Effect of high glucose on nitric oxide production and endothelial nitric oxide synthase protein expression in human glomerular endothelial cells
Effect of high glucose on nitric oxide production and endothelial nitric oxide synthase protein expression in human glomerular endothelial cells
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DOI：
10.1159/000073673
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发表时间：
2003-10-01
影响因子：
--
通讯作者：
Oite, T
中科院分区：
文献类型：
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作者：
Hoshiyama, M;Li, B;Oite, T
背景：高血压直接导致糖尿病肾病的发生。一氧化氮（NO）是一种有效的内皮源性血管扩张剂，已被认为参与肾血流、肾小球滤过率和系膜基质积聚的调节。已知人血管内皮细胞具有功能异质性，这促使我们首次研究了高糖暴露对人肾小球内皮细胞（HGECs）中NO生物利用度的影响。研究方法：通过Griess试验检测HGECs中亚硝酸盐的产生，在L-精氨酸（1 mM）或超氧化物歧化酶（SOD）（250 U/ml）存在或不存在的情况下，在不同的时间段（24，48和72 h）暴露于对照水平（5.5 mM）和高水平（15，30和60 mM）葡萄糖溶液中，检测NO的释放。此外，我们评估了葡萄糖对内皮型一氧化氮合酶（eNOS）在HGECs的表达的影响，通过蛋白质印迹。结果如下：在高糖暴露后，HGECs中产生的亚硝酸盐的最终水平以时间和浓度依赖性的方式显著降低。然而，蛋白质印迹分析显示，eNOS蛋白表达显着上调后12小时暴露于高浓度的葡萄糖（30 mM），在48小时达到峰值（基线水平的两倍增加）。高糖对NO生成的抑制作用可通过加入SOD而恢复。外源性L-精氨酸（1 mM）也可逆转高糖对HGECs NO生成的抑制作用。结论：高糖可增加eNOS蛋白表达，但最终导致NO释放减少。NO生物利用度的降低似乎与超氧化物的过度产生和L-精氨酸缺乏有关。这些发现为阐明葡萄糖升高导致NO和超氧化物之间失衡，导致内皮功能受损的机制的分子基础提供了重要线索。此外，通过给予L-精氨酸和摄入足够的抗氧化剂来恢复NO功能，表明对糖尿病肾病患者有潜在的支持性治疗。版权所有（C）2003 S. Karger AG，巴塞尔
Background: Hyperglycemia directly contributes to the development of diabetic nephropathy. Nitric oxide (NO), a potent endothelium-derived vasodilator, has been suggested to participate in the regulation of renal blood flow, glomerular filtration rate, and mesangial matrix accumulation. Human vascular endothelial cells are known to exhibit functional heterogeneity, this prompted us to do the first study of NO bioavailability in human glomerular endothelial cells (HGECs), in response to high glucose exposure. Methods: NO release was examined by detecting nitrite generation by the Griess assay in HGECs exposed to control-level (5.5 mM) and high-level (15, 30 and 60 mM) glucose solutions at various time periods (24, 48 and 72 h) in the presence or absence of L-arginine (1 mM), or superoxide dismutase (SOD) (250 U/ml). In addition, we evaluated the effect of glucose on the expression of endothelial nitric oxide synthase (eNOS) in HGECs by Western blotting. Results: Final levels of nitrite generated in HGECs were reduced significantly, in a time- and concentration-dependent manner, after high glucose exposure. However, Western blot analysis revealed that eNOS protein expression was significantly upregulated at 12 h after exposure to high glucose concentrations (30 mM), reaching a peak at 48 h (twofold increase over baseline levels). The inhibitory effect of high glucose on NO production was restored by the addition of SOD. Addition of L-arginine (1 mM) to external media also reversed the inhibitory effect of high glucose on NO production of HGECs as well. Conclusions: The present study demonstrated that high glucose increased eNOS protein expression, but decreased NO release finally. Decreased NO bioavailability seems to be associated with overproduction of superoxide and L-arginine deficiency. These findings provide an important clue in clarifying the molecular basis of the mechanisms by which elevated glucose leads to an imbalance between NO and superoxide, resulting in impaired endothelial function. In addition, restoration of NO function by both administration of L-arginine and adequate intake of antioxidants suggests a potential supportive treatment for patients with diabetic nephropathy. Copyright (C) 2003 S. Karger AG, Basel