Stretch-induced mesangial cell ERK1/ERK2 activation is enhanced in high glucose by decreased dephosphorylation.

Glomerular hypertension and hyperglycemia are major determinants of diabetic nephropathy. We sought to identify the mechanisms whereby stretch-induced activation of mesangial cell extracellular signal-regulated kinase 1 and 2 (ERK1/ERK2) is enhanced in high glucose (HG). Mesangial cells cultured on fibronectin Flex I plates in normal glucose (NG; 5.6 mM) or HG (30 mM), were stretched by 15% elongation at 60 cycles/min for up to 60 min. In HG, a 5-min stretch increased ERK1/ERK2 phosphorylation by 6.4 +/- 0.4/4.3 +/- 0.3-fold (P < 0.05 vs. NG stretch). In contrast, p38 phosphorylation was increased identically by stretch in NG and HG. Unlike many effects of HG, augmentation of ERK activity by HG was not dependent on protein kinase C (PKC) as indicated by downregulation of PKC with 24-h phorbol ester or inhibition with bisindolylmaleimide IV. In both NG and HG, pretreatment with arginine-glycine-aspartic acid peptide (0.5 mg/ml) to inhibit integrin binding or with cytochalasin D (100 ng/ml) to disassemble filamentous (F) actin, significantly reduced phosphorylation of ERK1/ERK2 and p38. To determine whether the rate of mitogen-activated protein kinase dephosphorylation is affected by HG, cellular kinase activity was inhibited by depleting ATP. Post-ATP depletion, phosphorylation of ERK1/ERK2 was reduced to 36 +/- 9/51 +/- 14% vs. 9 +/- 5/7 +/- 6% in NG (P < 0.05, n = 5). Thus stretch-induced ERK1/ERK2 and p38 activation in both NG and HG is beta(1)-integrin and F-actin dependent. Stretch-induced ERK1/ERK2 is enhanced in high glucose by diminished dephosphorylation, suggesting reduced phosphatase activity in the diabetic milieu. Enhanced mesangial cell ERK1/ERK2 signaling in response to the combined effects of mechanical stretch and HG may contribute to the pathogenesis of diabetic nephropathy.

Effect of high glucose on mesangial cell protein kinase C-delta and -epsilon is polyol pathway-dependent.

In diabetes mellitus, enhanced activity of mesangial cell protein kinase C (PKC) may contribute to nephropathy. The purpose of this study was to determine whether high glucose alters mesangial cell diacylglycerol-sensitive PKC-alpha, -beta2, -delta, and -epsilon content, cellular distribution, and activity through polyol pathway activation. Primary cultured rat mesangial cells (passage 10) were growth-arrested in 0.5% fetal bovine serum and cultured in 5.6 mM glucose (NG) or 30 mM glucose (HG) for 48 h, with or without the aldose reductase inhibitor tolrestat or ARI-509. PKC isoform content in total cell lysates, or cytosol, membrane (Triton X-soluble), and particulate (sodium dodecyl sulfate-soluble) fractions was analyzed by immunoblotting, and band density in HG was expressed as a percentage of corresponding NG values. In HG at 48 h, increased total PKC-alpha (222 +/- 17% of NG, P < 0.001), -beta2 (209 +/- 12%, P < 0.001), and -epsilon (195 +/- 19%, P < 0.001) were observed. L-Glucose had no effect on total PKC isoform content. HG caused increased membrane- and particulate-associated PKC-alpha (257 +/- 87 and 327 +/- 66%, respectively, P < 0.05), membrane-associated PKC-delta (143 +/- 10%, P < 0.05), and membrane-associated PKC-epsilon (186 +/- 11%, P < 0.001), with no change in cytosol contents. The HG effects were not mimicked by L-glucose. In NG or HG, PKC-beta2 was not detected in the cytosol fraction, and membrane and particulate association were unchanged with phorbol ester stimulation. Confocal immunofluorescence imaging revealed that in HG, PKC-alpha, -delta, and -epsilon translocate to the nucleus and plasma membrane. Total PKC activity measured by in situ 32P-phosphorylation of the epidermal growth factor receptor substrate increased from 18 +/- 1 pmol/min per mg cell protein in NG to 33 +/- 3 pmol/min per mg cell protein in HG (P < 0.002 versus NG). In NG, tolrestat and ARI-509 exposure caused increased PKC activity, enhanced accumulation of total PKC-alpha and -beta2, with no change in total or fractional recovery of PKC-delta or -epsilon. In HG, tolrestat and ARI-509 prevented the increase in total PKC-epsilon and membrane-associated PKC-delta and -epsilon. It is concluded that within 48 h of HG, enhanced mesangial cell PKC activity is associated with accumulation and cellular redistribution of diacylglycerol-sensitive PKC isoforms, and that increased PKC-epsilon content and membrane-associated PKC-delta and -epsilon are dependent on polyol pathway activation.

