Mechanism Of IL-17 And Related Chemokines In Adriamycin Induced Nephropathy In Mice And Podocytes

Background and objectives:Primary nephrotic syndrome(PNS) is one of the most common kidney disease in childhood. Its clinical symptoms are nephrotic range of proteinuria, hypoalbuminemia, edema and hypercholesterolemia.The pathogenesis of PNS is still unknown, and it is a multi-element disease.The renal pathological types of PNS go as follows:minimal change nephropathy syndrome (MCNS), focal segmental glomerular sclerosis (FSGS), mesangial proliferative glomerulonephritis (MsPGN), and membranous glomerulonephritis (MNGN),etc. The main pathological types of PNS were MCNS (77.1%) and FSGS (7.9%). Most children are sensitive to hormones.But a small number of children are dependent or resistant to hormones, and finally develop into the end-stage renal disease. Therefore, clarifying the pathogenesis of PNS, for its pathogenesis of drug intervention, can be opened for children with nephropathy new treatment approach.Interleukin 17 (IL-17)is a proinflammatory cytokine produced by activated CD4+T cell subsets (often called Th17). The IL-17 family consists of six family members, which are IL-17(also known as IL-17A), IL-17B, IL-17C, IL-17D, IL-17E, and IL-17F. The IL-17 receptor family consists of five members, which are IL-17RA, IL-17RB, IL-17RC, IL-17RD and IL-17RE. In recent years, domestic and foreign research on IL-17 in kidney disease focused on lupus nephritis, diabetic nephropathy, glomerulonephritis, renal injury and kidney transplant rejection. There are few studies on IL-17 in nephrotic syndrome.We know that T cells participate in the pathogenesis of PNS, and the cytokines of Th17 secretion involved in the immune mechanism of PNS is not clear. Some researchers suggest that IL-17 is likely to be a bridge between T cell activation and inflammation.Wang et al found that Th17/IL-17 promotes the development of PNS by down-regulating the expression of podocalyxin and inducing podocyte apoptosis. Liu et al reported that the underlying pathogenesis of MCNS is Th17/Treg imbalance. Recently, few studies have shown that IL-17 plays a major role in the pathogenesis of MCNS. Recent data suggest that rituximab can interfere with the pathogenesis of MCNS by inhibiting IL-17, affecting B cell depletion, or altering the cytoskeleton of podocytes.The main way to produce IL-17A is a chemokine receptor CCR6 on kidney of Th17 cell,combinating with CCL20 to reaction. IL-17 produces singal pathway through its combination with the receptor. In the inflammatory diseases, IL-17A may collect neutrophils and macrophages, resulting in producing polypeptide proteins, cytokines and chemokines (CXCL1, CXCL2, and CXCL5). CXCL1, CXCL2 can promote neutrophil's chemotaxis. IL-8 can also promote the neutrophil's chemotaxis in site of infection, and ultimately eliminate the foci of infection.CXC chemokine ligand 9(CXCL9) and CXCL10 can regulate Thl-related immune pathway.Pathogens, tumor antigens,interferon -? (IFN-?),and IL-12 at the intracellular induce ThO differentiate to Thl; Th1 secret IFN-y,IL-2,etc; Th2 secrete IL-4, etc; Therefore, we conclude that IL-17-associated chemokines are CCR6, CCL20, neutrophil-associated chemokines are CXCL1,CXCL2,IL-8, and Thl-associated chemokines are CXCL9, CXCL10, IL-12, IFN-y.Currently there are no reports about IL-17 and related chemokines in PNS at home and abroad.The PNS patients's main damage to the kidney is losing protein.PNS range proteinuria is due to glomerular filtration membrane permeability damage, and tubular reabsorption obstacles. Glomerular filtration barrier is composed of three layers:the endothelial cell layer, the glomerular basement membrane, and podocyte foot layer. The most characteristic of glomerular diseases are the molecular structure of slit diaphragm (SD) changing, and foot process restructuring (fusion and disappearance). The abnormal interaction between the GBM or podocyte-GBM, damaging SD area, altering actin cytoskeleton and associated proteins,which are the most important reasons resulting to foot effacement and proteinuria.The changing of podocyte's structure can lead to podocyte's damage,which results to the podocyte cell's loss. Foot cell deficiency is the starting of glomerulosclerosis.Mitogen-activated kinase(MAPK) is a functional pathway of the cellular stress activity,which plays an important role for cell growth, development, division,and apoptosis. There are four MAPK pathway in mammalian cells: regulate intracellular signaling enzymes (extracellular