A Negative Feedback Loop Involving TIR8/NF-?B Regulates IL-1?-induced Epithelial-myofibroblast Transdifferentiation In Tubular Cells

Objective Epithelial-myofibroblast transition(EMT)is one of the important reasons for the formation of renal interstitial fibrosis(RIF),and also an important indicator for the evaluation of renal function.It was found that Interleukin-1?(IL-1?),as an important inflammatory factor,can be involved in EMT by activating IL-1/IL-1 receptor(IL-1R)/NF-?B signaling pathway.Toll/IL-1R 8(TIR8),a member of the Toll-like receptor superfamily,has been identified as a negative regulator of IL-1R signaling and plays an important role in inhibiting renal inflammation and renal tissue remodeling.However,the function of TIR8 remains unknown in IL-1?-induced RIF.Therefore,the purpose of this study was to explore whether TIR8-mediated negative regulation of IL-1R/NF-?B signaling which is involved in EMT during RIF development and provide a new theoretical basis for improving the occurrence of RIF.Methods 1.Cytological studies(1)In order to investigate whether inflammatory factor IL-1? promotes the regulation of TIR8 expression by activating NF-?B,human renal tubular epithelial cells(HKC)were used as the cell model of the study,and the cells were treated with recombinant human IL-1?(10 ng/ml)and NF-?B activated inhibitors(10 ?M BAY 11-7082).The changes of TIR8,NF-?B(p65)and activated NF-?B(p-p65)were detected by Western blot(WB).(2)To investigate whether TIR8 expression negatively regulated IL-1?-induced NF-?B activation in HKC cells,respectively using the control lines of HKC which were stable transfected with green fluorescent protein gene(HKC/Sc)and the experimental cell lines of HKC which were stable transfected with TIR8 gene(HKC/shTIR8),the control lines of HKC which were instantaneous transfected with green fluorescent protein gene(HKC/vector)and the experimental cell lines of HKC which were instantaneous transfected with TIR8 expression plasmid(HKC/TIR8),WB were used to detect the effect of TIR8 expression in HKC cells on NF-?B activation.(3)To evaluate the role of TIR8 in IL-?-induced EMT in HKC cells,the morphological changes were recorded under an optical microscope,cell motility and migration were evaluated using scratch-wound and Transwell assays,respectively.WB and Immunofluorescence(IF)were used to detect the marker molecules related to EMT,such as transforming growth factor-?1(TGF-?1),E-cadherin,Vimentin and so on;2.Zoological studies(1)We established UUO-induced renal fibrosis animal models to identify whether a negative feedback loop involving NF-?B/TIR8 regulates in vivo IL-1?-induced EMT of tubular cells.Biochemical methods were used to detect changes in renal function.HE staining and Masson staining were used to detect the degree of renal injury and the degree of renal interstitial fibrosis.(2)Immunohistochemistry(IHC)analysis to detect levels of IL-1?,TIR8,p-p65 protein levels and the expression of EMT-related marker molecules of renal tissues.Results 1.TIR8 negatively regulates IL-1?-induced EMT in HKC Cells.(1)TIR8 expression was significantly and continuously increased in HKC cells by exposure to IL-1?,whereas p-p65 levels increased during 15 m of IL-1? induction and then decreased gradually;TIR8 expression was diminished following BAY 11-7082 treatment and relative to levels observed in DMSO group.(2)WB analysis showed p-p65 levels in HKC/shTIR8 cells increased both in the presence and absence of IL-1?,and p-p65 levels in HKC/TIR8 cells decreased significantly compared to the HKC and HKC/vector cells.(3)HKC/shTIR8 cells displayed significant hypertrophy and lost the cobblestone morphology than HKC/shSc cells in the presence of IL-l?;Both immunoblot analysis and immunofluorescence assay showed that IL-1? attenuated E-cadherin levels and upregulated vimentin levels in HKC cells;We observed higher levels of TGF-?1 in HKC/shTIR8 cells in both the presence and absence of IL-1?;TIR8 attenuation promote HKC cells migration and invasion in the absence of IL-1?.2.IL-1? Activates NF-?B and Promotes TGF-?1-mediated EMT Associated with TIR8 Downregulation in a UUO-induced Renal Fibrosis Model.(1)The levels of serum urea and 24-h urinary creatinine,as well as 24-h urinary urea nitrogen,were significantly increased in the UUO group relative to the normal Control group and Sham-operated group;The gross appearance of UUO group showed that there was a large amount of yellow-brown turbid-like fluid and renal parenchyma thinning and hydronephrosis in the kidney of the obstructed side;H&E staining showed normal morphological structures in renal tissue sections from both the Control and the Sham-operated groups;Moreover,kidney damage in the forms of tubular dilatation,epithelial cell necrosis,hemorrhage,and inflammatory cell infiltration were observed in renal tissues from the UUO group;Collagen deposition was significantly increased in obstructed kidneys based on Masson trichrome staining.(2)Compared to the Control and the Sham-operated groups,IHC analysis to detect high levels of both IL-1? and p-p65 in renal tissues from the UUO group,attenuated levels of TIR8 in renal tubular epithelial cells.IHC analysis showed decreases in E-cadherin levels,as well as increases in vimentin levels;Conclusion 1.IL-1? activation of NF-?B promotes the expression of TIR8 in HKC cells,TIR8 also inhibits IL-1?-induced NF-?B activation.2.Loss of TIR8 expression in renal tubular epithelial cells promoted EMT in IL-1?-induced tubular epithelial cells.3.TIR8 can be used as a potential target against RIF.