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Increased expression levels of tumor necrosis factor-α (TNFα) is involved in tubulointerstitial cell proliferation and apoptosis in obstructive renal injury. Two TNFα receptors (TNFRs), TNFR1 and TNFR2, are known to exist. On TNFα binding, TNFR1 recruits TNFR-associated death domain (TRADD), an assembly platform to mediate TNFR1 signaling. We investigated postreceptor TRADD regulation in rat kidneys with unilateral ureteral obstruction (UUO). Whereas UUO was associated with increased expression levels of TNFα, TNFR1, TNFR2, and TRADD mRNAs, it resulted in the marked decrease of TRADD protein levels (which appeared at day 1 and persisted thereafter) and a slight decrease in TNFR1 protein levels at days 7 and 14. Both ubiquitination and degradation of TRADD were increased in UUO kidneys, degradation of TRADD was stimulated by TNFα in HK-2 cells, and TRADD degradation was suppressed by proteasome inhibitor. Inhibition of TNFα by soluble TNFR2, etanercept, reduced significantly, although transiently, tubular and interstitial cell proliferation, fibronectin expression, and apoptosis in UUO kidneys, and also suppressed TRADD degradation. These data suggest that the decrease in TRADD resulting from enhanced ubiquitin-dependent degradation is involved in obstructive renal injury. Since TRADD is not incorporated into TNFR2-mediated TNFα signaling, the persistent decrease in TRADD, associated with a mild decrease in TNFR1 levels, may function, at least in part, to divert TNFα signals toward a TNFR2-mediated pathway in UUO kidneys.

The overexpression of transforming growth factor (TGF)-β and Smad-mediated intracellular TGF-β signaling in the kidney underlies the development of renal scarring from pathological matrix accumulation. However, changes in the Smad proteins during the progression of kidney disease are unclear. In this study, we investigated the regulation of Smad proteins in the glomeruli of rats with anti-thymocyte serum nephritis. We found that Smad2 protein decreased markedly in nephritic glomeruli, whereas no significant changes were observed in the levels of Smad3 and Smad4 proteins. In contrast, the level of Smad2 mRNA in nephritic glomeruli did not differ significantly from that in control glomeruli. Based on recent reports of the ubiquitin-mediated degradation of Smad2, we investigated the degradation and ubiquitination activity directed against Smad2 in glomerular extracts. Both the degradation and ubiquitination of Smad2 were markedly increased in glomerular extracts from rats with nephritis. We also found that Smurf2, a ubiquitin ligase for Smad2, was increased in the nephritic glomerular extracts. These data suggest that the decrease in Smad2 resulted from enhanced ubiquitin-dependent degradation of Smad2 mediated by Smurf2, and is involved in the regulation of Smad2-mediated TGF-β signaling in nephritic glomeruli.