The Study Of Effect And Molecular Mechanism Of Klotho In The Formation Of Renal Allograft Interstitial Fibrosis In Kidney Transplantation

Objective:Klotho is a newly discovered anti-aging gene,which is mainly expressed in kidney and brain tissue.In recent years,Klotho has been found to be an important cancer suppressor and kidney protective factor.However,the role of Klotho in the formation of renal fibrosis and its related mechanism are still unclear.The formation of interstitial fibrosis of transplanted kidney is an important pathological feature of chronic allograft dysfunction.This study aims to explore the role of Klotho in the formation of interstitial fibrosis of transplanted kidney and its related mechanism.Method:A 24-weeks rat renal transplant model with chronic allograft dysfunction(CAD)was established by orthotopic kidney transplantation from F344 donor rats to Lewis recipient rats.Serum creatinine and urea nitrogen were used to detect the transplanted renal function of rats in each group.The transplanted kidney tissues of rats at different time points were collected and embedded in paraffin,and then stained with Hematoxylin and eosin(HE)and Masson to observe the pathological morphology of transplanted kidney of rats in each group.The distribution and expression of Klotho protein in transplanted kidney tissues were detected by immunohistochemical staining.Total RNA and protein were extracted from transplanted kidney tissues of rats in each group,Klotho and markers of interstitial fibrosis were detected by real-time fluorescence quantitative polymerase chain reaction(qrt-PCR)and Western Blot(WB).Human renal tubular epithelial cell line(HK-2)were cultured in vitro,and intervened with human Transforming growth factor-β1(TGF-β1)at different time points.Total RNA and protein from cells of each group were extracted,Klotho and the markers of interstitial fibrosis were detected by qRT-PCR and WB respectively.Finally,the Klotho small interfering plasmid siRNA-Klotho was constructed.HK-2 cells were cultured and intervened with TGF-β1 and/or siRNA-Klotho by various time points.The cells were collected and the total RNA and protein were extracted.qRT-PCR and WB methods were used to detect the expression levels of Klotho,markers of interstitial fibrosis and key mediators of related cellular pathways in cells of each group.Results:We constructed a CAD model of allograft kidney transplantation with F344 rats as donor and Lewis rats as recipient,and examined the renal function of transplantation group(Allo group)and control group(Syn group).The pathological morphology of transplanted kidneys of the two groups was observed by HE and Masson staining.The results showed that the CAD model of allogeneic rat kidney transplantation was successfully constructed.The rusults of immunohistochemical staining showed that Klotho was widely distributed in transplanted renal tubules region of Syn group,and its expression was significantly higher than that of Allo group.Immunohistochemical staining of fibronectin showed that fibronectin was distributed in interstitial region of transplanted kidney in Allo group,and its expression was significantly higher than that in Syn group.The results of PCR and WB also showed that the expression of Klotho decreased significantly in Allo group,while the expression of fibronectin increased significantly in Allo groupIn the cell experiment,TGF-β1 significantly induced HK-2 cells to undergo epithelial-to-mesenchymal transition(EMT),and during this process,Klotho protein decreased significantly,and this process was time-dependent.After interfering HK-2 cells with Klotho small interfering plasmid siRNA-Klotho and TGF-β1,the EMT process in renal tubules was significantly enhanced.In this process,with the activation of β-catenin signaling pathway,β-catenin in cytoplasm was transferred to nucleus,and the expression ofβ-catenin in nucleus was significantly increased.Furthermore,the mRNA levels of matrix metalloproteinase-2(MMP-2)and MMP-9 were also significantly increased respectively.However,after using ICG001——a specific inhibitor of β-catenin,to intervene the EMT process of renal tubules,it was found that although the expression of Klotho protein decreased significantly,α-SMA protein was not overexpressed,thus the EMT process of renal tubules was significantly inhibited.Conclusion:We observed significant pathological features of renal allograft interstitial fibrosis in the CAD model of rat kidney transplantation,accompanied by a significant decrease in Klotho level,suggesting that Klotho may play an inhibitory role in the formation of renal allograft interstitial fibrosis.Cell experiments showed that Klotho may inhibit the EMT process of renal tubules through β-catenin signaling pathway,thus slowing down the formation of interstitial fibrosis of transplanted kidney and protecting transplanted kidney function.