Effect Of Intracellular Complement Activation In Podocytes On Immune Kidney Injury In Trichloroethylene-sensitized Mice

Background Trichloroethylene(TCE)is a volatile halogenated hydrocarbon organic solvent,which has been widely used in industry because of its good cleaning and defatting effects on metal parts.Some occupational workers exposed to TCE can lead to Occupational medicamentosa-like dermatitis due to trichloroethylene(OMDT).As OMDT progresses rapidly,the case fatality rate is high and the treatment period is long,which brings huge economic burden and health loss to the patients.Kidney injury is one of the important clinical manifestations in OMDT.For the pathogenesis of OMDT,studies found that in addition to delayed type IV allergy,complement system also plays an important role in it.Our previous study of animal experiments found that a large number of complement3(C3)fragments deposited on glomerular podocytes.Related studies have found that in a variety of cells including podocytes,C3 can be cleaved into biologically active C3 a and C3 b fragments through the intracellular cathepsin L(CTSL),resulting in the activation of intracellular complement to produce a series of biological effects.Objective This study was based on the model of trichloroethylene-sensitized mice,some mice were injected intraperitoneally with CTSL inhibitor.By detecting the expression of C3 fragments,CTSL,Nephrin in podocytes and renal function in diffierent groups of mice,to investigate the role of podocyte complement activation in immune kidney injury in trichloroethylene-sensitized mice.Methods The 120 specific pathogen-free female BALB/c mice were randomly divided into blank control group(n=15),solvent control group(n=15),TCE treatment group(n=45)and TCE+CTSL inhibitor treatment group(n=45).TCE sensitization model was established according to the mature method of our research group,the animals were euthanized at24 h,72h and 7d after the last challenge and the corresponding biological samples were collected.Serum levels of creatinine(CRE)and blood urea nitrogen(BUN)were measured by CRE and BUN commercial kits.Enzyme linked immunosorbent assay(ELISA)were used to detected serum level of cystatin C(Cys-C)and urinary podocyte marker protein Podocalyxin(PCX)expression level.The expression of C3,CTSL and podocyte marker protein Nephrin in kidiney were detected by immunohistochemistry.The expression of C3 and CTSL in podocytes was deteced by immunofluorescence.Western Blot was used to detected protein expression of C3,CTSL and Nephrin in kidney cortex.The ultrastructural damage of podocytes were observed by transmission electron microscope.Results1.Sensitization rate During the whole experiment,there was no obvious abnormality in skin of mice in blank control group and solvent control group.A part of mice in TCE and CTSL inhibitor treatment groups showed varying degree of erythema and edema in the skin.The sensitization rate of TCE treatment group and CTSL inhibitor treatment group were33.3%(15/45)and 31.1%(14/45),respectively.2.The detection results of kidney function There were no significant differences in the levels of CRE,BUN,Cys-C and PCX between the solvent control group and the blank control group(P>0.05).Comparedwith the solvent control group,the expression levels of Cys-C and PCX increased in TCE-sensitized 24 h group,the expression of all four indexes increased significantly in TCE-sensitized 72 h group and expression levels of CRE,Cys-C,PCX increased in TCE-sensitized 7d group,all the differences were statistically significant(P<0.05).Compared with the solvent control group,parts of indexes in CTSL inhibitor-sensitized groups at each time point also increased,but the increase extent was lower than that in TCE-sensitized groups at corresponding time points,all the differences were statistically significant(P<0.05).Compared with the solvent control group,the expression levels of all four indexes in the non-sensitized groups at each time point in each treatment group showed no significant difference(P>0.05).By testing four indexes of kidney function,we found that all indexes reached the peak at 72 h after the last challenge and then gradually recovered or decreased.Therefore,we only detected releated indexes at 72 h groups in the follow-up study.3.The expression of C3 fragments in kidney The results of immunohistochemistry showed that,C3 fragments were mainly expressed in the glomerrulus of TCE-sensitized 72 h group and were slightly expressed in CTSL inhibitor-sensitized 72 h group,there was no obvious expression in all other four groups.The results of immunohistochemistry scoring showed that the score of TCE-sensitized72 h group was significantly higher than other five groups(P<0.05).The results of immunofluorescene showed that,C3 fragments were mainly expressed on the glomerular podocytes of TCE-sensitized 72 h group and no significant expression of C3 fragments were observed in other five groups.The expression of i C3 b in kidney cortex was detected by Western Blot.There was no significant difference between the slovent control group,the blank control group and two corresponding non-sensitized groups(P>0.05).Compared with the solvent group,the expression of i C3 b in TCE-sensitized72 h group was significantly increased(P<0.05).Compared with TCE-sensitized 72hgroup,the i C3 b expression in CTSL inhibitor-sensitized 72 h group was significantly decreased,the difference was statistically significant(P<0.05).4.The expression of CTSL in kidney The results of immunohistochemistry showed that CTSL was expressed in all treatment groups,but the expression on glomerulus in TCE-sensitized 72 h group was significantly increased(P<0.05).The immunohistochemistry scoring showed that the score in TCE-sensitized 72 h group was significantly higher than other five groups(P<0.05).Immunofluorescence assay also found that CTSL was mainly expressed on podocytes in TCE-sensitized 72 h group.The results detected by Western Blot were consistent with the results of immunohistochemistry.5.The expression of Nephrin in kidney The results of immunohistochemistry showed that the expression of Nephrin was significantly down-regulated in TCE-sensitized 72 h group,there was no significant difference in other five groups(P>0.05).The immunohistochemistry score showed that the score in TCE-sensitized 72 h group was significantly lower than other five groups,the difference was statistically significant(P<0.05).The results detected by Western Blot were consistent with the results of immunohistochemistry.6.Ultrastructural damage of podocyte Transmission electron microscopy observation results showed that in the blank control group,the solvent control group and two corresponding non-sensitized groups,podocyte foot process is complete,the basement membrane thickness is uniform,no obvious organelles damage.In TCE-sensitized 72 h group,we observed that lots of podocyte foot process effacement,inhomogeneous thickening of the basement membrane,mitochondria vacuolar degeneration and crest rupture.In CTSLinhibitor-sensitized 72 h group,we can see parts of the foot process effacement but no other obvious abnormalities,the degree of injury than TCE-sensitized 72 h group were significantly reduced.Conclusions1.Intracellular complement in podocytes may be activated by CTSL in TCE-induced immune kidney injury in BALB/c mice.2.Intracellular complement activation in podocytes may involve in podocyte damage,making the foot process effacement,inhomogeneous thickening of the basement membrane,mitochondria vacuolar degeneration and crest rupture,further reducing the glomerular filtration function,which may play an important role in TCE-induced immune kidney injury.