| Objective: Diabetic nephropathy(DN) is one of the most important microvascular complications of diabetes mellitus(DM). The early changes in DN are characterized by the thickening of glomerular basement membrane and expanded extracelluar matrix, leading to glomerular hyperfiltration and microalbuminuria. DN becomes a leading cause of end-stage renal disease. Essential cells in glomerular, especially mesangial cells secrete many cytokine in which transforming growth factor(TGF)-β1 and vascular endothelial growth factor(VEGF) are key pathogenic factors in the early-stage of DN. TGF-β1 has been recognized as a modulator of ECM . The overexpression of TGF-β1 could lead to the expanded extracelluar matrix and stimulate the podocyte secreting VEGF. Pigment epithlium-derived factor(PEDF) is a multifunctional serine proteinase inhibitor. As a potent anti-angiogenic factor, the balance of PEDF and VEGF palys an important role in maintaining the homeostasis of the ocular vascular system. Although PEDF was first identifined in the eye, it is widely distributed in a variety of organs. In the present study abroad showed the low expression of PEDF in the kidney of diabetic rats, both in cortex and medulla. The cells culture shows PEDF blocked high-glucose-induced TGF-β1 overexpression in primary human mesangial cells. So we infer PEDF is a key pathogenic factor in DN. Rosiglitazone belongs to the thiazolidinediones (TZDs) which is a novel insulin sensitizer. The agent improves glomerular hyperfiltration, modulates the proliferation of mesangial cells and renal tubular epithelial cells, also reduces inflammation in kidney. Previous studies have shown that TZDs could decrease the expression of TGF-β1 and VEGF in a dose-dependent manner. We think it's the effect of anti- inflammation and vascular-protection in diabetes. As the low expression of PEDF in kidney of diabetic rats , its close relation with VEGF and decreasing the overexpression of TGF-β1 in kidney of DN, We infer rosiglitazone could downregulate TGF-β1 and VEGF by increasing the expression of PEDF. In this way, the agent improves glomerular hyperfiltration and inhibits extracelluar matrix expanding. Therefore rosiglitazone exerts its effect of renal protection. In the study, We established type 1 diabetic rats by a high-dose intraperitoneal injection of streptozotocin. The characteristics of the model were similar to human type 1 diabetes. The expression of PEDF and TGF-β1 were detected in order to discuss their roles in the pathogenesis of DN. At the same time, rosiglitazone was applied to investigate their effect of renal protection of DM.Methods: Male SD rats were randomly assigned into two groups: 14 in normal control group(NC) and the others in diabetes model group(MO). All the rats were fed with the normal diet. Hyperglycemia was induced by intraperitoneal injection STZ (55mg·kg-1) into MO group rats after 1 weeks on this diets . At 72h after injection, the rats whose blood glucose were higher than 16.7mmol/L were diabetic rats. Diabetic rats were randomly divided into two groups: diabetes control group (DM),diabetes group treated with rosiglitazone (5mg·kg-1·d-1) (RSG). The rats in RSG group were given rosiglitazone sodium after model- made, while the other two groups were given N.S in the same volume. Fasting blood glucose was measured before the experiment, 72h after injection and the final of research. Body weight and urinary albumin excretion (UAE) were measured after 12 weeks. Serum TG, TC, LDL-C, VLDL-C, HDL-C, BUN-C, SCr, AST, ALT and serum PEDF were observed after 12 weeks . Kidney specimens were prepared for H.E.-stained and immunohistochemistry. The protein expression and location of PEDF and TGF-β1 were obsreved by immunohistochemistry. The kidney tissue stucture was observed by light microscope. Mean glomerular area(MGA) was calculated by analysis system of image. Morever, PEDF and TGF-β1 expression in renal cortex were analysed by Western blot. All the data were dealt with SPSS 12.0.Results:1. 72h after injection in model group, fasting blood glucose was markedly higher than normal control group (P<0.01). After 12 weeks in DM group and RSG group, fasting blood glucose were markedly higher than normal control group(P<0.01, P<0.01), but there is no significantly difference between the two group.2. After 12 weeks, in DM, kidney mass and kidney mass/body mass markedly increased compared with NC (P<0.01,P<0.01). Compared with DM there was significant decrease in kidney mass and kidney mass/body mass in RSG (P<0.05,P<0.05).3. Serum TG, TC, LDL-C, BUN, Scr in DM group were increased compared with NC group (P<0.05,P<0.05,P<0.05,P<0.01, P<0.01).4. Compared with NC group, there was significant increase in urinary albumin excretion (UAE) in DM(P<0.01). Compared with DM, there was significant decrease in UAE in RSG (P<0.05).5. Compared with NC, the PEDF levels in the serum were a little increase in DM(P=0.15) and a little decrease in RSG(P=0.75). Compared with DM there were no significant decreases of the PEDF levels in the serum in RSG (P=0.19).6. Light microscopy shows: Compared with NC, Glomerular hypetrophy, mesangial expansion, thickening of glomerular basement membrance were observed in DM.7. Immunohistochemistry shows: PEDF was predominantly expressed in the glomeruli, along glumerular basement membrane. In the medulla, the PEDF signal was also detected at lower levels in the tubular basement membrane and interstitial tissue. Compared with NC, the expression of PEDF was markedly decreased in DM(P<0.01). Compared with DM there were significant increases in the expression of PEDF in RSG (P<0.05). TGF-β1 was predominantly expressed in the glumerular basement membrane and interstitial tissue. Compared with NC, the expression of TGF-β1 was markedly increased in DM(P<0.01). Compared with DM there were significant decreases in the protein expression of TGF-β1 in RSG (P<0.05).8. Immunohistofluorescence shows: PEDF was predominantly expressed in the glomeruli, along basement membrane and microwascular walls. TGF-β1 was predominantly expressed in the glumerular basement membrane and interstitial tissue.9. Compared with NC, protein expression of PEDF was markedly decreased in DM(P<0.01). Compared with DM there were significant increases in the protein expression of PEDF in RSG (P<0.05). Compared with NC, protein expression of TGF-β1 was markedly increased in DM(P<0.01). Compared with DM there were significant decreases in the protein expression of TGF-β1 in RSG (P<0.05).Conclusions1. The male SD rats were intrapenitoneally injected a high-dose STZ to severely destroyedβcells of pancreas islet, by which the rat model of type 1 diabetes mellitus can be established. continous hyperglycemia in this model could damaged the kidney of rats. So the model could be used to study pathogenesis of DN in human and the direct effect on kidney of agents.2. The protein expression of PEDF decreased markedly on renal tissue in diabetic rats while TGF-β1 was elevated markedly in renal tissue, which shows PEDF and TGF-β1 can play an important roles in the progression of DN.3. The protein expression of TGF-β1 was markedly decreased in renal tissue treated by rosiglitazone in diabetic rats. At the same time, the protein expression of PEDF was markedly elevated in renal tissue and also with the serum PEDF level in rosiglitazone-treated rats. The result implicates that rosiglitazone can obviously improve pathophysiological changes of DN by effecting inflammatory factor TGF-β1 and anti-angiogenic factor PEDF. So rosiglitazone has a direct renoprotective effect on the progression of DN through helping to regulate inflammation by modulating inflammatory factors and increasing the renoprotective cytokine, besides its improvement in insulin sensitivity and lipid metabolism.
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