Regulation Of TRPC6 On EMT In Kidney Fibrosis And The Underlying Mechanism

Background:Kidney fibrosis is generally confirmed to a significant role in chronic kidney diseases resulting in the end-stage kidney failure.Epithelial-mesenchymal transition（EMT）is an important molecular mechanism contributing to fibrosis.Generally speaking,EMT is the process that epithelial cells lose some original characteristics and gain the characteristics of mesenchymal cells.Tubular epithelial cells（TEC）,as the major component of kidney parenchyma,are often suffer ill-effects from different types of kidney injuries.Importantly,it has been reported that TEC,as a vital source of myofibroblasts,contribute to kidney fibrosis by EMT.TRPC6 is widely present in different kidney cell types,such as podocytes,glomerular mesangial cells and TEC.Mutations in TRPC6 would cause kidney dysfunction.In addition,it has been found that TRPC6 inactivation or inhibition is involved in the protective role of kidney fibrosis.However,it is not clear whether the protective effect of TRPC6 is associated with the EMT of TEC,which requires further study.Objective:In this study,129Sv Ev（WT）and TRPC6 knockout(TRPC6^-/-)mice were subjected to a unilateral ureteric obstruction（UUO）operation in vivo,and primary TEC from both TRPC6^-/-and WT mice were treated with TGF-β1 in vitro.The purpose of the research was to:investigate the regulation of TRPC6 on EMT of TEC in kidney fibrosis;illustrate the signal pathways involved in regulating EMT by TRPC6;elucidate the influence of TRPC6 on Na⁺/K⁺-ATPase and AQP1 of TEC.Methods:In the first part:The WT and TRPC6^-/-mice were conducted the operation of UUO.The fibrotic injuries after UUO were observed by the staining of HE,Masson and Sirius red.Besides,the kidneys after UUO were used to detect the expression of EMT molecular markers（E-cad,Cadh16,α-SMA and snail）,TRPC6,the proteins of signaling pathways（AKT,m TOR,ERK1/2）,the functional proteins（Na⁺/K⁺-ATPase and AQP1）by immunofluorescence and Western Blot.In the second part:The primary TEC were isolated and cultured from renal cortex of WT and TRPC6^-/-mice in vitro.After stimulation with TGF-β1 for 72 h,TEC were used to analyze the expression of EMT molecular markers（E-cad,Cadh16,α-SMA and snail）,TRPC6,the proteins of signaling pathways（AKT,m TOR,ERK1/2）,the functional proteins（Na⁺/K⁺-ATPase and AQP1）by immunofluorescence and Western Blot.Results:1,Obvious interstitial fibrosis occurred in the obstructed kidney after UUO.The primary TEC had apparent fibrotic changes after stimulation of TGF-β1.The expression of TRPC6 increased along with the activation of EMT in both UUO model and TGF-β1 treated primary cultured TEC.2,EMT was inhibited by deletion of TRPC6 in vivo and in vitro.3,In both UUO model and TGF-β1 treated primary cultured TEC,the signal pathways of AKT-m TOR and ERK1/2 were activated,and down-regulated by deletion of TRPC6.4,In both UUO model and TGF-β1 treated primary cultured TEC,the expression of Na⁺/K⁺-ATPase and AQP1 decreased,and was partially reversed by deletion of TRPC6.Conclusion:TRPC6 knockout may alleviate fibrosis injuries by inhibition of EMT through down-regulation of AKT-m TOR and ERK1/2 signal pathways to ameliorate kidney function.