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-I), 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 [3H]leucine and [3H]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-I and C-III 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 μM). In study B2, agents interfering with intracellular metabolism of excess glucose via the polyol pathway (14 μM 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 L/P 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.
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Original Articles|
March 01 1994
Mechanisms of Glucose-Enhanced Extracellular Matrix Accumulation in Rat Glomerular Mesangial Cells
Giuseppe Pugliese;
Giuseppe Pugliese
Department of Experimental Medicine, La Sapienza University
Rome
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Flavia Pricci;
Flavia Pricci
2nd Institute of Internal Medicine, La Sapienza University
Rome
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Francesco Pugliese;
Francesco Pugliese
2nd Institute of Internal Medicine, La Sapienza University
Rome
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Paolo Mene;
Paolo Mene
2nd Institute of Internal Medicine, La Sapienza University
Rome
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Luisa Lenti;
Luisa Lenti
Department of Experimental Medicine, La Sapienza University
Rome
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Domenico Andreani;
Domenico Andreani
2nd Institute of Internal Medicine, La Sapienza University
Rome
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Gianna Galli;
Gianna Galli
Department of Clinical Pathophysiology and 3rd Institute of Internal Medicine, University of Florence
Florence
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Alessandro Casini;
Alessandro Casini
Department of Clinical Pathophysiology and 3rd Institute of Internal Medicine, University of Florence
Florence
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Sauro Bianchi;
Sauro Bianchi
Department of Clinical Pathophysiology and 3rd Institute of Internal Medicine, University of Florence
Florence
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Carlo M Rotella;
Carlo M Rotella
Department of Clinical Pathophysiology and 3rd Institute of Internal Medicine, University of Florence
Florence
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Umberto Mario Di
Umberto Mario Di
Department of Experimental and Clinical Medicine, University of Reggio Calabria
Catanzaro, Italy
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Address correspondence and reprint request to Dr. Giuseppe Pugliese, Diabetes, Endocrinology and Metabolism (D.E.M.) Foundation, Largo Marchiafava 1, 00161 Rome, Italy.
Diabetes 1994;43(3):478–490
Article history
Received:
December 01 1992
Revision Received:
October 05 1993
Accepted:
October 05 1993
PubMed:
8314022
Citation
Giuseppe Pugliese, Flavia Pricci, Francesco Pugliese, Paolo Mene, Luisa Lenti, Domenico Andreani, Gianna Galli, Alessandro Casini, Sauro Bianchi, Carlo M Rotella, Umberto Mario Di; Mechanisms of Glucose-Enhanced Extracellular Matrix Accumulation in Rat Glomerular Mesangial Cells. Diabetes 1 March 1994; 43 (3): 478–490. https://doi.org/10.2337/diab.43.3.478
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