signal-regulated kinase, Erk)-1 and-2, c-Jun N-terminal enzyme (c-Jun N-terminal kinase, JNK), p38MAPK and cell within a single regulatory enzyme-5 (extracellular signal-regulated kinase,-5, Erk5/BMK1). Studies have confirmed that MAPK pathway plays an important role in acute and chronic inflammatory diseases.The expression of p38MAPK in anti-GBM nephritis increases, which influenced the inflammatory cells aggregation and the damage of tubular.In this study, the mouse model of nephrotic syndrome(MCNS) was induced by adriamycin (ADR).We observed the changes about the expression of IL-17 in renal tissues and peripheral blood concentration.The expression of IL-17-related chemokines CCR6/CCL20, neutrophils-associated chemokines CXCL1/CXCL2/IL-8 and Thl-associated chemokines CXCL9/CXCL10/IL-12 /IFN-y in renal tissues, were detected by immunohistochemistry. At the same time, IL-17 neutralizing antibody applied in mice was used to observe the changes of inflammation and renal function in model mice,so as to explore the proinflammatory mechanism of IL-17 in PNS. In this study, the molecular mechanism of IL-17 in renal podocyte inflammation and the correlation with MAPK signal pathway were studied by culturing kidney podocytes in vitro. We further explore the role of IL-17 and its related chemokines in the pathogenesis and progression of PNS in children, and provide a new theoretical basis for further prevention and treatment of PNS in children.Methods:1. Animal experiment:Balb/c mice were randomly divided into three groups: normal control group, model group (Adriamycin nephropathy), IL-17 neutralizing antibody group.10 mice in each group.The mice were given to intravenous injection of adriamycin induced adriamycin nephropathy model(MCNS).After one week,a group of adriamycin nephropathy mice were given to intraperitoneal injection of IL-17 neutralizing antibodies.After two weeks,the mice were sacrificed, and collected the specimens of blood,urine,kidney tissue.Using pyrogallol red colorimetric measured 24-hour urinary protein excretion, ELISA detected the levels of serum IL-17, Cys C, Kim-1,and NGAL. The expression of IL-17 in renal tissue was observed by HE staining and immunohistochemical staining.The mRNA expression of IL-17-related chemokines CCR6/CCL20, neutrophil-related chemokines CXCL1/CXCL2/IL-8,and Thl-related chemokines CXCL9/CXCL10/IL-12/IFN-? were detected by realtime fluorogenic quantitative PCR. Mouse renal podocytes were cultured in 1640-fold fetal bovine serum containing 10% fetal calf serum and 10 U/ml interferon-?.2, Kidney podocyte culture:The cells were then subcultured in a 33? incubator and then transferred to an interferon-free medium at 37? incubating the cells.So that the cells were differentiated after 2 weeks. The podocytes were seeded on plates and cultured in serum-free culture medium for 24 hours. Mouse kidney podocytes were co-incubated for 24 hours with recombinant mouse IL-17 protein 100 ng/ml. The expression of p38, Erk, p-p38 and p-Erk were detected by western blotting.After p38 and Erk inhibitors were added,the mRNA expression of neutrophil-associated chemokine IL-8 and Thl-associated chemokine IL-12 were measured by fluorescence quantitative PCR.Results:1.The light microscopy observations.Compared with the control group, model group:part of the Bowman's capsule adhesions, glomerular mesangial cell proliferation, matrix increase, tubular cavity portion appears protein casts, renal interstitial inflammatory cell infiltration; And IL-17 neutralizing antibody group was lighter than the model group.2. The electron microscopy observations.Compared model group with control group, glomerular morphology was normal, foot process had been fused widely.The lesion in IL-17 neutralizing antibody group was milder.3. The expression of IL-17 protein in renal tissue of each groupDAB staining showed that IL-17-positive cells were mostly cytoplasmic staining, and small amounts of IL-17 was expressed in normal renal tissue, mainly in the renal tubules, no expression in glomeruli.In model group, IL-17 was significantly expressed in kidney tubules,slightly in glomerulus; And IL-17 neutralizing antibody group were lighter than model group. Compared with the normal group, positive for the chromaticity of cells in model group and IL-17 neutralizing antibody group were significantly increased, P<0.05.Compared with model group and IL-17 neutralizing antibody group, positive for the chromaticity of cells in model group were significantly increased, P<0.05.4.The mRNA expression in mice kidney tissue of