Transforming growth factor-B (TGF-B) is a regulatory cytokine that is implicated in a variety of kidney diseases where it promotes extracellular matrix deposition and fibrosis. Paradoxically, TGF-B has a critical role in normal homeostasis and stabilizes and attenuates tissue injury through mechanisms that are not clearly understood. We previously reported that, in renal tubular epithelial cells, TGF-B1 is a potent inducer of a cytoprotective enzyme, heme oxygenase-1 (HO-1). The overall hypothesis of this proposal is that HO-1 induction is a cytoprotective response to TGF-B1mediated cellular injury in the kidney. The mechanisms underlying the induction of HO-1 by TGF-B as well as the effects of HO-1 expression and its products on the renal epithelial cellular responses to TGF-B1 are not known. In preliminary studies, we have observed that kidneys from HO-1-/- mice (age 24 weeks) show significantly higher fibronectin expression than age matched HO-1 () mice. In contrast, HO-1 induction is associated with decreased levels of fibronectin. Furthermore, bilirubin, 1of the products of the HO-1 catalyzed reaction, significantly lowers TGF-B1-mediated increases in fibronectin levels in renal tubular epithelial cells. Our studies also demonstrate that the inhibitory Smad, Smad7, blocks HO-1 induction by TGF-B1 in renal epithelial cells. The in vivo relevance of the inverse relationship between Smad7 and HO-1 was also observed in kidneys from TGF-p overexpressing mice. TGF-B1-mediated HO-1 induction is transcriptionally regulated and requires a cis-acting region between-9.1 and-9.4kb of the human HO-1 promoter and may involve Sp1-like sequences. This proposal will evaluate the biological role of HO-1 induction in response to TGF-B1 in vitro, using HO-1 deficient cells (primary cells derived from HO-1 -/-, +/+ mice and HO-1 knockdown using siRNA in HK-2 cells) as well as HO-1 overexpressing HK-2 cells (Aim I). HO-1 -/- mice and their littermates will be used to study the role of HO-1 and HO reaction products in the pathogenesis of fibrosis in a model for obstructive uropathy and renal fibrosis, the unilateral ureteral obstruction (DUO) model (Aim II). Finally, the molecular regulation of TGF-B1 -mediated HO-1 gene expression will be characterized (Aim III) and integrated with Aim 1 by testing the effects of overexpression or dominant negative expression of the protein/transcription factor identified on the cellular responses to TGF-B1. Studies to understand the molecular mechanisms involved in the induction of HO-1 by TGF-B1 are timely and relevant for efforts to fine tune endogenous HO-1 activity in renal injury. ? ? ?
