Deubiquitinase ubiquitin-specific protease 36 mitigates ischemia/reperfusion injury-induced renal fibrosis by regulating mitochondrial fission and fusion
Objective To investigate the role and mechanism of deubiquitinase ubiquitin-specific protease 36(USP36)during the process of ischemia/reperfusion-induced renal fibrosis.Methods A renal fibrosis model of ischemia-reperfusion injury(IRI)was established in vitro and in vivo.Western blotting was used to detect the expression of USP36,Mitofusin-2(MFN2),and fibrosis related proteins.Masson staining and Sirius Red staining were used to evaluate interstitial fibrosis in renal tissue.Mito Tracker stai-ning was used to observe mitochondrial fission and fusion.Co-immunoprecipitation was used to detect ubiq-uitination modification of MFN2 protein.T-test was used to compare the differences between two groups,and analysis of variance(ANOVA)was used to compare the differences between multiple groups.Results The USP36 expression in the control group in vivo and in vitro(1.001±0.125,0.994±0.113)was high-er than that in the model group(0.269±0.071,0.195±0.054),and the difference was statistically sig-nificant(t=11.42,14.23,P＜0.01).The expression of Collagen Ⅰ and α-SMA in the experimental ani-mal control group(1.012±0.070,1.008±0.067)was lower than that in the model group(2.899±0.187,2.265±0.209),and the difference was statistically significant(t=21.16,17.66,P＜0.01).The expression of Collagen Ⅰ and α-SMA in the cell control group(1.039±0.081,0.997±0.096)was lower than that in the model group(3.067±0.311,2.619±0.116),and the difference was statistically significant(t=16.36,18.58,P＜0.01).Massion and Sirious Red staining showed extracellular matrix deposition caused by IRI.The protein expression of Collagen Ⅰ and α-SMA in the animal experiment IRI+Vector group(3.689±0.460,2.257±0.197)was higher than that in the IRI+OE-USP36 group(1.889±0.314,1.525±0.103),and the difference was statistically significant(t=7.22,7.36,P＜0.01).The protein expression of Collagen Ⅰ and α-SMA in the H/R+Vector group(3.298±0.168,4.309±0.283)were significantly lower than that in the H/R+OE-USP36 group(1.409±0.127,1.530±0.176,t=20.06,18.67,P＜0.01).Overexpression of USP36 reduced extracellular matrix dep-osition induced by IRI.The expression of MFN2 protein in the IRI+Vector group and H/R+Vector group(0.276±0.063,0.247±0.105)was significantly lower than that in the IRI+OE-USP36 group and H/R+OE-USP36 group(0.789±0.201,0.746±0.105,t=8.45,9.27,P＜0.01).In the process of hypoxia and reoxygenation,overexpression of USP36 inhibited mitochondrial fission and promoted mito-chondrial fusion in renal tubular epithelial cells.USP36 promoted mitochondrial fusion by deubiquitinating MFN2 and inhibiting MFN2 protein-zymosomal degradation.Conclusion During IRI-induced renal fibro-sis,deubiquitinating enzyme USP36 inhibits MFN2 protein-enzymic degradation through deubiquitinating MFN2 protein modification,thereby stabilizing MFN2 protein,promoting mitochondrial fusion,and ulti-mately alleviating renal fibrosis.
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