Intracellular Complement Activation In Podocytes Mediates Immune Kidney Injury Through Endothelin-1 System In Trichloroethylene-sensitised Mice

BackgroundTrichloroethylene(TCE)is a volatile chlorinated solvent that has been widely used to degrease metal machinery and extract chemicals for about 100 years.In China,at least 20,000 new workers are threatened by TCE exposure every year.Workers exposed to TCE via skin and respiratory system can contract the occupational disease-like dermatitis due to TCE(OMLDT),which is characterised by fever,a generalised rash,and liver and kidney injury.Clinical data have suggested kidney injury as one of the main causes of death in OMLDT patients and seriously affects the prognosis and patient quality of life.Our previous work has reported inflammatory cell infiltration and pathological damage in the kidney after TCE sensitization;therefore,identifying the mechanisms of TCE induced kidney injury is very important for prevention and treatment of OMLDT.Studies have shown that OMLDT is an immune disease in which delayed allergic reaction(type IV hypersensitivity)causes skin lesions.Clinical and experimental studies reported that complement system also played an important role in multi-organ injuries induced by TCE.Complement system is activated through three major pathways:alternative(AP),classical(CP),and lectin(LP)pathways,which all lead to central component C3 cleavage into its bioactive fragments,C3a and C3b.Our previous research have found that C3 deposition increased significantly after TCE sensitisation,and the main deposition site was the glomerulus,indicating complement was activated locally in the kidney after TCE sensitisation.Cathepsin L(Cat L)is a ubiquitously expressed endopeptidase which can cleave C3 into bioactive C3a and C3b,and has been found on many cell types of glomerulus,suggesting that there is Cat L-mediated local complement activation in glomerulus.However,whether and how the local renal complement activation mediates TCE-induced kidney injury is largely unknown.Studies have confirmed that endothelin-1(ET-1)signalling is linked with complement activation in clinical cases and experimental models.ET-1 is a 21 amino acid peptide that was first isolated from cultured porcine aortic endothelial cells.Many studies have suggested ET-1 is an important pro-inflammatory factor which can be used as a trigger for complement activation to induce the secretion of inflammatory cytokines such as TNF-?,IL-1?,ICAM-1,and mediate the inflammatory kidney injury.Moreover,by binding to the endothelin receptor B(ET_BR)which is mainly located on renal endothelial cells,the ET-1 signalling system can mediate the impairment of e NOS system,lead to the endothelial dysfunction,and further aggravate TCE-induced immune kidney injury.ObjectiveTo explore functions of local complement activation in mediating immune kidney injury through endothelin-1 system after TCE sensitisation.Part one.Intracellular complement activation in podocytes mediates immune kidney injury by inducing endothelin-1 secretion and inflammation intrichloroethylene-sensitised miceMethodsThe TCE percutaneous positive sensitisation BALB/c mouse model was established by pretreating with or without the local complement activation antagonist,cathepsin L inhibitor(Cat Li).Deposition of C3 and C3a in the kidneys were detected by IHC and IF;The m RNA and protein expression of ET-1 were detected by RT-PCR,IHC,and ELISA;The protein expression of the inflammatory cytokines TNF-?,IL-1?,ICAM-1were detected by IHC;NF-?Bp65 and p38 MAPK pathway proteins in the kidneys were measured by western blot.Results1.Sensitization rateSensitisation rate were calculated as the number of redness or swelling/number of mice.There were no sensitised mice in either the blank control or vehicle control groups;however,there was a 40.00%(8/20)sensitisation rate in the TCE treatment group and a 35.00%(7/20)sensitisation in the TCE+Cat Li treatment group.2.Deposition of C3 and C3a in the kidneyDeposition of C3 and C3a in the kidney were detected by immunohistochemistry(IHC)and immunofluorescence(IF).There were no visible C3 and C3a deposition in the blank control mice,vehicle