The Role Of Endothelin 1 In Kidney Injury Of Patients With Occupational Medicamentosa-like Dermatitis Induced By Trichloroethylene

BackgroundSome workers exposed to trichloroethylene may suffer from hypersensitivity reactions,which mainly manifest as extensive skin damage,called occupational medicamentosa-like dermatitis induced by TCE（OMLDT）.In addition to severe skin damage,OMLDT may also be accompanied by liver,kidney and other organ damage.There are many studies on liver damage in OMLDT,but few on kidney damage,and the kidney damage mechanism is unclear.The kidney is one of the main target organs in patients with OMLDT except for skin damage.Podocytes and endothelial cells are the two major functional cells of the glomerulus.Whether there are any interactions between podocytes and endothelial cells in TCE-induced immune kidney damage is unknown.Endothelin-1（ET-1）is a cytokine released by endothelial cells.Overexpression of ET-1 can also promote the body’s inflammatory response and participate in the body’s injury process.Our previous studies found that ET-1 was overexpressed and promoted the expression of inflammatory factors such as IL-1βin TCE-sensitized mouse kidneys.In addition,ET-1 exerts different biological functions mainly by binding to its receptor.Studies have found that ET-1 can promote kidney inflammation and fibrosis by binding to endothelin receptor-type A（ET_AR）.Whether ET-1 is involved in TCE-induced kidney immune injury by combining with ET_AR and the kidney endothelial cell repair mechanism has not yet been investigated.Vascular endothelial-derived growth factor-A（VEGF-A）,a factor that promotes the repair of vascular damage and cardiovascular production,can promote the repair of endothelial cells and promote vascular homeostasis during the process of endothelial cell damage.Angiopoietin（Ang）is also a kind of protein family that regulates the pathophysiological process of blood vessels.Physiological functions include promoting angiogenesis,development,maturation,and remodeling in the process of pathological damage to blood vessels.Ang-1 can show anti-inflammatory activity by inhibiting tumor necrosis factor-α（TNF-α）.In addition,Ang-1 binding to tyrosine kinase receptor 2（Tie-2）can mediate phosphatidylinositol-4,5-bisphosphate-3-kinase（PI3K,phosphatidylinositol-3-kinase）/Akt and endothelial nitric oxide synthase（e NOS）activation,showing antiapoptotic activity and vascular protection functions.However,the relationship between ET-1,VEGF-A and Ang-1 is unknown.How ET-1,VEGF-A and Ang-1 mediate the repairment of podocytes and endothelial cells in TCE-induced kidney injury needs further research.ObjectiveThis study combined OMLDT patient studies with trichloroethylene-sensitized mouse model experiments to detect the serum levels of ET-1,Ang-1,Ang-2 and renal function in OMLDT patients to determine their relationship.Then,endothelin converting enzyme-1（ECE-1）inhibitors and endothelin receptor inhibitors were injected to study the role of ET-1 and VEGF-A in TCE-induced kidney injury and explore which endothelin receptors are involved in the regulation of angiogenin by detecting endothelial cell apoptosis,podocyte structural protein expression levels,and glomerular inflammation status.Finally,the interaction between glomerular endothelial cells and podocytes was clarified,which provides a theoretical basis for the treatment of OMLDT patient kidney injury and provides new ideas for the development of new clinical drugs.MethodsPart Ⅰ:Six OMLDT patients were enrolled in this study,their blood and urine were collected on the first day after admission and one month after receiving treatment.At the same time,blood and urine from 18 normal controls were collected from the hospital physical examination center.Serum levels of ET-1,creatinine（Cre）,urea nitrogen（UN）and urinary podocalyxin（PCX）between OMLDT patients and the control population were detected.In addition,a trichloroethylene-sensitized mouse model was established,and some mice were treated with the ECE-1 inhibitor CGS 35066 to observe the pathological changes of the mouse skin and kidney and to detect the serum Cre,UN and urine total protein（UTP）levels to reflect kidney damage in mice.Enzyme-linked immunosorbent assay（ELISA）was used to detect mouse serum ET-1 levels,and immunohistochemistry was used to detect mouse kidney ECE-1 and TNF-αexpression levels.Mouse glomeruli protein levels of ET-1,VEGF-A,glypican1,and syndecan1 were detected by western blot to reflect the expression of ET-1 in kidney endothelial cells and endothelial cell damage.Part Ⅱ:Use 102 mice to establish the trichloroethylene-sensitized mouse model,part of them were received specific inhibitors of endothelin receptor type-A or endothelin receptor type-A BQ123 or BQ788,then detect creatinine,urea nitrogen cystatin-C（Cys-c）and PCX to reflect glomerular injury;kidney TNF-αand monocyte chemotactic protein-1（MCP-1）were detected by immunohistochemistry.Immunofluorescence was used to locate