Wnt 5a/Ca²⁺ Mediates The Inflammatory Injury Of Renal Tubular Epithelial Cells In Trichloroethylene Sensitized Mice

BackgroundTrichloroethylene（TCE）is a volatile organic solvent generally used in industry as a cleaning agent for wool and fabrics and as a metal degreaser.Occupational trichloroethylene-induced dermatitis resembling medicamentosa（OMDT）.Although the exact mechanism is still unclear,patients with occupational medicamentosa-like dermatitis can experience severe and potentially fatal kidney damage.Tumor necrosis factor（TNF）-α,which can increase Wnt 5a expression levels,has been shown to be overexpressed in the kidneys of mice exposed to TCE.The activation of non-classical signaling pathways by Wnt 5a is a contributing mechanism to renal tubular inflammation in diabetic nephropathy,so it is important to discuss whether Wnt 5a contributes to the inflammatory injury brought on by TCE in mouse renal tubular epithelial cells.ObjectiveThe aim of this study was to investigate how the Wnt 5a/Ca²⁺pathway induces tubular epithelial cell injury in TCE-sensitized mice:in one treatment group,mice were injected with BOX5 to inhibit Wnt 5a expression in tubular epithelial cells,and then the activation of the Wnt 5a/Ca²⁺pathway in the kidneys of each group was examined to see whether Wnt 5a could mediate intracellular Ca²⁺elevation in tubular cells.In another treatment group,KN93 was injected intraperitoneally to inhibit the expression of Ca MKII in renal tubular epithelial cells,and then examined whether Ca MKII mediated the activation of NF-κB in each group of mice.Thus,the mechanism of Wnt5a/Ca²⁺pathway mediating inflammatory injury in renal tubular epithelial cells of TCE-sensitized mice was investigated.MethodsAccording to their body weight at the conclusion of the acclimatization feeding,85 BALB/c female mice were randomly assigned to 5 group.The group’s previous modeling approach was used to create the TCE percutaneous sensitization model.On days 17 and 19,100 l of phosphate buffer containing BOX5（1 mg/kg）and 100 l of phosphate buffer containing KN93（5 mg/kg）were intraperitoneally injected into the TCE+BOX5 group and the TCE+KN93 group,respectively.The mice’s backs were scored 24 hours after the last excitation.According to the back scoring,the mice were then split into groups that had sensitization-positive and negative reactions.After that,the mice were separated into groups that had undergone sensitization and those that had not based on the back scoring criteria.TCE and BOX5 combined treatment-sensitized positive and negative groups,TCE and KN93 combined treatment-sensitized positive and negative groups,and TCE-sensitized positive and negative groups were used to group the mice.48 hours after the last excitation,mice’s kidneys and sera were collected,and sera were used to identifyα1-microglobulin（α1-MG）andβ2-microglobulin（β2-MG）in mice.To detect pathological structural damage in the mouse kidney,one kidney was divided evenly into two parts along the axis,and the other kidney was prepared into paraffin sections.Double immunofluorescent staining was used to find ROR2 and p-Ca MKII co-localization with renal tubular epithelial cells,and immunohistochemistry was used to find the amount of Wnt 5a,PLC,and p65 deposited in the mouse kidney.Levels of PLC,p-Ca MKII,p-IκB/IκB,p65,IL-6,IL-1β,and TNF-αprotein expression were detected by Western Blot;real-time quantitative PCR for Camk2a,IL-6,IL-1β,and TNF-αgene levels.To find the expression of intracytoplasmic Ca²⁺,renal tubular epithelial cells from some kidneys were extracted.Results1.The sensitization rate was zero percent in the solvent control group and blank control group,but it was forty-one percent in the TCE group,eighteen percent in the TCE and BOX5 group,and forty-two percent in the TCE and KN93 group.2.The results of the mouse kidney pathology（HE）test revealed that the kidneys of the blank control group,solvent control group,TCE-sensitized negative group,TCE and BOX5 combined treatment-sensitized negative group had no detectable pathological damage.When compared to the solvent control group,the levels of AMG and BMG were significantly higher in the TCE-sensitized group（P<0.05）,but not in the TCE-sensitized negative group or the TCE and BOX5 combined treatment sensitized negative group（P>0.05）.In the TCE and BOX5 combined treatment sensitization positive group compared to the solvent control group,serum AMG and BMG expression levels were also elevated;however,their expression levels were lower than those of the TCE sensitization positive group（P<0.05）..3.In the blank control,solvent control,TCE-sensitized negative group,TCE and BOX5 combined treatment-sensitized negative group,the protein immunoblotting findings did not reveal any significant variations in the expression of IL-1β,IL-6,or TNF-α（P>0.05）.In contrast,the TCE-sensitized positive group had substantially higher amounts of these proteins than the solvent control group（P<0.05）.When compared to the TCE-sensitized positive group,the expression levels of these proteins were considerably lower in the TCE and BOX5 combined treatment-sensitized negative group positive group（P<0.05）.4.Immunohistochemical results showed that Wnt 5a was mainly deposited in the renal tubules in the TCE-sensitized positive group and moderately expressed in the TCE and BOX5 combined treatment-sensitized negative group positive group;no Wnt5a expression was detected in all other four groups.Immunofluorescence showed that ROR2 was