Hyperglycemia and the resultant generation of advanced glycosylation end products (AGEs) are major factors in the pathogenesis of diabetic nephropathy. The pro-fibrotic growth factor, TGF-b, is key to the development of diabetic glomerulosclerosis due to TGF-b's role in the stimulation of matrix protein synthesis and matrix assembly. Oxidative stress and protein kinase C (PKC) activity in response to high glucose/AGEs stimulates TGF-b expression. There is now compelling evidence linking hyperglycemia and AGEs to up-regulation of autocrine TGF-b activity, however, the molecular mechanisms regulating activation of the latent precursor form of TGF-b are not clear. We previously established that the platelet and matrix molecule, TSP, binds and activates latent TGF-b and showed that TSP-dependent activation of TGF-b occurs in physiologic situations. Thrombospondin may be involved in the pathogenesis of diabetic nephropathy, since TSP1 expression by mesangial cells is up-regulated by high glucose and TSP is a marker of early fibrosis in diabetic kidneys. On this basis, we propose that glucose regulation of oxidative mechanisms induce glomerulosclerosis via up- regulation of thrombospondin-stimulated TGF-b activation.
The specific aims are: 1) to determine whether TSP is involved in glucose-mediated stimulation of TGF-b activity in mesangial cells; and 2) to determine whether oxidative mechanisms are involved in TSP-dependent TGF-b activation regulated by high glucose and/or AGEs. Preliminary data show that TSP antagonist peptides block glucose stimulated autocrine TGF-b activity in mesangial cells, suggesting a role for TSP. These peptides antagonists provide us with a unique tool to study the role of TSP in activation of TGF-b in diabetic nephropathy. We propose to characterize the role of TSP in a) TGF-b regulation in both acute and chronic conditions, b) regulation of matrix synthesis and assembly, c) mesangial cell proliferation, and d) the role of glucose/AGEs and PKC in regulation of TSP expression and TGF-b activity, and TSP's distribution in diabetic rat kidneys. We also propose to examine the effects of nitric oxide (NO) donors on TSP mRNA, protein, and TGF-b in high glucose; to determine whether NO regulation of TSP involves PKC, and to assess the role of intracellular redox balance in TSP and TGF-b regulation. A long-term goal is the development of these TSP antagonists as a therapeutic tool to intervene in the critical step of TGF-b activation in order to ameliorate fibrosis. These studies are part of a larger initiative to understand the molecular mechanisms involved in fibrosis.