ET-1,Tie-2,Ang-2,ET_AR and Ang-1,Western blotting was used to detect kidney levels of ET-1,ET_AR,endothelin receptor-type B（ET_BR）,Ang-1,Ang-2,Tie-2,phosphor-Tie-2（p Tie-2）,PI3K,phosphor-AKT（p AKT）,e NOS,Bcl-2,Bad,phosphor-Bad（p Bad）,Fas,Fas-L,Bax,caspase-3,nephrin,podocin,MCP-1,and TNF-α.Transmission electron microscope was used to the observe the ultrastructure of podocytes.Cell apoptosis rate was detected by TUNEL kit.Real-time quantitative PCR was used to detect the Ang-1 and Ang-2 gene levels in the mouse kidney.Human serum Ang-1 and Ang-2 levels were detected to explore the relationship between endothelin 1 and angiogenin,and the relationship between angiogenin and apoptosis.ResultsPart Ⅰ:The levels of ET-1,UN and PCX in OMLDT patients serum were higher than those after treatment and the normal control group at the time of admission（P<0.05）.However,the serum Cre level of patients was not significantly increased compared to normal control group（P>0.05）.The nuclear pores of glomerular endothelial cells were clear,the nuclear membrane was intact,the cytoplasm was rich in organelles,and the structure of mitochondria and other organelles was normal.Compared with the solvent control group,the Cre,UN and UTP of the mice in TCE sensitized positive group were significantly increased,the glomerular mesangial cells proliferated,the basement membrane thickened,the capillary cavity was narrowed,and the endothelial cell cytoplasmic vacuoles like degeneration,the number of organelles such as ribosomes significantly reduced.Compared with the TCE sensitized positive group,the Cre,UN and UTP levels of the mice in the TCE+CGS 35066 sensitization-positive group were significantly reduced,and a small amount of mitochondrial mild vacuolar degeneration was seen in the endothelial cells,but the glomerular pathological damage was significantly reduced.The results of immunohistochemistry showed that there was a small amount of ECE-1 expression in the kidneys of the blank control group,solvent control group,TCE sensitization negative group,and TCE+CGS 35066 sensitization negative group,while the TCE sensitized positive group showed a large amount of ECE-1 expression.After treating with ECE-1 inhibitors,the expression levels of ECE-1 in renal tubules and glomeruli were decreased,but it was still higher than that in the solvent control group.Compared with the solvent control group,the serum and glomerular ET-1 levels of the mice in TCE sensitized positive group and the TCE+CGS 35066 sensitized positive group also increased,while the VEGF-A level was significantly downregulated,but compared to TCE sensitized positive group,ET-1decreased,VEGF-A and the endothelial cell framework proteins glypican1 and syndecan1 significantly increased in TCE+CGS 35066 sensitized group（P<0.05）.In addition,there was a small amount of TNF-αdeposition in the glomeruli in TCE+CGS35066 sensitized positive group,while TCE sensitized positive group mice had more obvious TNF-αdeposition.Part Ⅱ:The serum level of Ang-1 of patients at admission was 1172.25 pg/ml,which was significantly lower than 1,690.79 pg/ml after treatment and slightly lower than1272.85 pg/ml of the normal control population（P<0.05）.The average serum level of Ang-2 of the patients at admission was 932.26 pg/ml,which was significantly higher than the 267.07 pg/ml after treatment and the 446.18 pg/ml of the normal control population（P<0.05）.Serum levels of Ang-1/Ang-2 ratio of patients before treatment was 1.09,which was significantly lower than the ratio of Ang-1 to Ang-2 after treatment and the normal population（P<0.05）.The linear correlation analysis of Ang-1,ET-1 and BUN of OMLDT patients before treatment showed that there was a negative correlation between Ang-1 and ET-1 in the blood circulation and a negative correlation between BUN and Ang-1.However,the indexes of the mice in the TCE-sensitized positive group were significantly increased（P<0.05）.After intraperitoneal injection of the ET_AR inhibitor BQ123,the Cys-c,PCX,Cre and UN levels were significantly reduced,and renal function was significantly restored（P<0.05）.The results from immunohistochemical showed that compared with the blank control group,there was no obvious expression of TNF-αand MCP-1 among the mice in the solvent control group,TCE sensitized negative group,and TCE+BQ123 sensitized negative group.However,compared with the solvent control group,the levels of TNF-αand MCP-1 in the glomeruli of mice in the TCE sensitized positive group were significantly increased,but they recovered significantly after treating with BQ123.In the kidneys of the TCE+BQ123 sensitized positive group,there were only low levels of TNF-αand MCP-1 in the balls.Western blot results showed that the pro-apoptosis-related proteins Fas,Fas/L,Bax,Bad,and the effector molecules pro-caspase-3 and cleaved-caspase-3 were all significantly increased in the TCE sensitized positive group,the levels of apoptosis inhibitory proteins PI3K,p AKT,e NOS,p Bad,and Bcl-2 decreased