mainly expressed on renal tubular epithelial cells（TECs）in the TCE-sensitized positive group,while it was not significantly expressed in the other five groups（P>0.05）.Protein immunoblotting results showed that Wnt5a,ROR2 and FZD5 proteins were not significantly expressed in the blank control,solvent control,TCE-sensitized negative,TCE and BOX5 combined treatment-sensitized negative groups（P>0.05）.However,high expression of these proteins was detected in the TCE-sensitized positive and TCE and BOX5 combined treatment-sensitized positive groups.The expression levels of these proteins were significantly lower in the TCE and BOX5 combined treatment-sensitized positive group than in the TCE-sensitized positive group（P<0.05）.5.Both the solvent control group and the blank control group had no detectable PLC deposition,according to immunohistochemistry.However,the TCE-sensitized group experienced PLC deposition.By using immunofluorescence double staining to detect the co-localization of p-Ca MKII and CK18（a marker of renal tubular epithelial cells）,it was discovered that p-Ca MKII was only detectable in the renal tubular epithelial cells of TCE-sensitized mice,whereas p-Ca MKII was hardly expressed in the renal tubular cells of the other five groups.Protein immunoblotting was also used to identify PLC and p-Ca MKII expression in the kidney,and comparisons between the solvent control group,the blank control group,and the corresponding two negative groups revealed no statistically significant changes（P>0.05）.When compared to the solvent group,PLC and p-Ca MKII were considerably greater in the TCE-sensitized positive group（P<0.05）.When compared to the TCE and BOX5 combined treatment-sensitized positive group,PLC and p-Ca MKII expression was considerably reduced（P<0.05）.6.The effect of TCE on intracellular Ca²⁺([Ca²⁺]c)in mouse renal tubular epithelial cells was analysed and the results showed that[Ca²⁺]c was significantly higher in the TCE-sensitized positive group（P<0.05）.The level of[Ca²⁺]c was significantly lower in the TCE and BOX5 combined treatment-sensitized positive group compared to the TCE-sensitized positive group（P<0.05）.7.To find the protein expression of the renal NF-κB（p65）pathway,protein immunoblotting was used.Between all treatment groups,there were no discernible differences in the protein expression of IκBα（P>0.05）.However,these proteins were significantly increased in the kidneys of TCE-sensitized positive group mice（P<0.05）,and as predicted,the expression of p65 and p-IκBαwas significantly higher in the TCE and BOX5 combined treatment-sensitized positive group of mice compared to TCE-sensitized positive group mice（P<0.05）.p-IκBαand p65 expression was also not significantly different between the blank control and solvent control groups（P>0.05）.8.The kidneys of the blank control,solvent control,TCE-sensitized negative,TCE and KN93 combined treatment-sensitized negative groups exhibited no significant histological alterations or inflammatory cell infiltration,according to the renal pathology data.The TCE-sensitized positive group,however,showed substantial vacuolation of epithelial cells and inflammatory cell infiltration.After that,we used Elisa kits to test AMG and BMG in order to evaluate renal tubular function.The blank control,solvent control,TCE-sensitized negative and TCE and KN93 combined treatment-sensitized negative groups’expression levels did not differ significantly from one another（P>0.05）.As opposed to the solvent control group,the TCE-sensitized positive group’s AMG and BMG levels were noticeably greater（P<0.05）.When compared to the solvent control group,serum AMG and BMG levels were likewise higher in the TCE and KN93 combined treatment-sensitized positive group,although they were lower than in the CE-sensitized positive group（P<0.05）.9.Immunofluorescence double stainin of p-Ca MKII and CK18 in the kidney was discovered using immunofluorescence.Only the renal tubular cells of TCE mice were found to have p-Ca MKII.When compared to the TCE and KN93 combined treatment-sensitized positive group,the expression of p-Ca MKII was considerably lower（P<0.05）.Western blotting revealed that there was no significant difference in the expression of p-IκB,p65,IL-1β,TNF-α,and IL-6 between the blank control and solvent control groups（P>0.05）;however,these proteins were significantly elevated in the kidneys of the TCE-sensitized positive group（P<0.05）;additionally,the expression of p-IκBα,p65,IL-1β,TNF-α,and IL-6 in the kidneys of the TCE-sensitized positive group;The TCE and KN93 combined treatment-sensitized positive group’s kidneys had considerably decreased expression of p-IκBα,p65,IL-1β,TNF-α,and IL-6（P<0.05）.TNF-αand IL-6 expression was decreased in the kidneys of TCE and KN93 combined treatment-sensitized positive group compared to the TCE-sensitized positive,and this difference was significant（P<0.05）.Conclusions1.TCE sensitized renal tubular epithelial cell injury is caused by Wnt 5a binding to ROR2 and FZD5,p65 nuclear translocation,and the release of inflammatory cytokines.2.Increased Ca²⁺expression in renal tubular epithelial cells of mice exposed to TCE.3.Preconditioning with BOX5 or KN93 can stop the expression of inflammatory cytokines and lessen renal tubular epithelial cell damage.Our findings show that the Wnt 5a/Ca²⁺pathway is the primary cause of the renal tubular inflammation caused by TCE,which may have significant implications for the development of therapies for TCE-induced renal injury.