Bardoxolone ameliorates TGF-β1-associated renal fibrosis through Nrf2/Smad7 elevation

Transforming growth factor-beta (TGF-beta) is a potent pathogenic factor of renal injury through the upregulation of extracellular matrix (ECM) expression and facilitation of renal fibrosis. Nuclear factor erythroid 2-like 2 (Nfe2l2; Nrf2), a master regulator of antioxidant and detoxifying systems, is mainly controlled by the binding with cytosolic protein Kelch-like ECH-associated protein 1 (Keap1) and subsequent proteasomal degradation. The protective effect of Nrf2 on renal injury has been attributed to its antioxidant role, where it aids in coping with oxidative stress-associated progression of renal disease. In this study, we investigated the effect of Nrf2 activation on ECM production and TGF-beta/Smad signaling using Keap1-silenced MES-13 cells (a genetic glomerular mesangial cell model with Nrf2 overexpression). The TGF-beta 1-inducible expression of fibronectin and alpha-smooth muscle actin (alpha-Sma) was suppressed and Smad2/3 phosphorylation was blocked in Nrf2-high mesangial cells as compared with that in control cells. Notably, in these Nrf2-high mesangial cells, levels of TGF-beta 1 receptor 1 (T beta R1) were substantially diminished, and the protein levels of Smad7, an inhibitor TGF-beta 1/Smad signaling, were increased. Nrf2-mediated Smad7 elevation and its anti-fibrotic role in Keap1-silenced cells were confirmed by studies with Nrf2-or Smad7-silencing. As a molecular link for Smad7 elevation in Nrf2-high cells, the reduction of Smad-ubiquitination-regulatory factor 1 (Smurf1), an E3 ubiquitin ligase for Smad7, was notable. Silencing of Smurf1 increased Smad7 in the control mesangial cells; however, forced expression of Smurf1 repressed Smad7 levels in Keap1-silenced cells. Additionally, we demonstrate that bardoxolone (BARD; CDDO-methyl), a pharmacological activator of Nrf2, increased Smad7 levels and attenuated TGF-beta/Smad/ECM expression in MES-13. Moreover, in an aristolochic acid (AA)-mediated nephropathy mouse model, the renal expression of Nrf2 and Smad7 was elevated by BARD treatment, and AA-induced tubular necrosis and interstitial fibrosis were substantially ameliorated by BARD. Collectively, these results indicate that the Nrf2-Smad7 axis plays a key role in the protection of TGF-beta-induced renal fibrosis, and further suggest a novel molecular mechanism of beneficial effect of BARD on renal disease.