NAD(P)H oxidase and uncoupled nitric oxide synthase are major sources of glomerular superoxide in rats with experimental diabetic nephropathy
NAD(P)H oxidase and uncoupled nitric oxide synthase are major sources of glomerular superoxide in rats with experimental diabetic nephropathy
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DOI：
10.1152/ajprenal.00221.2004
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发表时间：
2005-06-01
影响因子：
4.2
通讯作者：
Kashihara, N
中科院分区：
文献类型：
--
作者：
Satoh, M;Fujimoto, S;Kashihara, N
糖尿病中活性氧（ROS）的增加可能是连接糖尿病血管并发症（包括肾病）的多种致病机制的共同途径。因此，评估氧化应激产生途径对于预测和预防糖尿病并发症非常重要。然而，糖尿病肾小球中ROS的产生机制尚不清楚。为了确定糖尿病肾脏中ROS产生的来源和机制，采用链脲佐菌素诱导大鼠糖尿病。6周后，用二氢乙胺衍生化学发光法评估，链脲佐菌素大鼠肾脏肾小球ROS生成增加。添加NADH或l -精氨酸可增加ROS的产生，添加二苯碘或n - g -硝基l -精氨酸甲酯可部分减少ROS的产生，表明NAD(P)H氧化酶和一氧化氮（NO）合成酶（NOS）是ROS的来源。real-time RT-PCR和Western blotting检测内皮细胞NOS (eNOS) mRNA和蛋白表达量显著升高（mRNA水平升高1.3倍，蛋白水平升高1.8倍）。然而，通过低温SDS-PAGE检测，糖尿病肾小球中二聚体形式的eNOS减少。用l -精氨酸灌注2′，7′-双乙酸二氯荧光素（ROS标志物）和二氨基诺达明- 4m AM （NO标志物）肾脏后，用共聚焦激光显微镜观察未偶联NOS对肾脏ROS和NO产生的影响。在糖尿病肾脏中，由于NOS解偶联导致ROS生成加速和生物可利用NO减少。四氢生物蝶呤（BH4）是eNOS的辅助因子，可以逆转eNOS二聚体形式的减少和肾小球NO的产生。我们的研究结果表明，NAD(P)H氧化酶和eNOS的解偶联通过BH4可用性的丧失介导肾小球ROS的产生。这些机制是治疗干预的潜在关键目标。
Increased production of reactive oxygen species (ROS) in diabetes may be a common pathway linking diverse pathogenic mechanisms of diabetic vascular complications, including nephropathy. Assessment of the oxidative stress production pathway is therefore important for the prediction and prevention of diabetic complications. However, ROS production mechanisms remain unclear in diabetic glomeruli. To identify the source and determine the mechanisms of ROS production in the diabetic kidney, diabetes was induced with streptozotocin in rats. After 6 wk, glomerular ROS production had increased in the streptozotocin rat kidney, as assessed by dihydroethidium-derived chemiluminescence. ROS production was increased by the addition of NADH or L-arginine and was partially reduced by the addition of diphenylene iodonium or N-G-nitro-L-arginine methyl ester, identifying NAD(P)H oxidase and nitric oxide (NO) synthase (NOS) as ROS sources. The mRNA and protein expression of endothelial NOS (eNOS), as measured by real-time RT-PCR and Western blotting, increased significantly (mRNA level, 1.3-fold; protein level, 1.8-fold). However, the dimeric form of eNOS was decreased in diabetic glomeruli, as measured by low-temperature SDS-PAGE. Production of renal ROS and NO by uncoupled NOS was imaged by confocal laser microscopy after renal perfusion of 2',7'-dichlorofluorescein diacetate (a ROS marker) and diaminorhodamine-4M AM (a NO marker) with L-arginine. Accelerated ROS production and diminished bioavailable NO caused by NOS uncoupling were noted in the diabetic kidney. Administration of tetrahydrobiopterin (BH4), a cofactor for eNOS, reversed the decreased dimeric form of eNOS and glomerular NO production. Our results indicate that NAD(P)H oxidase and uncoupling of eNOS contribute to glomerular ROS production, mediated by the loss of BH4 availability. These mechanisms are potential key targets for therapeutic interventions.