The chronic hyperglycemia in diabetes mellitus enhances the nonenzymic glycation of structural proteins possibly increasing the formation of highly reactive advanced glycation end products (AGE). These protein changes might be involved in tissue-damaging mechanisms leading to diabetic complications, including diabetic nephropathy. To simulate these events, an in vitro model, based on isolated human glomerular basement membrane (hGBM), has been developed. In this study we have investigated the extent of AGE formation and the binding changes induced by the nonenzymic glycation of hGBM. An enriched fraction of hGBM was isolated from normal human kidneys and glycated in vitro by incubation with glucose (500 mmol/l) at 37 °C for 10 days. The presence of AGE was investigated by two methods – spectrofluores-cence and the diazonium salt reaction – both specific for this type of chemical entity. The binding capacity of glycated hGBM was tested by a 10-day incubation with human insulin, albumin, immunoglobulin G and fibrinogen. Higher relative spectrofluorescence values at 440 nm emission (20.0 ± 2.0 vs. 12.5 ± 5.0) and higher absorbance values at 492 nm (0.798 ± 0.063 vs. 0.429 ± 0.228) indicated the presence of increased levels of AGE in glycated vs. native hGBM. Insulin and the three proteins were bound to hGBM in increased amounts after its glycation (p < 0.05). The results obtained in this in vitro model confirm that enhanced nonenzymic glycation of hGBM induces the formation of AGE and possibly, through these compounds, alters its physicochemical and binding properties. This reation might contribute to the mechanisms eventually leading to diabetic nephropathy.

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