LAUSR

23 Introduction: Renal fibrosis is one of the common pathologies of chronic kidney disease. 24 This study aimed to investigate the function of ubiquitin specific peptidase 49 (USP49) in 25 renal fibrosis and to explore the underlying mechanism. 26 Methods and materials: After analyzing the correlation between UPS49 and Smad2/3 27 pathways, we explored the effect of transforming growth factor-β1 (TGF-β1) on the 28 expression of USP49. Then, the USP49 knockdown and ectopic expression human kidney-2 29 (HK-2) cell lines were constructed to investigate the role of USP49 in fibrosis, by determining 30 the expression of epithelial-to-mesenchymal transition (EMT) markers (E-cadherin, α-SMA, 31 and vimentin), phosphorylated Smad2/3 (p-Smad2/3), and protein phosphatase 32 magnesium-dependent1A (PPMIA). Coimmunoprecipitation and ubiquitination analyses were 33 used to determine the direct interaction between USP49 and PPM1A. The 34 PPM1Aoverexpressed HK-2 cells were further introduced to evaluate the effects of USP49 on 35 fibrosis. The unilateral ureteral obstruction (UUO) rats were introduced to confirm the UPS49 36 function in renal fibrosis in vivo. 37 Results: USP49 was negatively correlated with Smad2/3 pathway, and TGF-β1 inhibited the 38 USP49 expression. In HK-2 cells, USP49 overexpression suppressed the activity of α-SMA 39 and p-Smad-2/3 and activated E-cadherin, vimentin, and PPMIA, whereas USP49 knockdown 40 displayed the reverse effects. USP49 could form a complex with PPM1A. USP49 positively 41 regulated PPM1A expression through deubiquitination. Moreover, the fibrotic effects of 42 USP49 knockdown were significantly attenuated with ectopic expression of PPM1A. The 43 anti-fibrotic effect was confirmed with low expressed USP49 and PPM1A in vivo. 44 Pr ep rin t Conclusion: USP49 might exert anti-fibrotic effects via regulating PPM1A/Smad2/3, and 45 USP49 might be an effective target for the treatment of renal fibrosis. 46