Regulation of Rat Glomerular Epithelial Cell Proteoglycans by High-Glucose Medium

In diabetic nephropathy the heparan sulfate proteoglycan (HSPG) content of the glomerular basement membrane (GBM) is reduced but the cellular mechanisms involved have not been studied. Glomerular epithelial cells (GEC) are thought to be the source of HSPG present in the GBM. In this study we examined if proteoglycan metabolism of the rat GEC in culture is dysregulated in a metabolic environment simulating diabetes. Following incubation for 8 days with a serum-supplemented medium containing 30 mM glucose and no added insulin, a significant increase in the overall synthesis of 35SO4-labeled molecules by the GEC was seen compared to control monolayers incubated with medium containing 5 mM glucose and insulin. Ion exchange chromatography revealed that 30 mM glucose did not alter the anionic charge density of proteoglycans, but significantly increased the amount of 35S-labeled low-anionic macromolecules in the medium; mannitol induced similar changes. Sepharose CL-4B chromatography, glycosaminoglycan analysis and immunoprecipitation of control cell layer proteoglycans demonstrated the presence of HSPG of hydrodynamic size, Kav 0.4, resembling rat GBM HSPG in size and antigenic nature. Incubation of GEC with 30 mM glucose resulted in a significant reduction (58%) in this HSPG species, an effect not seen with equimolar mannitol. Additionally, 30 mM glucose induced a significant increment in synthesis of a small HS species (Kav 0.71 on Sepharose CL-4B column) present in the cell layer. Our findings suggest that both osmotic and nonosmotic mechanisms are operative in dysregulation of glycopeptide metabolism by high-glucose medium and that reduced synthesis by the GEC may contribute to decreased content of GBM HSPG in diabetic nephropathy.