i r u b i n was performed simultaneously on EKTACHEM autoanalyzer. The analysis i s based on a modification of the c l a s s i c a l diazo reaction (97). A dual-wave length reflectance spectrophotometric method was used. Dyphylline and surfactant are used to dissociate unconjugated b i l i r u b i n from albumin. Unconjugated and conjugated b i l i r u b i n reacts with the diazonium s a l t [4-(N-carboxymethyl-sulfamyl)-benzene diazonium hexafluorophosphate] to produce a z o b i l i r u b i n which has an absorbance maximum around 520nM. Unconjugated and conjugated b i l i r u b i n bound to a cationic mordant (polymeric quaternary amine). As a res u l t of t h i s i n t e r a c t i o n , the absorbance peaks of the b i l i r u b i n fractions were shift e d about lOnms, and the molar extinction c o e f f i c i e n t s were s i g n i f i c a n t l y increased. G - Serum phosphate was done on EKTACHEM automatic analyzer. IV S t a t i s t i c a l Analyses. A l l data were assessed by Student's t - t e s t , both paired and unpaired. Accordingly, t h i s s t a t i s t i c a l test examined the difference between-group means at each period and between-period means i n each group. Correlations were determined by multiple l i n e a r regression analysis. -50A-RESULTS - 51 -I General Observations In both experiment I and experiment I I I , the animals kept on cholesterol-r i c h and cholesterol/protein-rich diets showed smaller weight gain at day 70. Compared to the CONT group (422 + 52.6)gms and HP group (363.3 + 24.9) gms, HC and HCHP groups gained (235 _+ 29.3)gms and (105 +_ 39.9) gms, respectively. A s i g n i f i c a n t reduction (p<0.01) was found i n HCHP group compared to HC group (Fig. 7), but no change found between CONT and HP groups. As shown i n (Table 6), the differences i n weight gain were f i r s t noticed at day 30. Both HC and HCHP groups had lower weight gain compared to the CONT group (p<0.05) and (p<0.01), respectively. These results suggested that keeping guinea pigs on cholesterol-rich or cholesterol/protein-rich diet does not affect the animals' a b i l i t y to gain weight during the f i r s t two weeks of the experiment. Addition of protein supplement to the cholesterol-rich diet affected weight gain more severely than cholesterol-r i c h diet alone (p<0.02). Animals made anaemic by acetylphenylhydrazine also showed a s i g n i f i c a n t reduction i n weight gain comparable to that found i n the HCHP group. They gained 171.7 +_ 55 gm compared to 370.1 +_ 50 gm i n the CONT group. Mortality rate i n experiment I I I was almost the same as i n experiment I. At day 70 of both experiments the HCHP group had 40% mortality compared to almost 10% mortality i n the HC group. There were no deaths among the control animals or i n those kept on any of the diets for 30 days. As depicted i n Fig. 8, the l i v e r was enlarged and yellowish i n the cholesterol-fed guinea pigs at day 70 compared to the animals of the CONT and HP groups. Table 7 summarizes the organ weight changes i n the ind i v i d u a l groups. The animals of the HC and HCHP groups had s i g n i f i c a n t enlargement Table 6 [Weight Gain (gm). Mean +_ SD] Group Day 5 Day 10 Day 30 (n = 5) (n = 5) (n = 5) CONT 60 + 81 136.7 + 16.9 353.3 + 49.9 HC 6 4 + 8 116 + 10.2 236 + 45.9 a HCHP 58 + 7.5 112 + 16 132 + 32.5 b n = Number of animals/group CONT = Control HC = High cholesterol HCHP = High cholesterol/protein a = S i g n i f i c a n t l y d i f f e r e n t from CONT (p<0.05) b = S i g n i f i c a n t l y different from CONT (p<0.01) 500 400 « 300 CD 100 0 Fig - 7 Weight Gain - 54 -in l i v e r size compared to the control groups (p<0.01). The spleen was enormously enlarged i n these two groups (Fig. 8). Almost ten-fold enlarge-ment was found i n the HC and HCHP groups compared to the normal-sized spleen i n the CONT and HP groups. Table 7 also shows the s i g n i f i c a n t increase i n weight of the kidney i n the HP group compared to other groups (p<0.05). There was no change i n the HC or HCHP groups. As shown i n Table 7, the only s i g n i f i c a n t change was present i n spleens of the HC and HCHP groups at day 30; there was a s i g n i f i c a n t increase (p<0.05) i n weight compared to the CONT group. There was a s i g n i f i c a n t increase i n the weight of the spleen and the l i v e r from day 30 to day 70 i n the HC and HCHP groups (p <0.01) (Table 7). - 55 -Figure 8: A severely enlarged spleen from the HCHP group compared to a normal-sized spleen from the CONT group (upper l e f t corner). The rig h t side of the figure shows enlarged yellowish pale l i v e r from the same animal compared to a normal l i v e r . ( A l l specimens were obtained at day 70.) - 56 -- 57 -Table 7 [Organ weight (gm). Mean _+ SD] Group Organ Day 5 Day 10 Day 30 *Day 70 (n = = 5) (n 5) (n = 5) Kidney 2.1 .1 2.2 + .2 2.6 + 3 2.1 + .6 CONT Liver 24.3 + 1.7 23.7 + 1.2 34.1 + 7.2 22.3 + 2.6 Spleen .8 + .1 .8 + .1 .74 + .1 .8 + .1 Kidney 2.3 +_ .1 2.1 +_ .1 2.3 + .3 2.3 + .4 1 HC Liver 25.9 + .7 25.8 + 1.9 40.2 + 4.2 59.5 + 4.51 Spleen .8 + .1 .8 .1 1.2 + .4° 9.5 + 7.3' Kidney 2.1 + .1 2.1 + .1 2.7 + .5 2.3 + .3 i HCHP Liver 25.9 + .7 25.6 + 1.0 35.6 + 8.2U 46.8 + 9.31Spleen .8 + .1 1.0 + .2 2.0 + 1.8° 9.5 + 6.61 Kidney 3.3 + .3 a HP Liver 21 + .9 Spleen .8 + .1 n = Number of animals/group * = Data from experiment I I I a = S i g n i f i c a n t l y d i f f e r e n t than other groups (p<0.05) b = S i g n i f i c a n t l y d i f f e r e n t than the control groups (p<0.01) c = S i g n i f i c a n t l y higher than the control groups (p<0.05) d = S i g n i f i c a n t l y higher than the control groups (p<0.01) - 58 -II Plasma L i p i d and Lipoprotein Changes In experiment I, plasma t o t a l cholesterol (TC) was s i g n i f i c a n t l y increased at day 30 i n both HC and HCHP groups compared to the CONT and HP groups (p <0.001) (Figure 9). As shown i n Figure 10, the increase i n plasma TC i n the HC and HCHP groups was already s i g n i f i c a n t at day 5. There was only a numerical, but not a s t a t i s t i c a l , difference i n plasma TC between HC and HCHP groups (Figures 9 and 10). The changes i n plasma TC after 5, 10, 30 and 70 days of the feeding are shown i n Figure 10. Only the increase between days 10 and 30 was not s t a t i s t i c a l l y s i g n i f i c a n t . The increase i n plasma free cholesterol (FC) generally paralleled that in TC (experiment I) except for a s i g n i f i c a n t increase i n the HCHP group over the HC group (p<0.01) at day 70 (Figure 11). As shown i n Table 8, the percentage of plasma free cholesterol (FC%) was s i g n i f i c a n t l y higher at both day 30 and day 70 i n the HC and HCHP than i n the CONT and HP (p<0.05). Plasma t r i g l y c e r i d e s were only measured i n experiment I. There was no s i g n i f i c a n t difference between the groups (Table 8). Plasma TC was s l i g h t l y elevated i n the CONT and HP groups at day 70 compared to day 0. As shown i n Table 9, plasma t o t a l phospholipids (TPL) were s i g n i f i c a n t l y increased i n the HC and HCHP groups at the day 70 of the experiment r e l a t i v e to the CONT group (p < 0.001). Once again the highest values were found i n the HCHP group versus HC group (p<0.01). This increase i n plasma TPL i n both groups was f i r s t noted at day 30 (p<0.02) compared to the CONT group (Table 9). Plasma cholesterol of HDL (HDL-C) as presented i n Table 9 was unchanged at days 5 and 10 i n a l l groups of the experiments. At day 30, HDL-C increased s i g n i f i c a n t l y i n the HC and HCHP groups compared to the CONT group. This Fig - 9 400 Plasma T. Cholesterol E 300 200 100 — — Control • • Hi Choi Hi Prot S Hi PrCh 0 Si I 0 30 70 Time (days) Concentration (mg/dl) — | \ 3 CO 4^  cn o o o o o o o o o o o I—I—I—I—r~I—I—I—I—I—I 09 - 61 -Table 8 [Plasma L i p i d P r o f i l e . Mean + SD] Group* Day 0 Day 30 Day 70 CONT %FC 32.9 + 4.7 27.2 + 4.4 29.8 + 5.3 n=6 TG(mg/dL) 73.3 + 31.8 72.3 + 23.5 65.3 + 14.6 HP %FC 28.4+8.2 27.6+5.6 30.8+11.8 n=6 TG 73.8 + 30.1 82.2 + 22.6 70.5 + 11.8 HC %FC 30.2 + 3.9 40.0 + 3.3 a 40.9 + 5.5 a n=6 TG 62.3 + 23.3 68.4 + 23.4 61.3 + 9.2 HCHP %FC 31.0 + 9.7 33.7 + 8.5a 46.3 + 12.0 a n=6 TG 69.2 + 29.4 71.8 + 28.7 60.3 + 29 *C0NT = Control HP = High Protein HC = High Cholesterol FC = % Free Cholesterol HCHP = High Cholesterol & High Protein TG = Triglycerides n = Number of animals/group a = S i g n i f i c a n t l y higher than the control groups ( p<0.05) Fig - 11 - 63 -increase was even more pronounced by day 70 i n both HC and HCHP groups. No si g n i f i c a n t change was detected between the HC and HCHP groups. No v i s i b l e band was detected at the alpha region on agarose gel electrophoresis at day 30 which looked indistinguishable from the CONT group (Figure 12). The alpha band appeared at day 70 i n both HC and HCHP groups (Figure 13). Other abnormalities i n electrophoretic p r o f i l e of plasma lipoproteins were as follows: the prebeta band i n the HC and HCHP group disappeared from the region where i t runs i n the CONT group (Figure 13). Only one beta band appeared on the electrophoresis gel of plasma from HC and HCHP groups at day 30 (Figure 12) while the CONT group shows both beta and prebeta bands. At day 70 there was an additional alpha-migrating band i n the HC and HCHP groups (Figure 13). Also, a slow broad beta band was found i n these two groups at day 70 compared to the control beta band. The slow beta could be possibly a combination of slow prebeta and beta bands (Figure 14). The slowness of the prebeta band was monitored i n experiment IV, where there was a gradual decrease i n the anodal movement of the prebeta band which ultimately blended with beta band (Figure 14). The e a r l i e s t change i n the pattern of electrophoretic mobility was noticed at day 10 i n the HC and HCHP groups and t h i s was limi t e d to the slowness of the prebeta band (Figure 14). The apoprotein p r o f i l e of the lipoproteins isolated by preparative ultracentrifugation i n the hydrated density ranges < 1.006 gm/ml and 1.063-1.21 gm/ml showed no sa l i e n t differences i n patterns of the HC and HCHP groups at day 70. The lipoprotein species isolated i n the hydrated density range of 1.063-1.21 gm/ml from these two groups showed a doublet protein band with an apparent molecular weight of > 31000 (Figure 15). This band was not observed i n the CONT group. This band(s) may represent apoprotein E. - 64 -Table 9 [Plasma L i p i d P r o f i l e . Mean +_ SD] Group Day 5 Day 10 Day 30 *Day 70 n CONT** n=12 HDL-TPL -C 6.5 63.2 + + I. 6 I I . 2 6.5 63.2 + + I. 6 I I . 2 6.5 63.2 + + I. 6 I I . 2 6.5 63.2 + + I. 6 I I . 2 HC HDL-•C 6 + 1 9.8 + 1.6 18.8 + 9.4 79.8 + 20. i e TPL 65 + 19 71 + 21 133 + 34.4 314.5 + 50.3e HCHP HDL-TPL •C 8.5 68 + + 3.4 15 11.4 75 + + 5.8 18 19.4 145.2 + + 6.7 7.8 68.4 475.2 + + 23.9e 79.4e n = Number of animals (5)/group/5, 10, and 30 day periods. The number of animals at day 70 was: HP(5), HC(6), HCHP(6) * = Data from experiment I I I HDL-C = Cholesterol of HDL (mg/dL) TPL = Total plasma phospholipids (mg/dL) e = S i g n i f i c a n t l y different than CONT (p<0.001) ** = The data of days 5, 10, 30 and 70 are combined due to the absence of s i g n i f i c a n t changes among the control animals. - 65 -Figure 12: An electrophoretogram on agarose gel of the CONT, HC, and HCHP groups at day 30. The CONT group reveals beta and prebeta bands while both HC and HCHP groups have only one darkly stained band runs i n the beta region. - 66 -HCHP CONT HC HCHP HC CONT t \ BETA PREBETA - 67 -Figure 13: An electrophoretic p r o f i l e on agarose gel of the HC, CONT, and HCHP groups at day 70. Both HC and HCHP groups reveal a clear alpha band. Prebeta band i n these two groups are absent, instead, there i s a darkly stained beta band which migrates slower than the beta band of the CONT group. Both beta and prebeta bands are clear i n the CONT group, while there i s no alpha band. o o o mm ALPHA PREBET BETA - 69 -Figure 14: Electrophoretic p r o f i l e of the CONT, HCHP, and HC groups at day 10 on an agarose gel. Compared to the CONT group which shows beta and prebeta bands, different stages of blending of the slow prebeta band into the beta band can be seen i n the HC and HCHP groups. The complete blending i s presented as a single dark thick band. - 70 -C O N T . +• C O N T . H C H P . H C H P . H C . H C . 4$ I m 1 I / t BETA PREBETA - 71 -I I I . Plasma LCAT A c t i v i t y Cholesterol feeding alone or with high protein supplement lowered plasma LCAT a c t i v i t y expressed by either FER or MER. However, the magnitude of the change differed with two different subtrates used. As shown i n Table 10, with an endogenous substrate, FER was remarkably decreased at both day 30 and 70 i n the HC and HCHP groups compared to the CONT and HP groups (p<0.01). However, there was no s i g n i f i c a n t change i n MER i n any of the four groups at the dif f e r e n t experimental periods. On the other hand, using an exogenous substrate, both FER and MER showed a s i g n i f i c a n t moderate decrease (p<0.05), at day 70 i n the HC and HCHP groups r e l a t i v e to the CONT group (Figure 16). As shown i n Table 10, the CONT and HP groups had a si g n i f i c a n t decrease i n FER (p<0.05), at day 70 compared to day 0. - 72 -Figure 15: Two lipoprotein fractions separated by preparative ultracentrifugation and resolved i n 10% SDS-polyacrylamide. The HCHP, HC and CONT groups are presented i n the two fractions. The same pattern of protein d i s t r i b u t i o n i s present i n the two fractions i n the HC and HCHP groups. In the fr a c t i o n with hydrated density of d = 1.063 - 1.21 gm/ml, both HC and HCHP groups have double bands with an apparent molecular weight > 31000. - 73 -d=1.063-1.21 g/ml d=<1.006 g/ml HCHP HC CONT CONT HC HCHP i t 92.5 K 66 K 45 K Fig - 16 E 200 r Lcat activity (MER) Exogenous substrate 160 CD *CD CD TS 120 h CD O «-f— C / ) 0) o E c DC UJ 80 40 0 Control Hi Choi Hi PrCh 70 Time (days) - 75 -Table 10 [LCAT A c t i v i t y Using Endogenous Substrate. Mean ^  SD] Group FERl CONT n=6 MER2 Day 0 10.5 + 1.1 31.4 + 6.4 Day 30 9.5 + 2.2 27.6 + 5.7 Day 70 7.7 + 1.0a 28 + 8.3 FER HP n=6 MER 10.7 + 3.4 33.8 + 13.8 8.6 + 1.5 25.3 + 6.2 6.8 + 0.8a 31.9 + 12.0 FER HC n=6 MER 13.1 + 2.5 24.9 + 4.9 1.0 + 0.3° 21.6 + 9.9 1.2 + 0.2 20.3 + 2.1 FER HCHP n=6 MER 11.4 + 1.5 28.3 + 5.9 1.0 + 0.3b 20.6 + 6.0 1.4 + 0.3 27.4 + 14.2 (1) FER = Fractional E s t e r i f i c a t i o n Rate (%/hr) (2) MER = Molar E s t e r i f i c a t i o n Rate [nmole (FC)/hr/ml plasma]. a = S i g n i f i c a n t l y d i f f e r e n t than the control groups (p<0.01) (CONT at day 70 i s compared to day 0) b = S i g n i f i c a n t l y less than the l e v e l at day 0 (p<0.05) - 76 -IV Haematological Changes 1. Complete Blood Count: Anaemia developed by day 70 i n the HC and HCHP groups (Table 11). There were s i g n i f i c a n t decreases i n erythrocyte count, haemoglobin concentration, and haematocrit at day 70 i n the HC and HCHP groups compared to the CONT and HP groups (p < 0.001). There was no sign of reduction i n any of these parameters i n any of these groups at day 30. Compared to day 0, the HP group at day 70 showed a s i g n i f i c a n t increase i n haematocrit (p<0.01). In experiment I I after treatment with acetylphenylhydrazine) there was a si g n i f i c a n t decrease at day 40 i n the erythrocyte count, haemoglobin concentration (p<0.01) and haematocrit (p < 0.001) compared to the values at day 0 (Table 12). Also, a remarkable increase i n the mean corpuscular volume of erythrocyte (p < 0.001) was found at day 40. This finding was i n accordance with the extensive r e t i c u l o c y t o s i s detected i n the treated group. Mean corpuscular haemoglobin was also s i g n i f i c a n t l y increased at day 40 i n these animals. 