Neddylation of RhoA impairs its protein degradation and promotes renal interstitial fibrosis progression in diabetic nephropathy.

Abstract

Diabetic nephropathy (DN) is a common and serious complication of diabetes, characterized by chronic fibro-inflammatory processes with an unclear pathogenesis. Renal fibrosis plays a significant role in the development and progression of DN. While recent research suggests that the neddylation pathway may influence fibrotic processes, its specific dysregulation in DN and the underlying mechanisms remain largely unexplored. This study identified the neddylation of RhoA as a novel post-translational modification that regulates its expression and promotes renal fibrosis in DN. We here demonstrated that two key components of the neddylation pathway-NEDD8-activating enzyme E1 subunit 1 (NAE1) and NEDD8-are significantly upregulated in human chronic kidney disease (CKD) specimens compared to healthy kidneys, implicating neddylation in CKD-associated fibrosis. Our findings further revealed that both pharmacological inhibition of neddylation using MLN4924 and genetic knockdown of NAE1 mitigate renal fibrosis in mouse models of streptozotocin-induced diabetes and unilateral ureteral obstruction (UUO). Immunoprecipitation-mass spectrometry (IP-MS) and subsequent function assays demonstrated a direct interaction between RhoA and NEDD8. Importantly, neddylation inhibition reduced RhoA protein expression, highlighting a potential therapeutic target. Additionally, a positive correlation was noted between elevated NEDD8 mRNA levels and RhoA mRNA expression in human CKD specimens. RhoA overexpression counteracted the antifibrotic effects of neddylation inhibition, underscoring its critical role in fibrosis progression. Mechanistically, we unveiled that neddylation enhances RhoA protein stability by inhibiting its ubiquitination-mediated degradation, which subsequently activates the ERK1/2 pathway. Collectively, this study provides novel insights into NAE1-dependent RhoA neddylation as a key contributor to renal fibrosis in DN. The NAE1 protein mediates RhoA protein hyper-neddylation and subsequent stabilization of the RhoA protein, which, in turn, contributes to the development of renal fibrosis and inflammation through an ERK1/2-dependent mechanism. Consequently, targeting neddylation inhibition represents a viable therapeutic approach for the treatment of renal fibrosis in DN.