title: Mesangial cell actin disassembly in high glucose mediated by protein kinase C and the polyol pathway.
abstract: 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.
authors.affiliation: Department of Medicine, University of Toronto, Ontario, Canada.
authors.forename: X
authors.initials: X
authors.lastname: Zhou
authors.forename: C
authors.initials: C
authors.lastname: Li
authors.forename: J
authors.initials: J
authors.lastname: Dlugosz
authors.forename: J
authors.initials: J
authors.lastname: Kapor-Drezgic
authors.forename: S
authors.initials: S
authors.lastname: Munk
authors.forename: C
authors.initials: C
authors.lastname: Whiteside
mesh_terms: D000199:Actins / Q000378:metabolism / Q000502:physiology*; D000818:Animals; D002478:Cells, Cultured; D005455:Fluorescent Antibody Technique; D005920:Glomerular Mesangium / Q000166:cytology / Q000378:metabolism*; D005947:Glucose / Q000494:pharmacology*; D007527:Isoenzymes / Q000378:metabolism; D008297:Male; D018613:Microscopy, Confocal; D008856:Microscopy, Fluorescence; D011108:Polymers / Q000378:metabolism*; D011493:Protein Kinase C / Q000378:metabolism / Q000502:physiology*; D051381:Rats; D017207:Rats, Sprague-Dawley
chemical_list: D000199:Actins; D007527:Isoenzymes; D011108:Polymers; C024617:polyol; D011493:Protein Kinase C; D005947:Glucose