| BackgroundOccupational Medicamentosa-like Dermatitis due to TCE(OMLDT)is an acute skin inflammatory disorder occurring in workers with occupational exposure to trichloroethylene(TCE).Severe visceral injuries including hepatitis and immune renal damage are the common complications of OMLDT,in which the immune renal damage caused by TCE seems to be later than the fatal hepatitis,but a central factor in treatment and prognosis of OMLDT patients.Clinical data showed that OMLDT patients often presented with a diffuse inflammation of both kidneys and an increase of serum uric acid and creatinine.This observation suggests that glomerular injury might be involved in the renal damage and is one of the main causes of the glomerular filtration dysfunction in OMLDT patients.Unfortunately,the precise mechanism of glomerular injury induced by TCE sensitization is still unclear.Studies found that the expression of cathepsin L(CTSL)was increased in a variety of immune and non-immune kidney disorders,especially glomerular diseases.Abundant CTSL mainly expressed in the glomerulus,resulting in the reorganization of podocyte skeleton,the damage of GFB integrity,the destruction of podocyte structure,and even the formation of proteinuria.CTSL inhibitors or gene knockout seems to be an effective way to protect the structural and functional integrity of glomerulus in various kidney diseases.Besides,studies also found that CTSL was increased in atopic dermatitis,suggesting that CTSL might also participate in the process of allergic reaction.In our previous study,we found an increase expression of CTSL in TCE-induced glomerulus damage in mice.However,the specific mechanisms of CTSL in the glomerular injury caused by TCE sensitization still need to be clarified.Recently,the mammalian target of rapamycin(mTOR)signaling was found to play a vital role in various renal diseases.The activation of mTOR signaling,together with the downstream p70S6 K and 4EBP1,could regulate cell growth and differentiation.Actually,the precise role of mTOR signaling in kidney depends on the active state and extent.On one hand,a proper activation of mTOR signaling could promote cell proliferation,induce podocyte compensatory hypertrophy,and maintain the normal glomerular function integrity.On the other hand,hyperactive mTOR signaling could also cause the loss of podocytes from GBM,the fusion of foot process,and even evolvement of proteinuria.However,whether mTOR signaling participates in TCE-induced allergic glomerular injury remains unknown.Therefore,the present study was taken out to explore the role of over-expression of CTSL and hyperactivation of mTOR signaling in TCE-induced allergic glomerular injury,and further to study the biological link between CTSL and mTOR signaling.We hope to enrich the pathogenesis of TCE-induced renal damage and provide the possible target of clinical treatment with OMLDT kidney damage.ObjectiveIn this study,we established the mouse model of OMLDT in presence or absence of a specific CTSL inhibitor/mTOR signaling inhibitor.Then,the structural and functional integrity of kidney,the protein markers of podocyte,the apoptosis of glomerular cell,the expression of CTSL,and the activation of mTOR signaling in the process of TCE sensitization were studied with or without the related specific inhibitors.We explored the possible role of CTSL and mTOR signaling in glomerular injury,in which CTSL might participate in the regulation of mTOR signaling activation and cause glomerular damage.The possible target of clinical treatment of OMLDT renal damage will be explored in this study.MethodsThe mouse model of skin sensitization induced by TCE was established with or without pretreatment of CTSL inhibitor or mTOR signaling inhibitor.Then,the renal histopathological changes were detected by H&E staining,PAS staining,and transmission electron microscopy(TEM).The renal function was detected via immunoturbidimetric assay.The expression of podocyte specific proteins including,nephrin,podocin,actinin-4,and integrin β1,were assessed by immunofluorescence test.The apoptosis of glomeruli cells were counted by TUNEL staining and the apoptosis-related proteins including Bax,Bcl-2,and Caspase-3 were detected by western blot.In rapamycin pretreatment+TCE group,the isolated glomerulus was also used to detect the phosphorylation of mTOR,p70S6 K,4EBP1 proteins and expression of related apoptotic proteins including Bax,Bcl-2,and Caspase-3.Finally,the phosphorylation of mTOR,p70S6 K,4EBP1 proteins in TCE treatment group and CTSL inhibitor pre-treatment group+TCE group were assessed to identify the regulation of CTSL on mTOR signaling.Results 1.Sensitization rateIn this study,a total of 42 BALB/c mice showed different degrees of skin erythema and edema.In TCE treatment group,the sensitization rate was 37.5% and 15 BALB/c mice showed sensitization positive reaction.In CTSL inhibitor pretreatment+TCE group,the sensitization rate was 32.5% and 13 mice showed sensitization positive reaction.In rapamycin pretreatment+TCE group,the sensitization rate was 35.0% and 14 mice showed sensitization positive reaction.No significant difference in the sensitization rate was found among the three above groups(P>0.05).However,it is worth noting that no mice with skin allerge score to 3 points were