MECHANISMS AND ACTIONS OF H2O2 PRODUCTION IN THE mTAL OF THE DAHL S RAT

DAHL S 大鼠脑中 H2O2 产生的机制和作用

• 批准号: 9091603
• 负责人: Allen W Cowley
• 金额: $ 44.17万
• 依托单位: MEDICAL COLLEGE OF WISCONSIN
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/9091603
• 关键词: Accounting; Animals; Automobile Driving; Back; Biological Models; Blood Pressure; Blood flow; Cell membrane; Computers; Coupled; Dahl Hypertensive Rats; Diffuse; Diffusion; Epithelial Cells; Event; Exhibits; Feeds; Fluorescent Probes; Fumarate Hydratase; Fumaric acid; Genes; Genetic Engineering; Homeostasis; Hydrogen Peroxide; Hypertension; Infiltration; Injury; Intake; Kidney; Knowledge; Lead; Limb structure; Malignant - descriptor; Malignant Hypertension; Mediating; Mitochondria; Molecular; NADPH Oxidase; Nitric Oxide; Organ; Perfusion; Pericytes; Phase; Play; Process; Production; Protocols documentation; Rattus; Rectum; Regulation; Renal Hypertension; Renal function; Research; Resistance; Role; Signal Transduction; Signaling Molecule; Sodium; Sodium Chloride; Staging; Superoxides; System; T-Lymphocyte; Techniques; Testing; Thick; Tissues; Transgenic Organisms; Tubular formation; Whole Organism; antioxidant therapy; blood pressure regulation; cellular imaging; design; enzyme pathway; feeding; fluorescence imaging; fluorophore; inhibitor/antagonist; interstitial; kidney medulla; neutrophil cytosol factor 67K; new therapeutic target; novel; novel strategies; null mutation; overexpression; pressure; protein expression; response; salt intake; salt sensitive; salt sensitive hypertension; vasoconstriction

Superoxide and nitric oxide production within the outer medulla (OM) of the kidney are known to play an important role in sodium homeostasis and in salt-sensitive hypertension and renal injury. Little is yet known about the highly reactive H2O2 molecule which is involved in these processes and may account for as much as 50% of the hypertension and renal injury observed in the Dahl salt-sensitive (SS) rat. We hypothesize that increased NaCI delivery to the medullary thick ascending limb (mTAL) of the OM, as occurs following an increase in NaCI intake, stimulates mitochondrial production of H2O2 in the mTAL epithelial cells that diffuses to the cell membrane and enhances the activity of NADPH oxidase leading to an overall increase of cellular levels of H2O2 (Aim 1). We propose that this H2O2 response is significantly amplified in SS rats by greater expression of the p67phox cytosolic subunit of NADPH oxidase compared to a salt-resistant control strain as explored in Aim 2. The contribution of p67phox to these events will be determined ufilizing SS rats with a ubiquitous null mutation in the p67phox gene and salt-resistant SS.13BN26 rats with mTAL-specific transgenic overexpression of p67phox. In Aim 3, studies will determine if the greater production of H2O2 in the mTAL of SS rats results in diffusion of H2O2 into the interstitial space of the surrounding vasa recta which results in pericyte-mediated vasoconstriction and reduction of medullary blood flow leading to the initial moderate rise of arterial pressure. Aim 4 will determine if the rise of blood pressure with salt intake provokes renal T-lymphocyte infiltration, excess p67phox and fumaric acid in the mTAL leading to greater H2O2 production and a progression from a mild to severe form of hypertension and renal injury. This is a highly collaborative protocol between Projects 1, 2 and 3 that will utilize a computer-controlled system to examine the consequences of the elevated renal perfusion pressure by protecfing one kidney from the hypertension while the other is exposed to the elevated blood pressure. Since technical limitations have impeded a thorough mechanistic understanding of the role of H2O2 in renal function and hypertension, a number of new fluorescent imaging approaches, a novel fluorescent probe that specifically detects mitochondrial changes of H2O2, and a novel inhibitor of mitochondrial H2O2 will be utilized to advance our understanding of this field. Several novel genetically engineered rat model systems have been developed to test several of the key hypotheses including an SS rat with a ubiquitous null mutation in the p67phox gene and a salt-insensitive SS.13BN26 in which p67phox is transgenically overexpressed only in the thick ascending limb of Henle. H2O2 appears to be an important signaling molecule in the OM of the kidney. If more effective antioxidant therapies are to be developed it will require knowledge of the expression and regulation of the key pathways and enzymes responsible for H2O2 formation and their functional relevance at the level of the tissue and whole organism. By identifying the two novel controllers of H2O2 production, one related to the mitochondria and the other to the p67phox gene, we anticipate identifying new therapeutic targets for hypertension.

已知肾脏外髓(OM)中产生的超氧化物和一氧化氮在其中起作用 在钠平衡、盐敏感型高血压和肾脏损伤中起重要作用。目前还知之甚少 关于高活性的过氧化氢分子，它参与了这些过程，可能解释了 Dahl盐敏感型(SS)大鼠的高血压和肾脏损伤的发生率为50%。我们假设 向OM的髓质粗大升支(MTAL)的NaCI释放增加，这发生在 NaCI摄入量的增加，刺激mTAL上皮细胞线粒体产生过氧化氢，并扩散到 并增强NADPH氧化酶的活性，导致细胞水平的全面提高 过氧化氢(目标1)。我们认为SS大鼠的这种H_2O_2反应通过更高的表达而显著放大 NADPH氧化酶的p67Phox胞浆亚基与耐盐对照菌株的比较 目的2.p67Phox在这些事件中的作用将通过利用SS大鼠普遍存在的空基因来确定 MTAL特异性转基因过表达p67Phox基因突变与耐盐SS.13BN26大鼠 P67Phox。在目标3中，研究将确定SS大鼠mTAL中更多的过氧化氢产生是否会导致 H_2O_2扩散到周围直肠血管的间质间隙，导致周细胞介导 血管收缩和髓质血流量减少导致动脉压开始适度升高。 目标4将确定盐摄入量引起的血压升高是否会刺激肾脏T淋巴细胞的渗透， MTAL中过量的p67Phox和富马酸导致更多的过氧化氢产生，并从轻微的 到严重形式的高血压和肾脏损伤。这是项目1、2之间高度协作的协议 3将利用计算机控制系统来检查肾脏升高的后果 通过保护一个肾脏免受高血压，而另一个肾脏暴露在升高的 血压。 因为技术上的限制阻碍了对过氧化氢在体内的作用的彻底的机械理解 肾功能与高血压，一些新的荧光成像方法，一种新的荧光探针 它可以特异性地检测线粒体过氧化氢的变化，而一种新的线粒体过氧化氢抑制剂将是 用来促进我们对这一领域的理解。几种新型基因工程大鼠模型系统 已经被开发来测试几个关键的假设，包括带有普遍存在的零突变的SS大鼠 在p67Phox基因和盐不敏感SS.13BN26中，p67Phox仅在 粗大的亨勒上肢。H_2O_2似乎是肾脏OM中的重要信号分子。如果 更有效的抗氧化剂疗法将被开发出来，这将需要了解表达和 H_2O_2形成的关键途径和酶的调节及其功能相关性 组织和整个有机体的水平。通过确定两个新的过氧化氢产生控制器，一个 一个与线粒体有关，另一个与p67Phox基因有关，我们预计会发现新的治疗靶点。 治疗高血压。