High glucose-induced mesangial cell altered contractility: role of the polyol pathway.

Glomerular mesangial cells cultured in high glucose conditions display impaired contractile responsiveness. It was postulated that glucose metabolism through the polyol pathway leads to altered mesangial cell contractility involving protein kinase C. Rat mesangial cells were growth-arrested for 24 h with 0.5% fetal bovine serum in either normal (5.6 mmol/l) or high (30 mmol/l) glucose concentrations or high glucose plus the aldose reductase inhibitor, ARI-509 (100 micromol/l). The reduction of cell planar surface area (contraction) in response to endothelin-1 (0.1 micromol/l), or to phorbol 12-myristate 13-acetate (50 pmol/l), was studied by videomicroscopy. In response to endothelin-1, mesangial cells in normal glucose contracted to 52+/-3% of initial planar area. In high glucose, the significantly (p < 0.05) smaller cell size and no contractile responsiveness to endothelin-1 were normalized with ARI-509. Membrane-associated diacylglycerol, measured by a kinase specific 32P-phosphorylation assay, in high glucose was unchanged after 3 h, but significantly increased (p < 0.05) after 24 h which was normalized with ARI-509. Protein kinase C activity, measured by in situ 32P-phosphorylation of the epidermal growth factor receptor substrate was: increased by 32% at 3 h of high glucose, unchanged by ARI-509; and decreased significantly (p < 0.05) at 24 h compared to cells in normal glucose, normalized by ARI-509. Total cellular protein kinase C-alpha, -delta and -epsilon, analysed by immunoblotting, were unchanged in high glucose at 24 h. Only protein kinase C-epsilon content was reduced by ARI-509 in both normal and high glucose. Therefore, high glucose-induced loss of mesangial cell contractility, diacylglycerol accumulation and altered protein kinase C activity are mediated through activation of the polyol-pathway, although no specific relationship between elevated diacylglycerol and protein kinase C activity was observed. In high glucose, altered protein kinase C function, or another mechanism related to the polyol pathway, contribute to loss of mesangial cell contractile responsiveness.

Altered expression and subcellular localization of diacylglycerol-sensitive protein kinase C isoforms in diabetic rat glomerular cells.