significantly（P<0.05）.However,after treating with ET_AR inhibitors,the levels of pro-apoptosis-related proteins were significantly reduced,and the levels of apoptosis-inhibiting proteins were significantly increased in TCE+BQ123 sensitized positive group（P<0.05）.The TUNEL staining results showed that the mouse glomeruli cells apoptosis rate was approximately 3%in blank control group,solvent control group,TCE+BQ123 control group,TCE sensitized negative group,and TCE+BQ123 sensitized negative group,while the apoptosis rate was approximately 17%in TCE sensitized positive group,which was significantly higher than that of the solvent control group（P<0.05）.After treating with BQ123,the apoptosis rate of glomerular cells in TCE+BQ123 sensitized positive group decreased,and the apoptosis rate was approximately 9%;the difference was statistically significant（P<0.05）.Western blotting was used to detect the expression levels of the glomerular podocyte cytoskeleton proteins podocin and nephrin.The results showed that compared with the blank control group,the solvent control group,the TCE sensitized negative group and the TCE+BQ123 sensitized negative group mouse glomerular podocin and nephrin expression levels were normal,but the TCE sensitization positive group was significantly downregulated（P<0.05）.Compared with the TCE sensitized positive group,the expression levels of podocin and nephrin in TCE+BQ123 sensitized positive group recovered,and the difference was statistically significant（P<0.05）.Transmission electron microscopy results showed that the glomerular podocytes of mice in the solvent control group had abundant intracytoplasmic organelles,a large number of rough endoplasmic reticulum,abundant ribosomes,a large number of mitochondria and normal structure.A large amount of vacuolar degeneration of mitochondria was seen in the cytoplasm of glomerular podocytes in the TCE-sensitized mice,and the cells were slightly swollen.In the TCE+BQ123 sensitization-positive group,a small amount of vacuolar degeneration of mitochondria was seen in the glomerular podocytes of mice,and the other structures were relatively normal.Real-time PCR was used to detect the gene levels of Ang-1/Ang-2 in the glomeruli of mice in each group.The results showed that compared with the solvent control group,the Ang-1 levels of the mice in the TCE-sensitized positive group did not increase.The level of Ang-2 was significantly increased,the ratio of Ang-1 to Ang-2 genes was significantly reduced（P<0.05）.However,the Ang-1 level of the mice in TCE+BQ123 sensitized positive group increased significantly,but the Ang-2 level did not increase significantly,the ratio of Ang-1 to Ang-2 genes increased（P<0.05）.Western blot results showed that compared with the solvent control group,the TCE-sensitized mice had a large amount of glomerular ET_AR expression,Ang-1 was significantly reduced,and Ang-2 was significantly increased（P<0.05）.There was no significant change in Tie-2 and p Tie-2（P>0.05）.After treating with inhibitor BQ123,the ET_AR level of the glomeruli of the mice in the TCE+BQ123 sensitized positive group decreased significantly,and the expression levels of Ang-1 and p Tie-2 increased,while Ang-2 decreased（P<0.05）.Compared with the solvent control group,the glomerulus of TCE-sensitized mice also had high expression of ET_BR,the expression level of Ang-1 was significantly reduced,and the expression of Ang-2 was significantly increased（P<0.05）.There was no significant change in Tie-2 and p Tie-2（P>0.05）.After treating with BQ788,compared with the TCE sensitized positive group,the ET_BR level of the glomerulus of the TCE+BQ788 sensitized positive group decreased,but the expression levels of Ang-1,Ang-2,Tie-2,and p Tie-2 were not significantly changed（P<0.05）.Immunofluorescence was used to locate ET-1,Tie-2,Ang-2,ET_AR and Ang-1 in the glomerulus,the results showed that ET-1,Ang-2,and Tie-2 are mainly expressed in endothelial cells,ET_AR and Ang-1 are mainly expressed in podocytes.Conclusions1.There was a large amount of ET-1 expressed in the kidneys of trichloroethylene-sensitized mice.Highly expressed ET-1 caused the expression of glycocalyx core proteins syndecan1 and glypican1 to decrease in endothelial cells.After treating with ECE-1 inhibitors,the expression level of ET-1 in mouse kidneys was downregulated,the level of inflammation was reduced,and the level of VEGF-A increased,which promoted the repair of glomerular endothelial cell damage and reduced TCE-mediated immune kidney damage.2.Serum Ang-1 expression level was downregulated and Ang-2 was upregulated in OMLDT patients.ET-1 was negatively correlated with Ang-1,and Ang-1 was negatively correlated with BUN.3.After treating with the ET_AR inhibitor BQ123,it was found that BQ123 can increase glomerular Ang-1 expression level and reduce Ang-2 level,inhibit glomerular cell apoptosis rate and reduce kidney damage.4.The ET_BR inhibitor BQ788 cannot increase Ang-1 expression level or reduce Ang-2level in TCE-sensitized mice kidney.