Zhou X, Hurst R D, Templeton D, Whiteside C I
Department of Medicine, University of Toronto, Canada.
Lab Invest. 1995 Sep;73(3):372-83.
Glomerular mesangial and epithelial cell structure and function are maintained by cytoskeletal protein organization and function. To determine whether the diabetic milieu alters filamentous (F-) actin assembly, the spatial distributions and content of F- and monomeric (G-) actin were analyzed in rat mesangial and glomerular epithelial cells (10 to 15 passages) cultured for 5 days in high (22.4 mM) or normal (5.2 mM) glucose and in cells of whole glomeruli isolated from streptozotocin-treated diabetic or normal rats.
Cells were labeled with the fluorescent probes rhodamine-phalloidin and FITC-DNase-1 specific for F- and G-actin, respectively. The average pixel intensities per cell were measured using dual channel confocal laser scanning microscopy (N = 60 cells per group). Total and G-actin were measured in mesangial cells by a spectrophotometric-based DNase-1 inhibition assay.
In response to endothelin-1, 0.1 microM, vasopressin 1.0 microM, or angiotensin II 1.0 microM, mesangial cells cultured in normal glucose displayed partial disassembly of F-actin characterized by decreased fluorescence intensity (microfilament bundle pattern changed to network) with no change in G-actin fluorescence. In high glucose, but not mannitol (22.4 mM), partial disassembly of F-actin and loss of response to the agonists were observed. In high glucose, the F-actin content (micrograms/mg cellular protein) was reduced significantly with no change in absolute G-actin compared with normal glucose exposure. The effect of high glucose on mesangial cell actin was reversed by returning the cells to normal glucose for 2 days, stimulation with insulin 2 micrograms/ml, or with a protein kinase C inhibitor. Mesangial cells in high glucose were smaller in planar area and exhibited loss of contractile response to endothelin-1 (0.1 microM) or vasopressin (1.0 microM) measured by videomicroscopy. High glucose-induced F-actin disassembly, possibly due to activated protein kinase C, could account for smaller cell size and lack of response to vasopressor agents. Glomerular epithelial cells cultured in normal glucose demonstrated F-actin disassembly and increased G-actin fluorescence intensity in response to A23187 (5 microM) or bradykinin (10 nM). When cultured in high glucose, but not mannitol, increased epithelial G-actin fluorescence and loss of F- and G-actin response to agonists were observed. Although stimulation with insulin reversed the high glucose effect on epithelial G-actin, F-actin remained unresponsive to agonists. The cells of glomeruli isolated from the diabetic rat displayed the same increase in G-actin, no change in F-actin fluorescence, and loss of response to agonist stimulation as observed in cultured epithelial cells.
These findings suggest that high glucose alters actin assembly in both glomerular mesangial and epithelial cells in vitro and in vivo, possibly contributing to cellular dysfunction in early diabetes.
肾小球系膜细胞和上皮细胞的结构与功能由细胞骨架蛋白的组织和功能维持。为了确定糖尿病环境是否会改变丝状（F-）肌动蛋白组装，对在高糖（22.4 mM）或正常糖（5.2 mM）环境中培养5天的大鼠系膜细胞和肾小球上皮细胞（传代10至15次）以及从链脲佐菌素处理的糖尿病大鼠或正常大鼠分离的整个肾小球细胞中F-肌动蛋白和单体（G-）肌动蛋白的空间分布和含量进行了分析。
分别用对F-肌动蛋白和G-肌动蛋白特异的荧光探针罗丹明-鬼笔环肽和FITC-DNase-1标记细胞。使用双通道共聚焦激光扫描显微镜测量每组60个细胞每个细胞的平均像素强度。通过基于分光光度法的DNase-1抑制试验测量系膜细胞中的总肌动蛋白和G-肌动蛋白。
在1.0 microM血管加压素、1.0 microM血管紧张素II或0.1 microM内皮素-1作用下，在正常葡萄糖环境中培养的系膜细胞显示F-肌动蛋白部分解聚，其特征为荧光强度降低（微丝束模式转变为网络状），而G-肌动蛋白荧光无变化。在高糖环境中，但不是在甘露醇（22.4 mM）环境中，观察到F-肌动蛋白部分解聚以及对激动剂的反应丧失。与正常葡萄糖暴露相比，在高糖环境中，F-肌动蛋白含量（微克/毫克细胞蛋白）显著降低，而绝对G-肌动蛋白无变化。将细胞放回正常葡萄糖环境2天、用2微克/毫升胰岛素刺激或用蛋白激酶C抑制剂处理可逆转高糖对系膜细胞肌动蛋白的影响。高糖环境中的系膜细胞平面面积较小，并且通过视频显微镜测量显示对0.1 microM内皮素-1或1.0 microM血管加压素的收缩反应丧失。高糖诱导的F-肌动蛋白解聚可能归因于活化的蛋白激酶C，这可能解释了细胞尺寸较小以及对血管加压剂无反应的原因。在正常葡萄糖环境中培养的肾小球上皮细胞在5 microM A23187或10 nM缓激肽作用下显示F-肌动蛋白解聚和G-肌动蛋白荧光强度增加。当在高糖环境中培养时，但不是在甘露醇环境中，观察到上皮细胞G-肌动蛋白荧光增加以及对激动剂的F-和G-肌动蛋白反应丧失。尽管胰岛素刺激可逆转高糖对上皮细胞G-肌动蛋白的影响，但F-肌动蛋白对激动剂仍无反应。从糖尿病大鼠分离的肾小球细胞显示出与培养的上皮细胞中观察到的相同的G-肌动蛋白增加、F-肌动蛋白荧光无变化以及对激动剂刺激的反应丧失。
这些发现表明，高糖在体外和体内均会改变肾小球系膜细胞和上皮细胞中的肌动蛋白组装，这可能是早期糖尿病细胞功能障碍的原因之一。
