TRIM27 Mediates Glomerular Endothelial Cell Injury In Lupus Nephritis By Activating FoxO1 Signaling Pathway

Objective:As one of the complications of systemic lupus erythematosus,lupus nephritis（LN）significantly increase the morbidity and mortality of patients,so it is particularly important to research its pathogenesis.In this study,lupus nephritis patients,MRL/Mp J-Faslpr/J（MRL/lpr）mice and HRGECs were used to explore the role and possible mechanism of TRIM27 in glomerular endothelial cells damage of lupus nephritis,which will further reveal the pathogenesis of lupus nephritis and provide experimental evidence for the exploration of targeted therapies.Methods:1.To detect the expression of TRIM27 in glomerular endothelial cells of lupus nephritis⑴The biopsy kidney tissue specimens of LN patients and normal renal tissue adjacent to neoplastic lesions were collected,IHC was used to detect the expression and localization of TRIM27 and CD31 in glomeruli cells.⑵IF was used to detect the expression of TRIM27 and CD31 in glomeruli of MRL/lpr mice.⑶Healthy human plasma（control group）and LN plasma（LN group）were used to incubated with HRGECs,WB and IF were used to detect the expression and localization of TRIM27 protein.2.To verify the role of TRIM27 in the damage of endothelial cell of LN⑴14-week-old female MRL/lpr mice were randomly divided into the MRL/lpr group,MRL/lpr+sh-TRIM27 group and MRL/lpr+sh-NC group,the matched MRL/Mp J were set as the control group.The MRL/lpr+Sh-TRIM27group was injected with 50μL recombinant adeno-associated virus（Sh-TRIM27-AAV）with a titer of 1.6×10¹¹μg in situ,the MRL/lpr+Sh-NC group was injected with the same dose of Sh-NC-AAV virus,the MRL/lpr group and the control group were injected with the same dose of saline.10weeks later after blood and urine samples were collected,and the mice were sacrificed to collect kidney tissues.WB and IHC were used to observe the transfection efficiency and silencing effect,WB was used to detect the expression of VCAM-1,ELISA was used to determine the content of VCAM-1 and SDC-1 in the supernatant,and total nitric oxide detection kit detected the level of nitric oxide.⑵HRGECs were randomly divided into control group,LN group,LN+Sh-TRIM27 group,and LN+NC group.HRGECs were infected with the virus when 50%density,after incubation with 10%healthy plasma or LN plasma for 12 hours,the cells and the supernatant were collected.WB was used to detect the expression of TRIM27,VCAM-1,p-FoxO1-Ser256 and FoxO1,ELISA was used to determine the content of VCAM-1 and SDC-1 in cell supernatant,IF was used to detect the expression of SDC-1 and F-actin,FITC-BSA was used to detect endothelial permeability,and the total nitric oxide detection kit was used to detect the total amount of nitric oxide.3.To further explore the relationship between FoxO1 and the damage of HRGECsHRGECs were randomly divided into control group,LN group,LN+WT-FoxO1 group and LN+NC group,the detection methods and indicators are the same as above.Results:1.TRIM27 is up-regulated in glomerular endothelial cells of lupus nephritis⑴IHC results showed that in the normal group,TRIM27 was mainly located in the nucleus of CD31-labeled glomerular endothelial cells,while in the glomeruli of LN patients,the expression level of TRIM27 in glomerular endothelial cells was significantly elevated,and located in both nucleus and cytoplasm.⑵IF results showed that compared with the control group,the expression of TRIM27 in CD31-labeled glomerular endothelial cells of MRL/lpr mice was significantly increased,and it was mainly located in the cytoplasm and nucleus.⑶WB and IF showed that the expression level of TRIM27 was significantly increased in LN group compared with control group,and position in both nucleus and cytoplasm.2.Knockdown of TRIM27 improved glomerular endothelial cell injury and glycocalyx loss in lupus nephritis⑴The results of animal experiments showed that compared with control mice,the expression of TRIM27,VCAM-1 and the level of NO were increased in the renal cortex of MRL/lpr mice,the expression of the endothelial glycocalyx core protein syndecan-1 protein in the glomerulus was significantly reduced,and the serum VCAM-1 and syndecan-1 levels were significantly increased.Sh-TRIM27-AAV effectively down-regulated the expression of TRIM27 in the renal cortex of MRL/lpr mice,and compared with the MRL/lpr group,expression of syndecan-1 was increased in the glomerular endothelial cells,was decreased in the serum of MRL/lpr+Sh-TRIM27-AAV group,the level of VCAM-1 and NO were decreased in the renal cortex of MRL/lpr+Sh-TRIM27-AAV group.⑵The results of cell experiments showed that compared with the control group,the HRGECs cells were arranged irregularly,the F-actin expression was reduced in the cells,TRIM27,VCAM-1 expression levels and NO levels were increased,and the syndecan-1 and VCAM-1 levels in supernatant and cell permeability of HRGECs were increased in the LN group.TRIM27-sh RNA-LV significantly knocked down the expression of TRIM27 in HRGECs.Compared with LN group,the disordered arrangement of the cytoskeleton in HRGECs of the LN+Sh-TRIM27 group was significantly improved,and the expression of syndecan-1 was increased,the levels of syndecan-1 and VCAM-1 in the supernatant,the permeability of endothelial cells and the content of NO in the cells were decreased.3.TRIM27 mediated glomerular endothelial cell injury of LN through FoxO1 pathwayWB showed that expression level of FoxO1 was decreased,p-FoxO1-Ser256 was increased significantly in HRGECs stimulated by LN plasma.And FoxO1 and p-FoxO1-Ser256 protein expression appeared the opposite change after knocking down TRIM27 expression.ELISA results showed that LN plasma increased the levels of syndecan-1 and VCAM-1 in the cell supernatant,while the WT-FoxO1 plasmid inhibited their expression.The results of WB,IF and transwell assay experiments showed that WT-FoxO1 abolished the up-regulation of VCAM-1,F-actin,NO and cell permeability induced by LN plasma.Conclusion:The upregulation of TRIM27 participated in glycocalyx loss and endothelial cell damage in lupus nephritis by activating the FoxO1 signaling pathway,which lead to damage of filtration membrane and acceleration of nephritis progression.