Connective Tissue Growth Factor Mediates Advanced Oxidation Protein Product-induced Epithelial-to-mesenchymal Transition In Human Proximal Tubular Epithelial Cells

BackgroundRenal fibrosis,which includes glomerulosclerosis and tubulointerstitial fibrosis,is the final common pathway of a wide variety of chronic kidney diseases(CKD).Notably,growing evidence suggests tubulointerstitial fibrosis is the most reliable indication of an irreversible loss of renal function.During tubulointerstitial fibrosis,numerous studies have indicated that tubular EMT plays a crucial role.The mechanisms underlying EMT are complicated,manifesting as diverse signaling pathways being activated or various secretory factors being induced in different pathologic state.Of note,our previous stdudy has found AOPPs could induce EMT in human renal proximal tubular epithelial cells through the induction of endoplasmic reticulum(ER)stress.The mechanisms of EMT are complicated,and whether other mechanisms underlying AOPPs induce EMT in tubular cells remain to be clarified.Substantial evidences show that CTGF represents a critical mediator of tubular EMT.CTGF can cooperate with other factors to provoke the production of extracellular matrix remodeling molecules including a vast amount of collagen.CTGF can be regulated by specific mediators and stress factors involved in kidney injury such as high glucose,cellular stress,TGF-?,and angiotensin ?.However,whether CTGF mediates AOPP-induced EMT in tubular cells is unclear.The Wnt/?-catenin signaling pathway is involved in multiple physiological and pathological processes.Once the heterodimeric receptor complex consisted of a frizzled and a low-density lipoprotein receptor-related protein(LRP)5/6 protein binds to Wnt ligands,the canonical Wnt signaling pathway actives,leading to a protein complex containing glycogen synthase kinase(GSK)-3 falls apart.As a result,?-catenin is stabilized and then it is translocated into the nucleus and binds to T cell factor/lymphoid enhancer-binding factor(TCF/LEF)to stimulate the transcription of Wnt target genes.Previous studies have shown the Wnt/?-catenin signaling pathway is aberrant in various adult kidney diseases.Importantly,CTGF has been shown to be a target gene of ?-catenin-TCF/LEF(TBE)transcription factor and TBE binding site is identified in the promoter region of the CTGF gene.Additionally,the mechanisms of CTGF induction depend on the types of cells,tissues and stress factors.Therefore,it is necessary to clarify the regulation mechanism of AOPP-induced CTGF in HK-2 cells.In this study,we focused on the Wnt signling pathway.ObjectivesIn the present study,we investigated whether AOPPs increased the expression of CTGF in human proximal tubular epithelial cell line(HK-2 cells),and whether CTGF was involved in AOPP-induced EMT in HK-2 cells.Furthermore,we determined the role of the Wnt/?-catenin signaling pathway in this process.MethodsHK-2 cells were incubated with AOPPs(200 ?g/mL)or albumin((200 ?g/mL)for indicated time(6,12,18,24,36h)and the expression of CTGF in these cells was detected by qPCR and Western blot.Then,we transfected CTGF siRNA or scramble siRNA into HK-2 cells and treated the cells with AOPPs,and Western blot,qPCR and immunofluorescence staining were used to detect the expression of E-cadherin,Vimentin and ?-SMA of those cells.In addition,HK-2 cells were treated with AOPPs or albumin for indicated time(6,12,18,24h)and the expression of p-GSK3?,GSK3? and ?-catenin in these cells was detected by Western blot.Lastely,HK-2 cells were incubated with albumin or AOPPs in the presence or absence of DKK-1,a specific Wnt antagonist,for 24 h.Then the protein expression of nuclear ?-catenin,p-GSK3?,CTGF,E-cadherin,Vimentin,and ?-SMA was examined.ResultsWestern blot and qPCR analysis showed that AOPPs but not control albumin induced CTGF overexpression.CTGF-specific siRNA transfection significantly reversed AOPP-suppressed expression of E-cadherin and AOPP-induced overexpression of Vimentin and ?-SMA as compared with scramble siRNA transfection.In addition,compared with the albumin-treated cells,the phosphorylation of GSK3? induced,the expression of nuclear ?-catenin upregulated,and the expression of cytoplasm ?-catenin downregulated in HK-2 cell after exposure to AOPPs.Moreover,AOPP-induced overexpression of nuclear ?-catenin and phosphorylation of GSK3? was reversed by DKK-1.AOPP-induced CTGF overexpression was attenuated when the cells were treated with both AOPPs and DKK-1.Lastly,DKK-1 reversed AOPP-induced suppression of E-cadherin expression and overexpression of Vimentin and ?-SMA in HK-2 cells.ConclusionIn conclusion,our results suggested that AOPPs triggered EMT in cultured human proximal renal tubular cells probably through CTGF induction.Furthermore,the CTGF expression induction involved in this process could be mediated by the Wnt/?-catenin signaling pathway.AOPPs commonly accumulate in patients with CKD and promote the development and progression of CKD.Therefore,our data may provide novel evidence for the treatment of CKD by targeting CTGF or the Wnt/?-catenin signaling pathway.