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.

Agency
National Institute of Health (NIH)
Institute
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Type
Research Project (R01)
Project #
5R01DK054624-02
Application #
2906310
Study Section
Special Emphasis Panel (ZDK1-GRB-4 (M1))
Program Officer
Meyers, Catherine M
Project Start
1998-08-14
Project End
2002-07-31
Budget Start
1999-08-01
Budget End
2000-07-31
Support Year
2
Fiscal Year
1999
Total Cost
Indirect Cost
Name
University of Alabama Birmingham
Department
Pathology
Type
Schools of Medicine
DUNS #
004514360
City
Birmingham
State
AL
Country
United States
Zip Code
35294
Zhou, Yong; Poczatek, Maria H; Berecek, Kathleen H et al. (2006) Thrombospondin 1 mediates angiotensin II induction of TGF-beta activation by cardiac and renal cells under both high and low glucose conditions. Biochem Biophys Res Commun 339:633-41
Wang, Shuxia; Skorczewski, Jim; Feng, Xu et al. (2004) Glucose up-regulates thrombospondin 1 gene transcription and transforming growth factor-beta activity through antagonism of cGMP-dependent protein kinase repression via upstream stimulatory factor 2. J Biol Chem 279:34311-22
Meek, Rick L; Cooney, Sheryl K; Flynn, Stephanie D et al. (2003) Amino acids induce indicators of response to injury in glomerular mesangial cells. Am J Physiol Renal Physiol 285:F79-86
Wang, Shuxia; Wu, Xing; Lincoln, Thomas M et al. (2003) Expression of constitutively active cGMP-dependent protein kinase prevents glucose stimulation of thrombospondin 1 expression and TGF-beta activity. Diabetes 52:2144-50
Wang, Shuxia; Shiva, Sruti; Poczatek, Maria H et al. (2002) Nitric oxide and cGMP-dependent protein kinase regulation of glucose-mediated thrombospondin 1-dependent transforming growth factor-beta activation in mesangial cells. J Biol Chem 277:9880-8
Patel, R P; Moellering, D; Murphy-Ullrich, J et al. (2000) Cell signaling by reactive nitrogen and oxygen species in atherosclerosis. Free Radic Biol Med 28:1780-94
Poczatek, M H; Hugo, C; Darley-Usmar, V et al. (2000) Glucose stimulation of transforming growth factor-beta bioactivity in mesangial cells is mediated by thrombospondin-1. Am J Pathol 157:1353-63