Effect of High Glucose on Mesangial Cell Protein Kinase C-d and -e 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-a, -b2, -d, and -e 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-a (222 6 17% of NG, P , 0.001), -b2 (209 6 12%, P , 0.001), and -e (195 6 19%, P , 0.001) were observed. L-Glucose had no effect on total PKC isoform content. HG caused increased membraneand particulate-associated PKC-a (257 6 87 and 327 6 66%, respectively, P , 0.05), membrane-associated PKC-d (143 6 10%, P , 0.05), and membrane-associated PKC-e (186 6 11%, P , 0.001), with no change in cytosol contents. The HG effects were not mimicked by L-glucose. In NG or HG, PKC-b2 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-a, -d, and-e translocate to the nucleus and plasma membrane. Total PKC activity measured by in situ P-phosphorylation of the epidermal growth factor receptor substrate increased from 18 6 1 pmol/min per mg cell protein in NG to 33 6 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-a and -b2, with no change in total or fractional recovery of PKC-d or -e. In HG, tolrestat and ARI-509 prevented the increase in total PKC-e and membrane-associated PKC-d and -e. 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-e content and membrane-associated PKC-d and -e are dependent on polyol pathway activation. Hyperglycemia is the most important factor causing the development of progressive diabetic nephropathy (1,2). High glucose stimulates the increased production and decreased degradation of extracellular matrix proteins by glomerular mesangial cells (3–7). Overexpression of glucose transporters in rat mesangial cells transfected with the human GLUT1 gene mimics the diabetic phenotype in a normal extracellular glucose milieu (8). High glucose alters mesangial cell signaling and gene expression through effector mechanisms likely involving glucose metabolism. The polyol pathway converts glucose to sorbitol by the action of aldose reductase (9,10). Oxidation of sorbitol to fructose is coupled to the reduction of NAD to NADH, which serves as an electron donor for reduction of dihydroxyacetone phosphate to sn-glycerol 3-phosphate and subsequent de novo synthesis of 1,2-diacyl-sn-glycerol (DAG) (11,12). Increased membrane-associated DAG may activate protein kinase C (PKC) isoforms, which contribute to the stimulation of proto-oncogene and matrix protein gene expression (13–15). We found that mesangial cells cultured in high glucose for 24 h demonstrate increased membrane-associated DAG, which is prevented by the aldose reductase inhibitor ARI-509 (16). In cultured mesangial cells, high glucose causes F-actin disassembly through a PKCand polyol pathwaydependent mechanism, which may contribute to loss of normal contractility to vasoconstrictor agents (17,18). In streptozotocin-induced diabetic rats, the aldose reductase inhibitor tolrestat prevents glomerular hyperfiltration, glomerular hypertrophy, mesangial cell hypocontractility, and increased urinary albumin excretion (19), suggesting a functional link between the polyol pathway and pathogenesis of early diabetic nephro-