control mice,TCE negative sensitisation and TCE+Cat Li negative sensitisation mice(P>0.05);However,the expression of C3 and C3a significantly increased in TCE positive sensitisation mice(P<0.05).Moreover,the IF results showed that the main deposition site of C3 and C3a were on the podocytes.Previous studies have shown that Cat Li is effective at antagonising local complement activation.Therefore,we used Cat Li to alleviate local renal complement activation in the TCE sensitised mice.After pre-treating with Cat Li,the distribution of C3 and C3a was less extensive in the TCE+Cat Li positive sensitisation group than in the TCE positive sensitisation group(P<0.05).3.Renal functionSerum Cr and BUN levels were not significantly different among the vehicle control,blank control,TCE negative sensitisation and TCE+Cat Li negative sensitisation groups(P>0.05);however,the levels of Cr and BUN increased significantly in TCE positive sensitisation group compared to those in vehicle control group(P<0.05).After pretreatment with Cat Li,these levels were lower in TCE+Cat Li positive sensitisation group than in TCE positive sensitisation group(P<0.05).To detect effects on glomerular filtration rate(GFR),serum Cys-C and urine microalbumin/creatinine(ACR)levels were quantified as glomerular filtration function.The data showed that there was no difference among vehicle control,blank control,TCE negative sensitisation and TCE+Cat Li negative sensitisation groups(P>0.05);however,glomerular filtration function was impaired with increased serum Cys-C and urine ACR levels in TCE positive sensitisation group(P<0.05).After Cat Li pretreatment,the glomerular filtration function was effectively ameliorated in TCE+Cat Li positive sensitisation group(P<0.05).4.Pathological examination of kidneyThe pathological examination showed no obvious histopathological changes in the kidneys between the vehicle control,blank control,TCE negative sensitisation group,and TCE+Cat Li negative sensitisation groups.However,inflammatory cell infiltration,cell swelling,and glomerular impairment were clearly observed in TCE positive sensitisation group.Although the inflammatory cell infiltration,cell swelling,and glomerular impairment were also observed in TCE+Cat Li positive sensitisation group,the extent of these effects was attenuated compared to that in TCE positive sensitisation group.5.The m RNA and protein expression of ET-1 in the kidneyReal-time q RT-PCR assays showed that the m RNA levels of ET-1 were not significantly different among blank control group,vehicle control group,TCE negative sensitisation group,and TCE+Cat Li negative sensitisation group(P>0.05).However,renal ET-1 m RNA levels increased significantly in TCE positive sensitisation mice compared to those in vehicle control mice(P<0.05),whereas Cat Li significantly reduced the m RNA levels of ET-1 by antagonising complement activation in TCE+Cat Li positive sensitisation mice(P<0.05).In addition,although the renal ET-1 m RNA levels of TCE negative sensitisation mice elevated slightly compared to those of vehicle control mice(P<0.05),however,the renal ET-1 m RNA levels of TCE positive sensitisation mice were still increased significantly than those of TCE negative sensitisation mice(P<0.05).Moreover,the protein expression of ET-1 in the kidney was measured by IHC and ELISA.The IHC and ELISA results showed that there were no difference in the protein levels of ET-1 among the blank control,vehicle control,TCE negative sensitisation,and TCE+Cat Li negative sensitisation mice(P>0.05).In addition,the protein levels of ET-1 were significantly reduced in TCE+Cat Li positive sensitisation mice than in TCE positive sensitisation mice(P<0.05).6.The protein expression of inflammatory cytokines in kidneyInflammatory and adhesion molecules such as TNF-?,IL-1?,and ICAM-1 were detected in IHC assays,which showed that there were no visible TNF-?,IL-1?,and ICAM-1 deposition in kidneys of the blank control,vehicle control,TCE negative sensitisation,and TCE+Cat Li negative sensitisation mice.However,more TNF-?,IL-1?,and ICAM-1 deposition was detected in TCE positive sensitisation mice.Although still visible,TNF-?,IL-1?,and ICAM-1 protein