This proposal seeks to continue studies concerning the pathogenesis of diabetic nephropathy through examination of the consequences of biochemical changes affecting the renal glomerulus and its extracellular matrix in diabetes. The studies described focus on one of the mechanistic postulates that has been advanced in recent years to explain renal and other complications of diabetes; namely, excess non-enzymatic glycation. The experiments to be conducted will examine the effect of glycation on interactive binding properties of matrix macromolecules. Particular attention will be given to determining differences in effect on ligand binding resulting from glycation of specific proteins in vitro versus in vivo, in order to assess whether the high level of glycation (and consequent functional effects) generally achieved in vitro translate to the in vivo situation. Fibronectin and laminin will be isolated from control and diabetic plasma an glomerular basement membrane (GBM), respectively, and heparan sulfate proteoglycan will be purified from control and diabetic glomeruli. Matrix proteins from control animals will be glycated in vitro and binding properties will be compared with those of unglycated control and of diabetic preparations. Tryptic digests of fibronectin also will be prepared to measure ligand binding activity in domains of control, diabetic, an in vitro glycated fibronectin, and to determine whether there are site specific differences in reactivity of lysine residues with glucose in euglycemic versus hyperglycemic states. Additionally, collagen binding and heparin binding protease resistant fragments will be glycated in vitro for analogous experiments to probe for differences resulting from in vitro vs. in vivo glycation. A series of experiments is designed to pursue recent findings concerning an interrelationship between excess nonenzymatic glycation of matrix proteins and activation of the polyol pathway. These studies will examine the level of glycation and of fluorescence in collagen digests from skin, lens capsule, and GBM of control, diabetic and aldose reductase inhibitor treated diabetic rats. Finally, monoclonal antibodies that specifically discriminate glycated epitopes of fibronectin will be produced and used for in situ immunofluorescent and immunoreactive studies to determine whether the glycated protein is deposited in the GBM preferentially. The results of these studies are expected to help define biochemical mechanisms underlying the initiation and evolution of diabetic nephropathy, a significant and growing biomedical concern.

Agency
National Institute of Health (NIH)
Institute
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Type
Research Project (R01)
Project #
2R01DK038308-03A1
Application #
3237584
Study Section
Pathology A Study Section (PTHA)
Project Start
1986-07-01
Project End
1994-06-30
Budget Start
1989-07-01
Budget End
1990-06-30
Support Year
3
Fiscal Year
1989
Total Cost
Indirect Cost
Name
University City Science Center
Department
Type
DUNS #
City
Philadelphia
State
PA
Country
United States
Zip Code
19104
Ziyadeh, F N; Han, D C; Cohen, J A et al. (1998) Glycated albumin stimulates fibronectin gene expression in glomerular mesangial cells: involvement of the transforming growth factor-beta system. Kidney Int 53:631-8
Ziyadeh, F N; Cohen, M P; Guo, J et al. (1997) RAGE mRNA expression in the diabetic mouse kidney. Mol Cell Biochem 170:147-52
Cohen, M P; Ziyadeh, F N (1996) Role of Amadori-modified nonenzymatically glycated serum proteins in the pathogenesis of diabetic nephropathy. J Am Soc Nephrol 7:183-90
Cohen, M P; Hud, E; Wu, V Y et al. (1995) Glycated albumin modified by Amadori adducts modulates aortic endothelial cell biology. Mol Cell Biochem 143:73-9
Cohen, M P; Hud, E; Wu, V Y et al. (1995) Albumin modified by Amadori glucose adducts activates mesangial cell type IV collagen gene transcription. Mol Cell Biochem 151:61-7
Cohen, M P; Sharma, K; Jin, Y et al. (1995) Prevention of diabetic nephropathy in db/db mice with glycated albumin antagonists. A novel treatment strategy. J Clin Invest 95:2338-45
Wu, V Y; Cohen, M P (1995) Evidence for a ligand receptor system mediating the biologic effects of glycated albumin in glomerular mesangial cells. Biochem Biophys Res Commun 207:521-8
Cohen, M P; Vasselli, J R; Neuman, R G et al. (1995) Treatment with acarbose, an alpha-glucosidase inhibitor, reduces increased albumin excretion in streptozotocin-diabetic rats. Gen Pharmacol 26:1355-61
Wu, V Y; Cohen, M P (1994) Receptors specific for Amadori-modified glycated albumin on murine endothelial cells. Biochem Biophys Res Commun 198:734-9
Cohen, M P; Hud, E; Wu, V Y (1994) Amelioration of diabetic nephropathy by treatment with monoclonal antibodies against glycated albumin. Kidney Int 45:1673-9

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