The Role Of PI3K/Akt Signal Pathway And TRPC6 Channel In Ang Ⅱ-induced Podocyte Injury

Objective:The normal activation of the phosphatidylinositol-3-kinase/protein kinase B（PI3K/Akt）signal pathway is very important for maintaining intact podocyte structure and function.Transient receptor potential cation channel protein 6（TRPC6）is one of many podocyte slit membrane proteins（SD）;however,the mechanism of the two in the process of podocyte injury induced by AngiotensinⅡ（AngⅡ）is uncertain.In this study,we explored the interaction between PI3K/Akt signal pathway and TRPC6 channel in the process of AngⅡ-induced podocyte injury.Methods:（1）Culture mouse glomerular podocytes in vitro:use DMEM medium supplemented with 1%penicillin-streptomycin and 10%fetal bovine serum（FBS）,the culture condition is 37℃and 5%CO₂,and the medium was changed every 24 hours.When the cell density reaches about 80%,the subculture is carried out.（2）Experimental grouping:The cells were divided into 8 groups according to the purpose of the study:（1）control group,（2）AngiotensinⅡgroup(10^-6 mol/L),（3）AngiotensinⅡ(10^-6mol/L)+Losartan group(10^-6 mol/L),（4）Losartan group(10^-6 mol/L),（5）AngiotensinⅡ(10^-6 mol/L)+LY294002(10^-5 mol/L)group,（6）LY294002 group(10^-5 mol/L),（7）AngiotensinⅡ(10^-6 mol/L)+SKF96365(4×10^-5 mol/L)group,（8）SKF96365group(4×10^-5 mol/L).（3）q RT-PCR and Western Blot were used to detect Akt,phosphorylated Akt（P-Akt）and transient receptor potential cation channel protein 6（TRPC6）m RNA and protein expression levels in MPC5 podocytes.（4）Hoechst 33258staining method was used to detect podocyte apoptosis.Results:（1）Compared with the control group,the expression of TRPC6 was significantly increased,the expression of Akt in podocytes of AngⅡgroup was not statistically different,p-Akt was significantly decreased,and the apoptosis of podocytes was significantly increased.（2）Compared with the AngⅡgroup,after SKF96365intervention,the expression of TRPC6 decreased significantly,the expression of Akt was not statistically different.The expression of p-Akt increased significantly,and the apoptosis of podocytes decreased significantly.（3）Compared with the AngⅡgroup,after the intervention of LY294002,the expression of TRPC6 has not changed,the phosphorylation level of Akt was significantly reduced,and the apoptosis of podocytes was significantly increased.（4）Compared with the AngⅡgroup,the expression of TRPC6 decreased significantly,there was no statistical difference in the expression of Akt after losartan intervention,the expression of p-Akt increased significantly,and the apoptosis of podocytes decreased significantly.Conclusion:AngⅡcan cause podocyte damage by activating the TRPC6 channel.The damage process may be caused by further inhibiting the activation of the PI3K/Akt signal pathway.Blocking the activation of TRPC6 channel helps to activate the PI3K/Akt signal pathway,thereby reducing podocyte apoptosis.