The Study On Active Vitamin D3 Regulating Macrophage Function And Phenotype Via TREM-1 In Diabetic Nephropathy

BackgroundDiabetic nephropathy(DN)is an inflammatory chronic disease.Macrophage accumulation is a vital feature in DN.Macrophage infiltration process mainly includes adhesion and migration in renal tissue.Imbalance of M1/M2 macrophages phenotype activation is a key point in diabetic nephropathy(DN).M1 macrophages promote tissue inflammation and fibrosis while M2 macrophages mediate tissue repair.Macrophages mainly exhibit M1 phenotype,whcih contribute to the inflammation and fibrosis in DN.Therefore,the key to solve the problem is to explore the mechanism of macrophages regulation and to find effective and feasible intervention drug.Recent investigations demonstrated that active vitamin D3 reduced albuminuria and improve renal function.The present study aimed to provide a mechanistic insight into the potential effects of 1,25-dihydroxyvitamin D3(VD)in vivo and in vitro high glucose induced macrophages regulation.MethodsExperimental rat model were established and random distributed into four groups as follows: NC,VD,DN and DN+VD(DN+ Calcitriol 0.1 ?g·kg-1·d-1 by gavages).Animals were sacrificed respectively at 18 w after treatment.Pathological changes in kidney tissue were detected and the expression of CD68,TREM-1,pSTAT-1 were acquired by Immunohistochemistry stain and Western Blot.In vitro,RAW 264.7 cells were treated by high glucose with or without 10-8 mol/L 1,25(OH)2D3.TREM-1 siRNA,STAT-1 siRNA and high expression plasmid of TREM-1 and STAT-1 activtor were explored to elucidate the underlying mechanism.The expression of TREM-1 and STAT-1 and the changes of macrophage phenotype were examined separately by Western Blot,immunofluorescence stain.The ability of macrophage in adhesion and migration were examined by adhesion experiment and migration assay.Results In vivo: In DN group,levels of BUN,Scr,24-hour urinary protein and glomerular mesangial matrix proliferation were significantly higher,and the expressions of nephrin,podocin were significantly decreased compared with NC groups(P<0.05).These above changes were significantly improved in VD group(P<0.05).In DN group,number of macrophage with CD68+ and expression of iNOS,TNF-?,TREM-1,p-STAT-1 protein were significantly increased in comparation with NC group(P<0.05),while above changes were markedly decreased in DN+VD group(P<0.05).In vitro: In order to explore the mechanism of macrophage infitration and phenotype regulation in high glucose condition,the RAW264.7 cells were cultured.The capacity of adhesion and migration in macrophage and the amount of iNOS,TNF-?,TREM-1,p-STAT-1 protein were obviously increased in HG group,compared with NC group(P<0.05).In order to further determine the regulatory effects of TREM-1 and STAT-1 on the ability of adhesion and migration and the M1/M2 phenotype transformation,TREM-1siRNA and STAT-1siRNA were used respectively.Compared with NTC groups,the number of macrophage adhesion and migration and the expression of iNOS,TNF-? were notably reduced after TREM-1 siRNA and STAT-1 siRNA intervention(P<0.05).While,TREM-1 siRNA did not affect STAT-1 expression.The above results demonstrated that TREM-1 and STAT-1 are involved in the macrophage adhesion and migration and M1/M2 phenotype regulation.In order to observe the effect of VD on macrophage in high glucose condition,the 1,25(OH)2D3 was used.Compared with HG groups,VD significantly decreased the amount of macrophage adhesion and migration and expression of iNOS,TNF-?,TREM-1,p-STAT-1 caused by high glucose(P <0.05).In order to explore the mechanism of VD on macrophage regulation in high glucose condition,high expression plasmid of TREM-1 and STAT-1 activtor were used.The effect that VD significantly decreased the amount of macrophage adhesion and migration and expression of iNOS,TNF-?,TREM-1,p-STAT-1 caused by high glucose were abolished when TREM-1 was overexpressed by TREM-1 plasmid and STAT-1 was activated by STAT-1 activator.Conclusion 1.Macrophage infiltration was increased in STZ-induced DN rats,especially M1 macrophage in renal tissue of DN rats.2.The up-regulation of STAT-1/ TREM-1 signaling pathway promoted the capacity of macrophage adhesion and migration and M1 macrophage transformation under high glucose concentration.3.VD can inhibit the capacity of macrophage adhesion and migration and and M1 macrophage transformation through the STAT-1/TREM-1 pathway under high glucose condition,eventually alleviating the progress of diabetic nephropathy.