HMGB 1 Acetylation Mediates Trichloroethylene-induced Immune Kidney Injury By Facilitating Endothelial Cell-podocyte Communication

Background Trichloroethylene(TCE)is a volatile organic solvent.After TCE exposure,a systemic disease characterized by a large area of skin and mucous membrane allergic reaction,accompanied by multiple organ damage,occurs in some occupational workers.The TCE-induced disease is known as Occupational Medicamentose-like Dermatitis due to TCE(OMDT).OMDT patients are often accompanied by immune liver injury,kidney injury and skin injury,among which kidney injury is one of the main causes of increased disease burden.Recent studies have shown that vascular endothelial cells not only regulate vascular tone and anticoagulation,but also have secretory functions.HighMobility Group Box 1(HMGB 1)can be released from activated endothelial cells after acetylation and then bind to receptor for advanced glycation endproducts(RAGE),which ultimately induce immune kidney injury.This makes endothelial cells potentially a key target and driver of the process of kidney injury.In addition,our studies found that the translocation of HMGB 1 mainly occurred in endothelial cells,and RAGE was mainly located in podocytes.Thus,HMGB 1 is likely to play a role in endothelial cellpodocyte crosstalk in TCE-induced kidney injury,and the underlying mechanism remains unclear.ObjectiveRenal function,endothelial cell injury,podocyte injury and their association with HMGB 1 and acetyl-HMGB 1(Ac-HMGB 1)in OMDT patients were explored.The sirtuin 1(SIRT 1)activator SRT 1720 and the RAGE inhibitor FPS-ZM 1 were used to establish a TCE-sensitized OMDT mouse model to further investigate the specific mechanism of HMGB 1 in endothelial cell-podocyte crosstalk in TCE-induced glomerular injury.MethodsHuman experiment: 17 OMDT patients,17 TCE-exposed controls and 17 TCEunexposed controls were enrolled in our study.Serum levels of creatinine(Cre)and blood urea nitrogen(BUN)were detected by sarcosine oxidase and urease method to reflect glomerular function.Serum endothelin-1(ET-1)level was detected by ELISA to reflect the activation of endothelial cells.Urine podocalyxin(PCX)level was detected by ELISA to reflect podocyte injury.Serum levels of SIRT 1,HMGB 1 and Ac-HMGB1 were detected by ELISA.Pearson correlation coefficient was used to test the correlation between BUN,ET-1,PCX and HMGB 1,Ac-HMGB 1.Animal experiment: 90 specific pathogen-free(SPF)BALB/c female mice were randomly divided into blank control group(n= 10),solvent control group(n= 10),TCE treatment group(n= 30),FPS-ZM 1 + TCE treatment group(n= 20),and SRT 1720 +TCE treatment group(n= 20).According to the subsequent skin score,TCE treatment group,FPS-ZM 1 + TCE treatment group and SRT 1720 + TCE treatment group were further divided into sensitization positive group and sensitization negative group.The glomeruli of some mice were extracted for western blot(WB).Hematoxylin-Eosin staining(HE)staining was used to detect renal pathological injury.Cre,BUN,cystatin C(Cys-C),ET-1,PCX,SIRT 1,HMGB 1 and Ac-HMGB 1 levels were detected by the same method as above.The location and level of ET-1 were detected by immunohistochemistry(IHC).The co-localization of SIRT 1 and PECAM-1,HMGB 1and PECAM-1,and RAGE and nephrin were detected by immunofluorescence(IF).Glomerular SIRT 1,HMGB 1,Ac-HMGB 1,RAGE,nephrin,podocin,synaptopodin,bax,bcl-2,cleaved caspase 3 levels were detected by WB.The interaction of HMGB 1and RAGE and the interaction of HMGB 1 and Ac-lysine were detected by immunoprecipitation(Co-IP).The apoptosis of podocyte was detected by TUNEL costaining.Podocyte injury was observed by transmission electron microscopy.Results1.In OMDT patients,serum Cre,BUN,ET-1,SIRT 1,HMGB 1,Ac-HMGB 1 and urine PCX protein levels were significantly increased.BUN was positively correlated with HMGB 1 and Ac-HMGB 1.ET-1 was positively correlated with HMGB 1 and AcHMGB 1.PCX was positively correlated with HMGB 1 and marginally correlated with Ac-HMGB 1(P=0.096).2.Sensitization rates of mice: The skin sensitization rate of blank control group and solvent control group were 0%.The sensitization rate of TCE treatment group was36.7%.The sensitization rate of TCE + FPS-ZM 1 treatment group was 35%.The sensitization rate of TCE + SRT 1720 treatment group was 40%.3.Renal function and pathological injury in TCE-sensitized mice: The expression levels of serum Cre,BUN and urine Cys-C in the TCE sensitization positive group were significantly increased.HE staining result showed that in TCE sensitized positive group,the lumen of the renal tubules was enlarged and vacuolated,the glomerular capillary loops were dilated,and glomerular mesangial cells were proliferated.4.Endothelial cell activation was found in TCE-sensitized mice: IHC results showed that ET-1 was mainly located on glomerular endothelial cells in the TCE sensitization positive group,and the expression level of ET-1 was significantly increased.ELISA results also showed that the serum ET-1 level of TCE sensitized positive group was significantly up-regulated.5.Translocation of HMGB 1 from endothelial cells was repressed by SIRT 1 via regulating the deacetylation of HMGB 1 in TCE-induced glomerular damage: IF results showed that SIRT 1 was mainly expressed in endothelial cells,and WB results showed that the expression level of SIRT 1 was significantly down-regulated in the TCE sensitization positive group.However,ELISA results showed that serum SIRT 1 level was significantly up-regulated in the TCE sensitization positive group.The results of IF,WB and ELISA showed that the cytoplasmic translocation of HMGB 1 occurred in endothelial cells in the TCE sensitization positive group,and the HMGB 1 level in the nucleus was significantly decreased,the HMGB 1 levels in the cytoplasm and serum were significantly increased.WB and ELISA results showed that the expression level of Ac-HMGB 1 was significantly increased in the TCE sensitization positive group.6.Ac-HMGB 1 and RAGE complex was regulated by SIRT 1 in TCE-induced glomerular damage: The results of immunoprecipitation showed that HMGB 1 protein was coprecipitated with RAGE protein,and had a strong interaction in the cytoplasm in TCE sensitized positive group.However,the interactions were significantly weakened by SRT 1720 treatment.We also found HMGB 1 was acetylated in the nucleus and cytoplasm in TCE sensitized positive group,but there was no statistically difference in acetylation level of HMGB 1 in the nucleus.7.Podocyte-specific markers proteins and membrane receptor RAGE were regulated by FPS-ZM 1 in TCE-induced glomerular damage: IF and WB results showed that RAGE localized on podocytes and RAGE protein level was significantly up-regulated in the TCE-positive group.WB and ELISA results showed that the protein expression levels of podocin,nephrin and synaptopodin in the glomerulus of TCE sensitized positive group were significantly decreased,and urinary PCX protein level was significantly increased.After FPS-ZM 1 treatment,the expression levels of podocin,nephrin and synaptopodin were increased,while the expression level of PCX was decreased.8.Podocyte apoptosis was inhibited by SRT 1720 and FPS-ZM 1 in glomerular injury induced by TCE: WB results showed that the expression levels of bax and cleavedcaspase 3 were significantly increased and the expression level of bcl-2 was significantly decreased in the TCE sensitization positive group,while the expression of bax and cleaved-caspase 3 was down-regulated and the expression of bcl-2 was upregulated after FPS-ZM 1 treatment.Electron microscopy results showed that podocyte foot processes disappeared and fused extensively in the TCE sensitized group,but podocyte injury was alleviated after SRT 1720 and FPS-ZM 1 treatment.The results of IF and TUNEL staining showed that podocyte apoptosis level was significantly increased in the TCE-sensitized positive group,but the podocyte apoptosis level was alleviated after SRT 1720 and FPS-ZM 1 treatment.ConclusionIn TCE-induced glomerular injury,HMGB 1 is acetylated,and nucleocytoplasmic translocation of HMGB 1 occurs in endothelial cells and is secreted extracellular to interact with RAGE,leading to podocyte apoptosis.Intervention in the upstream and downstream pathways of HMGB 1 partly blocks the cross-talk between endothelial cells and podocytes,that is,the activation of SIRT 1 upstream reduces the nucleo-cytoplasm translocation of HMGB 1,and the inhibition of RAGE downstream reduces the binding of HMGB 1 to RAGE,thus alleviating the injury of glomerular podocytes.