2. Erythrocyte Morphology and F r a g i l i t y : Numerous spiked-surface c e l l s (echinocytes) were seen i n the blood films of the HC and HCHP groups at day 30 (Figure 17). These echinocytes had a smaller size than normal c e l l s . P o i k i l o c y t o s i s , anisocytosis, and r e t i c u l o -cytosis were not evident at that time. Abnormalities i n size and shape of erythrocyte were more obvious at day 70. A population of target c e l l s (an erythrocyte with a dark centre surrounded by a haemoglobin-poor zone) was detected i n the HC and HCHP groups (Figure 18). These two animal - 77 -Table 11 [Complete Blood Count (CBC) and Erythrocyte Osmotic F r a g i l i t y . Mean _+ SD] Group Day 0 Day 30 Day 70 RBC1 count Hb 2 5.27 13.17 + +_ 0.19 0.54 5.37 + 0.33 13.7 + 0.7 5.43 + 0.23 14.42 + 1.1 CONT n=6 HTC3% MCV4 0.F5 41.4 79.2 62 + + + 1.7 2.7 4 43.4 + 1.9 80.7 + 3.4 61 + 2 44.4 + 2.6 81.7 + 2.9 63 + 3 RBC count 5.03 + 0.35 5.55 + 0.11 5.85 + 0.33 Hb 12.48 + 1.08 14.5 + 0.84 15.6 + 0.85 HP HTC% 40.4 + 3.2 45.4 + 1.5 48.2 + 3.6 n=6 MCV 80.8 + 2.9 82.0 + 1.6 82.8 + 1.8 O.F 58 + 7 62 + 5 66 + 2 RBC count 4.96 + 0.27 5.39 + 0.29 3.79 + 0.81 e Hb 13.12 + 1.01 13.4 + 0.78 7.9 + 2.14e HC HCT% 40.9 + 1.8 42.2 + 2.4 30.0 + 4.3 e n=6 MCV 82.7 + 1.5 78.1 + 0.8 84.2 + 3.8 56 + 5 b O.F 64 + 1 48 + 4 e RBC count 4.99 + 0.45 5.61 + 0.45 3.90 + 0.96e Hb 12.33 + 0.83 14.22 + 1.11 9.45 + 2.7 e HCHP HCT% 40.8 + 2.0 43.9 + 4.0 27.8 + 4.9e n=6 MCV 80.0 + 2.4 78.2 + 2.9 82.0 + 2.5 O.F 62 + 4 52 + 4° 57 + 4 (1) RBC count = RBC x 10 6/ml blood (2) Hb = Haemoglobin (gm/dL blood) (3) HCT% = Percentage of haematocrit (packed c e l l volume) (4) MCV = Mean Corpuscular Volume (femto l i t e r ) (5) O.F = Osmotic F r a g i l i t y x 10~ 3 (NaCl molar solution producing 50% haemolysis) b = S i g n i f i c a n t l y less than the values at day 0 (p<0.01) e = S i g n i f i c a n t l y different than the control groups (p<0.001) - 78 -Figure 17: A peripheral blood smear from an animal kept on a cholesterol-rich diet at day 30. A large number of echinocytes (spiked-surface erythrocytes) are present. (Wright-Giemsa Stain x 1000) Figure 18: A peripheral blood smear from a cholesterol-fed guinea pig revealing a target c e l l , few echinocytes, and stomatocyte. (Wright-Giemsa s t a i n x 1000). - 19 -- 80 -groups had another abnormal erythrocyte form, a hypochromic c e l l (HCC) (a bigger-than-normal erythrocyte with a remnant amount of haemoglobin located at the periphery of the c e l l ) . Morphologically, the HCHP group had more HCC than HC group (Figure 19). The number of echinocytes decreased by day 70 i n both groups. Reticulocytosis was also evident at day 70 i n the HC and HCHP groups, although i t s magnitude did not correspond to the degree of anaemia in these animals. However, re t i c u l o c y t o s i s was more marked i n the HCHP group than i n the HC group. As shown i n Table 11, erythrocyte osmotic f r a g i l i t y was s i g n i f i c a n t l y decreased at days 30 and 70 i n the HC group (p<0.001, <0.01, respectively) compared to the value at day 0. The animals of the HCHP group had a si g n i f i c a n t decrease only at day 30 (p<0.01) but not at day 70. 3. Erythrocyte L i p i d P r o f i l e : Table 13 summarizes the observed changes i n t o t a l cholesterol and phospholipids i n erythrocytes. A s i g n i f i c a n t increase i n cholesterol was found at days 30 and 70 i n the HC group compared to the CONT group and to the day 0 l e v e l (p<0.05). While the HCHP group showed the same increase at day 30, there was no s t a t i s t i c a l l y s i g n i f i c a n t change at day 70 compared to the CONT group. A negative i n s i g n i f i c a n t correlation was found between the increase i n erythrocyte cholesterol and the decrease i n osmotic f r a g i l i t y among the experiment groups ( r = -0.69, p> 0.1). I t appeared that increases i n erythrocyte TC and sp/pc were accompanied by the appearance of large numbers of echinocytes i n the peripheral blood, whereas the appearance of HCC was noticed when there was no s i g n i f i c a n t change i n TC and sp/pc. - 81 -Figure 19: A peripheral blood smear from the HCHP group at day 70. The echinocytes are diminished i n number, while the predominant erythrocyte form i s a hypochromic c e l l (HCC) (larger-than-normal erythrocyte with a fine rim of haemoglobin and a big pale center with a corrugated boundary). (Wright-Giemsa s t a i n x 1000). Figure 20: A peripheral blood smear from the HCHP group at day 70 showing a vacuolated monocyte. (Wright-Giemsa x 1000). - 82 -• t e a r * - 83 -4. Characterization of the Vacuolated White Blood C e l l s : Peripheral blood films from the HC and HCHP groups at day 70 stained with Wright-Giemsa st a i n showed vacuolated white blood c e l l s (WBC) with indented nuclei; unlike lymphocytes, these c e l l s have less compact nuclear chromatin, and the nuclei do not occupy the major part of cytoplasm, which seems agranular. Such c e l l was considered vacuolated when showing more than three cytoplasmic vacuoles (Figure 20). To i d e n t i f y the nature of the vacuole, blood films were fixed with formalin fumes and stained with O i l Red-0 (0R0). As i l l u s t r a t e d i n Figure 21, the vacuoles stained p o s i t i v e l y with 0R0. Further i d e n t i f i c a t i o n of these c e l l s was undertaken using nonspecific esterase s t a i n (NSE). As shown i n Figure 22, the cytoplasm of these c e l l s contained reddish brown dif f e r e n t - s i z e d p a r t i c l e s . Neutrophils, usually, show a mild positive reaction, but the extensiveness of the reaction and the morphological cha r a c t e r i s t i c s (as described above) suggest strongly that these c e l l s are monocytes laden with l i p i d p a r t i c l e s . These c e l l s were not seen i n peripheral f i l m of the HC group at days 5, 10 or 30. However, at day 70 these c e l l s with a 14.6% of peripheral blood monocytes were found i n the HC group. On the other hand, i n the HCHP group, lip i d - l a d e n monocytes (LLM) appeared with a 0.4% of peripheral blood monocytes at day 30, whereas at day 70 they showed a larger population of 31.2% of the monocytes. A positive correlation was found between the percentage of LLM among blood monocytes and the concentration of plasma TC at day 70 (r = 0.70, p<0.05). - 84 -Figure 21: A peripheral blood smear from a cholesterol-fed animal at day 70 depicting two monocytes i n the center of the f i e l d with intracytoplasmic Oil-Red 0 positive droplets (ORO-Stain x 1000). Figure 22: A peripheral blood smear from a cholesterol-fed animal at day 70. Two vacuolated NSE-positive monocytes are shown (NSE-stain x 1000). - 85 -- 86 -(AcetylphenylHydrazine (APH) - Induced Anaemia Experiment) Table 12 [Complete Blood Count. Mean + SD] Experi-mental Period RBCx 10 6 Hb(g/dL) HTC% MCVtfL 1) MCH(Pg2) Day 0 4.9 + 0.1 12.8 + 0.3 40.7 + 1.1 82.7 + 1.2 25.9 + 0.6 Day 40 3.9 + 0.2b 11.7 + 0.3b 35.9 + 0.9e 95.3 + 1.2e 30.7 + 0.9 (1) MCV (fl_) = Mean Corpuscular Volume (femtoliter) (2) MCH (Pg) = Mean Corpuscular haemolglobin (Picogram) n = 6 (number of the animals) b = S i g n i f i c a n t l y different than the values at day 0 (p<0.01) e = S i g n i f i c a n t l y d i f f e r e n t than the values at day 0 (p<0.001) - 87 -Experiment I Table 13 [Li p i d P r o f i l e of Erythrocyte. Mean + SD] Groups Day 0 TCl CONT n=6 SP/PC2 0.94 + 0.9 0.35 + 0.03 TC HP n=6 SP/PC 1.11 + 0.17 0.26 + 0.1 TC HC n=6 SP/PC 1.04 + 0.11 0.20 + 0.10 TC HCHP n=5 SP/PC 0.93 + 0.21 0.33 + 0.01 Day 30 Day 70 0.87 + 0.17 0.35 + 0.03 0.94 + 0.2 0.43 + 0.11 1.22 + 0.21 0.41 + 0.09 1.29 + 0.26 0.39 + 0.12 1.42 + 0.05a 0.57 + 0.11 a 1.77 + 0.5a 0.61 + 0.14a 1.61 + 0.37a 0.55 + 0.03a 1.39 + 0.32 0.38 + 0.04 (1) TC = Total Cholesterol (mg/mL packed RBC) (2) SP/PC = Spingomyelin/Phosphatidylcholine r a t i o a = S i g n i f i c a n t l y d i f f e r e n t than the control groups (p <0.05) - 88 -V Changes i n Liver Function: Liver function was assessed by measuring serum direct and in d i r e c t b i l i r u b i n , SGOT(AST), t o t a l protein, and albumin at different experimental periods (see "Methods"). The e a r l i e s t change was detected i n AST, namely, a si g n i f i c a n t increase (p<0.05) at day 5 of the experiment i n the HCHP group compared to the CONT group (Table 14). At day 10 both HC and HCHP groups showed a s i g n i f i c a n t elevation i n AST (p<0.01) compared to the CONT group. Such an increase with a higher l e v e l of significance was also present at day 30 and 70 i n the HC and HCHP groups. Interestingly, the HP group also showed a s i g n i f i c a n t increase i n AST at day 70 (p<0.02) compared to the CONT group. However, the CONT group showed a gradual increase i n AST throughout the experimental periods; the f i r s t s i g n i f i c a n t increase, compared to the day 0 l e v e l , was noticed at day 30 (p<0.01). The AST changes were the same i n the HCHP and HC groups. Indirect b i l i r u b i n increased s i g n i f i c a n t l y i n the HCHP group at day 70 compared to the HC group (p <0.002). Both groups also had an elevated direct b i l i r u b i n at day 70 compared to the CONT group. However, there was no s i g n i f i c a n t change between the HC and HCHP groups. Except for the day 70, no s i g n i f i -cant differences between the HC and HCHP groups were detected throughout the experimental periods. The increase i n in d i r e c t b i l i r u b i n ( i n the HCHP group) and direct b i l i r u b i n ( i n the HC and HCHP groups) appeared to be correlated with the severity of haemolysis i n both groups (see "Discussion" for further analysis of the data). Table 14 also shows the serum t o t a l protein and albumin levels at different periods. No change i n albumin was found between the groups at - 89 -Table 14 [Liver Function Tests at Different Periods. Mean + SD] Days Group I n d . b i l i D . b i l i SGOT(AST) TP Alb. n=13 CONT** .08 + .02 .09 + .02 100.4 + 49 4.6 + 0.3 2.3 + .1 5 n=5 HC .07 + .01 .08 .02 86.5 + 37.9 4.7 + .19 2.3 + .07 n=5 HCHP .09 + .02 .09 + .03 117.2 + 74.6 a 4.7 + .2 2.4 + .2 10 n=5 HC .08 + .03 .09 + .01 225 + 51 b 4.8 + .2 b 2.3 + .1 n=5 HCHP .09 + .02 .09 + .02 181 + 44.8 b 5.1 + .3 b 2.4 + .1 30 n=5 HC .09 + .01 .09 + .02 366.6 + 105 b 4.82 + • .2 b 2.4 + .1 n=5 HCHP .09 + .02 .09 + .03 316 + 83.4 b 5.03 + 2.5 .14 70* n=6 HC .12 + .05 .3 + .02 b 510 + 30 b 5.4 + .2 b 2.6 + .1 n=6 HCHP .5 .3 f .7 + .5 b 510 + 10 b 5.3 + .2 b 2.4 + .1 n=5 HP .09 + .02 .3 + .2 209 + 15 5.4 + .2 2.6 + .1 n = Number of animals/group * = Data from experiment I I I SGOT(AST) = Aspartate transaminase (IU/L) Ind. b i l i = Indirect b i l i r u b i n (mg/dL) D. b i l i = Direct b i l i r u b i n (mg/dL) TP = Total protein (gm/ L) Alb = Albumin (gm/ L) a = S i g n i f i c a n t l y d i f f e r e n t than CONT (p<0.05) b = S i g n i f i c a n t l y d i f f e r e n t than CONT (p<0.01) f = S i g n i f i c a n t l y d i f f e r e n t than HC at day 70 (p<0.002) ** = Data of days 5,10,30 and 70 are combined due to the absence of si g n i f i c a n t difference - 90 -a l l periods, whereas t o t a l protein i n the HC and HCHP groups was s i g n i f i -cantly increased (p<0.01) at days 10, 30 and 70 compared to the CONT group. Taken together, although hepatic injury was evident by the increase i n AST p a r t i c u l a r l y i n the HCHP and HC groups, hepatic f a i l u r e was unl i k e l y , as indicated by the normal l e v e l of albumin and normal conjugation response for the increase i n i n d i r e c t b i l i r u b i n i n the HCHP group. - 91 -VI Renal Function Changes i n blood urea nitrogen (BUN), serum creatinine (Cr), 24-hour urinary protein (UPr), creatinine clearance (Ccr) and haematuria are shown i n Table 15. 1. Urinary Findings: Twenty-four-hour urinary protein s i g n i f i c a n t l y increased at day 70 i n the HC and HCHP groups (p<0.001) compared to the CONT and HP groups (Figure 23). A markedly elevated UPr was found i n the HCHP group at day 70 compared to the HC group (p <0.001). Despite the tendency to show higher UPr than CONT group, the difference was not s t a t i s t i c a l l y s i g n i f i c a n t i n the HP group. Twenty-four-hour urinary protein was not measured before day 70 because the semiquantitative data obtained i n the preliminary experiments showed absence of s i g n i f i c a n t proteinuria. No s i g n i f i c a n t change could be detected i n Ccr at day 70 between the four groups, except for a tendency toward an increase i n the HP group . This was not the case with haematuria as an indicator for glomerular basement membrane damage. Semiquantitatively, HC group had at day 70 (1+) l e v e l of erythrocyte in urine compared to the (-) l e v e l i n the CONT and HP groups, while the HCHP group showed haematuria of (4+) l e v e l . This finding was po s i t i v e l y correlated (r = 0.73, p<0.01) with the mesangial area/glomerular area r a t i o . Again, no haematuria was detected i n the preliminary study (up to day 30). Fig - 23 - 93 -Table 15 [Kidney Function Tests at Day 70. Mean + SD] Group C c r 1 U.Prot. 2 BUN3 C r 4 RBC/urine 5 CONT 8.3 + 0.4 6.4 + 2.3 18 + 2.8 0.3 + 0.05 (-) (n=4) HP 10.2 + 4.9 13.7 + 6.3 38.7 4.5 e 0.27 0.09 (-) (n=5) HC 9.4 + 2.3 22.1 + 7.2e 36.6 + 2.79 0.24 + 0.05 ( + ) (n=6) HCHP 8.2 + 3.7 57.9 + 10.3 e 64 276 0.25 + 0.1 (4+) (n=6) (1) Ccr = Creatinine Clearance (mL/min) (2) U.Prot. = Twenty four-hour urinary protein (mg/24hr) (3) BUN = Blood Urea Nitrogen (mg/dL) (4) Cr = Serum creatinine (mg/dL) (5) RBC/Urine = Number of erythrocytes i n 24-hr urine sample (high-power f i e l d ) n = Number of animals/group e = S i g n i f i c a n t l y d i f f e r e n t than other groups (p<0.001) g = S i g n i f i c a n t l y different than CONT (p<0.02) - 94 -2. Serum Findings: As shown i n Tables 15 and 16, BUN was increased i n the HCHP group at days 5, 10, 30 and 70. This increase was s i g n i f i c a n t l y different from that i n HC and CONT groups (p < 0.001), while i n the HC group the increase was si g n i f i c a n t (p<0.02) only at day 70. Both HC and HCHP groups showed a si g n i f i c a n t increase i n BUN r e l a t i v e to the CONT group at day 70. However, t h i s was less than i n the HCHP group (p< 0.001). Serum creatinine (Tables 15 and 16) did not show a s i g n i f i c a n t change i n any of the groups throughout the different experimental periods. The HP, HC, and CONT groups showed a si g n i f i c a n t increase i n Cr at days 30 and 70 compared to that at days 0 and 10 (p <0.01), whereas there was no change i n t h i s parameter i n the HCHP group. Serum phosphate: No s i g n i f i c a n t change was found between the different experimental groups. VII Renal Tissue Alterations 1. Histopathology, histochemistry, histoimmunofluorescence,' and electron microscopy: H i s t o l o g i c a l sections of kidneys from the CONT group revealed normal glomeruli and renal tubules (Figure 24). The glomeruli had patent c a p i l l a r y lumina with a modest amount of mesangial matrix and c e l l u l a r i t y . The HP group had a si m i l a r normal kidney structure except for enlarged glomeruli. At day 70, the histopathological changes were variable i n the HC and HCHP groups. The increase i n mesangial matrix and c e l l u l a r i t y varied from glomerulus to glomerulus. Glomerular hype r c e l l u l a r i t y (Figure 25) i n the HC and HCHP groups was constituted by an increased number of mesangial and - 95 -Table 16 [Blood Urea Nitrogen and Serum Creatinine. Mean + SD] Group Day 5 CONT BUN (n=9) Cr 16.7 + 2.6 0.2 + .07 HC BUN (n=5) Cr 15.8 + 1.6 .3 + .07 HCHP BUN (n=5) Cr 28.2 + 7.66 .18 + .04 Day 10 Day 30 16.7 + 2.6 0.2 + .07 16.7 + 2.6 0.2 + .07 15.7 + 1.2 .15 + .02 20.8 + 1.3 .3 + .07° 32.5 + 5.8e .13 + .05 40.3 + 3.6e .2 + .07 n = Number of animals/group BUN = Blood Urea Nitrogen (mg/dL) Cr = Serum Creatinine (mg/dL) b = S i g n i f i c a n t l y d i f f e r e n t than days 5 and 10 (p<0.01) e = S i g n i f i c a n t l y d i f f e r e n t than other groups (p< 0.001) ** = Data of days 5,10,30 and 70 were combined due to the absence of s i g n i f i c a n t changes among the animals of the control group - 96 -in t r a c a p i l l a r y c e l l s - mostly mononuclears with few neutrophils. Intracyto-plasmic vacuoles were noted i n some i n t r a c a p i l l a r y and mesangial c e l l s ; few vacuoles were e x t r a c e l l u l a r l y distributed. The i n t r a c a p i l l a r y vacuolated c e l l s were