Protein kinase C (PKC) is implicated in the pathogenesis of diabetic nephropathy. This study was designed to identify the expression of diacylglycerol (DAG)-sensitive PKC-alpha, -betaII, -delta, and -epsilon isoforms in normal and diabetic rat glomerular cells and to determine the effects of high glucose and insulin on PKC isoform cellular compartmentalization and PKC activity. Diabetic rats treated with or without insulin and normal rats were examined 2 and 4 weeks after streptozotocin/vehicle injection. Renal cortical tissue immunogold-labeled with anti-PKC-alpha, -betaII, -delta, or -epsilon antibody was visualized by electron microscopy. From isolated glomeruli, total cell lysate and cytosol and membrane fractions were immunoblotted with the same anti-PKC isoform antibodies. PKC activity in isolated glomeruli was measured by 32P-phosphorylation of the epidermal growth factor (EGF)-receptor substrate. Immunogold labeling revealed expression of the four PKC isoforms by glomerular visceral epithelial, endothelial, and mesangial cells of both normal and diabetic rats. Immunoblot analysis of the diabetic rat glomeruli at 2 weeks demonstrated a significant increase in membrane-associated PKC-alpha, -delta, and -epsilon and a significant decrease in membrane PKC-betaII content compared with normal, which were similar at 4 weeks. Insulin treatment normalized membrane PKC isoform contents and caused a significant decrease in the cytosol content of PKC-alpha, -betaII, and -delta and total cellular PKC-alpha compared with normal. Although PKC activity in the cells of diabetic rat glomeruli was increased by 20% compared with normal, the difference did not reach statistical significance. In insulin-treated diabetic rat glomeruli, PKC activity was significantly decreased compared with non-insulin-treated diabetic rat glomeruli. In conclusion, DAG-sensitive PKC-alpha, -betaII, -delta, and -epsilon isoforms are all found in the three major glomerular cell types in rats, and the expression, compartmentalization, and activity are modulated independently by high glucose and insulin.

Mesangial cell actin disassembly in high glucose mediated by protein kinase C and the polyol pathway.

High glucose alters mesangial cell cytoskeletal structure and function. We postulated that high glucose causes mesangial cell filamentous (F) actin disassembly through a protein kinase C (PKC) mechanism involving the polyol pathway. Rat mesangial cells (passage < 10, N = 60/group) were growth-arrested and then cultured in glucose 5.6 mM (NG), 15 mM (MG) or 30 mM (HG) for 48 hours, with or without the aldose reductase inhibitor Tolrestat 0.3 mM. F and globular (G) actin were labeled with rhodamine-phalloidin and FTTC-DNase-1, respectively. Both fluorescence probes were imaged simultaneously in each cell using dual-channel confocal laser microscopy. In HG, F-actin disassembly was observed and measured by a 40% decrease in F-/G-actin fluorescence intensity ratio (no change in NG + mannitol 24.4 mM). In separate experiments, cells were labeled with BODIPY FL-bisindolylmaleimide, specific for most PKC isoforms, and fluorescence intensity/cell was measured. In NG, exposure to phorbol 12-myristate 13-acetate (PMA) 0.1 microM for 15 minutes caused perinuclear and nuclear translocation of PKC, and F-actin disassembly identical to observations in HG alone. In HG, total PKC fluorescence increased by 50% and PMA exposure for 24 hours normalized both the total PKC and F-/G-actin fluorescence ratio. In NG and HG, exposure (15 min) to PMA 0.1 microM increased PKC activity three to four times, measured by in situ 32P-phosphorylation of EGF-receptor substrate. By immunofluorescence and confocal imaging, diacylglycerol-sensitive PKC-delta was localized to the cytosol in NG, and after 15 minutes exposure to PMA, translocated to the perinuclear region and plasma membrane. In HG. PKC-delta immunofluorescence was significantly increased/cell, distributed in a cytoskeletal pattern and the intensity was glucose-concentration dependent (30 > 15 > 5.6 mM). In HG, exposure to PMA for 24 hours returned the PKC-delta fluorescence to the intensity and cytosolic pattern observed in NG, and simultaneously prevented F-actin disassembly. Tolrestat significantly reduced the total PKC and PKC-delta fluorescence intensity and F-actin disassembly observed in HG. Immunoblot confirmed increased PKC-delta in HG, which was normalized by Tolrestat. The immunofluorescence pattern of diacylglycerol-insensitive PKC-delta was unchanged in HG, with PMA or Tolrestat. We conclude that mesangial cell F-actin disassembly in high glucose is likely mediated through diacylglycerol-sensitive PKC isoforms, including PKC-delta and involves the polyol pathway.