The Effect Of TRPC6/NFAT2 On Glomerular Mesangial Cell Injury Induced By High Glucose

Objective: The mechanism of TRPC6(transient receptor potential canonical 6)and NFAT2(Nuclear factor of activated T cells 2)in diabetes are still in dispute.This study aims to investigate the effect of TRPC6/NFAT2 on glomerular mesangial cells injury induced by high glucose.Methods:1.Type 1 diabetes models: SPF male rats were molded as diabetes rats by injecting STZ intraperitoneally(soluted in 0.1 mol/L citrin with a p H of 3.5-4.5,55 mg/Kg).Glucose solution was provided in case of the death caused by low blood glucose.The NC group was injecting 0.1mol citrin(p H4.3-4.5).7 days later,the rats with blood glucose measured higher than 16.7mmol/L after starvation treatment for 12 h can be regarded as stable diabetes models.After modeling successfully;the rats were assigned to a control group,diabetic model group,and Ad-TRPC6-sh RNA treatment group.Rats were injected transfected Ad-TRPC6-sh RNA or Ad-null virus into pelvis and feeded for another 8 weeks,blood glucose and weights tested every 2 weeks.The weight of kidneys was measured and the ratio of kidney weight to body weight was calculated.Pathological changes and the level of collagen fibers in tissues can be observed by HE staining and Masson staining.the protein levels of TRPC6、Collagen IV、Fibronection were evaluated by Western Blot.2.(1)HBZY-1 cells were developed in Sterile culture room and divided into 2 groups.Normal group(NG 5.5 mmol/L glucose+19.5 mmol/L mannitol)and HG group(HG25mmol/L glucose)were treated for 48 h and 72 h respectively.Western Blotting and qRT-PCR were performed to evaluate the expression level of TRPC6、Fibronectin and collagen IV protein,and TRPC6 mRNA.(2)SiRNA-TRPC6 and siRNA-NFAT2 were used to transfect HBZY-1,cells were divided into 3 groups: control group(NG 5.5 mmol/L glucose+19.5 mmol/L mannitol),HG group(25mmol/L glucose +NC-siRNA)and siRNA group(25mmol/L glucose+siRNA).Western bolt was performed to evaluate the expression level of TRPC6,Collagen IV,fibronection and NFAT2 in HBZY-1 cells.Results: 1.In vivo:(1)HE results showed that no obvious structural difference and inflammatory cell infiltration;no obvious collagen sediment can be observed in all groups by masson stainning.(2)Compared with controls,fasting blood glucose(FBG),blood urea nitrogen(BUN),serum creatinine(Cr),the ratio of kidney weight to body weight significantly increased in STZ-induced diabetic rats.The increased levels of Cr in diabetic rats were reduced by Ad-TRPC6-sh RNA treatment(P<0.05).(3)The protein level of TRPC6、Collagen IV、Fibronection and NFAT2 were significantly increased in the diabetic rats..Ad-TRPC6-sh RNA directly inhibited the up-regulation of TRPC6、Collagen IV、Fibronection and NFAT2 protein level.2.In vitro :(1)Western Blot and qRT-PCR showed that the expression level of TRPC6 in HG group were apparently higher than control group(NG 5.5mmol/L glucose+mannitol19.5 mmol/L)(P<0.001).(2)The protein level of Collagen IV,Fibronection and NFAT2 of HG group were apparently higher than control group(P<0.001).(3)The protein and mRNA of TRPC6 level were apparently inhibited by siRNA-TRPC6(P<0.001).(4)the protein and mRNA level of TRPC6 were significantly increased in the HG group,which were inhibited by siRNA-TRPC6.(5)The protein level of Collagen IV,Fibronection and NFAT2 were significantly increased in the HG group,which were inhibited by siRNA-TRPC6.(6)the protein level of Collagen IV,Fibronection and NFAT2 were higher in HG group,siRNA-NFAT2 inhibited the up-regulation of Collagen IV,Fibronection and NFAT2 protein level(P<0.01).Conclusions:1.The protein level of TRPC6 and NFAT2 increased in diabetes rats kidneys.The inhibition of TRPC6 is beneficial for kidney function in type 1 diabetes.2.High glucose induced overexpression of collagen IV 、 NFAT2 、 TRPC6 and Fibronection,which can be abrogated by siRNA-TRPC6 through modulating NFAT2.The modulating on abnormal expression of TRPC6 may work as a novel target for diabetic kidney diseases treatment.