mononuclear leukocytes and endothelial c e l l s . No changes were noted i n the i n t e r s t i t i u m , tubules or small art e r i e s or a r t e r i o l e s except for an occasional intracytoplasmic vacuole i n a r t e r i o l a r endothelium. Mild mesangial expansion was detected i n both HC and HCHP groups at day 30, but there were no changes either at day 10 or 5. Occasional extramedullar haematopoiesis was found i n the glomeruli of the HCHP and HC group at day 70 (Figure 26). In the animals injected with APH, there were no sa l i e n t glomerular h i s t o l o g i c a l a l t e r a t i o n s . However, t h e i r renal tubular e p i t h e l i a l c e l l s were loaded with Perl's Prussian blue (PPb) positive p a r t i c l e s (Figure 27). Oil-red 0 (0R0) staining was dif f u s e l y positive i n glomeruli and fo c a l l y present i n tubules and a r t e r i o l e s of the HC and HCHP groups at day 70 (Figure 28 and 29). A positive 0R0 reaction, although variable was detected predominantly i n the glomeruli and some tubules at day 30 i n both HC and HCHP groups. Also some animals of these two groups showed mild staining for 0R0 at day 5 and 10. Frozen h i s t o l o g i c a l sections from the CONT group were negative for t h i s s t a i n . Proximal tubular e p i t h e l i a l c e l l s of the CONT group p o s i t i v e l y showed a strong diffuse cytoplasmic reaction for NSE s t a i n . However, most of the glomeruli i n the CONT group had no NSE-positive c e l l s . On the contrary, many glomeruli i n the HC and HCHP groups at day 70 contained moderate numbers of NSE-positive c e l l s (Figure 30). Some of these c e l l s were vacuolated. There was no s i g n i f i c a n t - 97 -Figure 24: A kidney section from the CONT group at day 70. A normal glomerulus i s depicted. (H&E x 250). Figure 25: A kidney section from the HCHP group at day 70. The glomerulus i s enlarged and hypercellular (H&E x 250). - 98 -- 99 -Figure 26: A kidney section from the HCHP group at day 70 stained with H&E. Note the presence of megakaryocytes (arrows) indicating extramedullary haematopoiesis (H&E x 400). Figure 27: A kidney section from a cholesterol-fed guinea pig at day 70 showing the presence of Perl's Prussian blue-positive deposits i n the e p i t h e l i a l c e l l s of the renal tubules. No positive reaction i s present i n the affected glomerulus (Perl's Prussian blue x 250). - 100 -- 101 -Figure 28: A frozen kidney section from a cholesterol-fed animal at day 70. The glomerulus i s f i l l e d with Oil-Red 0 positive multi-size p a r t i c l e s (0R0 Stain x 250). Figure 29: A frozen kidney section from the HCHP group at day 70 depicts renal tubules f i l l e d with Oil-Red 0 positive p a r t i c l e s which are found i n the e p i t h e l i a l c e l l s and inside the tubular lumen (0R0 Stain x 250). - 102 -- 103 -Figure 30: A glomerulus with NSE positive c e l l s i n the kidney of a cholesterol-fed guinea pig at day 70. One of these c e l l s appears i n the center with two intracytoplasmic vacuoles (arrow) (NSE x 400). - 104 -- 105 -Figure 31: A kidney section from the HCHP group at day 70 stained with PAS s t a i n . The mesangium i s expanded (PAS x 400). - 106 -- 107 -increase i n the number of intraglomerular NSE-positive c e l l s at day 30 i n either of HC or HCHP groups. More extensive PPb-positive deposits were found in the renal tubular epithelium of the HCHP group than i n the HC group at day 70. No such deposits were present at day 30. The PAS sta i n revealed an increase i n mesangial matrix i n the HCHP and HC groups compared to other groups at day 70 (Figure 31). No von Kossa-positive lesions were detected in either HC or HCHP groups. The histoimmunofluorescence study showed that there was no staining for guinea pig IgG in the kidneys of the HC and HCHP groups. The normal glomerular ultrastructure of a CONT animal i s depicted i n Figure 32. At day 70 i n both HC and HCHP groups (Figures 33-37), clear vacuoles were present inside i n t r a c a p i l l a r y monocytes, endothelial c e l l s and the mesangial c e l l s . Some of these mesangial c e l l s had the cytoplasmic cha r a c t e r i s t i c s of monocytes (lysosomes, rough endoplasmic reticulum and ve s i c l e s ) . The increase i n the mesangial matrix was variable i n the HC and HCHP groups. There were no electron dense deposits nor was there any si g n i f i c a n t change i n the glomerular basement membranes (GBM) i n the test groups. However, fusion of foot processes was evident. Some i n t r a c a p i l l a r y mononuclear c e l l s had large number of ribosomes but no rough endoplasmic reticulum, lysosomes, ve s i c l e s , or filaments. They were most l i k e l y lymphoid or haematopoietic c e l l s . 2. Morphometry: The data of the morphometric analyses of a l l groups throughout the different experimental periods are presented i n Table 17. Mesangial area/ glomerular t u f t area (MA/GTA) r a t i o was s i g n i f i c a n t l y higher i n the HC and - 108 -Figure 32: A glomerulus of a control animal at day 70. Capillary loops are widely patent and foot processes are i n t a c t . There was no increase i n mesangial matrix. (EM x 3000). Figure 33: A glomerular endothelial c e l l with multi-sized l i p i d droplets from a cholesterol-fed animal at day 70. There i s focal fusion of the foot processes. (EM x 4400). - 109 -- 110 -Figure 34: A glomerulus from a cholesterol-fed animal at day 70. Note the i n t r a c a p i l l a r y monocytes each of which contains some clear vacuoles. (EM x 3000). - I l l -- 112 -Figure 35: Ult r a t h i n section from kidney of the HC group at day 70 reveals the expanded mesangium. (EM x 3000). - 113 -- 114 -Figure 36: An electron micrograph reveals a vacuolated c e l l with some of the charac t e r i s t i c s of a monocyte i n the expanded glomerular mesangium i n a HCHP animal at day 70. (EM x 7000). Figure 37: Erythrophagocytosis i n the mesangium of an animal i n the HCHP group at day 70. Note a macrophage with at least three l i p i d droplets engulfing an erythrocyte. (EM x 7000). - 115 -- 116 -HCHP groups than i n the CONT and HP groups at day 70 (p < 0.001). The (MA/GTA) r a t i o in the HCHP group was higher than i n the HC group at day 70 (p<0.05). There was a good correlation between MA/GTA and the number of intraglomerular NSE-positive c e l l s (r = 0.719, p < 0.001) over the entire experimental period. At day 30, the MA/GTA r a t i o was s i g n i f i c a n t l y increased in the HC group compared to the CONT group (p<0.01). Also, t h i s r a t i o was higher i n the HCHP group than in the HC group at day 30 (p<0.01). No si g n i f i c a n t increase i n MA/GTA r a t i o was detected prior to day 30 i n a l l the groups. In both HC and HCHP groups, the number of mesangial c e l l s was s i g n i f i -cantly increased compared to the CONT and HP groups (p<0.01) at day 70 (Table 17). A s i g n i f i c a n t increase was also found at day 30 i n the HCHP group (p<0.001) and HC group (p<0.05). The MA/GTA r a t i o correlated i n d i v i d u a l l y with serum TC (r = 0.790, p<0.001), tissue TC (r = 0.792, p<0.001), and c o r t i c a l CE% (r = 0.409, p<0.01). However, with multiple regression analysis, only correlations of MA/GTA with tissue TC (r = 0.490, p<0.01) and NSE-positive c e l l s (r = 0.239, p<0.05) were s i g n i f i c a n t . The l e v e l of proteinuria correlated i n d i v i d u a l l y with tissue TC (r = 0.756, p<0.001), MA/GTA (r = 0.785, p<0.001) and NSE-positive c e l l s (r = 0.519, p<0.05). None of the l a t t e r three parameters showed s i g n i f i c a n t independent correlation with urinary protein, using multiple lin e a r regression analysis. The animals injected with APH did not show any morphometric change compared to the values found i n the animals who received normal diet and were not subjected to t h i s treatment (Table 18). - 117 -Table 17 [Kidney Morphometry at Different Experimental Periods. Mean +_ SD] Group Day MA/GTA 2MC 3NSE/glomerular section CONT 5- 1 0n=15 .16 + .05 10.3 + 1.5 3 0n=16 .17 + .04 10.1 + 2.7 .11 + .07 * 7 0n=48 .17 + .04 18 + 5 .45 + .07 HC (5-10)n=30 n=25 .17 .20 + + .03 .03 b 12.8 13 + + 1.9 2.8a .28 + .1 .41 + .27 * 7 0n=56 .28 + .08e 32 + 8 b 1.30 + 0.83 HCHP 5 - 1 0 n = 3 1 .15 + .04 11.5 + 2.8 .37 .+ .21 3 0n=24 .25 + .07b 19.8 + 2.9 e .32 + .19 * 7 0n=55 .32 + . l l e 39 + 17 b 1.90 + 0.61 HP * 7 0n=30 .18 + .03 21 + 7 .40 + 0.08 (1) Mesangial area/glomerular t u f t area (2) Number of mesangial cells/glomerular section (3) Number of (Non-specific esterase)-positive cells/glomerular section * = Data from Experiment I I I n = Number of Glomeruli Examined a = S i g n i f i c a n t l y different than CONT (p<0.05) b = S i g n i f i c a n t l y different than the control groups (p<0.01) e = S i g n i f i c a n t l y different than CONT (p<0.001) - 118 -3. Kidney L i p i d Analysis: The l i p i d content of whole-kidney tissue (TC, FC, CE%, TPL, TG) i n the various groups of animals at day 70 (experiment I) i s shown i n Table 19. The HC and HCHP groups showed a s i g n i f i c a n t increase (p < 0.05, < 0.01, respectively) i n TC and CE% compared to the CONT and HP groups. Except for the HCHP group, FC did not s i g n i f i c a n t l y increased i n other groups. S i m i l a r l y , TPL was increased at p<0.05 l e v e l i n the HCHP group r e l a t i v e to the CONT groups, while no s i g n i f i c a n t changes were found i n these groups, although there was a tendency to higher values i n the HC group. Representative pieces of renal cortex i n experiment I I I were analysed for l i p i d content. Cortex was chosen to exclude the possible l i p i d contribution by the medullary portion of the renal tubules. The l i p i d changes i n cortex paralleled those i n the whole-kidney tissue (experiment I ) . There was a 10-20% reduction i n the levels of the different l i p i d components - a finding which demonstrates that the bulk of the l i p i d content was in the cortex (Table 20). As summarized i n Table 20, l i p i d p r o f i l e of the renal c o r t i c a l tissue did not s i g n i f i c a n t l y change after 5 and 10 days on the respective diets. Both HCHP and HC groups had an increased percentage of CE r e l a t i v e to the CONT group (p<0.05) at day 30. On the other hand, although there was a trend toward an elevation i n FC and TPL i n the HC and HCHP groups, t h i s increase was not s t a t i s t i c a l l y s i g n i f i c a n t (p>0.1). Table 18 shows the l i p i d contents of the whole-kidney extract i n the APH-treated animals (experiment I I ) . No s i g n i f i c a n t change i n any of the l i p i d parameters (examined at day 40) was found compared to the levels i n the CONT group. - 119 -Table 18 APH-Induced Anaemia Experiment [Kidney L i p i d P r o f i l e (ug/mg protein) and Kidney Morphometry at Day 40. Mean + SD] n = 6 TC FC CE% PL TG 4.4 + 0.3 4.1 + 0.3 7 + 1.1 27.2 + 1.7 7.4 + 1.8 Total c e l l count (GC) Mesangial C e l l Count (MC) MA/GTA 32.3 + 7.9 15.3 + 5 0.14 + 0.03 N.B. Data are compared to those of the control group i n tables 17 and 20. - 120 -Table 19 [Kidney L i p i d P r o f i l e at Day 70. Mean + SD] Groups TCl FC2 CE%3 TPL 4 TG^ CONT 4.3 + 1.1 3.8 + 1.2 10 + 5.3 31.4 + 4.6 10.9 + 3.3 (n=6) HP 5.4 + 0.5 4.9 + 0.4 10.1 + 3.8 32 + 10.9 14.6 + 7.1 (n=6) HC 6.2 + 1.5 4.7 + 1.6 23.6 + 37.7 + 5.1 16.7 + 9.5 (n=6) HCHP 8.1 + 1.7 6.5 + 1.5a 20 + 5.3 d 40.5 + 6.73 8.8 + 3.7 (n=6) (1) TC = Total Cholesterol (ug/mg protein) (2) FC = Free Cholesterol (ug/mg protein) (3) CE% = Percentage of Cholesteryl Ester (4) TPL = Total Phospholipids (ug/mg protein) (5) TG = Triglycerides (ug/mg protein) a = S i g n i f i c a n t l y d i f f e r e n t than CONT (p< 0.05) b = S i g n i f i c a n t l y different than the control groups (p<0.01) - 121 -Table 20 [L i p i d P r o f i l e of Renal Cortex (ug/mg protein). At Different Periods. Mean + SD] Group Day TC FC CE% TPL CONT** (n=13) 3.0 + 0.5 2.7 + 0.5 10 + 5 20.6 + 6.1 5(n=5) 3 + .7 2.7 + .5 9.6 + 4.1 20 + 7.1 HC 10(n=5) 3.5 + .4 3.1 + .6 10.9 + 6.1 23 + 7 30(n=5) 4 + 1 3.15 + 1.1 21.1 + 9.8a 24 + 7.7 *70(n=6) 5.4 + 1.2 4.0 + 1.4 26.8 + 10.9° 29 + 6.2 5(n=5) 2.8 + .4 2.5 + .3 10.3 + 3.5 19.5 + 6 HCHP 10(n=5) 3.8 + .7 3.35 + .4 11 + 6 25 + 4.9 30(n=5) 5 +_ 1.2 3.93 + 1.5 20 + 7.ia 25 + 9 *70(n=6) 7 + 2 5.6 + 1.63 21 + 5.6b 31 I 7 3 HP *70(n=5) - 4 + 1.1 3.7 + 1.2 10.2 + 4 24.6 + 9 n = Number of animals/group * = Data from Experiment I I I TC = Total Cholesterol (ug/mg protein) FC = Free Cholesterol (ug/mg protein) CE% = Percentage of Cholesteryl Ester TPL = Total Phospholipids (ug/mg/protein) a = S i g n i f i c a n t l y d i f f e r e n t than CONT (p<0.05) b = S i g n i f i c a n t l y different than CONT (p<0.01) ** = Data of days 5,10,30 and 70 were combined due to the absence of si g n i f i c a n t changes among the animals of the control group. - 122 -VIII Renal de novo Cholesterol Synthesis As described i n the "Methods", two different procedures (assay of HMG COA reductase and incorporation of 3H-water into cholesterol) were used. The purpose of these experiments was to assess tissue cholesterol synthesis i n the control animals and compare i t with that i n the HP, HC, and HCHP groups. Table 21 summarizes the findings. In comparison to the CONT group, HMG COA reductase a c t i v i t y at day 70 was s i g n i f i c a n t l y decreased i n the HC group (p <0.001), while that of the HCHP group decreased less markedly (p<0.05). The HCHP group's de novo cholesterol synthesis was higher, by both methods, than that of the HC group (p<0.001). Kidneys from the HP group had lower HMG-COA reductase a c t i v i t y than the CONT or HCHP groups (p <0.001 and p<0.01, respectively). These findings were i n good agreement with the data from a separate experiment i n which the rate of incorporation of t r i t i a t e d water into cholesterol was measured. In general, there was a s i g n i f i c a n t positive correlation (r = 0.91, p<0.05) between the data obtained by the two methods. These findings indicated that cholesterol feeding suppressed de novo cholesterol synthesis i n renal tissue. This suggests that the bulk of the accumulated l i p i d (as described i n the previous section) i s derived from plasma. This observation was strongly supported by the poor correlation between the parameters of de novo synthesis and those of the c o r t i c a l l i p i d p r o f i l e . - 123 -Table 21 [de novo Cholesterol Synthesis i n renal tissue at Day 70] Group HMG-CoAR.1 T.H20 Inc. 2 CONT 40.8 + 2.7 (n=3) 82.9 + 6.3 (n=2) HC 29.9 + 4.1 (n=5) e 18.5 + 3.6 (n=3) HP 24.4 + 2.7 (n=3) e 42.5 + 3.9 (n=2) HCHP 36.7 + 2.5 (n=5) a 44.1 + 6.3 (n=3) (1) HMG-CoAR. = 13-hydroxy methyl g l u t a r y l COA reductase (Pmol/mg protein/min) (2) T.H2O inc. = T r i t i a t e d water incorporation i n cholesterol (nmol/gm/hr) * = Mean + SD n = Number of animals/group a = S i g n i f i c a n t l y different than CONT (p<0.05) e = S i g n i f i c a n t l y d i f f e r e n t than CONT (p<0.001) -123A-DISCUSSION - 124 -DISCUSSION y- 1^ General Observations: The groups of animals receiving a cholesterol-rich diet grew at an equal rate to that of the control group for 30 days. Afterwards they f a i l e d to maintain a normal weight gain. This f a i l u r e was quite marked at day 70. These data agree with those obtained by other investigators (66,77,98,99). However, i n none of the previous studies were the changes i n body weight recorded before day 60, except for Matin and Ostwald (1975) who reported that cholesterol-fed guinea pigs showed a s i g n i f i c a n t reduction i n weight gain at the end of a 33-day experiment (100). Although there was no marked change i n erythrocyte count at day 30, the present study and others (66,77) provided good evidence for the correlation between the decrease i n weight gain and the severity of anaemia p a r t i c u l a r l y a fter 60 days of feeding. In t h i s investigation marked anaemia was noticed at day 70. However, morphological changes i n erythrocytes were already present at day 30. Such changes might have the potential to decrease the functional efficiency of erythrocyte which could, i n