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）：内皮功能障碍有助于糖尿病的微血管和大血管并发症。内皮细胞对血管损伤的主要防御是内皮一氧化氮合酶（ENOS），它在存在最佳浓度的底物L-精氨酸和辅助因子四氢异物蛋白（BH4）的情况下会产生一氧化氮（NO）。糖尿病性肾病（DN）与eNOS功能障碍引起的进行性无缺陷状态有关，这种现象称为“解偶联”。最近在1型糖尿病的实验模型中报道了肾脏中NO中NO中NO的ENOS功能障碍和降低的生物利用度，并且最近的研究最终确定了ENOS敲除小鼠使糖尿病的发育发育和进步DN的最终研究确定了NO ENOS在肾脏中的保护作用。尽管这些研究确立了eNOS的保护作用，但在DN中灭活eNOS的机制尚未清楚地鉴定出来。如果要将上述观察结果转化为旨在重新配置eNOS的临床治疗方案，恢复没有生产并减少活性氧（ROS）生成，那么了解上述观察结果是必不可少的。我们的初步数据表明，在孤立的肾小球中，内皮和中膜完整，ENOS会产生ROS，而在高葡萄糖浓度（HG）或血管紧张素II（ANG II）的刺激下，NO量较低。 eNOS在肾小球内皮和细胞细胞中表达，酶在暴露于Hg或Ang II后会产生ROS，较少的NO。 HG和ANG II还会导致二氢叶酸还原酶的降低和精氨酸I蛋白水平的增加，这两种酶分别调节了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，在培养的肾细胞中导致细胞肥大和纤连蛋白表达。使用反义寡核苷酸治疗对NOX4氧化酶的抑制可减少1型糖尿病大鼠中糖尿病诱导的NOX4表达，肾脏ROS产生，肥大和纤连蛋白表达。我们的核心假设是，NOX氧化酶和NOX4产生的ROS在eNOS中脱离肾小球膜和内皮和糖尿病的肾脏中起着关键作用，从而导致消除Enos的保护作用，还可以使Enzyme的保护效果转化为eNzyme的生成，从而增强了pherogistal sefartial rosister rogistal rosister rogials rog ofer side sine for in a phlogistal sen conlogistal sen conlogistal sen ins ins ins ins ins ins of pherogistion sen ins ins ins ins ins ins ins consection。我们提出，NOX4或其他NOX氧化酶产生的ROS会导致锌硫酸盐簇中的半胱氨酸残基对eNOS活性必不可少，或者降低底物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外基质的积累和核因子 - 卡帕布激活中的作用将在体外和体内探索。公共卫生相关性：糖尿病和糖尿病性肾病是普通人群发病率的主要原因。一氧化氮生物利用度的氧化应激和减少会导致糖尿病并发症。但是，氧气自由基的确切来源和这些事件中涉及的过程尚未完全定义。我们希望确定特定的氧化剂来源将允许有针对性的治疗预防糖尿病性肾病，而我们的长期目标是将我们的发现转移到转化领域，以治疗特定的抗氧化剂治疗患者。