| BackgroundAs a key factor,Wnt signaling is involved in pathogenesis and progression of kidney diseases.However,the underlying mechanism is unknown.In this study,we detected the potential role of Wnt9a in tubular cell senescence and renal fibrosis.MethodsIn multiple chronic kidney diseases(CKD)and mouse models of adriamycin(ADR)nephropathy,unilateral ureteral obstruction(UUO),and ischemia-reperfusion,Wnt9a was tested by both immunohistochemistry(IHC)and western blotting(WB).The co-localization of Wnt9a and a biomarker for senescence,p16lnk4A,was also checked in sequential sections of IgA or ADR nephropathy.In a mouse model of unilateral ischemic/reperfusion injury(UIRI),ectopic overexpression or knockdown of Wnt9a was respectively performed through hydrodynamic-based gene delivery of vector encoding coding sequence(CDS)or interfering sequence.Tubular senescence,tubular injury and renal fibrosis were detected by IHC,WB and immunofluorescence(IF).To further confirm the effect of Wnt9a in aging-related renal fibrosis,2-month-old and 24-month-old mice were performed the surgery of UIRI,and a vector encoding the secreted form of Klotho was intravenously administrated to inhibit the expression of Wnt9a.In cultured human proximal tubular epithelial cells(HK-2),cell cycle and senescence-related gene were determined by overexpression of Wnt9a.In some cells,a small interference RNA(siRNA)was used to check the effects of knockdown of Wnt9a in aristolochic acid(AA)-induced cell senescence.The cell-cell communication between fibroblasts and tubular cells triggered by Wnt9a was also examined in cultured fibroblast cells(NRK-49F)and HK-2 cells.ResultsWnt9a was significantly upregulated in the kidneys of both human CKD and animal models,and mainly located in kidney tubules.The overexpression of Wnt9a was concomitant with development of tubular senescence,cell injury,and renal fibrosis,as detecting by p16Ink4A,Klotho,Kim-1 and fibronectin.The exogenous expression of Wnt9a dramatically promoted renal fibrosis,increased serum ereatinine and proteinuria,induced tubular cell injury and senescence.To the contrary,knockdown of Wnt9a evidently protected kidney function,renal fibrosis,tubular senescence and injury.Compared to young mice,Wnt9a expression was remarkably upregulated in an aging model of 24-month-old mice.The increasing of Wnt9a in aging mice was closely related to fibrosis.Using cultured cell line HK-2,we found Wnt9a induced cell cycle arrest in S-phase,the upregulation of senescence-related gene p16Ink4A,ARF,RB,p53 and p21 and senescence-associated chromatin modification,γ-HZAX.Through the use of ICG-001,an inhibitor of β-catenin signaling pathway,we proved that the expression of p53,p21 and TGF-β1 induced by Wnt9a were mainly via the activation of Wnt/p-catenin pathway.AA also induced tubular cell cycle arrest and senescence.However,siRNA to Wnt9a significantly inhibited AA-induced S-phase arrest,and senescence-related gene expression.Wnt9a could induce TGF-β1 expression of tubular cells,while TGF-β1 could promote Wnt9a secretion of fibroblasts,both of which establish a positive feedback loop between renal tubular cells and fibroblasts.Wit9a could also directly induce fibroblasts proliferation,or indirectly induce fibroblasts activation through conditioned-medium from Wnt9a treated tubular cells which can be partly blocked by the neutralizing antibody of TGF-β receptor Ⅱ.On the other hand,Wnt9a conditioned-medium from fibroblasts promoted senescence and EMT of tubular cells.ConclusionThese results suggest that Wnt9a accelerates renal fibrosis through induction of tubular senescence.Targeted inhibition of Wnt9a could be a therapeutic method to protect kidney diseases. |
