Role Of Hypoxia Induced Decreased Expression Of Syndecan-1 In Glomerular Endothelial Cell Injury And Its Mechanism

Objective The mechanism underlying acute ischemic glomerular endothelial cell injury is unclear.Our study is designed to explore the role and mechanism of Syndecan-1 in GEnC injury after ischemia/hypoxia treatment.Methods We first examine changes in the expression of Syndecan-1in GEnC after ischemia/hypoxia treatment using immumohistochemical staining and Western blot.Then the primary cultured GEnC were transfected with Syndecan-1 siRNA,and the GEnC were also treated with Cocl_2,VEGF,Dynasore,MDC,Nystatin or aPKC inhibitor separately based on the needs of experiments.Changes in cell apoptosis,cell viability and cell permeability were evaluated by FACS analysis,Western blot,CCK-8 analysis and Cell permeability analysis,respectively.Besides,Western blot was applied to detect changes in the expression of target genes of VEGF-VEGFR-2 signaling,total VEGFR-2,membrane VEGFR-2,Dab2,p-Dab2,PAR-3,aPKC and p-aPKC at protein levels.Real-time RT-PCR was used to examine changes in the expression of VEGFR-2,Dab2 and PAR-3 at mRNA levels.Immunoprecipitation was used to evaluate interaction between VEGFR-2 and Caveolin-1.Immunofluorescence staining and immunoprecipitation were applied to examine changes in the location as well as interaction between Syndecan-1and VEGFR-2.Last but not the least important,the expression of Dab2and PAR-3 in GEnC were down-regulated by transfected with Dab2siRNA or PAR-3 siRNA.And GEnC were also treated with VEGF as described before.Then changes in cell viability and cell apoptosis were evaluated by FACS analysis and CCK-8 analysis,respectively.Changes in the expression of VEGF-VEGFR-2 signaling target genes,membrane VEGFR-2 and total VEGFR-2 were also evaluated by Western blot.Result?1?Ischemia/hypoxia treatment could reduce Syndecan-1expression in GEnC both in vivo and in vitro;?2?Decreased expression of Syndecan-1 lead to injury and dysfunction of GEnC through preventing activation of VEGF-VEGFR-2 signaling;?3?Dynasore and MDC treatment could inhibit activation of VEGF-VEGFR-2 signaling,while nystatin treatment activate the expression of VEGF-VEGFR-2 signaling in GEnC;?4?Reduced expression of Syndecan-1 in GEnC lead to increased interaction between Syndecan-1 and Caveolin-1;?5?Syndecan-1 could co-localize and interact with VEGFR-2 in GEnC;?6?Decreased expression of Dab2 or PAR-3 lead to GEnC injury and dysfunction through inhibiting VEGF-VEGFR-2 signaling;?7?Down-regulation of Syndecan-1 could lead to significant increase in aPKC mediated phosphorylation of Dab2.Besides,aPKC inhibitor treatment could significantly activate the expression of VEGF-VEGFR-2 signaling in GEnC compared with control.ConclusionIschemia/hypoxia treatment result in decreased expression of Syndecan-1 in GEnC.Downregulation of Syndecan-1 lead to dysfunction of GEnC through inhibiting activation of VEGF-VEGFR-2signaling.We speculated that Syndecan-1 could regulate internalization of VEGFR-2 through aPKC mediated phosphorylation of Dab2,thus modulating expression of VEGF-VEGFR-2 signaling.Our study provide a new target for the therapy of acute ischemic glomerular endothelial cell injury,and this has important clinical value and practical significance.