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 involvi ng 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 wit h rhodamine-phalloidin and FITC-DNase-I, respectively. Both fluorescen ce probes were imaged simultaneously in each cell using dual-channel c onfocal 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 w ere labeled with BODIPY FL-bisindolylmaleimide, specific for most PKC isoforms, and fluorescence intensity/cell was measured. In NG, exposur e to phorbol 12-myristate 13-acetate (PMA) 0.1 mu M for 15 minutes cau sed perinuclear and nuclear translocation of PKC, and F-actin disassem bly identical to observations in HG alone. In HG, total PKC fluorescen ce 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 (1 5 min) to PMA 0.1 mu M increased PKC activity three to four times, mea sured by in situ P-32-phosphorylation of ECF-receptor substrate. By im munofluorecence and confocal imaging, diacylglycerol-sensitive PKC-del ta was localized to the cytosol in NG, and after 15 minutes exposure t o PMA, translocated to the perinuclear region and plasma membrane. In HG, PKC-delta immunofluorescence was significantly increased/cell, dis tributed in a cytoskeletal pattern and the intensity was glucose-conce ntration dependent (30 > 15 > 5.6 mM). In HG, exposure to PMA for 24 h ours returned the PKC-delta fluorescence to the intensity and cytosoli c pattern observed in NG, and simultaneously prevented F-actin disasse mbly. Tolrestat significantly reduced the total PKC and PKC-delta fluo rescence 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-zet a was unchanged in HG, with PMA or Tolrestat. We conclude that mesangi al cell F-actin disassembly in high glucose is likely mediated through diacylglycerol-sensitive PKC isoforms, including PKC-delta, and invol ves the polyol pathway.