Signaling Pathways in Pathogenesis of Renal Fibrosis

Project: Research project

Project Details


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.
Effective start/end date9/1/015/31/06


  • Medicine(all)