Study On The Effects And Mechanism Of Resveratrol In Epithelial-Mesenchymal Transition Of Renal Tubular Epithelial Cells Induced By Hyperuricemia

BackgroundHyperuricemia is an independent risk factor for chronic kidney disease,and it can lead to renal fibrosis.Recent data indicates that uric acid(UA)can induce tubular epithelial cell mesenchymal transition(EMT)through oxidative stress,inflammation and TGF-β1-related signaling pathways,in vivo and in vitro.Resveratrol(RSV)has been proven to have both anti-inflammatory and antioxidant effects.Numerous studies have demonstrated that IL-11 plays an important role in the development and progression of fibrosis.However the role and mechanism of Resveratrol and IL-11 in uric acid-induced EMT remains unclear.ObjectiveFirstly,to observe the effects of uric acid in inducing oxidative stress and inflammation on renal tubular epithelial cells;Secondly,to recognize the effects of RSV treatment on oxidative stress,inflammation and EMT;And finally,to explore the role of TGF-β1/IL-11 in renal tubular EMT induced by uric acid.Method1.Human proximal tubular epithelial cells(HK-2)were cultured in vitro,untill about 60%to 80%of Human HK-2 cells demonstrated positive growth and adhered to the wall.The renal tubular epithelial cells were treated with different concentrations of UA(0,300,500,700 and 1000 μmol/L)for 48 h,and the morphology of HK-2 was observed,SOD and ROS levels were measured,and the expression of TGF-β1,IL-11,ILK,NF-κB p65,pNF-κB p65,ERK-1/2,p-ERK1/2,α-SMA and E-cadherin were detected by Western blot(WB)assay.The expression of IL-1β mRNA and TNF-αmRNA was measured by q-PCR technique.2.HK-2 cells were divided into four groups:control group,uric acid group(700μmol/L UA),Intervention groupl(700 μmol/L UA+25 umol/L RSV)Intervention group 2(700 μmol/L UA+50 μmol/L RSV).After 48-hour culture.SOD and ROS levels were measured,and the expression of TGF-β1,IL-11,ILK,NF-κB p65,p-NF-κB p65,ERK1/2,p-ERK1/2,α-SMA and E-cadherin were detected by WB assay.The expression of IL-1β and TNF-α mRNA was detected by q-PCR assay.3.Under the condition of hyperuricemia,the expression of senescent proteins such as p16,p21 and HMGB1 were detected by WB assay.The expression of senescence-related proteins was detected by WB assay after pretreatment with Resveratrol to evaluate the degree of senescence.4.IL-11 was silenced by an RNA interference technique.The role of IL-11 in the EMT of renal tubular epithelial cells was observed and the expressions of EMT proteins were detected by WB assay.ResultsCompared with the control group,UA stimulated HK-2 cells had obvious morphological changes demonstrated in a time dependent manner,moreover their level of ROS and their gene expression of IL-1β and TNF-α was significantly up-regulated and meanwhile their SOD activity was decreased significantly.These were corroborated to the expression of EMT molecular markers including IL-K,alpha-SMA and E-cadherin.RSV attenuated UA-induced EMT of HK-2 cells by its anti-inflammatory and anti-oxidative roles demonstrated by the decreased rate and onset of senescence following UA induction.UA induction significantly activated TGF-β1/IL-11 signaling pathways in HK-2 cells,Furthermore,IL-11 knockout attenuated UA-induced EMT in HK-2 cells.Conclusions1.Hyperuricemia can induce oxidative stress and inflammatory reaction in renal tubular epithelial cells leading to cell senescence.It presents an important potential mechanism involved in the induction of tubule epithelial cell EMT and the development of tubulointerstitial fibrosis.2.Resveratrol can relieve renal tubular epithelial cell senescence,oxidative stress,inflammatory reaction and EMT induced by uric acid.3.IL-11 is involved in the mechanism of uric acid-induced EMT.As a result,the knockout of IL-11 can inhibit uric acid-induced EMT and therefore represents a potential therapeutic target for the treatment of uric acid-induced renal tubular epithelial cell injury.