part, contribute to the f a i l u r e to t h r i v e . Moreover, acetyl phenylhydrazine-induced anemia was also accompanied by a s i g n i f i c a n t decrease i n weight gain. Additional evidence for the possible correlation between haemolytic anaemia and the decrease i n weight gain comes from the data on protein supplementation. High protein supplements resulted i n increased severity of haemolysis i n the cholesterol-fed animals; these animals also gained the least weight. The dual effect of high protein supplementation i n cholesterol-fed animals on weight gain and the degree of haemolysis has not been previously thoroughly investigated. - 125 -However, i t has been reported that neither anaemia nor reduction i n weight gain of cholesterol-fed guinea pigs were improved by casein enrichment of diet (101). Another report pointed out that a casein-rich diet suppressed the a b i l i t y to gain weight i n rabbit (102). Our data suggest that high casein supplementation per se does not s i g n i f i c a n t l y affect the a b i l i t y to gain weight. In addition, since there was no detectable change i n either erythrocyte morphology or count i n the group receiving high protein diet alone (HP), i t i s tempting to attribute the f a i l u r e to thrive to haemolytic anaemia as a major cause i n both HC and HCHP groups. Other causes for the reduction i n weight gain i n cholesterol- or cholesterol/protein-fed animals can be postulated. At least one report has pointed out a reduction of food intake after cholesterol feeding i n guinea pigs (73), although the workers did not elaborate on that observation. Such a finding, again, may be attributed to anaemia which i s known to cause anorexia. Also, anorexia i n uraemic .rats fed high protein diet has been suggested as a cause for stunted growth (103). This p o s s i b i l i t y i s unlikely i n our animals, since they did not develop uraemia. In conclusion, the data presented i n t h i s study are i n favor of the causal relationship between haemolytic anaemia and f a i l u r e to t h r i v e . The l i v e r and spleen of the experimental groups were markedly affected by both cholesterol and cholesterol/protein feeding. A gradual increase i n the weight of l i v e r and spleen from day 30 to day 70 was noticed i n animals kept on these two dietary regimens. The l i v e r enlargement has been described i n several previous reports (23,66,77,99,101) and has been attributed to massive fatty i n f i l t r a t i o n . Macroscopically, the l i v e r appears pale yellow and greasy. Unlike an e a r l i e r report (66) we did not - 126 -observe necrotic areas under gross examination. The enormous enlargement of spleen can be explained by the destruction of the modified erthrocytes. The enlargement of spleen i n animals fed either a cholesterol-rich diet alone or with a protein supplement was noticed for the f i r s t time at day 30 concommitantly with the morphological changes i n erythrocytes. The early splenomegaly was modest and probably represented the early stages of erythrophagocytosis and extramedullary haematopoiesis. These findings are in agreement with other studies (66,77), although i n those studies the gross changes were not monitored i n the early stages of the experiment. A mortality rate of approximately 10% was recorded among the cholesterol-fed guinea pigs. E a r l i e r reports on such animals mentioned different mortality rates. Approximately 30% of experimental animals died within 8-10 weeks of cholesterol/fat feeding i n Ostwald and Shannon's study (66); other workers found that the animals became more susceptible to infection (77). On the other hand, Drevon and Hovig (1977) reported no deaths among albino guinea pigs kept on 1% cholesterol-rich diet (99). Mortality i n cholesterol-fed guinea pigs might be attributed to haemolytic anaemia which has been consistently reported i n t h i s experimental animal model. Consistent with t h i s i s the observation that the mortality rate was higher i n the group that received a high protein supplement i n addition to the cholesterol diet. In t h i s group, the haemolysis i s more severe suggesting that the differences i n the mortality rates between the two groups were related to the severity of haemolysis. However, another possible cause for mortality i n our animals i s in f e c t i o n , but on necropsy, no gross pathological lesions were detected i n the viscera of the dead animals. Although t h i s finding decreases the li k e l i h o o d of in f e c t i o n as a leading cause of death, i t does not exclude - 127 -such a p o s s i b i l i t y from contributing to the increased mortality rate. Thus, we conclude that anaemia was most l i k e l y the main cause of death with the p o s s i b i l i t y of in f e c t i o n as a contributory factor. I I . Plasma L i p i d Abnormalities 1. Effect of the cholesterol-rich diet: As i n other studies (98,67,104), the cholesterol-fed guinea pigs showed high levels of plasma TC and FC compared to the control group. A gradual increase i n plasma TC was found at days 5,10,30 and 70 of the experiment. E a r l i e r i t had been pointed out that cholesterol-fed guinea pigs develop hypercholesterolemia accompanied by the appearance of new lipoprotein species r i c h i n unesterified cholesterol (50,69,76). In these reports of the abnormal lipoproteins VLDL and LDL were the most thoroughly studied. I t has been suggested that hypercholesterolaemia might be brought about i n part by dietary cholesterol suppression of LDL receptors (105,106). This suggestion i s supported by the data i n cholesterol-fed rabbits (107) and dogs (108) which showed an elevation i n IDL and LDL due to down regulation of LDL receptors. In t h i s regard, Terpstra and Beynen (1984) reported that most of the cholesterol i n cholesterol-fed guinea pigs was found i n the LDL fraction (104). The appearance of abnormal LDL and VLDL was observed i n the hypercholesterol-anemic animals (discussed further, below, i n t h i s section). Moreover, i n agreement with previous findings (104, 108), HDL-cholesterol was increased i n our animals. Thus, our results confirm that cholesterol-rich LDL i s the main source of the elevated plasma TC. This may be due to suppression of LDL uptake i n addition to increased intake of cholesterol. However, since the percentage of plasma unesterified - 128 -cholesterol (FC%) i n the HC group was higher than i n the CONT group, the increase in serum FC and subsequently TC cannot be explained by merely the retention of lipoproteins i n c i r c u l a t i o n . Rather, the explanation should include the e s t e r i f i c a t i o n a c t i v i t y . Despite the possible i n t r a c e l l u l a r e s t e r i f i c a t i o n of FC in jejunum (109) and l i v e r (98) by acyl COA:cholesterol acyltransferase (ACAT), the FC% was high i n HC group. This observation suggests a reduction i n cholesterol e s t e r i f i c a t i o n i n plasma. Our data showed that LCAT a c t i v i t y (as FER) was variably affected depending on the type of substrate used for the assay. Using endogenous substrate (whole plasma), FER was remarkably reduced i n HC group, while there was no s i g n i f i c a n t change i n MER. This i s i n agreement with the findings of Ostwald et a l . (1979) who showed an eightfold reduction i n the rate of free cholesterol e s t e r i f i c a t i o n i n cholesterol-fed guinea pigs using autologous plasma i n the enzyme assay (101). Also, they demonstrated no reduction i n MER, a finding which was also confirmed i n Drevon's report (98). Our data and these reports indicate that absence of a s i g n i f i c a n t reduction i n MER could be due to an increased l e v e l of FC i n plasma lipoproteins. Such an increase i n FC has been described as an inh i b i t o r y factor for LCAT a c t i v i t y i n humans and rat plasma due to decreased phospholipids: FC r a t i o i n the lipoproteins (110). In t h i s regard, the issue of LCAT substrate i n guinea pigs i s of a special inte r e s t . Due to the v i r t u a l absence of HDL i n normal guinea pig (69), and the a b i l i t y of apo-Cl in VLDL to activate LCAT (44), i t was suggested that VLDL normally acts as a substrate for LCAT i n guinea pigs (109). However, a lipoprotein species with a hydrated density of 1.063-1.21 gm/ml was detected i n t h e i r c i r c u l a t i o n (97). In cholesterol-fed guinea pigs, both VLDL and t h i s - 129 -lipoprotein species are cholesterol-rich (69). Thus, neither of the two lipoproteins i s a good substrate for LCAT a c t i v i t y . This indicates that using an endogenous substrate (whole plasma) i n our study may not be the optimal way to measure LCAT a c t i v i t y , since free cholesterol-rich lipoproteins were found i n our guinea pigs (discussed below). Therefore we used an exogenous ( a r t i f i c i a l ) substrate to measure LCAT a c t i v i t y . This assay has also been generally accepted as an approximation of LCAT mass (111,112). Employing t h i s method, LCAT a c t i v i t y (FER and MER) was s i g n i f i c a n t l y reduced i n the HC group at day 70. Although the reduction was moderate, i t l i k e l y reflected a reduction i n the enzyme mass. This could be due to a massive fatty degenerative change of the l i v e r (113). On the contrary, a misleading estimate of the enzyme mass i s achieved by applying an endogenous-substrate assay. Our data on LCAT a c t i v i t y i n the CONT and HP groups using an endogenous-substrate assay showed a s l i g h t reduction i n FER in the absence of detectable l i v e r lesions. This observation indicates a poor correlation between the reduction i n FER and the enzyme mass, and suggests that the type of substrate used affected the enzymatic reaction. In fact, plasma TC at day 70 was higher i n the HP group than i n the CONT group (see the second part of t h i s section for further discussion), and was s l i g h t l y increased i n the CONT group at day 70 compared to day zero. A sim i l a r elevation i n plasma cholesterol with age has been previously reported (114-117). In summary, the s l i g h t increase i n FC% i n plasma at day 70 i s apparently due to an absolute increase i n plasma FC and a moderate reduction i n LCAT a c t i v i t y which might be due to the fatty degenerative change found i n the l i v e r of cholesterol-fed animals. We recommend using an a r t i f i c i a l substrate - 130 -for measurement of LCAT a c t i v i t y to obviate the v a r i a b i l i t y i n the nature of substrates i n autologus plasma. Different alterations were observed i n the lipoproteins of HC group at dif f e r e n t experimental periods. Plasma cholesterol of HDL (HDL-C) was gradually elevated from day 30 to day 70. This finding i s i n agreement with the data obtained by other groups (104,118). Terpstra and Beynen (1984) suggested that cholesterol feeding to guinea pigs increased the density of HDL which led to the appearance of HDLc p a r t i c l e s r i c h i n apo E (104). In t h i s regard, our data on the HC group show a protein fraction with a molecular weight close to that of apo E found i n the lipoprotein species with a hydrated density 1.063-1.21 gm/ml. The same finding has been described e a r l i e r (69,119). In the present investigation, the VLDL fraction showed the e a r l i e s t electrophoretic alterations i n the HC group. A slow prebeta band appeared by day 10 on agarose gel. This abnormally migrating prebeta has been described e a r l i e r i n cholesterol-fed guinea pigs (68,69), and recently i n rabbits, dogs, rat s , and monkeys fed cholesterol-rich diets (120). I t has an increased amount of apo E and a decreased l e v e l of C-apoproteins (119). These protein changes correlate with the concentration of plasma unesterified cholesterol (119). I t has been suggested that changing proportions of apoprotein and cholesterol content affects the net surface charge of VLDL and causes slow prebeta migration of t h i s lipoprotein (121). Our data which showed a gradual decrease i n the anodic mobility of the prebeta band throughout the different experimental periods supports the speculation that the surface charge of VLDL might be changed i n p a r a l l e l with the hyper-cholesterolaemic state. This argument seems to be i n accordance with the - 131 -reports that slowly migrating prebeta lipoprotein i s r i c h i n unesterified cholesterol (50,69,104). The electrophoretic mobility of LDL i n the HC group showed an interesting change. At day 70, a slowly migrating beta band was detected i n the HC group compared to CONT group. This electrophoretic behavior which was noticed i n a l l animals of the HC group paralleled the marked increase i n plasma TC. Puppione et a l (68) and Sardet et a l (69) previously reported a normally migrating beta band i n cholesterol-fed guinea pigs. This discrepancy may be due to a difference i n the proportion of cholesterol supplement in the experimental diet, since we used 2% cholesterol-rich diet versus the i r 1% f a t t y diet regimen. Our finding might be related to a high cholesterol content of LDL p a r t i c l e s determining t h e i r electrophoretic mobility. In conclusion, the cholesterol feeding to guinea pigs induced hypercholesterolemia which might be accounted for by the following mechanisms: f i r s t , intake of a cholesterol-rich d i e t . Second, decreased uptake of LDL due to cholesterol-mediated suppression of LDL-receptors. Third, a s l i g h t increase of the percentage of unesterified cholesterol i n plasma due to a moderate reduction i n LCAT a c t i v i t y . The increased plasma cholesterol might modify the electrophoretic migration of the different lipoprotein species. The relevance of the lipoprotein abnormalities to the h i s t o l o g i c a l changes w i l l be discussed i n the following sections. - 132 -2. Effect of the cholesterol/protein-rich d i e t : Enrichment of the cholesterol-rich diet with high protein supplements increased plasma TC and FC. At day 70 FC was higher i n the HCHP group than i n the HC group. This marked increase i s attributed, i n addition to the factors discussed i n the f i r s t part of t h i s section, to the protein supplement. I t has been found that rabbits fed cholesterol-free diets of carbohydrate and casein had suppressed hepatic LDL receptors which led to a decreased rate of LDL clearance from c i r c u l a t i o n (122). The hyper-cholesterolaemic effect of different types of protein has also been i n v e s t i -gated. I t has been pointed out that animal protein i s more effec t i v e than vegetable protein (102,123). The plasma cholesterol l e v e l i n rabbits fed a casein-rich diet was double that seen with a soybean diet (124). Further study of the hypercholesterolaemic effect of casein i n the rabbit model has suggested that cholesterol l e v e l increased i n LDL, IDL, and VLDL (125). The findings of Terpstra et a l . (1982) also indicated that the bulk of the increased serum cholesterol after feeding 40% casein-rich diet was i n the LDL fraction (118). In addition to the theory of the suppressive effect of casein on the LDL receptors, there are other possible explanations for the hypercholesterolaemic effect of casein. Van der Meer (1983) hypothesized that the hypercholesterolaemic effect of casein i s related to i t s phosphorylation state. This hypothesis implies that casein and i t s phosphopeptides compete with b i l e acids and/or b i l i a r y micelles to bind insoluble calcium phosphate increasing the a v a i l a b i l i t y of b i l e acids for l i p i d digestion and reabsorption (126). Recently, ' Van der Meer et a l . (1985) proved t h i s effect i n rabbits fed casein (127). They found that increased amount of dietary calcium i n h i b i t e d casein-induced - 133 -hypercholesterolaemia. Valhouny et a l . (1985) pointed out that rates of clearance of chylomicrons and VLDL were decreased i n rats fed a semipurified diet containing casein (128). Moreover, i t has been reported that hepatic secretion of lipoproteins i s greater with casein-based diets than with soy protein-based one (129). Contrary to these reports, dietary experiments i n chickens showed