The Effect of Activation of Peroxisome Proliferator-Activated Receptor On Modulating Renal Alterations in the Early Phase of Experimental Diabetic Nephropathy

Abstract

Nephropathy is one of the major clinical manifestations of microvascular disease in diabetic patients. It has been shown that hyperglycemia plays an essential part in diabetic microangiopathy. Early changes in diabetic nephropathy include glomerular hypertrophy, hyperfilteration, development of microalbuminuria, followed by development of glomerular basement membrane thickening, accumulation of mesangial matrix, and overt proteinuria which may lead to glomerulosclerosis and end stage renal disease (ESRD). This study explores new grounds for therapy of diabetic renal changes by investigating effects of the anti diabetic type 2 drug peroxisome proliferator-activated receptor-ã (PPAR ã) agonist Rosiglitazon on type 1 induced diabetic nephropathy. Objectives: To examine Rosiglitazone's role in reducing the progression of diabetic nephropathy and angiogenic renal alternations in the early phase of diabetic nephropathy induced by STZ in rats. This examination is mediated by kidney function tests, microscopic and macroscopic evaluations of the kidneys, and also via assessing angiogenic factors: Ang-1, Ang-2, their receptor Tie-2, and VEGF-A. In order to do this, the following tests were used: immunohistochemsitry for Ang-1, Ang-2, and Tie-2, western blot was used for markers Ang-2 and Tie-2 while ELISA test was done to examine the level of VEGF. Methods: Experimental Groups: Because different doses of Rosiglitazone were used, six experimental groups (n=8-14 for each) male wistar rats weighing average 280-300g were recruited for this purpose. Group I was non diabetic rats on buffer vehicle (i.e. control), group II was STZ induced diabetic rats for 4weeks [50mg/kg via intraperitoneal needle], Group III was STZ diabetic rats + Rosiglitazone at the dose of (1mg/kg/daily for 4weeks), and Group IV was STZ diabetic rats + Rosiglitazone at (3mg/kg/ daily for 4weeks), group V was STZ diabetic rats + Rosiglitazone at the dose of 5mg/kg/ daily for 4weeks while group VI was STZ diabetic rats + Rosiglitazone at the dose of 30mg/kg/ daily for 4weeks. All groups were taking standard rat chow and were subjected to the same living conditions. Kidney Function Tests: This study measured urine flow, urine as well as serum levels of creatinine and also assessed creatinine clearance (CCr), urinary albumin excretion, and BUN. Assessment of Renal Hypertrophy: Absolute and relative kidney weights were histologically and microscopically assessed for renal hypertrophy. Kidney tissue samples were embedded in 10% formalin and paraffin sections were studied with H&E to assess glomerular hypertrophy. Immunohistochemistry: The study examined the extent of angiogenesis in paraffin embedded kidney sections. Therefore, immunostaining of kidney sections with the following angiogenic protein antibodies were carried out: angiopioten-1 (ang-1), angiopioten-2 (ang-2), and their receptor Tie-2. The intensity of the staining was semiquantified and statistically analyzed. Western Blot: Western blot analysis was preformed using tissue sample extracts in order to measure angiogenic factors: ang-2, and Tie-2. Immunisorbent Assay (ELISA): ELISA was used to determine the level of the angiogenic marker: vascular endothelial factor (VEGF) in kidney tissue and plasma. Results: This study found that when RSG was administered in low doses (1mg/kg/daily for 4weeks, and 3mg/kg/ daily for 4weeks), the size of kidney significantly increased relative to the normal group (1mg: p=<0.001, 3mg: p=<0.005). Also, with the low doses of RSG, the size of the glomerulus grew significantly larger relative to the normal (1mg: p=<0.01, 3mg: p=<0.002) and to the diabetic untreated group (3mg: p=<0.01). On the other hand, RSG given in 5mg/kg/daily for 4weeks or the high dose of 30mg/kg/daily for 4weeks greatly decreased glomerualr size relative to the diabetic untreated samples (5mg: p=<0.002, 30mg: p=<0.0001) although not to the extent of the normal values. Also, when compared to the diabetic untreated results, these doses of 5mg and 30mg of RSG/kg/daily for 4weeks significantly decreased kidney size reaching almost normal levels (5mg: p=<0.002, 30mg: p=<0.01). Moreover, low doses of RSG also dramatically increased protein expression of the angiogenic factors: ang-1, ang-2, and tie- 2 more than the levels measured for both normal and the diabetic untreated samples. Furthermore, when evaluating the immunohistochemistry (IHC) results, 5mg and the high dose of 30mg RSG/kg/daily for 4weeks seemed to have greatly decreased angiogenic factors to almost normal. Furthermore, kidney function tests were highly improved when diabetic rats were given high doses of RSG (i.e. 30mg/kg/daily for 4weeks) as it decreased Ccr relative to the diabetic untreated group (p=<0.02). In addition to this, analysis of immunblots for ang-2 showed a significant increase in this angiogenic marker with the low doses of 1mg and 3mg given/kg/daily for 4weeks. The expression of ang-2 with the 5mg/kg/daily for 4weeks dose was lower than that of 1mg and 3mg RSG doses given per kg/daily for 4weeks, but was considerably higher than the normal group protein expression. On the other hand, the high dose of 30mg/kg/daily for 4weeks has significantly decreased the protein expression of ang-2. Tie-2 western blot analysis revealed that it generally increased with the different RSG doses but the significant result here is the noticeable increase of Tie-2 in the normal and 30mg RSG groups. Furthermore, VEGF is known to signify angiogenesis; and for that, ELISA of plasma VEGF was done but it didn’t yield productive results; and hence, tissue samples were used and the study found that the high dose of the drug (30mg/kg/daily for 4weeks) considerably decreased VEGF level when compared with the diabetic untreated group. Conclusion: The results of this study indicate that when RSG is given in high doses (e.g. 30mg/kg/daily for 4weeks), it can ameliorate diabetic nephropathy; precisely, the resulting angiogenesis and glumerular hypertrophy while low doses of RSG (1mg, 3mg both given per kg/daily for 4weeks) can worsen the condition.