The Role And Related Mechanism Of CXCR4 In Podocyte Indury

Chapter I:C-X-C Chemokine Receptor Type 4 Plays a Crucial Role in Mediating Oxidative Stress-Induced Podocyte InjuryOxidative stress plays a role in mediating podocyte injury and proteinuria.However,the underlying mechanism remains elusive.In this study,we investigated the potential role of C-X-C chemokine receptor type 4(CXCR4),the receptor for stromal cell-derived factor 1α(SDF-1α),in mediating oxidative stress-induced podocyte injury.In the mouse model of adriamycin nephropathy(ADR),CXCR4 expression was significantly and specifically induced inpodocytes as early as 3 days.This was accompanied by an increased upregulation of oxidative stress in podocyte,as demonstrated by malondialdehyde assay,nitrotyrosine staining and secretion of 8-hydroxy-2’-deoxyguanosine in urine,and induction of NOX2 and NOX4,major subunits of NADPH oxidase.CXCR4 was also induced inhuman kidney biopsies with proteinuric kidney diseases and colocalized with advanced oxidation protein products(AOPPs),an established oxidative stress trigger.Using cultured podocytes and mouse model,we found that AOPPs induced significant loss of podocyte marker Wilms tumor 1(WT1),nephrin,and podocalyxin,accompanied by upregulation of desmin both in vitro and in vivo.Furthermore,AOPPs worsened proteinuria and aggravated glomerulosclerosis in ADR.These effects were associated with marked activation of SDF-la/CXCR4 axis in podocytes.Administration of AMD3100,a specific inhibitor of CXCR4,reduced proteinuria and ameliorated podocyte dysfunction and renal fibrosis triggered by AOPPs in mice.In glomerular miniorgan culture,AOPPs also induced CXCR4 expression and downregulated nephrin and WT1.Our studies have also elucidated the mechanism by which oxidative stress upregulates CXCR4 expression in podocytes.In this study,we have uncovered that AOPPs activated ERK and p65 NF-κB signaling and blockade of these pathways abolishes CXCR4 expression in podocytes.It appears that ERK activation is the upstream event of p65 phosphorylation because the peak of ERK phosphorylation precedes the peak of p65 and inhibition of ERK activation abolishes p65 phosphorylation.All of the results suggest that oxidative stress induced CXCR4 expression through p-ERK/p-p65 activation.In turn,CXCR4 also induces NAPDH oxidase activation and production of ROS,thereby creating a vicious cycle of oxidative stress,CXCR4 induction,and podocyte injury.These results suggest that chemokine receptor CXCR4 plays a crucial role inmediating oxidative stress-induced podocyte injury,proteinuria,and renal fibrosis.CXCR4 could be a new target for mitigating podocyte injury,proteinuria,and glomerular sclerosis in proteinuric chronic kidney disease.Chapter II:Mechanism of C-X-C Chemokine Receptor Type 4 Mediated Podocyte Injury ActivationWe have demonstrated that chemokine receptor CXCR4 plays a crucial role in mediating oxidative stress-induced podocyte injury,proteinuria,and renal fibrosis.CXCR4 was identified as a novel therapeutic target for mitigating podocyte injury,proteinuria,and glomerulosclerosis in proteinuric chronic kidney disease.How CXCR4 mediates podocyte injury remainsambiguous.In the current study,we determined the mechanisms of CXCR4 mediated oxidative stress-induced podocyte injury.In cultured MPC-5 cells,active-β-catenin was markedly increased at 30 minutesafter incubation with the specific ligand of CXCR4,SDF-la.It was accompanied by β-catenin nuclear translocation.Interestingly,SDF-1α did not increase the expression of the Wnt ligandsin podocytes.These results suggest SDF-la/CXCR4 signal axis activates β-catenin signaling pathway in a Wnts-independent fashion.Meanwhile,using cell or mini-organ culture,we found that the small molecue compound,ICG-001 significantly preserved expression of multiple podocyte markers,such as Wilms tumor 1(WT1),nephrin,and podocalyxin.Several downstream targets of P-catenin such as MMP-7 and Snail1 were inhibited by ICG-001.These results indicate that SDF-1/CXCR4 signaling mediates podocyte injury through β-catenin activation.We found that,as a G protein-coupled protein receptor,CXCR4 activation recruited the scaffold protein β-arrestin 1 and eventually formed a CXCR4/β-arrestin 1/Src complex after stimulation with SDF-la in podocyte.This complex then phosphorylated Src,EGFR,ERK,and GSK3P in sequence.Conversly,silencingβ-arrestin 1 suppressed ERK and GSK3P phosphorylation and active-β-catenin induction in podocytes.Specificlly knockdown CXCR4 in podocyte inhibited the upregulation of β-arrestin 1,p-ERK,active-β-catenin,β-catenin and its downstream targets MMP7,Snail1 and PAI-1 in podocyte,reduced proteinuria and ameliorated podocyte dysfunction and renal fibrosis induced by ADR in mice.The activation of Src promoted EGFR transactivation and ERK activationand induced the phosphorylation of GSK3β,which resulted in the upregulation of β-catenin and its nuclear translocation.Intranuclear β-catenin combining to the transcription factors TCF/LEF activated the transcription of target genes,and induced the loss of podocyte marker and acquired new mesenchymal specific proteins,which resulting in podocyte dysfunction and proteinuria.These results indicate that CXCR4 activates β-catenin signaling via recruiting scaffolding protein β-arrestin 1.Thus targeting CXCR4/β-arrestinl/β-catenin may be a new therapeutic stretgy for clinical treatment of proteinuria.