Mechanism of eNOS Uncoupling in the Renal Microvasculature

肾微血管中 eNOS 解偶联的机制

基本信息

批准号：
8512710
负责人：
Yves Claude Gorin
金额：
$ 29.44万
依托单位：
UNIVERSITY OF TEXAS HLTH SCIENCE CENTER
依托单位国家：
美国
项目类别：
财政年份：
2010
资助国家：
美国
起止时间：
2010-07-01 至 2015-06-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8512710
关键词：
Adenovirus Vector Albumins Angiotensin II Antisense Oligonucleotide Therapy Antisense Oligonucleotides Arginine Biochemical Biological Availability Blood Pressure Blood Vessels Cells Clinical Complement Complex Complications of Diabetes Mellitus Consensus Sequence Consumption Creatinine clearance measurement Cysteine Data Diabetes Mellitus Diabetic Angiopathies Diabetic Nephropathy Dihydrofolate Reductase Down-Regulation Electron Transport Endothelial Cells Endothelium Enzymes Event Excretory function Experimental Models Exposure to Extracellular Matrix Fibronectins Functional disorder General Population Generations Genetic Transcription Glomerular Mesangial Cell Glucose Goals Health Hyperglycemia Hypertrophy Immunohistochemistry In Vitro Injury Insulin-Dependent Diabetes Mellitus Intervention Isoenzymes Kidney Kidney Diseases Kidney Glomerulus Knockout Mice Lead Lesion Measures Mediating Mediator of activation protein Mitochondria Mitochondrial DNA Morbidity - disease rate Mus NADP Nitric Oxide Nox enzyme Nuclear Oxidants Oxidases Oxidation-Reduction Oxidative Stress Pathogenesis Pathway interactions Patients Peroxonitrite Play Positioning Attribute Process Production Protein Isoforms Proteins Rattus Reactive Oxygen Species Regimen Renin-Angiotensin System Reporting Role Severities Small Interfering RNA Source Streptozocin Structure of glomerular mesangium Superoxides Testing Therapeutic Therapeutic Intervention Transgenic Animals Transgenic Mice Translating Urine Western Blotting Wild Type Mouse antioxidant therapy arginase cell injury cellular transduction cofactor connective tissue growth factor cytokine diabetic diabetic rat dimer glomerular endothelium human NOS3 protein in vivo kidney cell kidney cortex macrovascular disease mesangial cell mitochondrial dysfunction monomer overexpression oxidant stress oxidation prevent promoter protective effect protein expression research study respiratory protein response tetrahydrobiopterin traditional therapy transcription factor type I diabetic

项目摘要

DESCRIPTION (provided by applicant): Endothelial dysfunction contributes to microvascular and macrovascular complications of diabetes. A major defense of endothelial cells against vascular injury is endothelial nitric oxide synthase (eNOS), which generates nitric oxide (NO) in the presence of optimal concentrations of the substrate L-arginine and the cofactor tetrahydrobiopterin (BH4). Diabetic nephropathy (DN) is associated with a state of progressive NO deficiency due to eNOS dysfunction, a phenomenon referred to as "uncoupling". eNOS dysfunction and decreased bioavailability of NO in the kidney have recently been reported in an experimental model of type 1 diabetes and the protective role of NO generation by eNOS in the kidney has been conclusively established by recent studies showing that eNOS knockout mice made diabetic develop advanced lesions and progressive DN. While these studies established a protective role for eNOS, the mechanism(s) by which eNOS is inactivated in DN are not clearly identified. Understanding the mechanisms of uncoupling of eNOS in type 1 diabetes is essential if the above observations are to be translated to clinical therapeutic regimens aimed at recoupling of eNOS, restoring NO production and decreasing reactive oxygen species (ROS) generation during the course of DN. Our preliminary data show that in isolated glomeruli, where endothelium and mesangium are intact, eNOS generates ROS and lesser amount of NO upon stimulation with high glucose concentration (HG) or angiotensin II (Ang II). eNOS is expressed in glomerular endothelial as well as in mesangial cells and the enzyme generates ROS and less NO after exposure of the cells to HG or Ang II. HG and Ang II also cause a decrease in dihydrofolate reductase and an increase in arginase I protein levels, two enzymes that regulate availability of BH4 and L-arginine, respectively. Ang II and HG result in cell hypertrophy and fibronectin expression in cultured renal cells through generation of ROS via the NAD(P)H oxidase isoenzyme Nox4. Inhibition of Nox4 oxidase using an antisense oligonucleotide therapy reduces diabetes-induced Nox4 expression, renal ROS generation, hypertrophy and fibronectin expression in type 1 diabetic rats. Our central hypothesis is that the ROS generated by Nox oxidases and specifically Nox4 play a pivotal role in eNOS uncoupling in the glomerular mesangium and endothelium and in the kidney in diabetes, thereby resulting not only in the elimination of the protective effect of eNOS, but also converting the enzyme to a phlogistic mediator that further enhances ROS generation. We propose that ROS generated by Nox4 or other Nox oxidases result in oxidation of cysteine residues in the zincthiolate cluster essential for the activity of eNOS, and or decrease the levels of the substrate L-arginine and the cofactor BH4. The role of these pathways in hypertrophy, fibrogenic cytokine expression extracellular matrix accumulation and nuclear factor-kappaB activation will be explored in vitro and in vivo. PUBLIC HEALTH RELEVANCE: Diabetes and diabetic nephropathy are major causes of morbidity in the general population. Oxidative stress and decrease in nitric oxide bioavailability contributes to diabetic complications; however, the precise sources of oxygen radicals and the processes involved in these events are not completely defined. It is our hope that identifying specific sources of oxidants will allow targeted therapy to prevent diabetic nephropathy and our long-term goal is to move our findings into the translational arena to treat the patients with specific antioxidant therapy.