expression was significantly reduced in the TCE+Cat Li positive sensitisation mice than in the TCE positive sensitisation mice(P<0.05).7.NF-?Bp65 and p38 MAPK signalling pathway proteins in the kidneyNF-?Bp65 and p38 MAPK signalling pathway proteins in the kidney were measured by western blot.Interestingly,the protein expression of p38 and p65 showed no significant difference between all treatment groups(P>0.05).The expression of phosphorylated protein,P-p38 and P-p65 was also not significantly different between the blank control and vehicle control groups(P>0.05).However,these phosphorylated proteins significantly increased in kidneys of TCE positive sensitisation mice(P<0.05),and as expected,Cat Li significantly decreased P-p65 and P-p38 expression in TCE+Cat Li positive sensitisation mice compared to that in TCE positive sensitisation mice by antagonizing complement activation(P<0.05).Conclusions1.The intracellular complement system in the podocytes is activated after TCE sensitization.2.The intracellular complement activation in the podocyte promoted the secretion of ET-1 and the release of its related inflammatory cytokines,leading to the inflammatory kidney injury.3.P38 MAPK and NF-?Bp65 pathway played a regulatory role in the function of intracellular complement activation on mediating renal inflammatory injury.Part two.Endothelin system mediated renal endothelial cell dysfunction and immune kidney injury via ET1/ETBR signaling pathway in trichloroethylene-sensitised miceMethodsIn the study one,we have found that m RNA and protein expression of ET-1 increased significantly in the kidney of TCE positive sensitisation mice,indicating that the renal endothelin system was activated after TCE sensitization.ET-1 is mainly synthesized and secreted in endothelial cells,and exerts biological effects through ET_BR,the type B receptor of endothelin.Therefore,we further selected the specific antagonistic ET_BR inhibitor BQ-788 to investigate the effect of ET-1/ET_BR signaling pathway on TCE induced immune kidney injury.The activation of endothelin system,the function and histopathology of kidney,the damage of renal endothelial cells,the activity of nitric oxide synthase(NOS),especially the activity of endothelial nitric oxide synthase(e NOS),and the levels of nitric oxide in kidney tissue were all detected.Results1.Sensitization rate.Sensitisation rates were calculated as the number of redness or swelling/number of mice.There were no sensitised mice in either the blank control or vehicle control groups;however,there was a 38.89.00%(7/18)sensitisation rate in the TCE treatment group and a 33.33%(6/18)sensitisation in the TCE+BQ-788 treatment group.2.The m RNA and protein expression of ET-1 in the kidneysThe q RT-PCR assays showed that the m RNA levels of renal ET-1 were not significantly different among blank control,vehicle control,and TCE negative sensitisation group(P>0.05).However,renal ET-1 m RNA levels increased notably in TCE positive sensitisation mice compared to those in vehicle control mice(P<0.05);Moreover,the protein expression of ET-1 in the kidney was measured by ELISA.The results showed that there were also no difference in the protein levels of ET-1 among the blank control,vehicle control,and TCE negative sensitisation groups(P>0.05).However,the renal ET-1 protein levels were elevated significantly in TCE positive sensitisation mice compared with those in vehicle control mice(P<0.05).3.The renal function detectionSerum Cr and BUN levels were not significantly different among the vehicle control,blank control,TCE negative sensitisation and TCE+BQ-788 negative sensitisation groups(P>0.05);however,the levels of Cr and BUN increased significantly in TCE positive sensitisation group compared to those in vehicle control group(P<0.05).After pretreatment with BQ-788,these levels were lower in TCE+BQ-788 positive sensitisation group than in TCE positive sensitisation group(P<0.05).To detect effects on glomerular filtration rate(GFR),serum Cys-C and urine microalbumin/creatinine(ACR)levels were quantified as glomerular filtration function.The data showed that there was no difference among vehicle control,blank control,TCE negative