Sigirr Deletion Upregulates NF-κB And Is Involved In The Development Of Renal Interstitial Fibrosis In Mice With Chronic Kidney Disease

Objective CKD can be complicated by renal interstitial fibrosis（RIF）.IL-1βis a common pro-inflammatory,fibrogenic cytokine that increases the expression of TGF-β1 by binding to the IL-1 receptor IL-1R,which is dependent on My D88 and thus activates the NF-κB signaling pathway.TGF-β1 is an important inducer of epithelial-myofibroblast transition（EMT）,which promotes the development and progression of renal interstitial fibrosis.SIGIRR,as a negative regulatory molecule of IL-1β,can interact with IL-1R and other signaling molecules such as My D88,thereby inhibiting IL-β/IL-1R-mediated activation of NF-κB signaling pathway,and has an important role in suppressing the development of intrarenal inflammation.This paper focuses on the role and mechanism of Sigirr gene deletion mice in chronic kidney disease（CKD）complicated by renal interstitial fibrosis（RIF）.Method1.Animal model studies.（1）To verify whether the SIGIRR/NF-κB feedback mechanism is involved in CKD complicated by RIF and whether it is related to IL-1β-induced EMT,we used 0.2%adenine diet and normal diet to feed Sigirr^-/-mice and Sigirr^+/+mice for 12 weeks to construct a mouse chronic kidney damage and complicated by RIF.We used H&E staining to analyze the histopathological changes of the kidney and Masson staining to observe the degree of renal fibrosis.（2）Changes in the levels of IL-1β,NF-κB（p65）and its activated NF-κB（p-p65）as well as the expression of marker molecules associated with EMT in renal tissues were measured using immunohistochemistry（IHC）and WB.2.Cellular level studies（1）To investigate whether SIGIRR has a negative regulatory effect on IL-1β-activated NF-κB,mice renal tubular epithelial cells（m RTEC）were used as the cellular model for the study.The cells were treated with recombinant human IL-1β（10 ng/ml）using Sigirr^-/-mice primary culture of Sigirr^-/-m RTEC cell line(m RTEC/Sigirr^-/-)and Sigirr^+/+mice primary culture of Sigirr^+/+m RTEC cell line(m RTEC/Sigirr^+/+),respectively.Changes in NF-κB（p65）and its activated NF-κB（p-p65）were detected by Western blot（WB）.（2）To investigate the role of SIGIRR in IL-1β-induced The process of EMT occurrence,m RTEC/Sigirr^-/-and m RTEC/Sigirr^+/+cells were treated with IL-1β,and marker molecules associated with EMTogenesis,such as E-calmodulin（E-cadherin）,vimentin,were detected using WB.Results1.Animal models of chronic kidney disease complicated by renal fibrosis further confirm that Sigirr deficiency facilitates NF-κB activation and promotes interstitial kidney development in mice with chronic kidney disease.（1）The results of renal function tests showed that the levels of serum urea nitrogen and creatinine were elevated in WT and Sigirr^-/-mice in the hyperpurine fed group compared to WT and Sigirr^-/-mice in the normal chow diet group.H&E staining showed normal kidney tissue morphology and structure in the control group,while renal tubular dilatation,epithelial cell necrosis,and massive Masson staining showed that blue deposits were observed in the hyperadenine-fed group;compared with the control group,collagen deposits were significantly increased in the kidneys of the hyperadenine-fed mice.The collagen deposition was significantly increased in the kidney tissues of Sigirr^-/-mice compared to Sigirr^+/+mice.（2）IHC and WB results showed increased expression levels of IL-1βand downstream Myd88 and p-p65 in WT and Sigirr^-/-mice in the hyperpurine fed group compared to the normal chow fed group,and the increase in IL-1β,Myd88 and p-p65 in Sigirr^-/-mice was more Sigirr^+/+mice were more significant.E-cadherin levels were decreased and TGF-β1 as well as Vimentin levels were increased in WT mice and Sigirr^-/-mice in the high-purine fed group,and the decrease in E-cadherin and the increase in TGF-β1 and Vimentin were more pronounced in Sigirr^-/-mice than in Sigirr^+/+mice.2.SIGRR is involved in EMTogenesis through feedback regulation of IL-1β-induced NF-κB activation in m RTEC cells.（1）After IL-1βtreatment of cells,p-p65 levels in m RTEC/Sigirr^+/+cells showed a transient peak at 15 min and then started to decrease,decreasing to the initial level at 24h.（2）WB showed a more significant increase in p-p65 levels in m RTEC/Sigirr^-/-cells than in m RTEC/Sigirr^+/+cells.（3）WB results showed that E-cadherin,an epithelial cell marker molecule,was significantly decreased and Vimentin,a mesenchymal marker,was significantly increased in m RTEC/Sigirr^+/+cells after IL-lβtreatment,and the decrease of E-cadherin and the increase of Vimentin were more significant in m RTEC/Sigirr^-/-cells than m RTEC/Sigirr^+/+were more pronounced.Conclusion1.deletion of Sigirr facilitates IL-1β-induced activation of NF-κB signaling pathway in m RTEC cells.2.deletion of Sigirr gene facilitates the activation of IL-1β/NF-κB/TGF-β1 signaling pathway and promotes the development of renal interstitial fibrosis.3.SIGIRR could be a potential target for future prevention and treatment of RIF.