Effect Of TRPC6 On ROS Mediated Autophagy And Apoptosis In Renal Tubular Epithelial Cells And The Underlying Mechanism

Back ground:Renal ischemia reperfusion（IR）injury is the most common cause of acute kidney injury（AKI）,often occurres in the process of renal surgery,kidney transplantation,renal artery stenosis as well as extracorporeal shock wave lithotripter（ESWL）and often results in high mortality in clinical cases.Renal IR injury results in apoptosis and necrosis of renal tubular epithelial cells,which are parts of the major damages in AKI.Overproduction of reactive oxygen species（ROS）causes oxidative stress which is the main reason in the pathogenesis of IR injury.Recent studies indicate that ROS can activate autophagy in TEC,and inhibition of autophagy worsens renal tubular injury and renal function,supporting the renoprotective effects of autophagy on IR injury.Transient receptor potential channel 6（TRPC6）,as a non selective cation channel,plays an important role in various renal diseases,such as focal segmental glomerulosclerosis,transient proteinuria and diabetic nephropathy.More importantly,studies showed that TRPC6 expression was upregulated in IR injury,and that TRPC6also served as a downstream effector of ROS.However,the cellular responses of TRPC6-mediated calcium influx in renal IR injury have not been fully understood.At the same time,the relationship between autophagy and TRPC6,as well as their function in renal I/R injury,remain to be addressed.Objective:The purpose of this study was to:explore the regulatory effects of TRPC6 on autophagy and apoptosis of TEC;clarify the relationship between autophagy and apoptosis caused by ROS;and elucidate the related signal transduction pathway in regulating autophagy and apoptosis by TRPC6.Methods:In this study,H₂O₂ treated TEC isolated from wild type（WT）or TRPC6knockout(TRPC6 KO,TRPC6^-/-)mice were used to simulate damages caused by ROS in vitro.The changes of autophagy in TEC induced by ROS were detected by transmission electron microscopy,GFP-mRFP-LC3 fluorescence detection and Western blot.On the other hand,by using LDH,CCK and JC-1 kit,flow cytometry and Western blot,the changes of apoptosis in TEC were detected.At the same time,TRPC inhibitor SKF96365 and autophagy inhibitor CQ were used to investigate the relationship between TRPC6 and autophagy,as well as their role in the regulation of ROS mediated apoptosis in TEC.Results:1.After 1-2 days,renal tubular epithelial cells were climbed out from the isolated renal tubules,and filled after 5-7 days.The positive rate of CK-18 and E-cadherin in renal tubular epithelial cells was more than 99%.2.Primary RTE cells of TRPC6 knock out mice exhibit higher levels of autophagy compared to those of wild-type mice in normal and H₂O₂ treated conditions.TRPC6 inhibition promotes autophagic flux in primary renal TEC and HK-2 cells.3.Silencing or over-expressing TRPC6 in HK-2 cells can not only influence the basal autophagy level but also the H₂O₂ induced autophagy level.4.SKF96365 promoted autophagy flux in HK-2 cells and primary RTE cells.5.H₂O₂ treatment increases TRPC6 expression,and SKF96365 inhibits TRPC6mediated SOCE,inhibits H₂O₂ mediated collapse of the mitochondrial membrane potential and inhibits the activation of cleaved Caspase-3.6.Addition of CQ caused significantly cell apoptosis and counteracted the protective effect of SKF96365 and TRPC6 knock out.7.Primary RTE cells from TRPC6^-/-mice showed lower level of p-AKT,p-70s6k and p-ERK1/2 expression than their WT conterparts.TRPC6 knock out may activate autophagy and attenuates apoptosis by negatively regulated PI3K/AKT/mTOR and ERK signaling pathway.Conclusion:We demonstrated that TRPC6-mediated calcium entry inhibited autophagy in TEC through upregulating the PI3K/Akt/mTOR signaling pathway and the ERK pathway.Thus knock down of TRPC6 increased the level of autophagy and alleviated ROS mediated apoptosis.