Activation Of Bradykinin Receptor Induces TCE-mediated Immune Renal Tubular Injury By Regulation Of NF-?B Pathway

Trichloroethylene?TCE?is a volatile chlorinated vehicle which commonly used as degreaser,and extractant in occupational settings.A large number of workers suffer from TCE-related adverse health effects,involving skin,liver,and kidney,in a pathology defined as Dermatitis Medicamentosa-like of Trichloroethylene?ODMLT?.Clinical studies have classified ODMLT as T-cell-mediated delayed-type hypersensitivity/Type IV allergic reaction,however,recent research has noted Type IV allergic reactions cannot fully explain the multi-organ injuries induced by TCE,especially the renal injury.TCE induced renal injury is one of the leading causes of death from multi-organ injury in ODMLT patients.Based on previous findings,it has become more accepted that immune dysfunction could play a vital role in renal injury caused by TCE.In our previous study,it was found that TCE exposure resulted in kallikrein-kinin system?KKS?activation,including that of bradykinin?BK?and the bradykinin receptor?B1R/B2R?.How BK and its receptors acted in the etiology of the induced renal injury is not clear.Increasing evidence has revealed that B1R and B2R expression is involved in activation of the NF-?B pathway in osteoblasts,fibroblasts,and colonic epithelial cells.Because as a family of inducible factors,NF-?B plays a prominent and evolutionarily conserved role in the immune system,it is plausible that BK receptors might also possibly activate NF-?B signaling pathways and so aggravate TCE-induced injuries in the kidneys.Thus,this study explored any correlation between BK receptors and immune renal injury in TCE-sensitized mice by blocking B1R/B2R.We hope to perform more studies to ultimately define more clearly the mechanisms of immune toxicity in the kidney.From that,more effective therapeutic approaches to treating TCE-induced renal injury could then follow on.ObjectiveWe explored the mechanism of TCE-induced immunological renal injury in vivo and in vitro.First,the animal experiment was used to observe the expression of B1R/B2R receptor and NF-?B signaling pathway which mediated by inflammatory factors after TCE-sensitized renal injury,furthermore,pretreatmented with B1R/B2R receptor inhibitor to explore the changes in kidney damage in TCE sensitived mice.Secondly,through in vitro experiment,extraction of renal tubular epithelial cells from BALB/c mice in order to verify the mechanism of TCE induced immune renal injury.Methods1.In vivoHealthy specific pathogen-free female BALB/c mice?6⁸-week old,18²4 g?were housed?six/cage?under conditions of temperature?20²5°C?and humidity?50±5%?with a 12-h light/dark cycle.All mice were provided ad libitum access to standard rodent chow and filtered water.After 1 week of adaptation,all mice were randomly allocated into blank control group,vehicle control group,TCE-treated group,TCE+B1R antagonist-treated group,or TCE+B2R antagonist-treated group.A model of TCE-sensitized BALB/c mice was induced and allergic reactions in the skin were scored on a 4-point scale at 24 h after the challenge according to our previous study.In B1R/B2R antagonist-treated groups?B1RA/B2RA?,mice received an intraperitoneal injection of R715/HOE140 before the TCE challenge on Days 17 and 19.2.In vitroWe assessed the injury of renal tubular epithelial cell?RETC?from TCE induced mice by established a new method for extration and cultrue RETC.Through reperfusion from aortaventralis,the kidneys of mouse were digested by 1mg/ml collagenase and 0.25%trypsase.Labeling renal tubular epithelial cells with CK18 antibody by immunohistochemistry?IHC?and immunofluorescence?IF?.Results1.Sensitization rates and kidney:body weight ratio.After the final challenge,the results showed sensitization rates of 44.3%?31/70 mice?in the TCE group,40.5%?15/37 mice?in the B1RA pre-treated and 34.0%?18/53mice?in the B2RA pre-treated groups.The overall sensitization rate was 40.0%.No mice in the blank control or vehicle control groups exhibited erythema and edema.Measures of kidney:Body weight ratios were also performed in each group.The data indicated that the ratio increased significantly compared with that in the vehicle control group in the 72h TCE⁺and 72hB1RA⁺groups.However,the ratio of 72h B2RA⁺mice was significantly attenuated than the 72h TCE⁺and 72h B1RA⁺groups.2.Pathological changes in kidneys and renal function.There were no significant histo-morphological changes in the renal tissues from the blank control,vehicle control,and negative groups.In comparison,among the TCE⁺mice,cell swelling,loss of brush border of proximal tubular epithelial cells,cell necrosis were clearly evident.Compared with these TCE⁺mice,damage was significantly attenuated in the 72h B1RA⁺and B2RA⁺hosts.There was no significant difference between vehicle control and blank control,and these levels of serum BUN and Cr levels gradually increased through 24h and significantly peaked at 72h in the TCE⁺mice.Serum BUN levels did not significantly differ among any of the pre-treatment groups.Among the B2RA⁺/B1RA⁺mice,serum Cr levels were significantly lower than in 72h TCE⁺counterparts.Levels of?