In view of the importance of mesangial extracellular matrix (ECM) accumulation in the pathogenesis of diabetic glomerulosclerosis, we investigated 1) the effects of high glucose on ECM production by rat glomerular mesangial cells in culture (study A) and 2) the mechanisms underlying these effects, particularly the role of high sugar levels irrespective of intracellular metabolism (study B1) and of excess glucose disposal via the polyol pathway and associated biochemical alterations (study B2). Cells were cultured for 4 weeks, through six to eight passages, under the experimental conditions indicated below and, at each passage, the levels of fibronectin (FN), laminin (LAM), and collagen types I (C-l), III (C-III), IV (C-IV), and VI (C-VI) in media and cell extracts were quantified by an enzyme immunoassay. In study A, medium and cell content of matrix were assessed, together with [H-3]leucine and [H-3]thymidine incorporation into monolayers, polyol, fructose, and myo-inositol levels and the cytosolic redox state, in cells grown in high (30 mM) D-glucose or iso-osmolar mannitol versus cells cultured in normal (5.5 mM) D-glucose. FN, LAM, C-IV, and C-VI accumulation, but not C-l and C-lll accumulation, was increased by 30 mM glucose, but not by iso-osmolar mannitol, when compared with 5.5 mM glucose, starting at week 2 and, except for C-VI, persisting throughout the remaining 2 weeks, whereas no change was observed in the measured indexes of total protein synthesis and DNA synthesis/cell proliferation. At any time point, polyol levels were increased, whereas myo-inositol was reduced by high glucose; in cells grown under elevated glucose concentrations, the lactate/pyruvate (L/P) ratio, an index of the cytosolic redox state, progressively increased. In study B1, the effects of high D-glucose were compared with those of iso-osmolar concentrations of sugars that are partly or not metabolized but are capable of inducing nonenzymatic glycosylation, such as D-galactose and L-glucose, and of mannitol, which does not enter the cell. Both D-galactose and L-glucose, but not mannitol, partly mimicked D-glucose-induced ECM overproduction. Although D-galactose is metabolized via the polyol pathway and alters the cytosolic redox state, ECM changes induced by high galactose were not prevented by the use of an aldose reductase inhibitor (ARI), Alcon 1576 (14 mu M), In study B2, agents interfering with intracellular metabolism of excess glucose via the polyol pathway (14 mu M Alcon 1576) and associated changes in myo-inositol metabolism (1 mM myo-inositol) and cytosolic redox state (1 mM sodium pyruvate, which corrects glucose-induced polyol pathway-dependent increased NADH/NAD+) were added to cells cultured in 30 and 5.5 mM D-glucose. Alcon 1576 inhibited polyol pathway activity with decreasing efficacy during the 4-week period, whereas myo-inositol and pyruvate produced complete and persistent prevention of reduced myo-inositol levels and increased UP ratio, respectively. High glucose-induced ECM overproduction was transiently reduced by pyruvate and, to a lesser extent, by the ARI and myo-inositol. These results suggest that 1) high glucose selectively increases accumulation of basement membrane components and 2) multiple mechanisms seem to be operating in the pathogenesis of glucose-induced ECM overproduction, including elevated sugar levels per se, possibly via nonenzymatic glycosylation, and to a lesser extent, intracellular glucose metabolism via the polyol pathway and associated changes in myo-inositol metabolism and cytosolic redox state.
