Zhou X, Li C, Dlugosz J, Kapor-Drezgic J, Munk S, Whiteside C
Department of Medicine, University of Toronto, Ontario, Canada.
Kidney Int. 1997 Jun;51(6):1797-808. doi: 10.1038/ki.1997.247.
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.
高糖改变系膜细胞细胞骨架结构与功能。我们推测高糖通过涉及多元醇途径的蛋白激酶C（PKC）机制导致系膜细胞丝状（F）肌动蛋白解聚。将大鼠系膜细胞（传代次数<10，每组N = 60）生长停滞，然后在5.6 mM（正常葡萄糖，NG）、15 mM（中等葡萄糖，MG）或30 mM（高糖，HG）葡萄糖中培养48小时，同时添加或不添加0.3 mM醛糖还原酶抑制剂托瑞司他。F肌动蛋白和球状（G）肌动蛋白分别用罗丹明 - 鬼笔环肽和异硫氰酸荧光素 - 脱氧核糖核酸酶 - 1标记。使用双通道共聚焦激光显微镜在每个细胞中同时对两种荧光探针进行成像。在HG条件下，观察到F - 肌动蛋白解聚，并通过F - /G - 肌动蛋白荧光强度比降低40%进行测量（在NG + 24.4 mM甘露醇条件下无变化）。在单独的实验中，用对大多数PKC亚型特异的BODIPY FL - 双吲哚基马来酰亚胺标记细胞，并测量每个细胞的荧光强度。在NG条件下，暴露于0.1 μM佛波酯12 - 肉豆蔻酸酯13 - 乙酸酯（PMA）15分钟导致PKC向核周和细胞核转位，且F - 肌动蛋白解聚，与单独在HG条件下的观察结果相同。在HG条件下，总PKC荧光增加50%，暴露于PMA 24小时使总PKC以及F - /G - 肌动蛋白荧光比恢复正常。在NG和HG条件下，暴露（15分钟）于0.1 μM PMA使PKC活性增加三到四倍，通过表皮生长因子受体底物的原位32P磷酸化进行测量。通过免疫荧光和共聚焦成像观察到，对二酰基甘油敏感的PKC - δ在NG条件下定位于胞质溶胶，暴露于PMA 15分钟后，转位至核周区域和质膜。在HG条件下，每个细胞的PKC - δ免疫荧光显著增加，呈细胞骨架分布模式，且强度与葡萄糖浓度相关（30 mM > 15 mM > 5.6 mM）。在HG条件下，暴露于PMA 24小时使PKC - δ荧光强度和分布模式恢复至NG条件下观察到的水平，同时防止F - 肌动蛋白解聚。托瑞司他显著降低了HG条件下观察到的总PKC和PKC - δ荧光强度以及F - 肌动蛋白解聚。免疫印迹证实HG条件下PKC - δ增加，而托瑞司他使其恢复正常。在HG条件下，无论有无PMA或托瑞司他，对二酰基甘油不敏感的PKC - ε的免疫荧光模式均未改变。我们得出结论，高糖条件下系膜细胞F - 肌动蛋白解聚可能是通过包括PKC - δ在内的对二酰基甘油敏感的PKC亚型介导的，且涉及多元醇途径。
