Progression of kidney fibrosis and ubiquitin-proteasome degradation of inhibitory factors for Smad-TGF-p signaling

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We investigated the expression, degradation and ubiquitination of inhibitory Smad7 and Smad transcriptional co-repressors SnoN (ski-related novel gene N) and Ski (Sloan-Kettering Institute proto-oncogene) in the fibrotic kidneys of C57BL6/J mice with unilateral ureteral obstruction (UUO). We found the following results.1.Increased mRNA expression of TGF-p, type IV collagen, Smad7, SnoN, and Ski was detected by real time RT-PCR in the fibrotic obstructed kidneys, while western blot analysis revealed the decreases in Smad7, SnoN and Ski proteins.2.Increased degradation of endogenous Smad7, SnoN and Ski proteins was noted in the renal extracts prepared from the obstructed kidneys by degradation assay.3.Increased ubiquitination activity directed against exogenous recombinant Smad7, SnoN and Ski proteins was noted in the renal extracts prepared from the obstructed kidneys by ubiquitination assay.4.Immunodepletion of Smurf2 in extracts of obstructed kidneys resulted in reduced ubiquitination of Smad7 and SnoN, while no significant changes were noted in the ubiquitination of Ski.5.Increased expression of Smurf2 mRNA and protein was noted in the obstructed kidneys by real time RT-PCR and western blot analysis, respectively.6.These data suggest that the reduction of Smad7, SnoN and Ski proteins resulting from increased ubiquitin-dependent degradation is involved in the progression of tubulointerstitial fibrosis after obstructive injury. It was also demonstrated that the ubiquitination activity directed against Smad7 and SnoN, but not Ski, was at least partially mediated by Smurf2 in UUO-induced fibrotic kidneys.