that hypercholesterolaemia did not resul t from feeding casein or soybean alone but rather after supplementing the protein diets with 1% cholesterol (130). Our data do not agree with t h i s report, since there was a s l i g h t increase i n plasma t o t a l cholesterol i n the control HP group at day 70. However, we confirm that hypercholesterolaemia i s more pronounced with a mixture of casein/cholesterol-rich diet. Thus, from the available reports, i t seems that casein may aggravate hypercholesterolaemia induced by cholesterol-rich d i e t , mainly, by increasing fat absorption and decreasing LDL uptake through suppressing LDL receptors. Although plasma FC was higher i n the HCHP group than i n the HC group, no s i g n i f i c a n t difference was noted i n FC% between the two groups. To our knowledge, the effect of high protein on cholesterol e s t e r i f i c a t i o n i n plasma has not been investigated. Our results using either a r t i f i c i a l or endogenous substrate indicate that there was no s i g n i f i c a n t difference i n LCAT a c t i v i t y (FER and MER) between the HCHP and HC groups. The absence of such a difference i n enzyme a c t i v i t y using endogenous substrate might be attributed to the low s e n s i t i v i t y of the assay where differences at the low l e v e l could not be detected. A l t e r n a t i v e l y , due to the higher l e v e l of the FC i n HCHP group than HC group, i t might be expected to have lower FER i n the f i r s t one due to the adverse effect of FC on appropriateness of the substrate (discussed above). The s l i g h t reduction i n FER found i n the HP - 134 -group where there was a moderate increase i n plasma cholesterol supports t h i s argument. However, we based our conclusion, that no difference i n LCAT a c t i v i t y was found between HCHP and HC groups on the data obtained with the a r t i f i c i a l - s u b s t r a t e . No s i g n i f i c a n t differences i n HDL-C, protein composition, or electro-phoretic mobility were observed between the HCHP group and HC group. This issue has not been studied thoroughly by other investigators i n guinea pigs. Mol et a l . (1982) found no differences i n the density p r o f i l e and lipoprotein composition between groups of chicken receiving cholesterol-free diets containing either casein or soybean protein (130). Our data suggest that high casein supplementation did not a l t e r the chemico-physical properties of lipoprotein species i n the animals. This might be due to the s l i g h t hypercholesterolaemic effect of the casein-enriched diet found i n our study. In conclusion, high protein supplementation i n either CONT or HC groups had a hypercholesterolaemic effect which might be due to an increased fat absorption and/or decreased catabolism of lipoproteins, p a r t i c u l a r l y LDL. The high protein supplement did not further impair cholesterol e s t e r i f i c a t i o n compared to the HC group. In addition, no remarkable changes i n the lipoprotein composition and electrophoretic behavior could be attributed to the high protein supplement per se. I I I . Haematological Changes 1. Effect of cholesterol-rich diet: Similar to several other studies (23,66,67,69,77) the cholesterol-fed guinea pigs i n our study developed anaemia. Compositional, morphological and functional alterations i n erythrocytes w i l l be discussed i n an e f f o r t to - 135 -elucidate the mechanism(s) responsible for anaemia i n these animals. Already after t h i r t y days on the cholesterol-enriched diet erythrocyte content of cholesterol increased. This finding i s i n agreement with the data obtained by others (66,131). A role for the abnormal lipoproteins observed i n cholesterol-fed guinea pigs has been postulated i n delivering cholesterol to erythrocyte membranes (EM). Thus, the appearance of new species of HDL and changes i n LDL i n the plasma of these animals correlate with the severity of haemolysis (69). Our findings of the lipoprotein alterations (discussed i n the previous section) agree with these observations. In an i n v i t r o study, i t has been reported that cholesterol i s transferred mainly from HDL to EM when normal erythrocytes were incubated with hypercholesterolaemic plasma of cholesterol-fed guinea pigs (131). In the same way, cholesterol-enriched erythrocytes obtained from cholesterol-fed guinea pigs l o s t t h e i r cholesterol to plasma when they were incubated with normal plasma (131). These observations demonstrate that plasma i s the main source of cholesterol found i n excess i n EM. This effect can be accounted for by plasma lipoproteins contributing cholesterol to c e l l s either by receptor-mediated endocytosis (132) or by isolated surface transfer of free cholesterol. In addition to cholesterol transfer, i t has been well documented that there i s an eq u i l i b r a t i o n between plasma and erythrocyte phospholipids (133). Our findings of increased plasma t o t a l phospholipids i n the HC group suggest that part of phospholipids i n EM of t h i s group may originate from plasma i n an eq u i l i b r a t i o n process s i m i l a r to that described previously (131,133). This suggestion i s i n accordance with another observation i n cholesterol-fed guinea pigs of an increased l e v e l of phospholipids i n EM (66). In summary, the l i p i d content of EM found i n the HC group i s most l i k e l y derived from plasma. - 136 -Studies of physical properties of l i p i d s i n membranes have shown that incorporation of cholesterol molecules into the l i p i d bilayer increased order in the hydrocarbon region which decreases water permeability (134). Also, i t has been suggested that insertion of cholesterol molecules into the phospolipid array of EM f a c i l i t a t e s a closer f i t and interaction between the long acyl groups of the adjacent l i p i d molecules (135). This a l t e r a t i o n causes a contraction of membrane surface referred to as a "condensation e f f e c t " (135). This effect might explain the s l i g h t decrease i n mean corpuscular volume (MCV) of erythrocytes i n the HC group at day 30. Other studies have reported that the increased cholesterol content of EM leads to an increase i n l o c a l v i s c o s i t y and r i g i d i t y of membranes (136). Also, increased membrane deformity and fragmentation were reported i n these c e l l s (137). Relevant to these observations i s the finding that the increase i n erythrocyte cholesterol content was accompanied by the appearance of spiked erythrocytes (69). We found echinocytes to be the predominant erythrocyte form i n the peripheral blood at day 30 i n the HC group. Interestingly, Owen et a l . (1985) reported that echinocyte form developed i n seconds when normal erythrocytes were incubated with plasma from jaundiced patients having abnormal HDL (138). They mentioned that formation of these c e l l s did not involve cholesterol transfer, but rather attachment of the abnormal HDL which contains apo E to s p e c i f i c membrane receptors on erythrocytes. Our findings do not exclude the p o s s i b i l i t y of echinocytes developing through a mechanism sim i l a r to that described by-Owen et a l . In any event, t h i s mechanism does not substitute for the cholesterol-mediated mechanism which we adopt. Whatever the mechanism, we found these c e l l s accompanied by a decrease i n erythrocyte f r a g i l i t y , a finding which has been reported by - 137 -several investigators (131,135,137). A si m i l a r decrease i n osmotic f r a g i l i t y has been described i n patients with abetalipoproteinemia who presented with acanthocytes i n t h e i r blood (136). Again, t h i s could be attributed to the "condensation e f f e c t " which decreases membrane permeability (135). The deformed erythrocytes on entry of the blood through the splenic cords are either engulfed and destroyed by the splenic macrophages on t h e i r f i r s t pass or their spikes are amputated f i r s t ( p i t t i n g process) to be engulfed on the following process (139). We speculate that p i t t i n g o f f the echnicoyte spikes could affect EM permeability and lead to s p i l l i n g out some of the haemoglobin into plasma. These hypochromic c e l l s (HCC) with corrugated membrane (on microscopy) appeared at day 70 i n our study. Such membrane corrugation could indicate the s i t e s of the " p i t t i n g process". In accordance with our speculation, the splenic "remodeling" or "conditioning" of "spikey" erythrocytes has been reported by several investigators (141,140,141). We suggest that HCC are osmotically unstable c e l l s due to th e i r defective membrane which could be subjected to intravascular haemolysis. Thus, these c e l l s might have a shorter l i f e span than normal c e l l s . In summary, hypercholesterolaemia affected erythrocyte composition -namely, increased cholesterol content of EM. Cholesterol enrichment of erythrocytes might, i n part, render t h e i r membranes r i g i d (echinocytes) and thus highly susceptible to splenic erythrophagocytosis. I t seems that haemolytic anaemia i n cholesterol-fed guinea pigs occurs both i n t r a and extravascularly. By day 70 vacuolated white blood c e l l s were found i n the peripheral blood of the HC group. Using 0R0 and NSE stains, i t was shown that these - 138 -c e l l s were lipid- l a d e n monocytes (LLM). The presence of these c e l l s has e a r l i e r been reported i n hyperlipidaemic rats (142;143). The appearance of lipophages i n peripheral blood has been an in t r i g u i n g subject i n the studies of atherogenesis and atherosclerotic plaque regression. Gerrity (1981) presented histochemical evidence that macrophages detected i n swine atherosclerotic plaques were blood-borne monocytes (144). He showed an important role for these c e l l s i n scavanging l i p i d deposits i n the a r t e r i a l intima. Gerrity was able to demonstrate a "monocyte clearance system" i n which the c i r c u l a t i n g monocytes penetrate the a r t e r i a l intima to be laden by l i p i d ; these foam c e l l s migrate back into the blood stream by crossing the a r t e r i a l endothelium (145). Later, these observations were supported by Faggiotto et a l . (1984) i n a sequential follow-up study of atherogenesis i n hypercholesterolaemic non-human primates. In t h i s model, after three months on a cholesterol-enriched d i e t , the endothelial continuity was interrupted over the lipid- l a d e n macrophages i n atherosclerotic plaques. The exposure of the foam c e l l s to the c i r c u l a t i n g blood may lead to th e i r appearance i n blood (146). In the present study, c i r c u l a t i n g LLM were not seen before day 70, although hypercholesterolaemia was noticed i n the HC group as early as day 5 of the cholesterol-rich diet regimen. There i s some analogy to the observations of Faggiotto et a l . who showed a time-dependent appearance of these c e l l s i n c i r c u l a t i o n . Interestingly, unlike t h e i r findings, we did not observe any detectable atheroma i n the aortas of the guinea pigs during the 70-day experiment. This suggests that there may be a different source of the c i r c u l a t i n g LLM i n our animals (other than a r t e r i a l fatty l e s i o n s ) . We hypothesize that these c e l l s might originate from the reticuloendothelial system. Data of other investigators as well as our own (as discussed i n the - 139 -following paragraphs) support t h i s hypothesis. E a r l i e r , Ostwald and Shannon found a s i g n i f i c a n t increase i n the l i p i d content of the spleens of cholesterol-fed guinea pigs (66). Also, i t has been noticed that t h i s l i p i d accumulation was distributed i n the splenic i n t e r s t i t i a l tissue and inside macrophages; the hepatic sinusoidal macrophages were also depicted as lipophages (77). Moreover, as discussed above, erythrocytosis and the " p i t t i n g process" of the spikes of echinocytes was most l i k e l y caused by the macrophages of splenic cords. Since echinocytes have l i p i d - r i c h membranes, splenic macrophages would have been overloaded with l i p i d materials. Based on a l l these observations we postulate that l i p i d - l a d e n splenic macrophages might be released from the l i n i n g of the splenic cords and gain access to the c i r c u l a t i o n . Another possible source of fat-loading macrophages of the reticuloendothelial system i s plasma lipoproteins. In an i n v i t r o study, peritoneal macrophages and monocyte-derived macrophages have been shown to accumulate massive amounts of s t e r o l when incubated with chemically modified lipoproteins or with beta migrating VLDL isolat e d from hypercholesterolaemic animals (147-150). Beta migrating VLDL isolated from hypercholesterolaemic rabbit plasma has been shown to react with s p e c i f i c membrane receptors on macrophages (147). We found beta migrating VLDL at day 70 when a s i g n i f i c a n t number of LLM was detected i n c i r c u l a t i o n . However, beta migrating VLDL was also demonstrated at day 30 without LLM presence i n peripheral blood. This might suggest that loading macrophage with t h i s abnormal lipoprotein i s a time-dependent process. F i r s t , macrophage must be overloaded with l i p i d and then released from i t s tissue of o r i g i n into the c i r c u l a t i o n . In addition to beta-VLDL, HDLC has also been implicated i n delivery of cholesterol to different tissues i n the body. Andersen and Dietschy (1981) - 140 -demonstrated preferential uptake of cholesterol from HDL by rat adrenal (151). The preferential degradation of the cholesterol moiety of HDL was also reported i n different c e l l s by other investigators (152,153). In our experiment a remarkable increase i n the percentage of c i r c u l a t i n g LLM was paralleled by the appearance of an alpha-migrating band upon agarose gel electrophoresis. Based on the above reports and our observations, i t i s tempting to suggest that alpha-migrating lipoproteins might be another source of macrophage l i p i d . In summary, i t i s unlikely that i n our experimental model c i r c u l a t i n g LLM originated from an atherosclerotic l e s i o n . We suggest that these c e l l s are macrophages of the reticuloendothelial system loaded with l i p i d materials and released into the c i r c u l a t i o n . The source(s) of t h i s l i p i d material might be, i n part, the l i p i d - r i c h membranes of echinocytes, and beta-migrating VLDL and/or alpha-migrating lipoprotein or a l l of these. Our study showed a concomittant increase i n the percentage of LLM i n the peripheral blood with the number of glomerular NSE-positive c e l l s . This suggests a possible role for LLM i n the pathogenesis of the glomerulo-s c l e r o t i c lesion found i n the cholesterol-fed guinea pigs. In conclusion, anaemia found i n the cholesterol-fed guinea pigs may be explained as follow: cholesterol feeding to these animals led to an increase i n EM cholesterol content which accounts for many of the changes i n erythrocyte morphology. The decrease i n erythrocyte osmotic f r a g i l i t y was most l i k e l y attributed to the increase r i g i d i t y of EM (morphologically shown as echinocytes). Echinocytes were either cleared d i r e c t l y from blood c i r c u l a t i o n by erythrophagocytosis i n spleen or t h e i r spikes were removed i n a " p i t t i n g process" by splenic macrophages, where they became susceptible to - 141 -intravascular haemolysis. We speculate that splenic macrophages, after being loaded with l i p i d materials from different sources, were released into c i r c u l a t i o n and were detected as LLM. 2. Effect of Cholesterol/Protein-Rich Diet: The effect of high protein supplementation on dietary cholesterol-induced anaemia has not been investigated previously except for the work of Ostwald et a l . (1971). They reported that neither 20% nor 30% casein-rich diets protected cholesterol-fed guinea pigs from anaemia (101). In our study there were several haematological changes i n the animals fed the cholesterol/protein-rich diet (HCHP group). As discussed