Activation Of Kallikrein Kinin System Induces TCE-mediated Immune Renal Injury By Upregulation Of NF-κB

Background Trichloroethylene trichloroethylene(TCE) is widely used for cleaning agent, degreasing agent, bactericide in industry, low concentration of long-term exposure to TCE can cause "TCE induced medicamentosa like dermatitis"(Occupationnal dermatitis Medicamentosa-like of trichloroethylene and ODMLT) and accompanied by severe multiple organ damage. What‘s more, kidney damage caused by renal failure also become the main cause of death in patients with ODMLT. The usage of trichloroethylene in industry is very huge and can not be banned in short term. With the continuous development of industry, crowd occupational exposure to trichloroethylene becomes more and more huge, so the trichloroethylene induced renal injury mechanism is particularly important in the prevention and treatment of patients with ODLMT. A large number of previous clinical cases show that delayed type allergic reaction can not fully explain the mechanism of renal injury caused by TCE. In addition,the previous studies in our research group found that there was kallikrein-kinin system( KKS) and the activation of nuclear factor kappa B activation in TCE sensitized mice kidney injury and their activation can aggravate renal injury in the TCE sensitized mice. The foreign experimental results suggest that the activation of KKS on cell damage may up regulate expression of nuclear factor NF-kappa B, investigating the effect of cytokines, thus aggravating kidney inflammation. In this experiment, through the establishment of mature TCE sensitized mouse model to study the KKS activation mechanism of serious damage to the kidney using KKS inhibitor PKSI-527.Through establishing trichloroethylene sensitized mouse model, inhibit the activation of the kallikrein kinin system by intraperitoneal injection of KKS inhibitor PKSI-527,specificity and correlation indexes of the KKS and NF kappa B were detected to fathem the mechanism of sensitized mice model in immune renal injury.ObjectiveMethod In our lab, after adaptive feeding after a week, 40 mice were randomly divided into blank control group(a total of five), solvent control group(total 5 TCE treatment groups(a total of 15), PKSI-527+TCE treatment group(a total of 15)(on the basis of TCE processing each mouse received KKS blocker PKSI-52750mg/kg) 40 healthy female 6-8 weeks BALB / c mice were selected. In 24 hours after the last challenge were observed and recorded back in experimental mice subject areas of skin reactions and in accordance with the toxicity of chemicals to identify technical specifications"scores, the TCE treatment components for TCE sensitive positive group and TCE was not caused by the sensitive group, PKSI-527+TCE group points for TCE PKSI-527+induced sensitive positive group and PKSI-527+TCE did not cause sensitive group.Serum were collected and serum Cr, BUN levels were detected in the First Affiliated Hospital of Anhui Medical University Department of biochemistry; under sterile conditions from mouse kidney tissue, a cryopreservation to 80 degrees refrigerator,another placed in 10% poly formaldehyde solution fixed, for hematoxylin staining to observe the pathological changes in the kidney of mice, immunofluorescence was used to detect the mouse PK expression, real-time quantitative PCR detection of renal p65,Tnf-α, IL-1, IL-6, level of IL-17 gene expression, immune blotting assay in mice kidney phosphorylation of p65 protein expression levels.Results1 sensitization case There was no erythema or edemathe in blank control group and solvent control group.Erythema and edema was appeared in 6 mice among 15 TCE treated mice, which the sensitization rate was 42.86% while in PKSI-527 treated group is 26.67%.2 The results of pathological examination of renal injury Blank control group and solvent control group mice kidney does not appear pathological changes; compared to the solvent control group mice, kidney juxtaglomerular apparatus malformation hyperplasia and tubular structure changes, the epithelial cells of renal tubules appeared dissolution phenomena in TCE induced sensitive positive mice.Compared with TCE sensitized mice,kidney juxtaglomerular hyperplasia and renal tubular abnormalities were significantly reduced, significantly improve the pathological changes in PKSI-527+TCE sensitized group.3 The dectection result of kidney function Renal function monitoring results results show that compared with the blank control group, the expression of serum Cr, BUN level protein level in solvent control group has no significant change(P > 0.05). Compared with the solvent control group, the expression of serum Cr, BUN level protein level were significantly increased in TCE sensitized group and TCE+PKSI-527 sensitized group(p<0.05). Compared with the relevant negative group, the expression of serum Cr, BUN level protein level were significantly increased in TCE sensitized group and TCE+PKSI-527 sensitized group(p<0.05). Significant reducing of the expression of serum Cr, BUN level protein level in PKSI-527 sensitized group was appeared compared with TCE sensitized group(P < 0.05).4 KKS system correlation Index expression Q-RT PCR results show that compared with the blank control group, the expression of PK protein level in solvent control group has no significant change(P > 0.05).Compared with the solvent control group, the expression of PK protein level weresignificantly increased in TCE sensitized group and TCE+PKSI-527 sensitized group(p<0.05). Compared with the relevant negative group, the expression of PK protein level were significantly increased in TCE sensitized group and TCE+PKSI-527 sensitized group(p<0.05).Significant reducing of the expression of PK protein level in PKSI-527 sensitized group was appeared compared with TCE sensitized group(P <0.05).5 NF-κB signal activation index expression Q-RT PCR result shows that show that compared with the blank control group, p65 gene level in solvent control group has no significant change(P > 0.05). Compared with the solvent control group, p65 gene levels were significantly increased in TCE sensitized group and TCE+PKSI-527 sensitized group(p<0.05). Compared with the relevant negative group, p65 gene levels were significantly increased in TCE sensitized group and TCE+PKSI-527 sensitized group(p<0.05).Significant reducing of p65 gene in PKSI-527 sensitized group was appeared compared with TCE sensitized group(P < 0.05).Western Blot results show that compared with the blank control group, the expression of P-P65 protein level in solvent control group has no significant change(P > 0.05). Compared with the solvent control group, the expression of P-P65 protein level were significantly increased in TCE sensitized group and TCE+PKSI-527 sensitized group(p<0.05). Compared with the relevant negative group, the expression of P-P65 protein level were significantly increased in TCE sensitized group and TCE+PKSI-527 sensitized group(p<0.05).Significant reducing of the expression of P-P65 protein level in PKSI-527 sensitized group was appeared compared with TCE sensitized group(P < 0.05).Q-RT PCR results show that compared with the blank control group, Tnf-α,Il-1β,Il-6,Il-17 level in solvent control group has no significant change(P > 0.05). Compared with the solvent control group, Tnf-α,Il-1β,Il-6,Il-17 levels were significantly increased in TCE sensitized group and TCE+PKSI-527 sensitized group(p<0.05). Compared with the relevant negative group, Tnf-α,Il-1β,Il-6,Il-17 levels were significantly increased in TCE sensitized group and TCE+PKSI-527 sensitized group(p<0.05).Significant reducing of Tnf-α,Il-1β,Il-6,Il-17 in PKSI-527 sensitized group was appeared compared with TCE sensitized group(P < 0.05).6 Inflammatory cytokine expressionConclusion1 A certain dose of TCE can cause sensitization effect and kidney damage in the skin of BALB/c mice.2 kidney damage in TCE induced sensitization mice may be caused by the activation of NF-κB cells signaling system after the activation of KKS and then increses the expression of inflammatory factors.