RESUMO
We previously found that polydatin could attenuate renal oxidative stress in diabetic mice and improve renal fibrosis. Recent evidence shows that NADPH oxidase 4 (Nox4)-derived reactive oxygen species (ROS) contribute to inflammatory and fibrotic processes in diabetic kidneys. In this study we investigated whether polydatin attenuated renal fibrosis by regulating Nox4 in vitro and in vivo. In high glucose-treated rat glomerular mesangial cells, polydatin significantly decreased the protein levels of Nox4 by promoting its K48-linked polyubiquitination, thus inhibited the production of ROS, and eventually decreasing the expression of fibronectin (FN) and intercellular adhesion molecule-1 (ICAM-1), the main factors that exacerbate diabetic renal fibrosis. Overexpression of Nox4 abolished the inhibitory effects of polydatin on FN and ICAM-1 expression. In addition, the expression of Connexin32 (Cx32) was significantly decreased, which was restored by polydatin treatment. Cx32 interacted with Nox4 and reduced its protein levels. Knockdown of Cx32 abolished the inhibitory effects of polydatin on the expression of FN and ICAM-1. In the kidneys of streptozocin-induced diabetic mice, administration of polydatin (100 mg·kg-1·d-1, ig, 6 days a week for 12 weeks) increased Cx32 expression and reduced Nox4 expression, decreased renal oxidative stress levels and the expression of fibrotic factors, eventually attenuating renal injury and fibrosis. In conclusion, polydatin promotes K48-linked polyubiquitination and degradation of Nox4 by restoring Cx32 expression, thereby decreasing renal oxidative stress levels and ultimately ameliorating the pathological progress of diabetic renal fibrosis. Thus, polydatin reduces renal oxidative stress levels and attenuates diabetic renal fibrosis through regulating the Cx32-Nox4 signaling pathway.
Assuntos
Conexinas/metabolismo , Fibrose/tratamento farmacológico , Glucosídeos/uso terapêutico , Rim/efeitos dos fármacos , NADPH Oxidase 4/metabolismo , Transdução de Sinais/efeitos dos fármacos , Estilbenos/uso terapêutico , Animais , Diabetes Mellitus Experimental/complicações , Diabetes Mellitus Experimental/metabolismo , Diabetes Mellitus Experimental/patologia , Fibronectinas/metabolismo , Fibrose/etiologia , Fibrose/metabolismo , Fibrose/patologia , Molécula 1 de Adesão Intercelular/metabolismo , Rim/metabolismo , Rim/patologia , Masculino , Camundongos Endogâmicos C57BL , Estresse Oxidativo/efeitos dos fármacos , Ratos Sprague-Dawley , Ubiquitinação , Proteína beta-1 de Junções ComunicantesRESUMO
Epithelial-to-mesenchymal transition (EMT) plays an important role in diabetic nephropathy (DN). Ubiquitin-specific protease 9X (USP9X/FAM) is closely linked to TGF-ß and fibrosis signaling pathway. However, it remains unknown whether USP9X is involved in the process of EMT in DN. Our previous study has shown that connexin 43 (Cx43) activation attenuated the development of diabetic renal tubulointerstitial fibrosis (RIF). Here, we showed that USP9X is a novel negative regulator of EMT and the potential mechanism is related to the deubiquitination and degradation of Cx43. To explore the potential regulatory mechanism of USP9X, the expression and activity of USP9X were studied by CRISPR/Cas9-based synergistic activation mediator (SAM) system, short hairpin RNAs, and selective inhibitor. The following findings were observed: (1) Expression of USP9X was down-regulated in the kidney tissue of db/db diabetic mice; (2) overexpression of USP9X suppressed high glucose (HG)-induced expressions of EMT markers and extra cellular matrix (ECM) in NRK-52E cells; (3) depletion of USP9X further aggravated EMT process and ECM production in NRK-52E cells; (4) USP9X deubiquitinated Cx43 and suppressed its degradation to regulate EMT process; (5) USP9X deubiquitinated Cx43 by directly binding to the C-terminal Tyr286 of Cx43. The current study determined the protective role of USP9X in the process of EMT and the molecular mechanism clarified that the protective effects of USP9X on DN were associated with the deubiquitination of Cx43.