The Role Of C-C Chemokine Receptor Type 2 In IgA Nephropathy

Objective: IgA nephropathy(IgAN)is the most common primary glomerulonephritis.Its pathological features are IgA1 deposition and mesangial cell proliferation in the mesangial area.The immunological pathogenesis of IgA nephropathy is one of the main research directions at present.CCR2(CC chemokine receptor 2,CCR2)is a receptor for monocyte chemoattractant protein-1(MCP-1),a G-protein coupled receptor superfamily,which is specific receptor for MCP-1.MCP-1 specifically binds to its receptor CCR2,brings the signal into target cells,chemotaxis and activates monocytes/macrophages,promotes the development of inflammation and.MCP-1 exerts a wide range of physiological and pathological effects.In this study,children with IgA nephropathy,IgA animal models,and cultured mesangial cells were used to detect CCR2 signal expression in glomerular cells of IgA nephropathy,using various experimental techniques from the whole to cell and molecular levels.Receptor antagonist RS102895 was used to interfere with IgA nephropathy model rats and cultured mesangial cells in vitro,and observed the role of CCR2 signaling in IgA nephropathy,IgA deposition,mesangial cell proliferation and inflammatory response,which provide a new idea for the pathogenesis of IgAN.Methods: Part?---CCR2 expression in kidney tissue of children with IgA nephropathy Blood,urine and kidney biopsy samples were collected from 15 children with IgA nephropathy in our department from July 2014 to September 2017.The normal renal tissue samples from 8 patients in the control group were obtained from children without pathological changes.Blood and urine of 12 children in control group were taken from the children for the healthy physical examination in our hospital.Renal specimens were stained with HE staining,Masson staining,PAS staining and PASM staining.Immunohistochemistry and immunofluorescence were used to detect IgA1 deposition,as well as the expression of CCR2,MCP-1,IL-6,TNF alpha,IL-17,IL-1,CD89 and CD71.Part ?---CCR2 expression in the kidney tissue of rat model with IgA nephropathy Using the combination of LPS+BSA+CCl4 as a modeling method,a 6-week-old Wistar male rat was treated to establish a rat model of IgA nephropathy.The IgA nephropathy model group was randomly divided into three groups: IgA nephropathy group(IgAN-Model),CCR2 blocker group(RS102895),and olmesartan group(Olmesartan).Model rats were administrated with the CCR2 antagonist,RS102895 and olmesartan.Pathological staining,such as HE staining and Masson staining,as well as immunofluorescence,immunohistochemistry,Western blotting and quantitative PCR,were used to detect pathological changes,such as mesangial proliferation and IgA deposition in the kidney of model rats,as well as the expression of CCR2,MCP-1,TNF alpha,IL-6,IL-17 and CD71;After the treatment of RS102895 and olmesartan in model rat,pathological changes,and the expression changes of CCR2,MCP-1,TNF alpha,IL-6,IL-17 and CD71 were detected.Part ?---Effect of CCR2 signal on proliferation of mesangial cells Peripheral blood was collected from 15 children with IgA nephropathy and healthy children,and IgA1 was purified.Human mesangial cells were cultured and identified in vitro,IgA1 derived from children with IgA nephropathy was used to culture human mesangial cells in vitro.BrdU incorporation assay and CCK-8 were used to detect the proliferation of mesangial cell,and Western blotting was used to detect the expression of CCR2 in mesangial cells.After the treatment of CCR2 antagonist,RS102895,inflammatory factors,including MCP-1,IL-6 and TNFalpha expression,as well as the phosphorylation levels of p-JNK,p-NF-?B,p-P38 MAPK and p-ERK1/2 in the cultured mesangial cells were detected.Results: Part ?---CCR2 expression in kidney tissue of children with IgA nephropathy The expression of CCR2 in renal tissue of children with IgA nephropathy was significantly increased compared with the control group,and the positive CCR2 signal was concentrated in glomerular mesangial cells.The Lee pathological grade was positively correlated with the signal intensity of CCR2,but not related with IgA deposition intensity.The expression of MCP-1,TNF alpha,IL-6 and IL-17 was significantly increased in renal tissues of children with IgA nephropathy compared with the control group.The results suggested that the CCR2 signaling pathway may be involved in the pathogenesis and development of IgA nephropathy.Part ?---CCR2 expression in the kidney tissue of rat model with IgA nephropathy Glomerular IgA deposition and mesangial cell proliferation were detected in the model rats.CCR2 signal was expressed in glomeruli and renal tubules of model rats.CCR2 positive signal was significantly increased in IgA nephropathy model rats compared with the control group.After the treatment of CCR2 antagonist,glomerular IgA deposition and mesangial cell proliferation were significantly reduced in model rats.The expression of MCP-1,TNF alpha,IL-6 and IL-17 in renal tissue of model rats was significantly increased.After the treatment of CCR2 antagonist,the expression of above positive signals,as well as mesangial proliferation and inflammatory response,were significantly reduced in renal tissue of model rats.Our reaults suggest that CCR2 signaling pathway may be involved in the development of IgAN model.Part ?---Effect of CCR2 signal on proliferation of mesangial cells IgA1 derived from children with IgA nephropathy promotes the proliferation of mesangial cells in vitro.IgA1 derived from IgAN children significantly increased CCR2 expression.CCR2 antagonist RS102895 inhibited the proliferation of mesangial cells induced by IgAN IgA1.The expression of MCP-1,IL-6 and TNF alpha was significantly increased in the mesangial cells of IgAN IgA1 group.IgA1 derived from children increased the phosphorylation levels of p-JNK,p-NF-?B,p-P38 MAPK and p-ERK1/2 in mesangial cells induced by IgAN IgA1 was significantly increased.After the treatment of CCR2 antagonist RS102895,the increased expression of MCP-1,IL-6 and TNFalpha,as well as the phosphorylation levels p-JNK,p-NF-?B,p-P38 MAPK and p-ERK1/2 were inhibited.These results suggested that CCR2 is involved in mesangial cell proliferation induced by IgAN IgA1.Conclusion:(1)The expression of CCR2 in glomerular mesangial cells was significantly increased in children with IgA nephropathy.Lee pathological grade was positively correlated with the positive signal intensity of CCR2.(2)Antagonizing CCR2 signal can alleviate renal pathological changes in IgA model rats,and reduce the expression of inflammatory factors,in the renal tissue of the children with IgA nephropathy and IgA nephropathy model rats,as well as the cultured mesangial cells in vitro.The phosphorylation level of JNK,NF-?B,P38 MAPK and ERK1/2 also reduced in the cultured mesangial cells in vitro after the treatment of CCR2 antagonist.(3)CCR2 is involved in mesangial cell proliferation and inflammatory response in children with IgA nephropathy.