Nonenzymatically glycated albumin (Amadori adducts) enhances nitric oxide synthase activity and gene expression in endothelial cells.

Hyperglycemia is considered to induce diabetic nephropathy through nonenzymatic glycation of proteins. Since hyperfiltration is likely to be the mechanism initiating the glomerular lesions, we investigated the effects of Amadori glucose adducts in serum albumin on the production of vasoactive mediators, including nitric oxide (NO) and eicosanoids, by endothelial cells (EC). Amadori adducts of glycated albumin induced a dose-response increase in NO synthase activity of murine endothelioma cells, up to 16.4 +/- 2.1-fold increase of basal values (P < 0.0001) at concentrations of 35 mg/ml mimicking physiological serum albumin concentration, and 4.6 +/- 0.8-fold increase at 17 mg/ml (P < 0.001). The effect was still detectable with glycated albumin 1.7 mg/ml, which approaches its estimated concentration in diabetic serum (1.6 +/- 0.3-fold increase, P < 0.05) The phenomenon was reproducible in human umbilical vein endothelial cells, though to a lesser extent, and further studies on murine EC were employed. The mRNA encoding for inducible NO synthase was overexpressed in EC incubated with Amadori adducts of glycated albumin in comparison to native albumin. Glycated albumin induced increased mRNA expression and synthesis of TNF-alpha. The stimulatory effect induced by glycated albumin on NO synthase activity was almost completely inhibited by anti TNF alpha antibodies. 3H-thymidine incorporation by EC was significantly inhibited when cells were grown in presence of glycated albumin (P < 0.001), and the phenomenon was abolished by the coincubation of the NO competitive inhibitor L-NAME. The early glycosylation products increased thromboxane production (P < 0.001), while prostaglandin E2 synthesis was unaffected. These data indicate that Amadori products of glycated albumin modulate NO synthase activity and eicosanoid balance in EC. These effects may be relevant to the hemodynamic changes in the early phases of diabetic nephropathy and in the lasting progression to sclerosis.