Expression And Clinical Significance Of ROBO4/ARF6 Signal Pathway In The Glomerulus Of Patients With Diabetic Nephropathy

Background and Objectives:Diabetic kidney disease is a major cause of end-stage renal disease due to severe renal microvascular complications caused by diabetes.Drugs that decrease blood glucose,lower blood pressure,or inhibit the actions of the hormone angiotensin can delay,but not eliminate,the onset of DKD,especially for early micro proteinuria.Therefore,the pathogenesis of early DKD is still needed to be further studied.Recently,the study found that the glomerular permeability may be an important reason for the initiation of DKD micro albumin urine.There are many factors that affect the glomerular permeability,but it is not clear what kind of common pathway or pathways are involved.We have known that high glucose and advanced glycation end products,endothelial source of no production decreased,inflammation,and oxidative stress and other factors can promote glomerular permeability,but in high glucose conditions GEnCs endothelial channel opening and closing key regulatory mechanism has not been determined,not to mention therapy target of glomerular permeability.Therefore,it is urgent to explore the mechanism.ROBO4/ARF6 signaling pathway plays an important role in the regulation of vascular endothelial function and endothelial cell migration in recent years.Protein ROBO family is a class of transmembrane proteins,including 4 members in mammals: ROBO1,ROBO2,ROBO3 and ROBO4,only ROBO4 specifically expressed in vascular endothelium.In ROBO4/ARF6 signaling pathway,ROBO4 has the function of regulating and stabilizing the barrier function of vascular endothelium to prevent excessive vascular leakage,while ARF6 has the effect of increased vascular endothelial permeability and increased vascular leak.The main mechanism is as follows: Robo4 activates GIT(an inhibitor of ARF6),and inhibites ARF6 function,and further inhibits actin cytoskeletal dynamics and endothelial cell prominent activity induced by Rac and slows neovascularization and reduces vascular permeability and angiogenesis.Recent research shows that,in diabetic retinopathy retinal endothelial cell model,activation of robo4 can alleviate retinal endothelial cell high permeability.Since the ROBO4 with vascular stabilization and the role in retinal endothelial cells to confirm,then we have reason to speculate ROBO4/ARF6 signaling pathway in DKD is also involved in glomerular endothelial cell high permeability,and then participate in the progression of glomerular vascular pathological injury and proteinuria.Methods:1.Clinical Data: 30 type 2 diabetic patients with biopsy-proven diabetic nephropathy,who were hospitalized at the department of nephrology of xinqiao hospital from September 2012 to February 2015,were recruited.All these patients met the World Health Organization criteria for type 2 diabetes mellitus,and renal biopsy consistent with the diagnosis of diabetic nephropathy and exclusion for other concomitant renal diseases.According to Mogensen' clinical classification standards and Tervaert'spathological staging criteria,the patients were divided into early,middle and late group,respectively.DKD patients in early,middle and late stages of the 10 cases,of which 20 cases were male and female 10 cases;The renal biopsy specimens of 10 healthy control subjects were taken from the hospital renal cell carcinoma were identified as normal renal tissue,male 7 cases,female 3 cases.Clinical parameters were collected from patients with renal biopsy and recent history of renal cell carcinoma.The eGFR(the estimate glomerular filtration rate,e GFR)according to the Cockcroft Gault formula determined that eGFR [ml /(min /1.73 m2)] = [(140-age(years)] x weight(kg)/ 72× SCR(mg / dl)gender(male = 1 women 0.85).2.Detection indicators and methods2.1 The expression of ROBO4 and ARF6 were detected by immunohistochemistry.3.Results interpretation methodImmunohistochemical staining of Slit2 and ARF6 was semi-quantitated using Image-Pro Plus software,version 6.0(Media Cybernetics,Inc.,Rockville,MD,USA),and at least five fields of view were selected on each section and photographed.Integrated Optical density were calculated using the following formula: IOD?=?percentage of positive cells?×?mean intensity.Mean IOD were obtained for the various fields of view in each section.For all stainings,all sections were analysed in a blinded fashion using a 400-fold magnification.All biopsies had to have at least five glomeruli and the same number of cortical fields for evaluation.For every biopsy the local nephropathologist evaluated the severity of diabetic nephropathy in a blinded fashion.Negative controls were run by replacing the primary antibody with PBS.4.Statistical analysisSPSS 21 software was used for statistical analysis,Data were presented as mean value±SD,between the four groups were compared using one factor analysis of variance,further two two compared the use of LSD-t test,correlation analysis using Pearson method.P<0.05 difference was statistically significant.Results:1.Clinical and laboratory data of patientsIncipient,Manifest and Advanced DKD group of 10 cases,a total of 30 cases,including 20 males and 10 females,there was no significant difference in the age between the groups.DKD group HbA1 c,24h urinary albumin,serum creatinine level and e GFR were higher than the normal control group;with the increase of DKD pathological stage,Hb A1 c,24h protein urine,serum creatinine levels were gradually increased,while e GFR gradually decreased.2.Expression of ROBO4 and ARF6 in glomerular tissue of each groupImmunohistochemistry showed that ROBO4 in GEnCs is rich in expression in the control group,while ARF6 expression is less;Robo4 mainly in GEnCs expression,and ARF6 are expressed in GEnCs and renal tubular epithelial cells.Compared with the normal group,ROBO4 positive intensity was significantly lower in DKD group and with pathological staging aggravated,ROBO4 positive strength gradually decreased(P < 0.05);on the contrary,with DKD pathological stage of exacerbation,ARF6 in early DKD,positive interim strength is obviously increased,only in the later middle group increased amplitude has declined(P < 0.05),suggesting that there is low expression of ROBO4 and high expression of ARF6 in DKD.3.Correlation of ROBO4,ARF6 and clinical parameters of glomerular endothelial cells in patients with DKDCorrelation analysis results showed that ROBO4 expression had no significant correlations with sex and age.ROBO4 expression had a negative correlation with 24 h urine protein(r=-0.840,P < 0.01),serum creatinine(r =-0.689,P < 0.01)and Hb A1c(r=-0.660,P < 0.01),and positive correlation with e GFR(r=0.589,P < 0.01).ARF6 expression expression had no significant correlation with serum creatinine and e GFR(P > 0.05),and a positive correlation with 24 h urine protein(0.603,P < 0.01)and glycosylated hemoglobin(r = 0.582,P < 0.01).Conclusions:1.GEnCs has low expression of Robo4 and high expression of ARF6 in DKD,suggesting that ROBO4/ARF6 signaling pathway may be involved in the pathogenesis of glomerular endothelial cell permeability and micro albumin urine.