Ubiquitin Specific Protease 14 Invoved In Renal Interstitial Fibrosis Through Regulating Mitophagy

Objectives:chronic kidney disease（CKD）is a global health problem that seriously endangers the quality of life of patients and imposes a great burden on families and society.Renal fibrosis is one of the important manifestations of advanced CKD.Many kidney diseases will eventually develop into renal fibrosis,and there is no effective treatment to prevent the progression of renal fibrosis.Therefore,it is necessary to further study the pathogenesis of renal fibrosis in order to provide new ideas for the treatment of renal fibrosis.Renal tubular epithelial cells play an important role in the pathogenesis of renal tubular fibrosis.Damaged renal tubular epithelial cells can undergo phenotypic transformation,switching the epithelial phenotype to a mesenchymal phenotype,leading to tubular inflammation and fibrosis.Mitochondrial damage in the proximal renal tubules is one of the initial factors for the progression of fibrosis.Mitophagy can remove dysfunctional mitochondria and regulate the generation of new mitochondria to rebalance the number and function of mitochondria to maintain the optimal overall mitochondrial function.Previous studies have shown that mitophagy has a protective role in renal fibrosis.ubiquitin specific protease 14（USP14）is a proteasome-associated ubiquitin-specific protease,which is involved in the regulation of mitophagy.IU1 is a specific small molecule inhibitor of USP14.IU1 treatment can reduce neuronal damage caused by ischemic stroke and alleviate lung injury caused by ventilator,which provides a new possibility for clinical treatment.At present,there is no study on whether USP14 is involved in the process of renal fibrosis.The present study aims to 1)characterize the altered expression of USP14 in fibrotic kidney tissues.2)To explore the effect of USP14and its inhibitor IU1 on extracellular matrix component fibronectin in renal tubular epithelial cells（HK-2）by constructing an in vitro fibrosis cell model induced by transforming growth factor-β₁（TGF-β₁）.FN),collagenⅠ（COL-1）and mitophagy.3)To explore the effect of USP14 specific inhibitor IU1 on renal function,fibrosis and mitophagy in mice with ffolic acid induced renal fibrosis in vivo.Methods:1.To determine the expression of USP14 in renal fibrotic tissues:clinical samples were stained with immunohistochemistry to determine the expression of USP14.To explore the effects of USP14 and its inhibitor IU1 on renal tubular epithelial cell fibrosis and mitophagia by establishing an in vitro TGF-β₁-induced fibrosis cell model:to clarify the changes of USP14 in TGF-β₁-induced fibrosis cell model;The effect of USP14 up-regulation on the expression of extracellular matrix proteins was investigated using USP14 overexpression plasmid.USP14-si RNA was used to investigate the effect of USP14 down-regulation on the expression of extracellular matrix proteins.The effect of USP14 inhibitor IU1 on extracellular matrix protein expression and mitophagy was investigated.Mitophagy inhibitor was used to investigate whether the effect of IU1 on extracellular matrix protein was caused by enhanced mitophagy.Dynamin-related protein1（DRP1）was knocked down to explore whether the change was dependent on DRP1.To investigate the effect of IU1 on mitophagy and mitochondrial membrane potential in HK-2 cells.To establish a mouse model of renal interstitial fibrosis induced by folic acid（FA）,and to investigate the effect of IU1 on renal function and renal interstitial fibrosis,mitophagy and mitochondrial quality in mice.Results:The expression of USP14 was increased in human renal interstitial fibrosis tissues（P<0.05）.The expression level of USP14 was increased in the renal fibrosis cell model induced by TGF-β₁（P<0.05）.Overexpression of USP14 up-regulated the protein expressions of FN and COL-1 in the renal fibrosis cell model induced by TGF-β₁（P<0.05）.USP14 knockdown down-regulated the protein expressions of FN and COL-1 in the renal fibrosis cell model induced by TGF-β₁（P<0.05）.USP14 inhibitor IU1down-regulated the expression of FN and COL-1 protein in the renal fibrosis cell model induced by TGF-β₁by activating mitophagy,and the effect was affected by DRP1（P<0.05）.The application of IU1 rescued the loss of mitochondrial membrane potential in renal tubular epithelial cells induced by TGF-β₁.The expression of USP14 in the renal tissue of FA-induced renal interstitial fibrosis mice was increased（P<0.05）.USP14inhibitor IU1 could improve the renal function of FA-induced mice（P<0.05）,alleviate the renal fibrosis of FA-induced mice（P<0.05）,reduce the protein expression levels of COL-1 and FN in FA-induced renal tissue of mice（P<0.05）,and enhance the level of mitophagy（P<0.05）and improve mitochondrial quality.Conclusions:USP14 expression is increased in human renal interstitial fibrosis tissues,TGF-β₁-induced renal tubular epithelial cells,and folic acid induced mouse renal interstitial fibrosis tissues.At the cellular level,inhibition of USP14 can reduce the expression of FN and COL-1 and improve mitochondrial quality in a cell model of renal fibrosis induced by TGF-β₁by activating mitophagy.In vivo,inhibition of USP14activated mitophagy,alleviated FA-induced renal interstitial fibrosis,and improved mitochondrial quality in mice.