Signaling Pathways in Pathogenesis of Renal Fibrosis

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DESCRIPTION (Applicant's abstract): Diabetic arteriopathy, diabetic nephropathy, and many forms of progressive renal disease are all characterized by excessive deposition of collagen IV. The lack of effective therapies for these forms of chronic tissue injury are in large part due to a lack of understanding of basic mechanisms of fibrogenesis. Although TGF-13 1 has emerged as the dominant determinant of collagen IV expression and is considered an essential fibrogenic cytokine, the intracellular signaling pathways elicited by TGF-B 1 and the mechanism by which TGF-J3 1 stimulates transcription of the collagen IV genes in pathobiologic states is not known. The central hypothesis to be tested in this application is that TGF-B 1 increases transcription of the collagen IV genes by triggering several functionally distinct intracellular signaling pathways, involving the Smad proteins and one or more of the mitogen activated protein kinase cascades. In Specific Aim 1, functional characterization of the collagen IV promoter and flanking regions will be completed. Deletion constructs of chimeric collagen IV promoter-CAT vectors will be used to define sequences which function as orientation-specific activator regions that direct transcription of the a1(IV) and a2(IV) collagen genes. Nuclear proteins that bind these critical regulatory regions will be defined by gel mobility shift assays. In Specific Aim 2, the role of TGF-B 1 elicited signaling pathways on collagen IV transcription, steady state mRNA expression, and protein production will be ascertained. Rat mesangial cells and aortic smooth muscle cells will be transfected with constitutively active or dominant negative Smad constructs and with constructs to constitutively activate the ERK, iNK, and p38 signaling pathways; the role of these interventions on basal and TOP-B 1 stimulated collagen IV expression will be ascertained. Potential sites of interaction between the Smad and MAPK signaling pathways will be defined. Based on these studies, transfection analysis and gel mobility shift assays will be employed to define sequence elements within the collagen N promoter or flanking region that confer a transcriptional response to TGF-beta 1(TGF-B1, Specific Aim 3). Finally, mesangial cells and vascular smooth muscle cells isolated from TGF-B 1 knockout animals will be used to determine whether activation of the Smad and/or MAP kinase signaling cascades can activate transcription of the collagen IV genes in a TGF-B 1-independent manner. Delineation of the collagen IV signaling pathway will reveal basic cellular mechanisms of enhanced fibrogenesis underlying progressive tissue injury, and may provide the rational basis for the design of new pharmaco-therapeutic interventions targeted to specific identified signaling steps. If successful, these interventions may retard progression of tissue injury to end-stage disease.