| Objective:To explore the possible mechanisms of podocyte apoptosis in type 2 diabetic nephropathy which AngII,TRPC6 were involved in through detecting the levels of AngⅡ,TRPC6,cleaved-caspase-3 in renal tissue of type 2 diabetic mellitus(T2DM)rats and MPC5.Methods:40 SPF,male Wistar rats were randomly selected.30 of them were treated with high-fat,high-sugar diet and intraperitoneal injection of low-dose streptozotocin(STZ)to establish model rats of T2 DM.The T2 DM models were randomly divided into diabetes group(DM),valsartan treatment group(ARB).The rest rats were grouped as normal control group(NC).After 12-week-intervention,blood and urine samples were collected to determine the rat tail systolic blood pressure(SBP),fasting blood glucose(FBG),cholesterol(CH),triglyceride(TG),urinary albumin excretion rate(UAE),creatinine clearance rate(Ccr)and other biochemical indexes.The pathological changes of renal tissue and the ultrastructure of podocytes were observed by light microscope and electron microscope respectively.We detected the expression of TRPC6 in renal tissue by western-blot and qRT-PCR,and the levels of AngⅡ、cleaved-caspase-3 was detected by western-blot.The mouse podocytes(MPC5)were divided into normal glucose group(NG),hypertonic group(HM),high glucose group(HG),valsartan group(VAL),SAR7334 group(SAR).The apoptosis rate of podocytes was determined by flow cytometry,the levels of AngⅡ,cleaved-caspase-3 was detected by western-blot,and the expressions of TRPC6 in podocytes were detected by western blot and qRT-PCR.Result:1.Compared with NC group,the levels of SPB,FBG,CH,TG,UAE and Ccr of DM group were significantly higher.The levels of UAE and Ccr in ARB group were significantly lower than that in DM group,the difference was statistically significant(P<0.05).Although SBP,FBG,CH,TG were lower than that in DM group,the differences were not statistically significant.2.The enlarged glomeruli,expanded capillary loops,mesangial hyperplasia and thickened basement membrane were observed in DM group by light microscope.Compared with the NC group,the structures of podocytes in DM group were disordered,with fusion and disappearance of foot process,diffuse thickening of basement membrane.The podocyte fusion rate and basement membrane thickness were significantly higher than those in NC group(P<0.05).The above pathological changes were alleviated with the intervention of AngⅡ receptor blocker.3.The levels of AngⅡ,TRPC6 and cleaved-caspase-3 in renal tissues of DM group were significantly higher than those in NC group(P<0.05).And the levels of TRPC6 and cleaved-caspase-3 were significantly lower with the intervention of AngⅡ receptor blocker than those in DM group(P< 0.05).4.High glucose made the podocyte apoptosis rate significantly higher than that of NG group(P< 0.05),while the apoptosis rate had no significant difference between NG and HM group.The podocyte apoptosis rate of VAL group and SAR7334 group were significantly lower than that of HG group(P<0.05).5.Compared with NG group,the expression levels of AngⅡ,TRPC6,cleaved-caspase-3 in HG group were significantly higher(P<0.05),while there were no significant differences between HM group and NG group.The expressions of TRPC6,cleaved-caspase-3 after blocking AngⅡ with valsartan were significantly lower than those of HG group(P<0.05).The expressions of AngⅡ and cleaved-caspase-3 with the intervention of TRPC6 inhibitor were also significantly decreased than those of HG group(P<0.05).Conclusion: AngⅡ,TRPC6 are involved in the process of podocyte apoptosis in type 2 diabetic nephropathy,and high glucose may induce podocyte apoptosis by upregulating the expression of AngⅡ/TRPC6. |
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