i n the previous part of the section HCC (hypochromic c e l l s ) appeared at day 70 possibly as a re s u l t of the " p i t t i n g process" of spikes of echinocytes i n splenic cords. HCC were seen i n a higher percentage i n the HCHP group than i n the HC group. This might be due to an increase i n the rate of the " p i t t i n g process" which could be attributed to an increase i n the degree of erythrocyte deformity. To discuss t h i s hypothesis the plasma l i p i d p r o f i l e i n the HCHP group should be examined. In t h i s group a higher l e v e l of mainly free cholesterol concentration was found as compared to the HC group. Having considered the equilibrium state between plasma and EM cholesterol (131), i t seems l i k e l y that EM i n the HCHP group had accepted more cholesterol than those of the HC group. The increase i n the l i p i d content of EM i s one factor determining the deformity of erythrocytes. These changes might lead to more p i t t i n g of the spikes of echinocytes. Supporting t h i s speculation i s the finding that the cholesterol content of EM i n the HCHP group was comparable to that i n the CONT group at day 70 - 142 -where HCC were the predominant erythrocyte form i n peripheral blood. We believe that such a reduction i n the EM cholesterol content was most l i k e l y due to the p i t t i n g of the echinocyte spikes by splenic macrophages. Unlike i n the HC group, erythrocyte osmotic f r a g i l i t y i n the HCHP group was within normal CONT-group l e v e l at day 70. Since osmotic f r a g i l i t y i s inversely related to the amount of cholesterol i n EM, t h i s f i t s with the normal cholesterol content of EM. Thus, with predominance of HCC i n the peripheral blood, there are at least two p o s s i b i l i t i e s which may be accounting for the elevation of osmotic f r a g i l i t y from the l e v e l found i n the HC group to the CONT-group l e v e l : f i r s t , a decreased amount of erythrocyte cholesterol leads to decreased r i g i d i t y and increased permeability of EM (134,136). Second, the " p i t t i n g process" of the echinocyte spikes most l i k e l y leads to decrease osmotic s t a b i l i t y of erythrocyte, hence we speculate that HCC are vulnerable c e l l s with shortened l i f e span. In general, although there was no s i g n i f i c a n t difference i n erythrocyte count, haemoglobin concentration, and haematocrit between the HCHP and HC groups, we believe that haemolysis was more severe i n the HCHP group for the following reasons: f i r s t , the presence of HCC i n a larger percentage i n the HCHP group than i n the HC group. This might indicate that the animals of the HCHP group had more vulnerable erythrocytes (see above) which are l i k e l y to be subjected to intravascular haemolysis. In t h i s regard, our results described HCC as bigger-than-normal c e l l s with a remnant of haemoglobin at the c e l l periphery. However, there was no s i g n i f i c a n t difference i n haemoglobin concentration between HCHP and HC groups. This might be due to compensatory r e t i c u l o c y t o s i s which appeared i n a larger degree i n the HCHP - 143 -group. Second, serum i n d i r e c t b i l i r u b i n was higher i n the HCHP group than i n the HC group at day 70. This observation indicates either a deterioration in l i v e r function ( p a r t i c u l a r l y the conjugating a b i l i t y ) or a higher degree of haemolysis. In fact, there was no s i g n i f i c a n t difference i n l i v e r function values between HCHP and HC groups, besides, a s l i g h t l y higher l e v e l of serum direct b i l i r u b i n was found i n the HCHP group (see the following section for further discussion). Thus, i t i s more l i k e l y that increased haemolysis accounted for the elevated serum i n d i r e c t b i l i r u b i n i n the HCHP group. In summary we speculate that greater l i p i d enrichment of EM i n the HCHP group than that i n the HC group might be the potential cause for the increased rate of " p i t t i n g process" which led to the appearance of more percentage of HCC i n c i r c u l a t i o n . For at least two reasons we believe that the degree of haemolysis was higher i n the HCHP group than i n HC group. In the f i r s t part of t h i s section, i t was postulated that the ci r c u l a t i n g LLM i n the HC group might originate from macrophages i n the reticuloendothelial system. Further we speculated that the l i p i d content of these c e l l s could be from at least two sources - the l i p i d - r i c h membranes of echinocytes and plasma lipoproteins. The findings observed i n the HCHP group support t h i s argument. There was a higher percentage of LLM found i n the HCHP group compared to the HC group at day 70 ( t h i s correlated well with the percentage of HCC). Since the percentage of HCC i n the c i r c u l a t i o n , as discussed above, might be considered an ind i r e c t indicator for the rate of " p i t t i n g process", i t seems l i k e l y that the appearance of LLM i s related to the " p i t t i n g process". Moreover, since plasma FC i n the HCHP group at day 70 was higher than i n the HC group, and since there was a paralleled increase i n the plasma FC to the increase i n the percentage of LLM i n the - 144 -c i r c u l a t i o n , plasma l i p i d may be another possible source for the l i p i d content of LLM. In conclusion, high protein supplementation of the cholesterol-fed animals led to more severe haemolysis than that observed i n animals on the cholesterol-rich diet alone. We speculate that the rate of the " p i t t i n g process" of echinocyte spikes by spleen macrophages was higher i n the HCHP group than i n the HC group. We believe t h i s might be related to the EM overloading with, mainly, cholesterol derived from plasma since plasma FC was higher i n the HCHP group than i n the HC group. Also, HCC may be the echinocyte remnant after the " p i t t i n g process". Hence, the high number of HCC i n c i r c u l a t i o n i n the HCHP group was concomittant with lower erythrocyte cholesterol and higher f r a g i l i t y than i n the HC group. IV. Hepatic Changes While the gross and histopathological changes i n the l i v e r s of cholesterol-fed guinea pigs have been previously described i n d e t a i l (23,66,77,99), these investigations have not included l i v e r function tests. In the present study, serum direct and in d i r e c t b i l i r u b i n , asparate transaminase (AST), albumin, and t o t a l protein l e v e l were monitored throughout the experimental periods. The animals of the HC and HCHP groups had severe gross l i v e r fatty i n f i l t r a t i o n which increased steadily from day 30 to day 70 of the experiment. In humans, the exact pathogenesis of fatty l i v e r i s not f u l l y understood but i t seems to be due to any interference with the diff e r e n t steps i n fat metabolism which can lead to fat accumulation i n hepatocytes (154). In the fa t t y - d i e t experiments, fatty l i v e r i s most l i k e l y due to the high l e v e l of chylomicron remnants i n plasma - 145 -which reach hepatocytes. Cholesterol i s stored after being e s t e r i f i e d by ACAT (155). This i s applicable to the fatty l i v e r found i n the HC and HCHP groups i n our experiments. Regarding c e l l u l a r changes, aminotransferases are known as indicators of hepatocellular damage. They are released from hepatic c e l l s when c e l l u l a r damage occurs or when there i s an increase i n c e l l membrane permeability (156). In t h i s study, an increase i n AST was noticed as early as 10 days i n both HC and HCHP groups. The AST a c t i v i t y showed a marked increase by day 70. Beynan et a l . (1985) demonstrated an increase i n serum AST and alanine amino transferase (ALT) i n rats on a cholesterol-rich diet (157). Also, hyperlipaemic obese rats have increased AST a c t i v i t y (158). On the other hand, Laitnen et a l . (1982) reported that a cholesterol-free diet l a b i l i z e d the membranes of rat hepatocytes and f a c i l i t a t e d release of AST i n blood (159). However, th e i r conclusion was based on the observation that AST a c t i v i t y did not change during the 6-week experiment of cholesterol-rich diet feeding while i t increased after withdrawal of cholesterol from the diet . L i k e l y , the c e l l u l a r injury i n the Laitnen et a l . experiment had already been induced by l i p i d deposits during the cholesterol-feeding period; thus, withdrawal of cholesterol could not preclude release of AST i n serum. Based on these reports and our findings, i t seems that there i s a good correlation between plasma cholesterol and serum AST i n cholesterol-fed guinea pigs. However, t h i s correlation should be viewed with caution, since i t r a r e l y , i f ever, exists i n patients with hypercholesterolaemia without hepatocellular lesions. Thus, i t would be more appropriate to examine the correlation between the hepatic fatty i n f i l t r a t i o n and serum AST. As discussed above, there i s a causal - 146 -relationship between hypercholesterolaemia i n cholesterol-fed animals and l i v e r lesions. I t i s t h i s relationship that results i n a positive correlation between AST levels and plasma cholesterol i n these animals. Our results (as mentioned above) indicate that an elevated l e v e l of AST i n serum was detected before the gross hepatic fatty i n f i l t r a t i o n . This might be attributed to the microscopic changes occurring prior to day 30 of the experiment and not evident macroscopically. In addition to the data derived from animal studies, there are some reports i n human pathology supporting our findings. In one study of patients with fatty l i v e r , a s l i g h t elevation of serum aminotransferases has been demonstrated (160). Also, i n Reye's syndrome where there i s fatty degeneration of l i v e r , the serum AST i s high (161). Interestingly, there was also a gradual mild increase i n AST i n serum of the CONT and HP groups. Although there was a s l i g h t elevation i n plasma cholesterol i n these two groups toward day 70 of the experiment, i t i s unlikely that t h i s leads to hepatocellular damage. However, i t has been reported that hepatocytes show a reduced a b i l i t y to synthesize cholesterol with aging (162,163). Furthermore, i n aged r a t s , physiological regeneration of hepatic c e l l s i s reduced compared to that i n young animals (164). These findings suggest a defective c e l l membrane due to the aging process which leads to leakage of the intracytoplasmic enzymes. Our interpretation of these reports i s supported by the findings of Nagy et a l . (1982) who pointed out that aging may cause deterioration of c e l l u l a r functions by a l t e r i n g the c e l l membrane structure and function (165). Thus the changes i n AST i n the CONT and HP groups could perhaps be attributed to the age-related c e l l membrane changes. - 147 -In summary, fatty i n f i l t r a t i o n of the l i v e r i n both groups kept either on cholesterol-rich diet alone or cholesterol/protein-rich diet might be brought about by hypercholesterolaemia with fa t t y i n f i l t r a t i o n of the l i v e r . The increase i n serum i n d i r e c t (unconjugated) b i l i r u b i n i n both HC and HCHP groups at day 70 correlated well with the severity of haemolysis. Also, serum i n d i r e c t b i l i r u b i n was higher i n the HCHP group than HC group. As discussed i n the previous section, the increase i n t h i s parameter i n our animals, most l i k e l y , r e f l e c t s the degree of haemolysis more than the hepatic a b i l i t y of conjugation. This interpretation i s supported by the r e l a t i v e increases of direct (conjugated) b i l i r u b i n found i n the HC and HCHP groups. Moreover, at day 70, serum direct b i l i r u b i n i n the HCHP group was higher than i n HC group. These two observations indicate that the conjugating a b i l i t y of hepatocytes suffered no major deterioration and was responding to increased l e v e l s of serum i n d i r e c t b i l i r u b i n . However, intrahepatic cholestasis might occur due to the extensive fatty degeneration found at day 70. In any event, t h i s does not rule out the fact that the hepatic conjugating system i s s t i l l e f f e c t i v e at day 70. Thus, the changes i n both direct and in d i r e c t b i l i r u b i n found i n the HC and HCHP groups, most l i k e l y , were related to the haematological rather than hepatic abnormalities. Albumin concentration i n serum was not reduced i n the HC and HCHP groups throughout the experimental period. This indicates that the synthetic a b i l i t y of the l i v e r was not affected (see further discussion on the l i v e r as a synthetic organ, below). On the other hand, t o t a l protein was increased i n both HC and HCHP groups at days 10,30 and 70. This finding might be attributed to an increase i n the production of immunoglobulins by the c e l l s of immune system i n a response to retained antigens i n the - 148 -c i r c u l a t i o n . In t h i s regard, several observations have suggested that Kupffer c e l l s may f a i l to sequester antigens absorbed from the gut, either because of functional impairment or as a re s u l t of shunting of portal blood due to the hepatic lesion (166-168). These two p o s s i b i l i t i e s are unlikely at least at the experimental periods prior to day 70. Nonetheless, Kupffer c e l l a b i l i t y for sequestering c i r c u l a t i n g antigens might be affected at day 70 due to the marked fatty degenerative lesion detected i n the l i v e r of the HC and HCHP groups. Another potential source of increasing t o t a l protein i n serum i s the acute phase reactant proteins which could be increased due to the hepatic changes found i n the HC and HCHP groups. Liver function tests were performed to answer a c r i t i c a l question -namely, was the decrease i n hepatic function severe enough to cause secondary renal f a i l u r e as i n the hepato-renal syndrome? The hepatorenal syndrome i s an incompletely explained renal f a i l u r e i n patients with l i v e r disease i n the absence of c l i n i c a l , laboratory, or anatomical evidence of other known causes of renal f a i l u r e (169). The renal lesions have been described with b i l i a r y c i r r h o s i s (170). The renal lesion has no correlation with the presence or absence of proteinuria (169). There i s a reduction i n glomerular f i l t r a t i o n rate and renal plasma flow. Our findings exclude the presence of any type of hepatic c i r r h o s i s . Moreover, the glomerular f i l t r a t i o n rate was not reduced i n any of the experimental groups. In agreement with our data, Papper (1983) stated that although there are examples of animal l i v e r disease, none of them i s accompanied by renal f a i l u r e (171). Further, our results indicate that the synthetic a b i l i t y of l i v e r i n the HC and HCHP groups was retained (the albumin l e v e l i n serum was normal). Also, urea production was increased compared to the CONT group - 149 -pa r t i c u l a r l y i n the HCHP group. Moreover, the conjugating a b i l i t y of the l i v e r i n the HC and HCHP groups was not severely affected. These data indicate that the p o s s i b i l i t y of involvement of the hepatic lesion i n induction of renal changes i s u n l i k e l y . V. Renal Structural and Functional Alterations 1. Effect of cholesterol-rich diet: a - H i s t o l o g i c a l Changes: A p r o l i f e r a t i v e s c l e r o t i c glomerular lesion was induced i n the guinea pigs kept on cholesterol-rich diet for seventy days. The main h i s t o -pathological features i n t h i s group encompassed moderate glomerular c e l l u l a r i t y and expansion of mesangial areas, i n addition to the presence of NSE-positive c e l l s and ORO-positive deposits i n the glomerular tissue. In agreement with our findings, French et a l . have previously reported s i m i l a r observations i n cholesterol-fed guinea pigs (23). On the other hand, Drevon and Hovig (1977) did not find any s i g n i f i c a n t h i s t