Empagliflozin Protects Proximal Renal Tubular Cells Injury Induced By High Glucose Via Regulation Of Hypoxia–Inducible Factor-1Alpha

Background: Evidence from both animal and human studies clearly supports the renal beneficial effects of empagliflozin,a sodium glucose cotranspoter 2(SGLT2)inhibitor but the mechanism in which it exerts its effect is not well understood.In this study we investigated the capability of empagliflozin(emp)on reducing hyperglycemia-induced renal proximal tubular epithelial cells injury and we evaluated if the renoprotecitve effect of empagligliflozin associate with HypoxiaInducible Factor-1?(HIF-1?).Methods: Human kidney cell lines(HK-2 cells)cultured in 5.5Mm normal glucose(NG),high glucose(HG)25mM,with or without empagligliflozin(50nM,100 nM,and 500nM)treatment under normoxia condition for 72 hours,Western blot was performed to evaluate the expression of SGLT2,induction of HIF-1?,glucose transporter-1,TGF-?1/Smad3 pathway and epithelialmesenchymal transition(EMT)markers(?-SMA,E-Cadherin).In addition,HK-2 cell lines cultured in normal glucose,high glucose with or without empagliflozin(50nM,100 nM,500nM)treatment under normoxia,cell proliferation measured at 24 hours,48hours and 72 hours using Cell Counting Kit-8 assay,Optical density value detected at 450 nm.Results: High glucose exposed HK-2 cells significantly increased expression of SGLT2(P <0.0001),however empagliflozin treatment decreased SGLT2 expression in dose dependent manner(P < 0.01).HG blunted expression of HIF-1? in HK-2 cells,addition of empagligliflozin treatment signifcantly induced HIF-1 expression in dose dependent manner(at 100 nM P < 0.05,95%CI-0.5918 to-0.002338,at 500 nM P <0.05,95%CI-0.6631 to – 0.07367).HG increased TGF-?1 expression in HK-2 cells(P < 0.001),and emp treatment significantly decreased expression of TGF-?1 in dose dependent manner(P < 0,0001).Empagligliflozin treatment at 500 nM significantly decreased expression of PhosphorylatedSmad3 induced by high glucose in HK-2 cells(P <0.01).High glucose significantly increased extracellular matrix proteins collagen IV and fibronectin expression compared to NG(P <0.0001).Empagligliflozin significantly decreased expression of Col IV and FN in dose dependent manner(P< 0.01).Empagligliflozin treatment at100 nM and 500 nM significantly decreased expression of ?-SMA in HK-2 cells induced by high glucose(P<0.05).Furthermore,emp treatment in dose dependent manner significantly reversed downregulation of E-Cadherin in HK-2 cells induced by high glucose(emp at 100 n M P< 0.001,emp at 500 nM P < 0.0001).In addition,emp treatment at 100 nM significantly increased expression of glucose transporter 1 protein in HK-2 cells(P <0.05).The cell proliferation of HK-2 cell induced by HG was significantly inhibited by empagliflozin treatment(P < 0.0001).Conclusion: Empagliflozin treatment attenuated proximal renal tubular cells injury induced by high glucose.Induction of HIF-1? expression by empagliflozin may play an essential role in the protection of high glucose induced proximal renal tubular epithelial cells injury.