USP9X deubiquitinates connexin43 to prevent high glucose-induced epithelial-to-mesenchymal transition in NRK-52E cells.

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.

The Therapeutic Effects of the Chinese Herbal Medicine, Lang Chuang Fang Granule, on Lupus-Prone MRL/lpr Mice.

Systemic lupus erythematosus (SLE) is a chronic autoimmune disease that leads to severe multiorgan damage. Lang Chuang Fang (LCF) is a Chinese herbal medicine that is clinically prescribed for treating SLE. In this study, we examined the therapeutic effects of LCF granule on lupus-prone MRL/lpr mice. Female mice were randomly separated into six groups, and LCF treatment groups received LCF granule at the dosage of 0.97 g/kg/d, 1.95 g/kg/d, and 3.90 g/kg/d, respectively. Here, we found that, compared to the MRL/lpr mice, both the spleen coefficient and thymus coefficient were reduced in the LCF granule-treated mice. There was a marked downregulation in CRP and anti-dsDNA autoantibody and an evident upregulation of CH50 in LCF granule-treated mice. LCF granule treatment also obviously reduced the proteinuria, BUN, and SCr levels in MRL/lpr mice at the dosage of 0.97 g/kg/d, 1.95 g/kg/d, and 3.90 g/kg/d, indicating that LCF granule alleviated the renal injury of MRL/lpr mice. Furthermore, LCF granule decreased p65 NF-κB levels and increased Sirt1 and Nrf2 levels in the kidney tissues of MRL/lpr mice, which might elucidate the beneficial effects of LCF on lupus nephritis. In conclusion, this study demonstrates that LCF granule has therapeutic effects on lupus-prone MRL/lpr mice.

AGEs-RAGE system down-regulates Sirt1 through the ubiquitin-proteasome pathway to promote FN and TGF-ß1 expression in male rat glomerular mesangial cells.

We previously demonstrated that advanced glycation-end products (AGEs) promote the pathological progression of diabetic nephropathy by decreasing silent information regulator 2-related protein 1 (Sirt1) expression in glomerular mesangial cells (GMCs). Here, we investigated whether AGEs-receptor for AGEs (RAGE) system down-regulated Sirt1 expression through ubiquitin-proteasome pathway and whether Sirt1 ubiquitination affected fibronectin (FN) and TGF-ß1, 2 fibrotic indicators in GMCs. Sirt1 was polyubiquitinated and subsequently degraded by proteasome. AGEs increased Sirt1 ubiquitination and proteasome-mediated degradation, shortened Sirt1 half-life, and promoted FN and TGF-ß1 expression. Ubiquitin-specific protease 22 (USP22) reduced Sirt1 ubiquitination and degradation and decreased FN and TGF-ß1 expression in GMCs under both basal and AGEs-treated conditions. USP22 depletion enhanced Sirt1 degradation and displayed combined effects with AGEs to further promote FN and TGF-ß1 expression. RAGE functioned crucial mediating roles in these processes via its C-terminal cytosolic domain. Inhibiting Sirt1 by EX-527 substantially suppressed the down-regulation of FN and TGF-ß1 resulting from USP22 overexpression under both normal and AGEs-treated conditions, eventually leading to their up-regulation in GMCs. These results indicated that the AGEs-RAGE system increased the ubiquitination and subsequent proteasome-mediated degradation of Sirt1 by reducing USP22 level, and AGEs-RAGE-USP22-Sirt1 formed a cascade pathway that regulated FN and TGF-ß1 level, which participated in the pathological progression of diabetic nephropathy.