Proteoglycans (PG) are strategically located along the glomerular basement membrane (GBM) where they are in part responsible for the charge barrier to plasma ultrafiltration. GBM from both humans and rats with diabetes mellitus contains a decreased content of heparan sulfate (HS) PG. The pathogenesis of this abnormality is poorly understood. Decreased PG synthesis, synthesis of a biochemically abnormal molecule, or abnormal interaction of PG with other GBM molecules in diabetes could explain a decreased GBM HS PG content. We have previously shown that glomerular PG synthesis and hydrodynamic size (both HS and chondroitin/dermatan sulfate (CS/DS) PGs and glycosaminoglycan (GAG) side chains) were similar in normal and streptozotocin-induced diabetic rats. However, GBM from diabetics were missing a rapidly synthesized and released pool of HS, pointing to the possibility that PG interaction with GBM may be perturbed by diabetes. Indeed, isolated glomeruli from diabetic rats had a decreased proportion of a unique heparin- displaced cell surface HS PG. Thus, abnormal interaction of cell surface PGs with GBM may be involved in the pathogenesis of the diabetic nephropathy. PGs on cell surfaces may be important links between the tissue and the intracellular cytoskeleton, through interaction with tissue glycoproteins such as fibronectin, laminin, and type IV collagen, all molecules to which PG binding has been demonstrated. We hypothesize that PG-matrix interaction is abnormal in diabetes and perturbs binding of HS to GBM with a breakdown in the charge barrier to plasma ultrafiltration. In addition, altered interactions between cell surface PGs and extracellular matrix macromolecules may alter cellular functions communicated by PGs to the intracellular environment through the cellular cytoskeleton. We propose to study the association between glomerular cell surfaces and PGs using visceral epithelial and mesangial cells in culture isolated from human kidneys. We will compare the biochemical characteristics of cell surface PGs with those present in cytoskeletal preparations from these cells. We will attempt to show that PGs associated with cell surfaces interact with extracellular matrix glycoproteins causing changes in cell behavior (adhesion, spreading) and that these interactions may be altered by glycosylation. Antibodies will be produced to human PGs from the cells in culture and used to characterize the glomerular PGs and to co-localize PGs and the cytoskeleton by immunofluorescent and electron microscopy. The morphologic effects upon PG localization of agents which disturb the cytoskeletal architecture will also be examined.
|Klein, D J; Cohen, R M; Rymaszewski, Z (1995) Proteoglycan synthesis by bovine myocardial endothelial cells is increased by long-term exposure to high concentrations of glucose. J Cell Physiol 165:493-502|
|Fioretto, P; Keane, W F; Kasiske, B L et al. (1993) Alterations in glomerular proteoglycan metabolism in experimental non-insulin dependent diabetes mellitus. J Am Soc Nephrol 3:1694-704|
|Faassen, A E; Schrager, J A; Klein, D J et al. (1992) A cell surface chondroitin sulfate proteoglycan, immunologically related to CD44, is involved in type I collagen-mediated melanoma cell motility and invasion. J Cell Biol 116:521-31|
|Moran, A; Brown, D M; Kim, Y et al. (1991) Effects of IGF-I and glucose on protein and proteoglycan synthesis by human fetal mesangial cells in culture. Diabetes 40:1346-54|
|Klein, D J; Oegema Jr, T R; Fredeen, T S et al. (1990) Partial characterization of proteoglycans synthesized by human glomerular epithelial cells in culture. Arch Biochem Biophys 277:389-401|
|Klein, D J; Brown, D M; Kim, Y et al. (1990) Proteoglycans synthesized by human glomerular mesangial cells in culture. J Biol Chem 265:9533-43|
|Klein, D J; Brown, D M; Moran, A et al. (1989) Chondroitin sulfate proteoglycan synthesis and reutilization of beta-D-xyloside-initiated chondroitin/dermatan sulfate glycosaminoglycans in fetal kidney branching morphogenesis. Dev Biol 133:515-28|