o l o g i c a l changes i n kidneys of the same experimental model (99). This might be due to guinea pig s t r a i n differences and/or dietary regimen var i a t i o n . Nonetheless, there has been another report which disagreed with Drevon and Hovig's observations; i t demonstrated, i n addition to the glomerular l e s i o n , haemosiderin i n the proximal convoluted tubules of cholesterol-fed guinea pigs (77). Our results confirmed the presence of Perl's Prussian-blue positive (PPb) materials i n the e p i t h e l i a l c e l l s of the c o r t i c a l tubules. The PPb-positive materials l i k e l y represent the reabsorbed dinners of haemoglobin molecules by tubular c e l l s during an intravascular haemolytic process (139). The histochemical studies demonstrated expanded mesangia i n the affected glomeruli i n the HC group. A previous report (23) i s i n agreement with t h i s - 150 -finding. In t h i s study, the u l t r a s t r u c t u r a l findings i n kidneys of the HC group at day 70 are si m i l a r to those of French et a l . (78). These authors reported fusion of foot processes of the glomerular e p i t h e l i a l c e l l s and intravascular erythrophagocytosis. In addition to these observations, we detected di f f e r e n t sized l i p i d droplets inside the cytoplasm of the endothelial c e l l s . L i p i d was noted i n mesangial c e l l s and i n f i l t r a t i n g monocytes. The morphometric assessment of the glomerular les i o n , i n t h i s study, showed a s i g n i f i c a n t increase i n MA/GTA r a t i o and MC i n the HC group at day 70 compared to the CONT group. Although a small but s i g n i f i c a n t increase i n MA/GTA r a t i o was observed at day 30, there were no s i g n i f i c a n t changes i n these parameters at days 5 and 10. No previous study has dealt with a morphometric assessment of glomerular lesions found i n cholesterol-fed guinea pigs. In summary, the cholesterol-rich diet induced a glomerulosclerotic lesion i n guinea pigs. This lesion included mesangial c e l l u l a r p r o l i f e r a t i o n , mesangial expansion, monocyte/macrophage i n f i l t r a t i o n of the glomerulus and the mesangium and l i p i d deposits. These h i s t o l o g i c a l changes led to an increase i n mesangial area. b. Renal functional alterations : 24-hour urinary protein (UPr) s i g n i f i c a n t l y increased at day 70 i n the HC group compared to the CONT group. Changes i n renal function have not been monitored i n previous studies of cholesterol-induced kidney diseases i n animals. However, i n humans, proteinuria has been reported with hyperlipidaemia and hypoalbuminaemia i n cases of nephrotic syndrome (10). - 151 -Also, In LCAT deficient patients, proteinuria i s common (1). In both these situations there are quantitative and q u a l i t a t i v e lipoprotein changes accompanied by variable degrees of glomerular changes i n some cases. The proteinuria can be either glomerular or tubular. The deterioration of the tubular reabsorptive c a p a b i l i t y due to protein overload i n glomerular f i l t r a t e has been mentioned previously (172,173). Our study did not demonstrate marked tubular disease i n either of the HC or HCHP groups. Thus, proteinuria found i n the HC group seems due, mainly, to a decrease i n glomerular permselectivity. Mild haematuria was noticed i n the HC group at day 70. These findings are i n agreement with those reported by French et a l (23). They found erythrocyte aggregations i n different portions of renal tubules. Haematuria in the cholesterol-fed animals could be attributed to the factors which cause damage of GBM. The mechanism of glomerular damage i s discussed i n the t h i r d part of t h i s section. However, there i s always a p o s s i b i l i t y of confusion i n tracing the o r i g i n of haematuria. There are, at le a s t , three possible causes of haematuria: glomerular damage, i n t e r s t i t i a l i n j u r y , and any injury to the post-renal urinary t r a c t . In the present study, there was no evidence of t u b u l o i n t e r s t i t i a l injury. Since lower urinary t r a c t injury i s sporadic, i t i s unlikely that t h i s would be a cause of haematuria i n HC since a l l animals demonstrated t h i s change. Thus glomerular damage i s the most l i k e l y cause of the haematuria. The levels of both UPr and haematuria i n the HC and HCHP groups at day 70 (see the "effect of cholesterol/protein-rich d i e t " i n t h i s section), correlated well with the number of the glomerular NSE-positive c e l l s and - 152 -MA/GTA r a t i o . This suggests a direct relationship between renal s t r u c t u r a l a l t e r a t i o n and functional deterioration. Blood urea nitrogen (BUN) was moderately increased i n the HC group at day 30 and became higher at day 70. This gradual increase i s unlikely to be accounted for by impairment of glomerular f i l t r a t i o n since creatinine clearance was normal. I t might be attributed to haemolytic anaemia and decreased weight gain. I t i s known that urea synthesis increases i n cases of tissue breakdown and decreased protein synthesis (174). Serum creatinine l e v e l i n the HC group did not increase s i g n i f i c a n t l y throughout the experimental period, indicating a lack of serious renal functional impairment (the creatinine clearance was normal i n the HC group). However, the serum creatinine levels were i n p a r a l l e l to those of weight gain i n both groups. This observation suggests that changes i n serum creatinine were related to the changes i n body muscle mass. Such a correlation has been established e a r l i e r (175). As mentioned above, creatinine clearance i n the HC group at day 70 was unchanged from that i n the CONT group. Such findings indicate that the glomeruloproliferative lesion was not severe enough to decrease GFR. 2. Effect of Cholesterol/Protein - Rich Diet:  a. H i s t o l o g i c a l Changes: No available reports have described the combined effect of cholesterol- and protein-rich diets on the progression of the glomerular lesion i n experimental animals. Our data revealed a si m i l a r pattern of histopathological changes i n both HC and HCHP groups. However, the renal changes were more pronounced i n the HCHP group than i n the HC group. - 153 -The strength of the Perl's Prussian-blue positive reaction was more prominent i n the tubular e p i t h e l i a l c e l l s of the HCHP group at day 70 than i n the HC group. Since t h i s change i s probably due to the renal tubular reabsorption of dimers of haemoglobin molecules, i t i s consistent with greater haemolysis observed i n the HCHP group. In the HCHP group there was an increase i n the glomerular NSE-positive c e l l s compared to the HC group at day 70. Further, there was a s i g n i f i c a n t but weak correlation between the number of the glomerular NSE-positive c e l l s and MA/GTA r a t i o found among the groups throughout the experimental period. These observations suggest a causal relationship between NSE-positive c e l l s and the mesangial expansion i n the HC and HCHP groups (see the t h i r d part of th i s section for detailed discussion). b. Renal functional alterations: The UPr and haematuria were markedly higher i n the HCHP group at day 70 than i n the HC group and other control groups. This l i k e l y r e f l e c t s a greater glomerular damage i n the HCHP group. Supporting t h i s conclusion i s the presence of higher MA/GTA r a t i o i n the HCHP group than i n the HC group. Serum BUN was markedly higher i n the HCHP group than i n the HC, HP and CONT groups. In addition to haemolytic anaemia and decreased weight gain, the higher BUN l e v e l i n the HCHP group compared to the other groups i s l i k e l y due to the high protein supplement, res u l t i n g i n more amino acids being metabolized to urea i n the l i v e r (176). This i s the l i k e l y explanation for the HP group's increased l e v e l of serum BUN. In conclusion, addition of high protein supplement to a cholesterol-rich diet aggravated the renal functional and st r u c t u r a l abnormalities found i n - 154 -the animals kept on a cholesterol-rich diet alone. The major abnormalities consisted of proteinuria and haematuria. Previously, i t has been suggested that glomerulosclerosis i n cholesterol-fed guinea pigs might be induced by haemolytic anaemia (23). Our data showed no Perl's Prussian blue-positive deposits i n the glomerular lesions in the HCHP and HC groups. Moreover, after induction of haemolytic anaemia by acetylphenylhydrazine i n j e c t i o n s , there was no glomerular change. However, there was a massive i n f i l t r a t i o n of Perl's Prussian blue-positive material i n the e p i t h e l i a l c e l l s of renal tubules, a finding consistent with haemolytic anaemia. These data make the proposed glomerulosclerotic role of haemolysis very u n l i k e l y . However, by diff e r e n t mechanisms, glomerular lesions were reported i n s i c k l e c e l l disease (177) and malaria (178). The normal serum phosphate i n a l l groups and the absence of tissue c a l c i f i c a t i o n indicate that difference i n diet phosphorus ( i n casein-rich diet) cannot account for the glomerular lesions found i n the HCHP group. The absence of glomerular lesions i n the HP group also supports t h i s conclusion. The next section deals with the biochemical-histological interactions which might take place i n kidney of t h i s animal model. 3. Kidney L i p i d Content: Origin and C e l l u l a r Interaction In kidney tissue, CE% was s i g n i f i c a n t l y increased i n the animals kept on a cholesterol-rich diet for 30 and 70 days. No s i g n i f i c a n t changes found at days 5 and 10 of the experiment. Previous studies dealing with cholesterol-fed rabbits (179) and guinea pigs (109,99,155) have also shown increased cholesterol content of kidney tissue. Our results confirm the - 155 -observations of Drevon and Hovig (99) i n that there was no s i g n i f i c a n t increase i n the content of kidney FC, TPL and TG i n the cholesterol-fed guinea pigs. However, we noticed a tendency to an increase i n FC and TPL in these animals. In contrast, FC content i n kidney tissue was s i g n i f i c a n t l y increased in the HCHP group at day 70 of our study compared to the CONT group. What i s the source of the increased l i p i d content i n kidney tissue? In general, there are three possible sources of tissue l i p i d : f i r s t , d i rect delivery from plasma, second, cell-mediated delivery of l i p i d , and t h i r d , kidney cholesterol de novo synthesis. Regarding the f i r s t source, l i p i d s could be delivered either s e l e c t i v e l y or as a part of an intact lipoprotein to the c e l l s of different tissues (10,24,147,180). Cholesterol flux between surfaces of lipoproteins and c e l l s has been reported (180) such that free cholesterol i s delivered to peripheral c e l l s from abnormal cholesterol-loaded lipoproteins. Moreover, preferential degradation of cholesterol of HDL by different c e l l s has been demonstrated by several investigators (151-153). In our study, alpha-migrating lipoprotein was detected at day 70 in the HC and HCHP groups and HDL-C was increased, as wel l . This might suggest a si m i l a r mechanism of cholesterol degradation by glomerular endothelial c e l l s . This speculation i s supported by the presence i n the HC and HCHP groups of slowly migrating VLDL found a lipoprotein species which was shown to be cholesterol-rich VLDL (see discussion, section I I ) . More evidence i n support of t h i s concept i s derived from the findings of the present study that revealed some of the endothelial c e l l s of the glomerular c a p i l l a r i e s contained clear vacuoles, presumably l i p i d . Nevertheless, the presence of these droplets i n the l i n i n g of glomerular c a p i l l a r i e s could - 156 -also indicate incorporation of intact lipoproteins into the endothelial l i n i n g . Other workers have addressed t h i s p o s s i b i l i t y , too. Iverius (1972) stated that VLDL and LDL could bind to polyanionic glycosaminoglycans of the GBM, such that these lipoproteins might a l t e r the permeability of the glomerular barrier (24). Later, Moorhead et a l . (1982) included these findings in t h e i r hypothesis of the role of l i p i d i n chronic progressive glomerulo-tubulo-interstitial disease (10). Thus, the increased l i p i d content of glomerular tissue might be s e l e c t i v e l y derived from cholesterol-r i c h lipoproteins and/or binding of intact lipoproteins to GBM and glomerular endothelial c e l l s . Delivery of l i p i d to kidney tissue could be further increased by blood-borne monocytes which i n f i l t r a t e glomerular mesangium and protein-rich dietary supplementation (discussed l a t e r i n t h i s section). The other source of the fatty accumulation i n kidney tissue i s c e l l -mediated delivery of l i p i d . Monocyte-macrophage and erythrocyte are the most l i k e l y c e l l s which could be implicated i n t h i s process. Kreisberg et a l . (1979) pointed out that monocytes participate d i r e c t l y i n inducing glomerular injury through release of l y t i c enzymes that a l t e r GBM (181). This effect could promote the lipid-induced damage of GBM and enhance permeation of intact lipoproteins into mesangium and to renal tubules. The c i r c u l a t i n g LLM might have contributed to the l i p i d delivery to the mesangium. Nevertheless, we could not provide conclusive evidence that c i r c u l a t i n g LLM i n f i l t r a t e the mesangial tissue where i t appears as NSE-positive c e l l s . In other words, although LLM might i n f i l t r a t e GBM to the mesangium as other monocytes do, i t i s unlikely that LLM can be the main source of the intraglomerular NSE-positive c e l l s . This conclusion i s - 157 -j u s t i f i e d by our observation that not a l l the intraglomerular NSE-positive c e l l s were li p i d - l a d e n . Thus, i t i s unlikely that c i r c u l a t i n g LLM are an important source of the glomerular l i p i d . The i n f i l t r a t i n g blood-borne monocyte into glomerular mesangium might engulf the permeating lipoprotein species. In t h i s regard, Goldstein et a l (1980) demonstrated a high a f f i n i t y binding s i t e on the surface of mouse peritoneal macrophages which recognizes beta-VLDL isolated from hyperlipidaemic dogs (147). They reported macrophage uptake of the beta-VLDL, LDL, and HDLc; however, beta-VLDL was the most e f f i c i e n t stimulator of cholesterol e s t e r i f i c a t i o n inside macrophage. In agreement with t h i s report, _in vivo studies demonstrated that accumulation of beta-VLDL i n the blood stream of cholesterol-fed animals i s associated with cholesteryl ester deposition i n macrophages i n a variety of tissues (182,183). Since we found beta-VLDL and cholesterol-rich alpha migrating lipoprotein i n the HC and HCHP group at day 70, we speculate that intramesangial monocyte-macrophage might take up these abnormal lipoproteins by a mechanism sim i l a r to that described above. In b r i e f , monocytes could mediate l i p i d accumulation i n the glomerular mesangium mainly by aggravating damage of GBM which enhances f i l t r a t i o n of intact lipoproteins. Also, blood-borne monocytes could i n f i l t r a t e into mesangium and engulf the f i l t e r e d lipoproteins. The other possible c e l l u l a r source of the accumulated kidney l i p i d i s the erythrocyte membrane (EM). The importance of t h i s source i s suggested by two observations reported i n our study: f i r s t , the EM was cholesterol r i c h i n the HC and HCHP groups, and second, the presence of intraglomerular erythrophagocytosis. The l a t t e r phenomenon was a rare observation even i n the HCHP group. Moreover, there was no indication of intramesangial - 158 -haemolysis i n both HC and HCHP groups. These findings argue against EM as an important source of the l i p i d accumulated i n kidney tissue. The t h i r d possible source of l i p i d i n kidney tissue i s de novo cholesterol synthesis. The present study