Mechanisms of Glucose-Enhanced Extracellular Matrix Accumulation in Rat Glomerular Mesangial Cells

Abstract

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 microM).(ABSTRACT TRUNCATED AT 400 WORDS)

Redox state-dependent and sorbitol accumulation-independent diabetic albuminuria in mice with transgene-derived human aldose reductase and sorbitol dehydrogenase deficiency

“…AR inhibitor normalised not only the diabetic decrease in GSH concentrations, but also the diabetic increase in L/P ratios even in the presence of transgenic hAR. The increased L/P ratio of diabetic mice in our study is consistent with a report showing an increase in L/P ratios in rat glomerular mesangial cells exposed to glucose [41]. Because SDH-deficiency did not improve the diabetic increase in UAE, our study did not analyse GSH concentrations and L/P ratios in kidneys of SDH null or hAR-Tg:SDH null mice.…”

The diabetic milieu modulates the advanced glycation end product-receptor complex in the mesangium by inducing or upregulating galectin-3 expression.

“…Preliminary data showing unchanged rates of proliferation and apoptosis, unchanged patterns of matrix protein and cytokine production, and unchanged expression of mesangial cell-specific markers (Thy1.1, vimentin, and smooth muscle actin) indicated that these cells do not undergo detectable phenotypic changes at advanced passages in culture. Cells were seeded in 35-or 100-mm culture dishes (Falcon; Becton Dickinson, Lincoln Park, NJ) and grown to confluence in Dulbecco's modified Eagle's medium (Sigma, St. Louis, MO) supplemented with 17% fetal bovine serum and 2 mmol/l L-glutamine and antibiotics (all obtained from Flow Laboratories, Irvine, Scotland, U.K.) at 37°C in a 95% air and a 5% CO 2 humidified atmosphere (3,21). Cells were exposed to one of the following 2 sets of conditions: 1) media containing high glucose (HG) levels (30 mmol/l) as compared with those containing iso-osmolar mannitol (M) (24.5 mmol/l + 5.5 mmol/l glucose) or media containing normal glucose (NG) levels (5.5 mmol/l) for 1-4 weeks, or 2) glycated bovine serum albumin with AGE formation (BSA-AGE) as compared with glycated BSA in which AGE formation was prevented by aminoguanidine (AM) (BSA-AM) or with nonglycated or native BSA, all previously prepared and either coated onto the culture dish or added to the culture media, for 4 days (3).…”

“…In vitro studies. Glomerular mesangial cells were isolated from human and rat kidneys and characterized as previously reported (3,21). Because the Gal-3 expression pattern was shown to vary with cell aging (i.e., increased basal and reduced serum-stimulated levels) (22), cells between the 3rd and the 15th passages (for human mesangial cells [HMCs]) and between the 5th and the 27th passages (for rat mesangial cells [RMCs]) were used in these experiments.…”

Effect of high glucose on formation of extracellular matrix components by cultured rat heart endothelial cells

“…This difference in tissue response may reflect the constellation of cells present in myocardial capillaries (endothelial cells and pericytes) and glo-meruli, which contain epithelial cells in addition to endothelial and mesangial cells; previous studies from our laboratory have indicated that only the latter two cell types form type VI collagen, while all three synthesize type IV. Most previous investigations on the effect of glucose on collagen synthesis by cells in culture have focused on type IV and have found an approximately twofold increase in this protein, whether the elevated glucose was present during the entire growth period of endothelial [17,26] or mesangial [27][28][29] cells or when exposure of confluent glomerular epithelial, mesangial or endothelial cells was only for short periods of time [3,6]. The influence of elevated glucose on type VI collagen has previously been studied only with rat glomerular mesangial cells and in that system the extent of stimulation was similar for both types VI and IV collagens, even though marked differences were observed in other aspects of the response, with type VI reflecting more rapidly both increased and normalized glucose levels, and only type IV being enhanced by addition of the glucose metabolite pyruvate or by the presence of aldosterone or IGF-1 [3].…”