The Role Of Complement C3 And C3a Receptor In The Immune Kidney Injury Of Trichloroethylene Sensitized Mice

Background Trichloroethylene(TCE)is a common metal detergents,lipids extraction agent,paint thinner,widely used in electronics,electroplating,pharmaceutical and other industries.It may cause ODMLT in occupational exposure population.ODMLT can show multi-system and multi-organ injury and some patients may occuer severe renal injury,which is a serious threat to the health of the occupational population.It is found that the complement system plays an important role in the pathogenesis of ODMLT except delayed type IV allergic reaction.In the early animal studies,we found that there was a complement system activation and C3 a deposition in kidney injury.C3 a combined with C3 a R has very important physiological functions,such as expansion of blood vessels,chemotaxis of inflammatory cells,regulation of inflammatory response and so on.The domestic and foreign research found that the C3a-C3 a R system plays an important role in a variety of kidney damage.Therefore,to study the function of the complement systemin the renal injury of TCE-sensitized mice has great significance for pathogenesis of ODMLT.Objective To study the effect of complement system in the kidney immune injury of TCE-sensitized mice by using C3 a receptor specific antagonist C3 a RA(SB290157)and detect the expression of C3 and C3 a R in the kidney,provide the basis for the pathogenesis of kidney injury in ODMLT.Method A total of 42 specific pathogen free(SPF),6-8 week female mice BALB/c,weight range of 17.1-24.7(20.3 ± 1.7 g).A week later of adaptive feeding,according to the principle of randomization of the experimental animal were divided into blank control group(5),solvent control group(5),TCE treatment group(16)and TCE+C3aRA treatment group(16).BALB/c mice induced by trichloroethylene,at 24 h after the last challenge,the mice whose skin score ?1were divided into sensitized group and the other were divided into non sensitized group according to the skin sensitization test score.Three days after the last challenge,mice were sacrificed and serum and renal were harvest.The paraffin sections and frozen sections were prepared by using renal tissue.The renal function of CRE and BUN levels were detected by CRE and BUN kits;The histological damage of renal was observed by HE stain;The expression of inflammatory cytokines were detected by immunohistochemstry;The reverse transcription of C3?C3aR and inflammatory cytokines were detected by Real-time RT-PCR;The expression of podocyte protein Podocin was detected by Immunofluorescence;The expression of C3 a R?Podocin were detected by Western Blotting.Results(1)sensitization case: The mice were in good condition and no significant difference in weight change between groups.No obvious damage in the skin was observed in blank control group and solvent control group.The sensitization rate of TCE treatment group was 7/16(43.75%),and the sensitization rate of TCE+C3aRA group was 6/16(37.5%).(2)The results of renal pathological: No significant pathological changes were observed in the renal of blank control group,solvent control group and the non-sensitized group.There were obvious inflammatory cell infiltration,renal tubular structure change and renal tubular epithelial cell lysis in renal of TCE sensitized group.After treatment with C3 a RA,the inflammatory cell infiltration in the kidney of sensitized mice was significantly reduced,the renal tubular injury was alleviated,and the renal injury was significantly decreased.(3)The determination results of renal function: There was no significant difference in CRE and BUN levels between the solvent control group and the non-sensitized group.CRE and BUN in TCE sensitized group and TCE+C3aRA sensitized group were significantly higher than in the solvent control group and the non-sensitized group.However,after C3 a RA treatment,the CRE and BUN levels in TCE+C3aRA sensitized group were significantly lower than the TCE sensitized group.(4)The determination results of C3 a R in renal tissue: Compared with the blank control group and the solvent control group,there was no significant difference in the transcription level of C3 a R in the TCE non sensitized group.Compared with the blank control group and the solvent control group,there was significantly increased in the transcription level of C3 a R in the TCE sensitized group.The results of Western Blotting also showed that the expression of C3 a R protein was lower in blank control group,solvent control group and TCE non-sensitized group,the expression of C3 a R protein was significantly increased in TCE sensitized group.(5)The determination results of C3 in renal tissue: Compared with the blank control group and the solvent control group,there was no significant difference in the transcription level of C3 in the TCE non-sensitized group.There was significantly increased in the transcription level of C3 in the TCE sensitized group.(6)The determination results of IL-1??TNF-? in renal tissue: Compared with the blank control group and the solvent control group,there was no significant difference in the transcription level of IL-1? ? TNF-? in the TCE non-sensitized group.The transcription level of IL-1? ? TNF-? in TCE sensitized group and TCE+C3aRA sensitized group were significantly higher than in the solvent control group and the non-sensitized group.However,after C3 a RA treatment,The transcription level of IL-1??TNF-? in TCE+C3aRA sensitized group were significantly lower than the TCE sensitized group.The results of Immunohistochemistry also showed that there was no obvious expression of IL-1??TNF-? in the blank control group and solvent control group.There was a significant expression of IL-1??TNF-? in TCE sensitized group and TCE+C3aRA sensitized group.Compared with TCE sensitized group,the expression level of IL-1??TNF-? was decreased in TCE+C3aRA sensitized group.(7)The determination results of Podocin protein in renal tissue: The expression of Podocin protein was normal in the blank control group and solvent control group.Compared with blank control group and solvent control group,the expression of Podocin protein in the renal of TCE sensitized group and TCE+C3aRA sensitized group were significantly decreased.The expression of Podocin protein in TCE sensitized group was significantly lower than in the TCE+C3aRA sensitized group.The results of Western Blotting also showed that the expression of Podocin protein in TCE+C3aRA sensitized group was significantly higher than in the TCE sensitized group.Conclusions(1)The expression of complement C3?C3aR was significantly up-regulated in the kidney of TCE sensitized mice,and there was a phenomenon of local complement activation in TCE sensitized mice.(2)Complement C3 a R signal increased the immune kidney injury in TCE sensitized mice by up regulating the secretion of IL-1? and TNF-?.(3)The expression of Podocin protein was down regulated in the immune kidney injury of TCE sensitized mice.(4)Complement C3 a R signal could have an effect on the expression of Podocin protein in TCE sensitized mice.