描述（由申请人提供）：内皮功能障碍导致糖尿病的微血管和大血管并发症。内皮细胞抵抗血管损伤的主要防御机制是内皮一氧化氮合酶 (eNOS)，它在最佳浓度的底物 L-精氨酸和辅因子四氢生物蝶呤 (BH4) 存在下产生一氧化氮 (NO)。糖尿病肾病 (DN) 与 eNOS 功能障碍导致的进行性 NO 缺乏状态有关，这种现象称为“解偶联”。最近在 1 型糖尿病实验模型中报道了肾脏中 eNOS 功能障碍和 NO 生物利用度降低，并且最近的研究已最终确定了肾脏中 eNOS 生成 NO 的保护作用，表明 eNOS 敲除小鼠使糖尿病发展为晚期病变和进行性 DN。虽然这些研究确立了 eNOS 的保护作用，但 eNOS 在 DN 中失活的机制尚不清楚。如果要将上述观察结果转化为旨在在 DN 过程中重新偶联 eNOS、恢复 NO 产生并减少活性氧 (ROS) 产生的临床治疗方案，那么了解 1 型糖尿病中 eNOS 解偶联的机制至关重要。我们的初步数据表明，在分离的肾小球中，内皮和系膜完整，eNOS 在高浓度葡萄糖 (HG) 或血管紧张素 II (Ang II) 刺激下产生 ROS 和少量 NO。 eNOS 在肾小球内皮细胞和系膜细胞中表达，在细胞暴露于 HG 或 Ang II 后，该酶会产生 ROS 和较少的 NO。 HG 和 Ang II 还会导致二氢叶酸还原酶减少和精氨酸酶 I 蛋白水平增加，这两种酶分别调节 BH4 和 L-精氨酸的可用性。 Ang II 和 HG 通过 NAD(P)H 氧化酶同工酶 Nox4 产生 ROS，导致培养的肾细胞中细胞肥大和纤连蛋白表达。使用反义寡核苷酸疗法抑制 Nox4 氧化酶可减少 1 型糖尿病大鼠中糖尿病诱导的 Nox4 表达、肾脏 ROS 生成、肥大和纤连蛋白表达。我们的中心假设是，Nox 氧化酶（特别是 Nox4）产生的 ROS 在糖尿病肾小球系膜和内皮以及肾脏中的 eNOS 解偶联中发挥关键作用，从而不仅导致 eNOS 的保护作用消除，而且将酶转化为炎性介质，进一步增强 ROS 的产生。我们认为，Nox4 或其他 Nox 氧化酶产生的 ROS 会导致硫醇锌簇中对于 eNOS 活性至关重要的半胱氨酸残基的氧化，和/或降低底物 L-精氨酸和辅因子 BH4 的水平。这些途径在肥大、纤维化细胞因子表达、细胞外基质积累和核因子-κB 激活中的作用将在体外和体内进行探索。公共卫生相关性：糖尿病和糖尿病肾病是普通人群发病的主要原因。氧化应激和一氧化氮生物利用度的降低会导致糖尿病并发症；然而，氧自由基的确切来源以及这些事件所涉及的过程尚未完全确定。我们希望，识别特定的氧化剂来源将能够进行靶向治疗来预防糖尿病肾病，我们的长期目标是将我们的发现转化为转化领域，以特定的抗氧化疗法治疗患者。