found in either CTSL inhibitor pretreatment+TCE sensitization positive group or rapamycin pretreatment+TCE sensitization positive group.The skin of mice in the blank control group and the solvent control group were completely normal without obvious changes.2.Occurrence of kidney damageThe occurrence of kidney damage in the present study was assessed by pathological examination.In TCE treatment group,the incidence of kidney damage was 35.0%,with 14 mice showing different degrees of renal pathological changes.In CTSL inhibitor pretreatment+TCE group,the incidence of kidney damage was 12.5%,with 5 mice showing different degrees of pathological changes.In rapamycin pretreatment+TCE group,the incidence of kidney damage was also 15.0%,with 6 mice showing different degrees of pathological changes.There were significantly different in the incidence of renal injury among different groups according to χ2 test.The incidence of kidney damage in both CTSL inhibitor pretreatment+TCE group and rapamycin pretreatment+TCE group were both lower than TCE treatment group(P<0.05).3.Body weight and skin temperature of miceOverall,the mice weight kept stable during the whole experiment and there was no obvious gain or loss of weight in mice of TCE sensitization positive group.Further,the weight difference(body weight at the end of the experiment-body weight at the beginning of the experiment)in all mice was calculated.The statistical analysis showed that no significant difference of the weight difference was found among the blank control group,vehicle control group,TCE sensitization positive group and TCE sensitization negative group(P>0.05).Besides,considering that the OMLDT patients often have high fever before eruption,we measured the skin temperature of mice at sensitization stage and challenge stage and calculated the difference value of skin temperature(skin temperature at challenge stage-skin temperature at sensitization stage).The statistical analysis showed that the skin temperature at challenge stage of TCE sensitization positive mice was higher than that at sensitization stage in various degrees.According to the statistical analysis of the skin temperature difference in mice,we found that the skin temperature difference of TCE sensitization positive mice was significantly higher than that in the blank control group,solvent control group and TCE sensitization negative group(P<0.05).This result is in agreement with the clinical findings of OMLDT patients.4.Pathological staining of mice kidneysH&E and PAS staining results showed that edema of glomerular cells,the thickened mesangial matrix and glomerular basement membrane with proliferation and widening in mice of TCE sensitization positive group.These pathological changes were alleviated obviously in both mice of CTSL inhibitor pretreatment+TCE sensitization positive group and rapamycin inhibitor pretreatment+TCE sensitization positive group.The kidney structure was normal with orderly arranged renal cells in the blank control group,vehicle control group and relevant TCE sensitization negative group.5.Ultrastructural changes in mice glomerulusThe results of transmission electron microscopy showed that the integrity of glomerular filtration barrier was destroyed in TCE sensitization positive group.Besides,the thickened glomerular basement membrane was uneven,fused podocyte foot processes,mitochondria edema and vacuolation degeneration were also found in the TCE sensitization positive group.These ultrastructural damages of glomerulus in CTSL inhibitor pretreatment+TCE sensitization positive group and rapamycin inhibitor pretreatment+TCE sensitization positive group were significantly improved,with maintaining the basic structural integrity.No obvious ultrastructural changes were found in the blank control group,vehicle control group,and relevant TCE sensitization negative group.6.Renal function in miceThe data showed that the expression levels of both BUN and Cre in TCE sensitization positive group were significantly higher than those in TCE sensitization negative group(P<0.05).Compared with TCE sensitization positive group,the expression levels of BUN and Cre were significantly lower in CTSL inhibitor pretreatment+TCE sensitization positive group and rapamycin inhibitor pretreatment+TCE sensitization positive group(P<0.05).No significant difference of the expression levels of BUN and Cre was found among the blank control group,vehicle control group and relevant TCE sensitization negative group(P>0.05).7.Over-expression of CTSL in mice podocyteAccording to the results of immunofluorescence,we presented that CTSL was mainly expressed in glomerular podocytes,by using podocin as a marker of podocyte.We found that the expression of CTSL in TCE sensitization positive group was significantly higher than that in the blank control group,vehicle control group and TCE sensitization negative group(P<0.05).Compared with TCE sensitization positive group,the CTSL expression level was significantly lower in CTSL inhibitor pretreatment+TCE sensitization positive group(P<0.05).8.CTSL inhibitor ameliorates the loss of podocyte protein markers during TCE sensitizationThe results of immunofluorescence showed that the fluorescence intensity of nephrin,podocin,actinin-4 and integrin β1 were significantly various in