Mycophenolic Acid May Delay Allograft Fibrosis by Inhibiting Transforming Growth Factor-β1-Induced Activation of Nox-2 Through the Nuclear Factor-κB Pathway

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Abstract
Background. We evaluated the role of renal tubular Nox-2 in the pathogenesis of epithelial-to-mesenchymal transition (EMT) in kidney allografts. Methods. We examined this question in the human kidney allografts with interstitial fibrosis and tubular atrophy not otherwise specified (IFTANOS), in the Fisher to Lewis rat transplant model, and in the in vitro model of transforming growth factor-β1-induced EMT in normal rat kidney epithelial cells (NRK52E). Results. We first demonstrated that Nox-2 and α-smooth muscle actin (SMA) were increased in renal tubules from kidney transplant recipients on calcineurin inhibitors, mycophenolic acid (MPA), and prednisone with IFTANOS, suggestive of EMT (n=6). Next, we examined Nox-2 expression and fibrogenesis in syngeneic transplants, allogeneic transplants treated with MPA 40 mg/kg per 24 hr, and untreated allogeneic transplants for 6 months (n=14 in each group). Immunofluorescent and immunohistochemical studies for Nox-2, α-SMA, and E-cadherin showed that similar to patients with IFTANOS, rat allografts had greater tubulointerstitial staining for Nox-2 and α-SMA. MPA therapy prevented these changes. Immunoblot analyses examining Nox-2 signaling (phospho-nuclear factor [NF]-κB), redox signaling (phospho-smad2), and fibrosis (α-SMA and fibronectin) demonstrated that MPA treatment prevented the up-regulation of Nox-2, inhibited p-NF-κB and p-smad2, and down-regulated α-SMA and fibronectin levels. Finally, we examined Nox-2 signaling in vitro and confirmed that MPA inhibited phospho-NF-κB, Nox-2, phospho-smad2, and α-SMA during transforming growth factor-β1-induced EMT of NRK52E cells while reducing Nox-2, vimentin, and fibronectin mRNA levels. Conclusions. MPA may down-regulate Nox-2 activation and EMT through the NF-κB pathway in the tubular epithelial cells, suggesting a novel role for this drug independent of its immunosuppressive properties.