IL-17-related chemokines CCR6/CCL20mRNA in each group.Compared with normal control group, the mRNA expression of IL-17-related chemokines CCR6/CCL20 in model group and IL-17 neutralizing antibody group were increased, P<0.05.The mRNA expression of IL-17-related chemokines CCR6/CCL20 were more increased in model group than that in IL-17 neutralizing antibody group,P<0.05.5.The mRNA expression in mice kidney tissues of neutrophil-related chemokine CXCL1/CXCL2/IL-8 in each group.Compared with normal control group, the mRNA expression of neutrophil-related chemokine CXCL1/CXCL2/IL-8 in model group and IL-17 neutralizing antibody group were increased, P<0.05.The mRNA expression of neutrophil-related chemokine CXCL1/CXCL2/IL-8 were more increased in model group than that in IL-17 neutralizing antibody group,P<0.05.6.The mRNA expression in mice kidney tissues of Thl-related chemokine CXCL9/CXCL10/IL-12/IFN-? in each group.Compared with normal control group, The mRNA expression of Thl-related chemokine CXCL9,CXCL10,IL-12, IFN-yin model group and IL-17 neutralizing antibody group were decreased, P<0.05.The mRNA expression of Thl-related chemokine CXCL9/CXCL10/IL-12/IFN-y were more decreased in model group than that in IL-17 neutralizing antibody group P<0.05.7. Changes in the concentrations of serum IL-17 in each group.Compared with normal control group, the concentration of serum IL-17 in model group and IL-17 neutralizing antibody group were increased, P<0.05.The concentration of serum IL-17 was more increased in model group than that in IL-17 neutralizing antibody group, P<0.05.8.Changes in the concentrations of serum KIM-1, NGAL, Csy C in each group.Compared with normal control group, the concentrations of serum KIM-1, NGAL, CsyC in model group and IL-17 neutralizing antibody group were increased, P<0.05.The concentrations of serum KIM-1, NGAL, CsyC were more increased in model group than that in IL-17 neutralizing antibody group, P <0.05.9.Mouse kidney podocytes p38, Erk and p38, Erk phosphorylation protein levelsCompared with the normal group, Erk, p-Erk, P38, p-P38 protein expression levels in IL-17 stimulation group were increased, especially the p38 and p-p38 were significantly increased.9. After applying Erk inhibitor and p38 inhibitor, the mRNA expression of neutrophil-related chemokine IL-8.Comparison with the normal group,the mRNA expression of neutrophil-related chemokine IL-8 in IL-17 stimulation group, p38 inhibitor group, and Erk inhibitor group were significantly increased,P<0.05.Compared with IL-17 stimulation group, the mRNA expression of IL-8 in p38 inhibitor group and Erk inhibitor group were decreased, P<0.05. The mRNA expression of IL-8 in P38 inhibitor group was significantly higher increased than that in Erk inhibitor group, P<0.05.11.After applying Erk inhibitor and p38 inhibitor, the mRNA expression of Thl-related chemokine IL-12.Comparison with the normal group, the mRNA expression of Th1-related chemokine IL-12 in IL-17 stimulation group was significantly decreased;p38 inhibitor group and Erk inhibitor group were significantly higher,P <0.05.Compared with IL-17 stimulation group,p38 inhibitor group, and Erk inhibitor group were significantly increased,P<0.05.P38 inhibitor group was significantly higher than Erk inhibitor group, P<0.05.Conclusions:1. In the adriamycin-induced nephropathy model mice, IL-17 expression in renal tissue were abnormally increased, allowed neutrophil recruitment, involved in the inflammatory process. A small amount of IL-17 had a role in maintaining the normal renal function.2. In the adriamycin-induced nephropathy model mice, the concentration of serum IL-17 significantly increased, and suggestted that IL-17 can promote the inflammatory process in ADR mice.But it could also induce Thl cell differentiation decreased.A large amount of IL-17 in ADR mice could lead to serious kidney damage.3. The mice were injected intraperitoneally and IL-17 neutralizing antibody was capable of reducing the concentration of serum IL-17, reducing neutrophil recruitment, and inhibiting Thl cell differentiation decreasing to improve the renal function.4. IL-17 mights regulate inflammatory cytokines of mouse kidney podocytes via MAPK pathway5. IL-17 was able to activate p38 and Erk signaling pathway, and p38 signaling pathway was major in this process.6.IL-17 promoted neutrophil recruitment, inflammatory response, changed the balance of T cells into Th1/Th2 differentiation of natural CD4+T cells,and induced the differentiation into Th2. inhibiting their differentiation into Thl.