demonstrates suppression of de  novo cholesterol synthesis i n the HC group. This finding agrees with those reported by several other investigators (184-186). Brown and Goldstein (1979) demonstrated that increased cholesterol uptake suppresses synthesis of endogenous cholesterol at the l e v e l of HMG-COA reductase (184). Also, HMG-COA reductase a c t i v i t y i s reduced i n fibroblasts cultured i n an LDL-rich medium (185). Furthermore, using t r i t i a t e d water incorporation into cholesterol as a means to measure the rate of cholesterol synthesis, i t has been demonstrated that the rate of t r i t i a t e d water incorporation was reduced in cholesterol-fed rats (166). Our findings i n the cholesterol-fed animals confirm the previously reported suppressive effect of cholesterol feeding on de novo cholesterol synthesis. However, protein supplementation of the cholesterol-rich diet resulted, i n our study, i n an increase i n the de novo synthesized cholesterol compared to the l e v e l found i n the HC group ( a l b e i t s i g n i f i c a n t l y less than that i n the CONT group). This r e l a t i v e increase i n de novo synthesis might be due to the increase i n glomerular c e l l u l a r p r o l i f e r a t i o n (requiring more membrane synthesis) found i n the HCHP group compared to the HC group at day 70. Interestingly, the HP group showed a reduced rate of de novo cholesterol synthesis compared to the CONT group at day 70. This finding might be explained, i n part, by the hyper-cholesterolaemic effect of protein which was shown i n experiment I and agreed with findings of other investigators (102,122,123,124,125). Also, concomittant with the s l i g h t increase i n plasma cholesterol i n the HP group, - 159 -we found, in the chemical analysis of kidney l i p i d , a tendency to an increase i n kidney cholesterol content. These findings indicate that suppression of de novo cholesterol synthesis occured due to a r e l a t i v e increase i n cholesterol delivery to kidney tissue. However, we believe that other mechanism(s) besides the one we propose might be involved to account for the l e v e l of reduction i n kidney. Thus, further investigation i s required to explain t h i s observation. Our data indicate the increase i n the l i p i d content of kidney tissue found i n the HC and HCHP groups i s unlikely to be synthesized in s i t u , except for that needed for mesangial c e l l u l a r p r o l i f e r a t i o n . Altogether, the present findings suggest that the accumulated kidney l i p i d content found i n the HC and HCHP groups i s of plasma o r i g i n . Our observations backed by data of other investigators suggest that plasma l i p i d could be delivered to glomerular tissue either as separate components (e.g. cholesterol) or as intact lipoproteins. Our results show a s i g n i f i c a n t correlation between the l i p i d content of kidney tissue and the degree of mesangial expansion. This finding i s consistent with a previous report which suggested that binding of plasma lipoproteins to the glomerulus may stimulate production of matrix by mesangial c e l l s (187). On the other hand, the early work of French et a l indicated no correlation between the presence of fatt y deposits i n glomeruli and the degree of glomerulosclerosis (23). This seems to be due to the fact that they derived t h e i r correlation from q u a l i t a t i v e assessment of both l i p i d deposits and h i s t o l o g i c a l lesions. However, no s i g n i f i c a n t difference at day 30 i n the intraglomerular NSE-positive c e l l s was detected between HCHP and HC groups. This indicates that a glomerular p r o l i f e r a t i v e lesion - 160 -could be induced by accumulated l i p i d materials i n absence of a s i g n i f i c a n t increase i n the number of NSE-positive c e l l s . There i s no direct evidence in the l i t e r a t u r e which supports a mitogenic role for l i p i d i n the development of glomerulosclerotic lesions. However, i n a recent e d i t o r i a l , Kashgarian (1985) stated that proteinaceous macromolecules engulfed by mesangial c e l l s by endocytosis may activate these c e l l s to pr o l i f e r a t e and/or produce matrix (188). This suggestion may provide at least a p a r t i a l explanation of our finding. High protein supplement induces hypercholesterolemia (102,118,122,123,124,125) and increases GFR (31,189,190) and glomerular plasma flow (191). Thus, at day 30 when there was no s i g n i f i c a n t increase i n intraglomerular NSE-positive c e l l s i n the HCHP group compared to HC group more l i p i d deposition could be induced i n the HCHP group by virtue of the two mentioned effects of protein. The si g n i f i c a n t increase i n MA/GTA i n the HCHP group compared to the HC group at day 30 was most l i k e l y due to the increase of l i p i d deposition i n the HCHP group compared to the HC group. In addition to the s i g n i f i c a n t increase i n the kidney l i p i d content i n both HC and HCHP groups which correlated well with MA/GTA r a t i o , there was a si g n i f i c a n t correlation at day 70 between the degree of monocytic i n f i l t r a t i o n and the MA/GTA r a t i o . Since there were no s i g n i f i c a n t changes in the l i p i d contents between days 30 and 70, i n neither the HC nor the HCHP groups, i t i s reasonable to conclude that monocytic i n f i l t r a t i o n augmented the lipid-induced glomerular lesion. The involvement of the monocyte i n the pathogenesis of glomerulosclerosis i s consistent with the results of previous investigations. In humans, using NSE sta i n and/or electron microscopy, Monga et a l . (1985) reported monocyte i n f i l t r a t i o n of glomeruli - 161 -i n various types of glomerulonephritis (192). Also, recently, the involvement of monocyte i n various types of experimental glomerulonephritis has been reviewed (193). In t h i s regard, a histochemical study has shown that monocytes constituted the major proportion of the c e l l s i n glomerular crescents i n rabbit and sheep (194). Furthermore, i n an i n v i t r o study, i t has been suggested that blood monocytes might play a role i n the pathogenesis of mesangial c e l l p r o l i f e r a t i o n i n glomerulonephritis (195). Recently, Ferrario et a l . (1985) reported a good correlation between proteinuria and intraglomerular monocytic i n f i l t r a t i o n (196). We have demonstrated that the accumulated l i p i d material found i n kidney in the HC and HCHP groups originated mostly from plasma. I t has been reported that rats fed a high-protein diet had higher glomerular plasma flow than that found i n rats kept on low-protein diet (190). I t i s possible that in the e a r l i e r stages of our experiment (prior to the development of glomerulosclerosis), plasma flow was increased i n the HCHP group, r e s u l t i n g i n more lipoprotein p a r t i c l e s delivered to the glomeruli. However, we could not provide conclusive evidence for such an increase i n the plasma flow. On the other hand, our findings i n the HP group indicate that no glomerular lesions were induced i n the absence of abnormal lipoproteins. This suggests that a synergistic effect of increased plasma flow and abnormal lipoproteins in mesangial tissue induces a glomerular p r o l i f e r a t i v e lesion i n the HCHP group. Alterations i n glomerular haemodynamics due to high-protein diet have been implicated i n the pathogenesis of mesangial injury (197). However, the mechanism i s s t i l l unknown. Raij et a l . (1983) provided experimental evidence which suggested that alterations i n glomerular haemodynamics act - 162 -s y n e r g i s t i c a l l y with mesangial entrapment of macromolecules (198). Their findings are i n agreement with our hypothesis that increased glomerular plasma flow leads to increased delivery of plasma lipoproteins to the glomerular tissue where the abnormal lipoproteins are trapped. The basis of our understanding of the mechanism of aggravating cholesterol-induced glomerulosclerosis by high protein supplement can be explained by the following scenario: In the cholesterol-fed guinea pigs, the increase i n plasma l i p i d s led to an increase i n renal tissue l i p i d which was accompanied by increased mesangial expansion. The i n i t i a t i o n of the glomerular lesion by l i p i d seems to be aggravated by the presence of monocyte-macrophage. The blood-borne monocytes i n f i l t r a t e the glomerular mesangium and participate i n induction of a glomerular l e s i o n by an as-yet-unidentified mechanism (200,201). However, i t has been proposed that the monocyte stimulatory effect on mesangial p r o l i f e r a t i o n could be due, i n part, to enhancement of endogenous mesangial c e l l prostaglandin E production (202). Once the mesangial c e l l s are stimulated by a mitogenic factor(s) released by i n f i l t r a t i n g monocyte-macrophage, mesangial c e l l s p r o l i f e r a t e and produce another growth factor (203). In t h i s regard, Lovett et a l . (1986) p u r i f i e d and characterized a protein derived from cultured glomerular mesangial c e l l s . They found that t h i s cytokine has a close resemblance to in t e r l e u k i n 1 produced by macrophage and acts as an autocrine or paracrine growth factor (203). A sim i l a r finding has been reported by the same group showing a mesangial cell-derived thymocyte-activating factor which induces thymocyte p r o l i f e r a t i o n and enhances production of interleukin-2 by peripheral lymphocytes (204). These workers have suggested that the l o c a l release of - 163 -cytokine by glomerular mesangial c e l l s might be an important factor i n enhancing mesangial p r o l i f e r a t i o n and matrix expansion. Thus, monocyte i n f i l t r a t i o n into glomerular mesangium may induce an accelerated propagation of mesangial c e l l s exerted by a cascade of actions started by a monocyte-derived growth factor which stimulates mesangial c e l l p r o l i f e r a t i o n . Since i t has been demonstrated that cultured mesangial c e l l s produce different connective tissue components (205), we believe that the expanded mesangial matrix i n our study i s due to p r o l i f e r a t i n g mesangial c e l l s . Supporting t h i s conclusion, the magnitude of the PAS-positive lesions we noticed i n the HC and HCHP groups was i n accordance with the values of MA/GTA r a t i o s . We did not study the chemotactic mechanism which induces monocytic i n f i l t r a t i o n into the mesangial tissue. However, dif f e r e n t chemotactic factors might be implicated, among them the platelet-derived growth factor (PDGF) (199) and a factor produced by endothelial c e l l s previously exposed to beta VLDL (206). These need to be studied i n d e t a i l . The high-protein supplementation might lead to increase renal plasma flow prior to the development of glomerulosclerosis i n the HCHP group (as discussed above). This effect could cause more delivery of blood monocytes to the glomerular tis s u e , a suggestion which i s supported by the presence of a s i g n i f i c a n t correlation between mesangial expansion and the number of intraglomerular monocytes. With t h i s i n mind together with the presence of greater l i p i d deposition i n the glomerular tissue of the HCHP group compared to the HC group, one could explain the aggravating effect of the high protein diet on the cholesterol-induced glomerular le s i o n . - 164 -VI. General Conclusions and Postulated Mechanisms of Anaemia and  Glomerulosclerosis Cholesterol feeding induced haematological and renal changes i n guinea pigs which are related to plasma l i p i d and lipoprotein abnormalities. Cholesterol may transfer from cholesterol-laden lipoproteins to erythrocyte membrane driven by i t s concentration gradient. Cholesterol-enriched erythrocytes acquires echinocyte morphology which would, l i k e l y , be subjected to phagocytosis and the " p i t t i n g process" by splenic macrophages. The net sequalae of these two processes are extra- and intravascular haemolytic anaemia. S i m i l a r l y , cholesterol could s e l e c t i v e l y transfer from the cholesterol-laden lipoproteins to glomerular tissue; i n addition, these lipoproteins might bind to the glomerular endothelial l i n i n g and GBM. The blood-borne monocyte-macrophages i n f i l t r a t e d into the mesangial tissue could stimulate mesangial c e l l u l a r p r o l i f e r a t i o n by an as-yet-unidentified mitogenic factor, possibly interleukin-1. There i s evidence that p r o l i f e r a t i n g mesangial c e l l s may release a growth factor which could amplify the c e l l u l a r p r o l i f e r a t i o n and production of c e l l u l a r matrix. Enrichment of the cholesterol-rich diet by a high protein supplement aggravated the hypercholesterolemia which, apparently, worsened haemolysis and glomerulosclerosis. In addition to the hypercholesterolaemic e f f e c t , the high protein supplement may have increased delivery of lipoproteins and monocytes to the glomeruli by increasing glomerular plasma flow prior to the development of glomerulosclerosis. The increase i n monocyte i n f i l t r a t i o n may be partly responsible for the augmented mesangial expansion seen i n the HCHP group. However, i n the early stage (day 30), no s i g n i f i c a n t correlation between intraglomerular monocytes and MA/GTA r a t i o was detected; - 165 -thus, the mesangial lesion found i n the HC and HCHP groups at that time was mainly attributed to the l i p i d effect on mesangial c e l l s . I t i s these findings that permit our conclusion that cholesterol-induced glomerulo-s c l e r o s i s could be accounted for by both l i p i d and monocytic i n f i l t r a t i o n into mesangial tissue. The s i g n i f i c a n t correlation between proteinuria and haematuria and the glomerular changes indicate a good relationship between the st r u c t u r a l and functional alterations found i n the HC and HCHP groups. Our data did not support the concept of a relationship between either haemolytic anaemia or abnormalities of l i v e r function and the glomerulosclerotic l e s i o n , nor was the lesion an immune-mediated one. New Findings i n t h i s study: 1. The sequential studies at 5, 10, 30, and 70 days showed that: a - The l i p i d deposits i n the glomerular tissues of cholesterol-fed guinea pigs i n i t i a t e the glomerulo-sclerosis. The presence of abnormal lipoproteins i n plasma i s a prerequisite for these deposits, b - The monocytic i n f i l t r a t i o n into the glomerular tissue follows the l i p i d deposits and augments the glomerulosclerotic process. We postulate that a process resembling atherosclerosis i s taking place at the c a p i l l a r y l e v e l . 2. The high protein suppplementation aggravated the cholesterol-induced glomerulosclerosis, most l i k e l y by elevating plasma cholesterol. In addition, monocytic i n f i l t r a t i o n into l i p i d - i n f i l t r a t e d glomeruli was increased i n animals on high cholesterol diet with protein supplement. - 166 -3. We ruled out haemoloysis as a possible cause of the glomerulosclerotic lesion found i n cholesterol-fed guinea pigs. 4. Lipid-laden monocytes were observed i n the c i r c u l a t i o n of cholesterol fed guinea pigs. The number of these c e l l s increased after a high-protein supplementation. VII. Further Work Several aspects of our findings need to be further investigated i n greater d e t a i l : 1. Confirmation, i n i n v i t r o and may be i n vivo studies, that mesangial c e l l p r o l i f e r a t i o n could be induced by l i p i d materials i n the absence of monocytes or other c e l l u l a r types which can release mitogenic factors (e.g. p l a t e l e t s ) . 2. Investigation of the possible chemotactic factors which might account for the monocytic i n f i l t r a t i o n into the mesangial tissue. 3. Studying the isolated effect of each lipoprotein species (from normal and hyperlipidaemic animals) and free cholesterol on the induction of mesangial c e l l p r o l i f e r a t i o n . 4. 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