The Role Of GSK3β In TGF-β1 Induced Tubular Epithelial Cells Injury And Potential Mechanism

Background Renal fibrosis is the common pathological outcome of maniflod chronic kidney diseases(CKD),characterized by glomerular sclerosis,interstitial fibrosis(IF),and tubular atrophy.The degree of tubulointerstitial fibrosis(TIF)is highly associated with renal function and the prognosis of patients.Therefore,inhibiting the development of TIF plays an important role in alleviating the progression of chronic kidney disease.In recent years,more and more studies have shown that renal tubular epithelial cells play an instrumental role in the development of renal fibrosis.When tubular epithelium are injured,the tubular epithelial cells undergo dedifferentiation,loss of brush border,cell adhesion molecules E-cadherin and tight junction protein ZO-1,de novo expressing vimentin.At the same time,the expression of extracellular matrix such as fibronectin(FN),and the secretion of profibrotic cytokines such as connective tissue growth factor(CTGF)and plasminogen activator inhibitor-1(PAI-1)are increased.In addition,during injury,some of the tubular epithelial cells will re-enter the cell cycle for proliferation to help to replace cells lost to apoptosis or necrosis,and some cells may arrest in either G1 or G2.However,recent studies pinpointed that when the tubular epithelial cells are arrested in the G2/M phase,CTGF,TGF-β1,collagen Type Ⅰ and type Ⅳ expression are increased.These cytokines and extracellular matrices will act on surrounding myofibroblasts,further promoting the formation of extracellular matrices.However,there are currently no effective treatments for the development of renal fibrosis.Transforming growth factor β1(TGF-β1)pathway is the main pathway for the development of renal fibrosis.When TGF-β1 is activated,the SMAD complex forms and binds to the transcriptional coactivator CBP/P300 to initiate transcription.CBP/P300 was originally identified as a partner of the c AMP-response element binding protein(CREB).When TGF-β1 signaling pathway is activated in TECs,the cells will undergo dedifferentiation,increased profibrotic cytokines like CTGF,PAI-1,and extracellular matrix synthesis,as well as cell cycle arrest.Glycogen synthase kinase(GSK3β)is a serine/threonine kinase that plays a critical role in the injury and repair of renal tubules in acute kidney injury.The role of GSK3β of renal tubular epithelial cells in the development of renal fibrosis has not been reported.Therefore,in our study,we explored the function of GSK3β on renal tubular interstitial fibrosis by using TGF-β1-induced tubulointerstitial fibrosis model,and sought a new target for the prevention and treatment of CKD.Objective To explore the role of GSK3β in TGF-β1-induced renal tubular epithelial cell injury and potential mechanism.Method 1.To explore the role of GSK3β in TGF-β1-induced renal tubular epithelial cell injury The cells were treated with TGF-β1 in the presence or absence of different doses of GSK3β inhibitors(TDZD-8 and Li Cl),and the expression of E-cadherin,Vimentin,Fibronectin,CTGF,PAI-1 and p H3 were detected by Western Blot.Immunofluorescence was used to detect the localization and expression of E-cadherin,ZO-1,Vimentin,Fibronectin and p H3.2.To explore the mechanism of GSK3β involved in TGF-β1-induced renal tubular epithelial cell injury(1)Detection of the activity(phosphorylation)of CREB under different active conditions of GSK3β The cells were divided into 6 groups: control,TGF-β1,TGF-β1+S9A,TGF-β1+KD,TGF-β1+Li Cl,TGF-β1+TDZD-8,TGF-β1+Forskolin,and Western Blot detected the expression of p-CREB and CREB.(2)Detection of TGF-β1-induced injury of renal tubular epithelial cells during CREB-specific activation The cells were further divided into 4 groups: control,TGF-β1,Forskolin,TGF-β1+ Forskolin.Western Blot was used to detect the expression of E-cadherin,Vimentin,Fibronectin,CTGF,PAI-1 and p H3.Immunofluorescence was used to detect the localization and expression of E-cadherin,Vimentin,Fibronectin and p H3.Result 1.Under the function of TGF-β1,the expression of E-cadherin in renal tubular epithelial cells was decreased,inversely,the expression of Vimentin wad increased,and the expression of Fibronectin,CTGF,PAI-1 and p H3 are increased.When GSK3β inhibitor was used at the same time,the effect of TGF-β1 was alleviated,(the difference was statistically significant,P<0.05).2.When GSK3β is inhibited,the activity of CREB is increased,that is,the expression of p-CREB is increased.3.When CREB is specifically activated,TGF-β1-induced renal tubular epithelial cell damage was alleviated.Conclusion GSK3β plays a crucial role in TGF-β1-induced dedifferentiation of renal tubular epithelial cells,increased expression extracellular matrix,profibrogenic cytokine secretion,and cell cycle arrest.The potential mechanism is that GSK3β regulates the transcription of TGF-β1 signaling by regulating the activity of CREB.