sensitisation and TCE+BQ-788 negative sensitisation groups(P>0.05);however,glomerular filtration function was impaired with increased serum Cys-C and urine ACR levels in TCE positive sensitisation group(P<0.05).4.The pathological examination of kidneysThe pathological examination showed no obvious histopathological changes in the kidneys between the blank control group,vehicle control group,TCE negative sensitisation group and TCE+BQ-788 negative sensitisation group.However,the inflammatory cell infiltration,cell swelling,and vacuolar lesion were clearly observed in the kidneys of TCE positive sensitisation mice.After pretreating with BQ-788,these renal pathological damage was effectively alleviated in TCE+BQ-788 positive sensitisation group.5.Expression and localization of ETBR in the kidneysCo-localization of ETBR and CD31(markers of endothelial cells)in the kidney was detected by immunofluorescence double-labeling.The results showed that ET_BR was mainly expressed in the renal endothelial cells.6.The renal endothelial cell function detection6.1 The m RNA and protein expression of VCAM-1 in the kidneysThe m RNA and protein levels of renal VCAM-1 showed no significant difference between the blank control group,vehicle control group,TCE negative sensitisation group and TCE+BQ-788 negative sensitisation group;However,the renal VCAM-1m RNA and protein levels increased significantly in TCE positive sensitisation mice compared to those in the vehicle control group;After BQ-788 pretreatment,the m RNA and protein levels of VCAM-1 decreased significantly in TCE+BQ-788positive sensitisation group.6.2 The m RNA and protein expression of ICAM-1 in the kidneysThe m RNA and protein expression of renal ICAM-1 increased significantly in TCE positive sensitisation mice(P<0.05);After pretreatment with BQ-788,the renal ICAM-1 m RNA and protein levels decreased significantly in TCE+BQ-788 positive sensitisation mice compared with those in TCE positive sensitisation mice(P<0.05).7.Activity of nitric oxide synthase(NOS)in the kidneysThe results showed that the renal NOS activity were not different significantly between the blank control group,vehicle control group,TCE negative sensitisation group and TCE+BQ-788 negative sensitisation group(P>0.05);However,the activity of NOS increased significantly in TCE positive sensitisation mice(P<0.05);After pretreating with BQ-788,these levels were lower in TCE+BQ-788 positive sensitisation group than in TCE+group(P<0.05).8.The m RNA and protein expression of e NOS in kidneysThe renal e NOS m RNA and protein levels showed no significant difference among the vehicle control group,blank control group,TCE negative sensitisation group,and TCE+BQ-788 negative sensitisation group(P>0.05);However,the m RNA and protein levels of e NOS increased significantly in TCE positive sensitisation mice(P<0.05);After pre-treatment with BQ-788,the e NOS m RNA and protein levels in TCE+BQ-788 positive sensitisation mice decreased significantly than those in TCE positive sensitisation mice(P<0.05).9.NO levels in the kidneysThe data showed that there were no significant difference in the renal NO levels between blank control group,vehicle control group,TCE negative sensitisation group and TCE+BQ-788 negative sensitisation group(P>0.05).However,the NO levels in kidneys increased significantly in TCE positive sensitisation group compared to those in vehicle control group(P<0.05).After pre-treatment with BQ-788,the renal NO levels decreased significantly in TCE+BQ-788 positive sensitisation group than that in TCE positive sensitisation group(P<0.05).Conclusions1.The endothelin system in the kidney was activated after TCE sensitization;2.ET_BR was mainly expressed in renal endothelial cells;3.By binding to ET_BR on the surface of renal endothelial cells,the endothelin system can inhibite the activity of NOS,reduce the m RNA and protein level of e NOS,and reduce the release of NO,and ultimately mediate the renal endothelial cell dysfunction,aggravated the immune renal injury;4.By inhibiting ET_BR,BQ-788 significantly alleviated endothelin-mediated renal endothelial cell dysfunction and improved immune renal injury in TCE-positive sensitisation mice.