Ha Tae-Sun
Department of Pediatrics, College of Medicine, Chungbuk National University, Cheongju, Korea.
Nephrology (Carlton). 2006 Oct;11(5):435-41. doi: 10.1111/j.1440-1797.2006.00668.x.
To investigate the molecular basis for the phenotypic alterations of glomerular epithelial cells (GEpC, podocytes), involving cytoskeletal changes especially on alpha-actinin-4 as a candidate regulating the barrier to protein filtration and the podocyte actin cytoskeleton.
To examine the effects of glucose and advanced glycosylated end-products (AGE) on alpha-actinin-4, the author cultured rat GEpC on AGE- or BSA-coated plates under normal (5 mmol) and high glucose (30 mmol) conditions and examined the distribution of alpha-actinin by confocal microscope and measured the change in alpha-actinin-4 production by western blotting and reverse transcription-polymerase chain reaction.
Confocal microscopy indicated that alpha-actinin-4 moved from the peripheral cytoplasm to inner actin filament complexes in the presence of AGE and high glucose. These changes might be related to the fusion of microvilli of cell surface examined by electron microscopy. In western blot analysis, AGE significantly decreased the amount of alpha-actinin by 28.1%. Furthermore, the combination of high glucose and AGE decreased the amount of alpha-actinin more significantly by 53.6% compared with that of the control. The mRNA expression for alpha-actinin-4 was not changed with high glucose or AGE-coated surfaces; however, when added, the combination of high glucose and AGE significantly decreased the expression of alpha-actinin-4 mRNA by 15.7% compared with that of the control.
The author suggests that both high glucose and AGE (either individually or in combination) induce the cytoplasmic translocation and the combination suppresses the production of alpha-actinin-4 at the transcriptional level with post-translational modification and these in vitro changes may explain the cytoskeletal changes of GEpC in diabetic conditions.
研究肾小球上皮细胞（GEpC，足细胞）表型改变的分子基础，涉及细胞骨架变化，尤其是作为调节蛋白质滤过屏障和足细胞肌动蛋白细胞骨架的候选蛋白α-辅肌动蛋白-4。
为检测葡萄糖和晚期糖基化终产物（AGE）对α-辅肌动蛋白-4的影响，作者将大鼠GEpC在正常（5 mmol）和高糖（30 mmol）条件下培养于AGE或牛血清白蛋白（BSA）包被的平板上，通过共聚焦显微镜检查α-辅肌动蛋白的分布，并通过蛋白质印迹法和逆转录-聚合酶链反应测量α-辅肌动蛋白-4产量的变化。
共聚焦显微镜显示，在AGE和高糖存在的情况下，α-辅肌动蛋白-4从外周细胞质转移至内部肌动蛋白丝复合物。这些变化可能与电子显微镜检查的细胞表面微绒毛融合有关。在蛋白质印迹分析中，AGE使α-辅肌动蛋白的量显著减少28.1%。此外，与对照组相比，高糖和AGE联合作用使α-辅肌动蛋白的量减少更为显著，达53.6%。α-辅肌动蛋白-4的mRNA表达在高糖或AGE包被表面时未发生变化；然而，当高糖和AGE联合作用时，与对照组相比，α-辅肌动蛋白-4 mRNA的表达显著降低15.7%。
作者认为，高糖和AGE（单独或联合）均诱导细胞质转位，且联合作用在转录水平抑制α-辅肌动蛋白-4的产生，并伴有翻译后修饰，这些体外变化可能解释糖尿病状态下GEpC的细胞骨架变化。
