The Role Of Robo4/ARF6 Signaling Pathway In Human Renal Glomerular Endothelial Hyperpermeability Induced By High Glucose

ObjectiveThe endothelial barrier is a structure that has a restrictive permeability between the blood and the vascular endothelium,and it is of great significance in maintaining the stability of glomerular tissue microenvironment.High permeability of endothelium is an important pathogenesis of early diabetic nephropathy,diabetic retinopathy and diabetic vascular disease.But the mechanism of glomerular hyperpermeability of diabetic nephropathy is largely unknown,therefore,we need to explore its mechanism.Slit family members were originally discovered as axon guidance molecules that mediate repulsive signalling mechanisms in the central nervous system.ROBO4 is an endothelial-cell-specific ROBO protein that can bind to Slit-2.The Slit-2�ROBO4 signalling pathway regulates endothelial permeability and maintains the integrity of the vascular network by inhibiting cytokine-mediated vasculogenesis and hyperpermeability.Slit proteins are secreted by glial cells and other tissues.ROBO4 is predominantly expressed in endothelial cells,including embryonic endothelium and tumour vascular endothelium,and is structurally different from the other ROBO proteins.Whether and to what extent ROBO4 expression is altered in the diabetic kidney vasculature is not known.ADP-ribosylation factor(Arf)is the small GTPase belonging to the Ras superfamily.Arf6 predominantly localizes to the plasma membrane and endosomal compartments,and plays important roles in endocytosis of the plasma membrane,exocytosis,endosomal recycling,cytokinesis,and actin cytoskeleton reorganization.It is known that Slit2�Robo4 signalling promotes vascular stability by blocking Arf6 activity.The activated ARF6 can induce vascular leak through disruption of endothelial adherens junctions.The ability of the stabilizing receptor ROBO4 to prevent leak and pathologic angiogenesis is dependent on its ability to recruit the GTPase activating protein,GIT1,to inactivate ARF6.Since Robo4 has an abilitity to decrease the permeability of vascular cells,we hypothesize that it may suppress the hyperpermeability of glomerular responses to HG by blocking Arf6 activity.We therefore investigated the expression of the ROBO4 and activated ARF6 protein in the primary cultures of human renal glomerular endothelial cells,and whether Over-expression of ROBO4 and blocking ARF6 can reduce permeability of Human Renal Glomerular Endothelial cell in high glucose medium.MethodsPart?: The influence of high glucose medium on the expression of ROBO4,activated ARF6 protein and cell permeability in glomerular endothelial cellsHRGEC,the 2nd to 5th generation cells were used for the experiment.The cells were divided into five groups : low glucose group,high glucose 12 h group,high glucose 24 h group,high glucose 48 h group,and high glucose 72 h group.GTP-ARF6 pull down to obtain activated ARF6.The levels of ROBO4 and activated ARF6 protein were detected by Western blot.The endothelial permeability was measured by the efflux of fluorescein isothiocyanate-dextran(FITC-Dextran)permeated through the monolayer endothelial cells using Transwell cell model system.Part II: The effect of Over-expression ROBO4 on the protein level of activated ARF6 and permeability of human renal glomerular endothelial cell in high glucose mediumHRGEC that were infected with recombinant lentiviruses encoding Robo4 were cultured in high glucose or low glucose medium in vitro.The cell viability after lentivirus transfection was measured by CCK8 assay.GTP-ARF6 pull down to obtain activated ARF6.The protein levels of Robo4 and activated Arf6 in each group were detected by western blotting.The endothelial permeability was measured by efflux of fluorescein isthiocyanate-dextran(FITC-Dextran)permeated through the monolayer endothelial cells using Transwell cell monolayer cell model system.Part?: The effect of SecinH3 on the protein level of Robo4 and activated ARF6,and permeability of Human Renal Glomerular Endothelial cell in high glucose mediumSecinH3 was dissolved in organic solvent DMSO and the corresponding experimental concentration was set at 5 ?mol / L,10 ?mol / L and 20?mol / L.The cells were divided into six groups: low glucose control group,high glucose control group,high gl?cose + DMSO group,high glucose + SecinH3 5 ?mol / L group,high glucose + SecinH3 10 ?mol / L group,high glucose + SecinH3 20 ?mol / L group.GTP-ARF6 pull down to obtain activated ARF6.The levels of ROBO4 and activated ARF6 were detected by Western blot and the endothelial permeability was measured by FITC-Dextran and Transwell cell model system.ResultsPart?:Compared with the low glucose group,the expression of Robo4 was increased obviously after 12 h,but declined after 24 h(P<0.05),reaching to minimun which was lower than the low glucose group(P<0.05)after 72 h.On the contrary,the expression of ARF6 was increased from 12 h(P<0.05)and the increase reached to the maximum after 72 h.The expression of ROBO4 was decreased and the expression of ARF6 increased gradually with the time of high glucose stimulation,and the expression of ROBO4 was the lowest in 72 hours after high glucose stimulation.Furthermore,the vascular permeability was increased gradually after 24h(P<0.05),reaching to the maximun after 72 h.Part II:Compared with the empty vector group,over-expression of Robo4 significantly inhibited the expression of ARF6 and the endothelial permeability induced by high glucose.Part?:SecinH3 dose-dependently diminished the increase of protein level of activied ARF6 and the elvated endothelial permeability induced by high glucose.Conclusion1.High glucose increased HRGEC permeability in a time-depentant manner.The effect may be mediated through downregulation of ROBO4 protein and upregulation of actived ARF6 protein.2.Over-expression of Robo4 could significantly enhance the barrier functions of HRGECs in high glucose medium,and the effect may be mediated through downregulation of actived ARF6 protein.3.SecinH3 dose-dependently diminished the high glucose-induced endothelial hyperpermeability,and the effect may be mediated through blocking ARF6 and upregulateing ROBO4 protein expression.4.Our study identifies the restoration of ROBO4 and inhibition of ARF6 as treatment strategies for diabetes-induced glomerular hyperpermeability.