different groups.The fluorescence intensity of nephrin,podocin,actinin-4 and integrin β1 in TCE sensitization positive group were significantly lower than that in the blank control group,vehicle control group or TCE sensitization negative group(P<0.05).Compared with TCE sensitization positive group,the fluorescence intensity of nephrin,podocin,actinin-4 and integrin β1 were significantly higher in CTSL inhibitor pretreatment+TCE sensitization positive group(P<0.05).No significant difference in the fluorescence intensity of nephrin,podocin,actinin-4 and integrin β1 were found among the blank control group,vehicle control group,and relevant TCE sensitization negative group(P>0.05).9.Activation of mTOR signaling in glomerular injury caused by TCE sensitizationWe detected the expression of mTOR signaling related proteins,including p-mTOR,p-p70S6 K,and p-4EBP1 in mice glomerulus.The results showed that the expression levels of p-mTOR,p-p70S6 K,and p-4EBP1 in TCE sensitization positive group were significantly higher than those in the vehicle control group and TCE sensitization negative group(P<0.05).Compared with TCE sensitization positive group,the expression levels of p-mTOR,p-p70S6 K,and p-4EBP1 in rapamycin pretreatment group+TCE sensitization positive group were also significantly down-regulated(P<0.05).No significant difference in the expression levels of p-mTOR,p-p70S6 K,and p-4EBP1 were found among the blank control group,vehicle control group and relevant TCE sensitization negative group(P>0.05).10.Rapamycin reduced TCE-induced apoptosis of glomerular cellsTUNEL staining showed that the counts of glomerlar apoptotic cells in TCE sensitization positive group was significantly higher than that in the blank control group,vehicle control group,and TCE sensitization negative group(P<0.05).Compared with TCE sensitization positive group,the counts of glomerlar apoptotic cells was significantly reduced in rapamycin pretreatment+TCE sensitization positive group(P<0.05).Besides,the counts of glomerlar apoptotic cells in rapamycin pretreatment+TCE sensitization positive group was significantly higher than that in the blank control group,solvent control group,and relevant TCE sensitization negative group(P<0.05).There was no significant difference of apoptotic cells counts was found among the blank control group,vehicle control group,and relevant TCE sensitization negative group(P>0.05).Western blot results showed that the expression levels of Bax and Caspase-3 in TCE sensitization positive group were significantly higher than that in the blank control group,vehicle control group and TCE sensitization negative group(P<0.05).Compared with TCE sensitization positive group,the expression levesl of Bax and Caspase-3 in glomerulus was significantly lower in rapamycin pretreatment+TCE sensitization positive group(P<0.05).On the contrary,the expression of Bcl-2 in TCE sensitization positive group was significantly lower than that in the blank control group,vehicle control group and TCE sensitization negative group(P<0.05).Compared with TCE sensitization positive group,the expression of Bcl-2 in glomerulus was significantly higher in rapamycin pretreatment+TCE sensitization positive group(P<0.05).There was no significant difference of Bax,Bcl-2 and Caspase-3 expression among the blank control group,vehicle control group,and relevant TCE sensitization negative group(P>0.05).11.CTSL inhibitor down-regulated mTOR signaling activation in TCE-induced glomerular injuryThe results showed that the expression levels of p-mTOR,p-p70S6 K and p-4EBP1 in CTSL inhibitor pretreatment+TCE sensitization positive group were significantly lower than those in TCE sensitization positive group(P<0.05).Compared with the vehicle control group,the expression levels of p-mTOR,p-p70S6 K,and p-4EBP1 were not significant different in CTSL inhibitor pretreatment+TCE sensitization positive group(P>0.05).12.Rapamycin unworked on the overexpression of CTSL in TCE-induced glomerular injuryThe qRT-PCR results showed that the level of CTSL mRNA in TCE sensitization positive group was higher than blank control group,vehicle control group,and TCE sensitization negative group(P<0.05).Compared with TCE sensitization positive group,the level of CTSL mRNA in CTSL inhibitor pretreatment+TCE sensitization positive group was markly reduced(P<0.05),while the level of CTSL mRNA in rapamycin pretreatment+TCE sensitization positive group was unchanged(P>0.05).No obvious difference was found among blank control group,vehicle control group,and relevant TCE sensitization negative group(P>0.05).Conclusions 1.In glomerular injury caused by TCE sensitization,an increase expression of CTSL was involved in the damaged integrity of podocyte structure and function.2.In glomerular injury caused by TCE sensitization,activation of mTOR signaling participated in apoptosis of glomerular cells caused by TCE allerge.3.Pharmacological inhibition of CTSL expression could effectively improve the damage of podocyte structure and function.4.Pharmacological inhibition of mTOR signaling reversed TCE-induced apoptosis of glomerular cells.5.CTSL could regulate the activation of mTOR signaling and participate in the glomerular damage induced by TCE sensitization. |
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