The Mechanism And Therapy Of Wnt/?-catenin Signaling Regulates Kidney Fibrosis

Chapter I Macrophage derived Wnts contribute to kidney fibrosisBackgroundActivation of Wnt/?-catenin signaling plays a pivotal role in the pathogenesis of many forms of chronic kidney diseases(CKD).Wnt ligands are induced in a wide variety of kidney resident cells as well as infiltrated cells including macrophages.However,the relative contribution of Wnts from different sources in CKD progression is poorly understood.To address this issue,we utilized genetic approach by blocking Wnt secretion via conditionally knockout of Wntless(Wls),a cargo receptor that is obligatory for Wnt trafficking and secretion.MethodsWe used unilateral ureteral obstruction(UUO)model at 0 day,3 days and 7 days to test macrophage activity by Western blot and immunofluorescence.We generated Wls conditional knockout mice in macrophage by crossing Lyz-Cre mice and Wls-floxed mice.Control and macrophage-specific Wls knockout mice were subject to renal unilateral ureteral obstruction(UUO),and then kidney fibrotic lesions and renal inflammation were assessed at 7 days after UUO.Mice were also subjected unilateral ischemia reperfusion injury(UIRI),kidney fibrosis and Wnt/?-catenin signaling activity was analyzed.Bone marrow-derived macrophages were prepared and treated with human recombinant protein Wnt1,Wnt3a and Wnt9a,M1 and M2 related markers were analyzed by Western blot and real time PCR.Bone marrow derived macrophages from WT and KO mice were cultured and analyzed to assess their migration and polarization.Conditional medium from WT and KO M1 macrophage were used to stimulate fibroblast,and fibrotic and proliferation-related proteins were analyzed by Western blot and immunofluorescence staining.ResultsM1 macrophage related marker such as B7-2 and TNF-? were increased in the kidney of UUO mice at 3 days and 7 days.M2 macrophage related marker mannose receptor was upregulated only at 7 days,whereas arginase 1 was increased initially at UUO 3 days but declined at UUO 7 days.Macrophage-specifc ablation of Wls in mice was confirmed by Western blot analysis of bone marrow-derived macrophage.ELISA showed that the secretion of Wntl and Wnt3a protein were largely blocked in the macrophages of KO mice compared with WT controls.The Lyz-Wls-/-mice were phenotypically normal.Ablation of Wls in macrophage attenuated renal fibrotic lesion at 7 days after UUO,accompanied by reduction of fibrosis-related protein,such as fibronectin and ?-SMA,?-catenin,PAI-1 and Snail1.Pro-inflammatory cytokines such as TNF-? and MCP-1 in KO mice were significantly inhibited,compared to the controls.Similarly,in model of UIRI,kidney fibrosis and Wnt/?-catenin activity were also attenuated in the KO mice compared with the WT controls.In vitro,bone marrow-derived macrophage(BMDM)from KO mice displayed a decreased migration and polarization,compared with BMDM from WT mice.In addition,conditional medium derived from BMDM of Lyz-Wls-/-mice exhibited a reduced fibroblast activition and fibronectin expression,compared to the controls.ConclusionThese results suggested that blockade of Wnt secretion from macrophage alleviates kidney injury in UUO and UIRI models.Macrophage-derived Wnts play an important role in mediating macrophage migration and polarization,as well as in promoting fibroblast proliferation and kidney fibrosis.Chapter ? Klotho-derived peptide 6(KP6)ameliorates kidney fibrosis by targeting Wnt/?-catenin signalingBackgroundAging is an independent risk for the development and progression of chronic kidney disease.Klotho is an novel antiaging protein that highly expressed in tubular epithelium of normal adult kidney.Membrane Klotho is the co-receptor for fibroblast growth factor-23(FGF-23),a bone-derived hormone that plays a critical role in phosphate homestasis.Klotho is an endogenous Wnt antagonist that physically binds to Wnt ligands and inhibits Wnt signaling.Loss of Klotho in aging and chronic kidney disease leads to Wnt/?-catenin activation.We sought to identify the peptide that can imitate Klotho protein to block Wnt/?-catenin signaling,and to evaluate its therapeutic efficacy in the pre-clinical setting.MethodsA series of overlapping peptides corresponding to the sequences of human Klotho protein were synthesized and screened for their ability to inhibit Wnt signaling in human kidney tubular epithelial cells(HKC-8).The candidate Klotho-derived peptide was further evaluated for its efficacy in ameliorating kidney fibrosis in mouse models of unilateral ureteral obstruction(UUO)and ischemia-reperfusion injury(IRI).ResultsThrough screening a peptide mini-library,we identified the Klotho-derived peptide 6(KP6)that specifically blocked Wnt-induced gene expression in HKC-8 cells.Incubation of HKC-8 cells with KP6 suppressed the Wnt-triggered ?-catenin activation.KP6 also inhibited Wnt-induced fibronectin and collagen I gene expression in HKC-8 cells.Co-immunoprecipitation revealed that KP6 could compete with Klotho to interact with Wntl ligands,and blocked Wntl interacted with LRP6.Intravenous injection of KP6 resulted in its selective accumulation in the injured kidney,but not in other major organs such as liver,heart and lung.We found that delivery of KP6 dramatically inhibited ?-catenin activation and ameliorated kidney fibrosis in mouse models of UUO and UIRI.KP6 blocked myofibroblast activation,inhibited fibronectin,collagen I expression and reduced renal matrix accumulation.ConclusionThese studies identify a novel Klotho-derived peptide that specifically inhibits Wnt/p-catenin signaling.By blocking a key fibrogenic signaling,KP6 could be a novel and effective agent for the treatment of fibrotic kidney disease.