X-C Motif Chemokine Receptor 4 Aggravates Renal Fibrosis Through Activating JAK/STAT/GSK3?/?-catenin Pathway

BackgroundRenal fibrosis is the ultimate common pathological outcome of various types of chronic kidney disease(CKD).However,the underlying mechanisms are not determined.To date,there are not completely effective therapeutics to renal fibrosis.Hence,to better understand the underlying mechanisms and identify therapeutic targets of renal fibrosis would be of great importance to CKD.Recently,some reports indicate C-X-C motif chemokine receptor 4(CXCR4)plays a crucial role in various types of CKD.It was reported in our previous study that CXCR4 up-regulation in injured podocytes is responsible for oxidative stress.Another report shows genetic ablation of CXCR4 from macrophages or tubular cells attenuates renal fibrosis.However,the underlying mechanisms of renal tubular cell injury need to be elucidated.In this study,we investigated the potential relationship between CXCR4 and ?-catenin in renal tubular cell injury and fibrosis,and explored the underlying mechanism.MethodsWe established CKD mice models of unilateral ureteral obstruction(UUO),bilateral ischemia-reperfusion injury(IRI),and unilateral IRI(UIRI),and collected the kidneys of CKD patients.First,we detected the changes of the protein and gene expression of CXCR4 and ?-catenin in CKD kidney and the relationship between them.Then CKD mice were injected intraperitoneally with AMD3100,a specific inhibitor of CXCR4,or were quickly injected through the tail vein to carry the CXCR4 encoding gene into the kidney and then intraperitoneally injected with ICG-001,a small molecule inhibitor of ?-catenin.Therefore,we investigated the expressions of CXCR4,fibrosis indicators,GSK3?,?-catenin and its downstream target genes from these model kidneys by a variety of techniques including Western bloting,immunohistochemical stainin,immunofluorescence,real-time fluorescence quantitative polymerase chain reaction,Masson trichrome staining,periodic acid-Schiff staining,sirius red staining,and functional indicators.In vitro,human proximal renal tubular epithelial cell line(HKC-8)was transfected with the overexpression plasmid or SiRNA or treated with cytokine to confirm the effect of GSK3?/?-catenin signalling on renal fibrosis and to explore its upstream mechanism.Finally,the upstream mechanism of GSK/?-catenin was verified in CKD mouse models in vivo.ResultsThe expressions of both CXCR4 and ?-catenin were time-dependently up-regulated,and predominantly localized in renal tubular cells and both of them were largely colocalized in CKD mice.SDF-1? expression was aslo elevated time-dependently.The mRNA expression levels of CXCR4 and MMP-7,an important downstream target of ?-catenin,were significantly up-regulated in CKD mice in a time-dependent fashion,and there was a significant positive correlation between them.CXCR4 was predominantly localized in renal tubular cells in human diseased kidneys.In CKD mice,renal tubules were injured and interstitial collagen deposition increased,and the protein expression levels of fibronectin,a-SMA,collagen I,vimentin were evidently up-regulated,and GSK3? was inhibited,?-catenin and its downstream target genes such as PAI-1,Snail1,and MMP-7,were dramaticly elevated,and the expression of E-cadherin that maintains normal epithelial integrity was obviously down-regulated.But the changes were all distinctly inhibited by treatment with AMD3100.Further inhibition of ?-catenin retards CXCR4-aggravated renal fibrosis in UUO.Similarly,CXCR4/?-catenin axis plays a critical role in tubules cell injury-induced fibrosis.Administration of SDF-1? induced the phosphorylation of JAK2/STAT3 and JAK3/STAT6 in a time-dependent manner.Ectopic expression of CXCR4,STAT3 or STAT6 could all significantly decrease the mRNA level of GSK3?.Furthermore,treatment with SDF-1? induced the colocalization of ?-catenin with STATS or STAT6 and translocation into nucles.Morerover,siRNA-mediated inhibition of STAT3 or STAT6 blocked GSK3?/?-catenin induced fibrosis.In vivo blockade of CXCR4 inhibited the activation of JAK/STAT,and gene knockdown of CXCR4 blocked renal fibrosis through inhibition of JAK/STAT/GSK3?/?-catenin pathway.ConclusionThese results uncover a novel mechanistic linkage between CXCR4 and ?-catenin activation in renal fibrosis in association with JAK/STAT/GSK3? pathway.Our studies also suggest that targeted inhibition of CXCR4 may provide better therapeutic effects on renal fibrosis by inhibiting multiple downstream signalling cascades.