Activation of ERK in renal fibrosis after unilateral ureteral obstruction: Modulation by antioxidants

Abstract

A recent in vitro model of oxidative stress-induced renal fibrosis demonstrated that activated or phosphorylated extracellular signal-regulated protein kinase (pERK) played a role in apoptosis of renal fibroblasts, but not tubular epithelium where it promoted cell growth and survival. The present study utilized an in vivo model of renal fibrosis after unilateral ureteral obstruction (UUO) to examine the relationship between pERK, apoptosis, proliferation, and differentiation in renal fibroblast and tubular epithelial cells, in comparison with the in vitro results. UUO was induced in rats for 0 (controls, untreated), 6, and 24 hours, 2, 4, and 7 days (N= 4), and tissue analyzed for fibrotic characteristics using microscopy and special stains, Western immunoblots and reverse transcription-polymerase chain reaction (RT-PCR). Controls and UUO animals were also treated with vitamin E, N-acetylcysteine (NAC), or fluvastatin to assess any antioxidant effect on attenuation of fibrosis and pERK expression. Azan stain and alpha-smooth muscle actin (alpha-SMA), collagen III, and fibronectin expression confirmed development of UUO-induced fibrosis. Oxidative stress markers heme oxygenase-1 (HO-1) and 8-hydroxy-2'-deoxyguanosine (8-OHdG) confirmed oxidative stress at all UUO time points. Tubular epithelial and interstitial mitosis and apoptosis were significantly increased over controls at 2 to 7 days after UUO (P < 0.01). The pERK/ERK ratio increased significantly at 1 to 7 days of UUO in comparison with controls (three- to fivefold, P < 0.05). There was a significant spatiotemporal correlation between pERK and tubular epithelial proliferation (P < 0.001). pERK occasionally colocalized with apoptotic cells (dual labeling) in the interstitium but not in the tubular epithelium. Fluvastatin was the only treatment that attenuated fibrosis (decreased alpha-SMA, fibronectin, tubular epithelial apoptosis) and it also significantly decreased expression of 8-OHdG at 2 and 7 days (P < 0.05). It was associated with decreased pERK at 7 days, compared with UUO alone (P < 0.05). Promotion of tubular epithelial proliferation and survival, and interstitial cell apoptosis, may minimize renal fibrosis after UUO. In the present study, both were linked spatially and temporally with increased pERK expression. Fluvastatin treatment attenuated UUO-induced fibrosis via an antioxidant and pERK-related mechanism.