This competitive renewal continues to focus on the regulation of TGF-beta and Bmp7 in chronic and acute renal disease. Given that TGF-beta promotes renal interstitial fibrosis, whereas Bmp7 is thought to protect against this effect, the potential for developing novel therapeutic agents that inhibit TGF-beta or enhance Bmp7 activity within the extracellular space must be pursued. During the previous funding period, we cloned a novel gene, kcp, that encodes a large protein with 18 repeated cysteine rich domains and binds to both TGF-beta and BMPs. The KCP protein affects receptor-ligand affinities, Smad phosphorylation, and Smad mediated reporter gene expression. Furthermore, a kcp null allele was generated and the kcp-/- mice used in two independent models of renal injury. KCP enhances Bmp7 signaling within the extracellular space, while inhibiting TGF-beta and Activin signaling. Consistent with this interpretation, kcp-/- mice proved to be hypersensitive to developing renal interstitial fibrosis in the unilateral ureteral obstruction model and showed decreased recovery and increased scarring in an acute nephrotoxicity model. The current application will test the ability of KCP to relieve TGF-beta mediated disease, examine the downstream effects of TGF-beta signaling in renal proximal tubule cells, and characterize the molecular mechanisms of Bmp signal enhancement by KCP. Both in vivo and in vitro methods will be employed to test whether exogenously applied or transgenic KCP can reduce the severity of renal disease. The mechanism of TGF-beta action will be addressed by direct identification of genes transcriptionally activated or repressed by Smad2/3 proteins. The effects of KCP on TGF-beta and BMP target genes will be assayed in proximal tubule cell cultures, whole kidney cultures, and whole animals. In addition, we will address the mechanism of KCP mediated regulation of signaling in a novel way. Preliminary data demonstrates that KCP1 can be found in intracellular, endocytotic vesicles, and that these vesicles are prominent after stimulation of cells with Bmp7. Our hypothesis is that vesicular KCP1 enhances the stability of receptor mediated endocytotic vesicles allowing for continued signaling from intracellular Bmp receptors through activation of Smad1. If this is correct, the paradigm of BMP and TGF-beta regulation by secreted factors will be dramatically shifted. These studies will directly examine the potential for KCP1 mediated protection against chronic renal disease and will lay the foundation for developing novel anti-fibrotic agents.
Renal interstitial fibrosis is a common denominator among many chronic renal diseases and can lead to insufficiency and failure. Often the result of obstruction, injury, or inflammation, fibrosis is stimulated by cytokines, such as TGF-beta, which promote myofibroblast proliferation, migration, and secretion of extracellular matrix. Other related cytokines, the BMP proteins, are thought to be protective. The effects of these extracellular proteins are mediated by a variety of mechanisms that control ligand-receptor interactions. We have identified a novel mechanism of TGF-beta suppression through interactions with secreted ligand binding proteins. How these interactions can be manipulated for therapeutic effects needs to be investigated and is the subject of this proposal.
Showing the most recent 10 out of 14 publications