₁MG and?₂MG displayed the same trends as for serum Cr among the various groups,except,there was no significant difference between B1RA⁺and TCE⁺mice.3.B1R and B2R expression on kidney.B1R and B2R expression on kidney was evaluated by double label IF with CK18.The analyses found that B1R and B2R were mainly expressed on tubular epithelial cells in the kidneys.Compared to the vehicle control,B1R and B2R expression increased significantly in the 72h TCE⁺mice,and this trend was not shown in 72h TCE^-mice.4.Kim-1,Lipocalin-2 and NF-?B p65 deposition by IHC.Using IHC analyses,tubular injury was assessed by examining Kim-1 and Lipocalin-2deposition in tubular epithelial cells.There was no visible Kim-1 and Lipocalin-2deposition in the kidneys from blank control and vehicle control mice.In comparison,deposition of each protein was significantly greater in samples from the 72h TCE⁺mice.Expressions of each were significantly lowered in the B1RA⁺and B2RA⁺mice than their TCE⁺counterparts.Nuclear NF-?B p65 translocation,mainly observed in nucleus of renal tubular epithelia cells,was increased from control mice levels in the 72h TCE⁺mice.Compared with these TCE⁺mice,NF-?B p65 levels was significantly attenuated in the 72h B1RA⁺and B2RA⁺hosts.There were no significant differences in expression of either protein between the TCE-,B1RA-,and B2RA-hosts.5.TCE induced renal NF-?B signaling pathway activation.At 72h time point after final challenge,effects of B1RA and B2RA pre-treatment on TCE-induced renal NF-?B signaling activation were also analyzed.Renal p-I?B levels markedly increased and I?B significantly decreased in 72h TCE⁺mice compared with levels in vehicle control mice.Moreover,the levels of nuclear NF-?B p65 subunits in the kidneys were significantly elevated in 72h TCE⁺mice.In contrast,B1RA and B2RA pre-treatments attenuated TCE-induced I?B phosphorylation and inhibited the reduction in renal I?B?levels caused by TCE.Nonetheless,there was no significant difference between B1RA⁺and B2RA⁺group.In addition,B1RA and B2RA pretreatment significantly inhibited TCE induced translocation of nuclear NF-?B p65 subunit.It was noteworthy that there existed significant difference between the two antagonist groups.6.NF-?B p65 and Inflammatory mRNA levels in kidney.There was no significant difference in NF-?B p65 mRNA expression between the blank and vehicle control groups.Compared with the vehicle control,renal NF-?B p65 mRNA levels were increased in 72h TCE⁺and B1RA⁺and B2RA⁺mice,though the latterpre-treatments each attenuated the overall TCE-induced up-regulation of NF-?B p65mRNA.Nevertheless,there was no significant difference between B1RA⁺and B2RA⁺group renal NF-?B p65 mRNA levels.It was also seen that pre-treatment with B1RA/B2RA effectively down-regulated the expression of il-1?,tnf-?and il-17mRNA compared to those in the 72h TCE⁺mice,and there were significant difference between B1RA⁺and B2RA⁺group.Further,only B2RA pre-treatment caused significant reductions in il-1?mRNA levels compared with levels in the 24h TCE⁺mice.7.Established a effective method for extration RETC.Through reperfusion from aortaventralis,the kidney of mouse was digested by collagenase and trypsase.Labeling renal tubular epithelial cells with CK18 antibody by IHC and IF.8.Megalin expression on tubular epithelial cell.The IF analyses found that megaline mainly expressed on tubular epithelial cells.Compared to the vehicle control,megalin expression decreased significantly in the 72h TCE⁺mice,and this trend was not shown in 72h TCE^-mice.9.The level of b1r/b2r mRNA on RETC.There were no significant difference in b1r/b2r mRNA expression between the blank and vehicle group.Compared with the blank and vehicle group,b1r and b2r mRNA levels were increased in TCE sensitized mice and decreased in 72h TCE^-mice.10.NF-?B signaling pathway protein on RTEC.The levels of p-I?B markedly increased and I?B significantly decreased in 72h TCE⁺RETC,compared with vehicle control mice.Moreover,the levels of nuclear NF-?B p65subunits in the RETC were significantly elevated.In contrast,B1RA and B2RA pre-treatments attenuated I?B phosphorylation and inhibited the reduction I?B levels in RETC,and the levels of nuclear NF-?B p65 subunits were also attenuated as p-I?B.Nonetheless,there was no significant difference in I?B and nuclear NF-?B p65 between B1RA⁺and B2RA⁺groupConclusions1.Sensitized dose of trichloroethylene can induced immune injury including skin and kidney damage.2.Renal tubular epithelium cell damage in TCE-sensitized mice that shed light on the excessive activation of KKS,especially B1R and B2R,and in part via NF-?B signaling pathway activation.This latter activation caused release of inflammatory cytokines that further aggravated TCE-induced immune injury.3.We first assessed the injury of renal tubular epithelial cell?RETC?from TCE induced mice and verify our hypothesis by established a new method for extration RETC.