Salt-sensitive hypertension: Role of renal superoxide

描述(由申请人提供)：异常粗大的升肢(THAL)盐重吸收与几种形式的高血压有关，包括盐敏性高血压。我们报道了NO抑制盐的重吸收，而O2-刺激盐的重吸收。NO的减少和O2-的增加都与盐敏感和其他形式的高血压有关。高盐饮食可增强流经TALS的管腔血流。我们已经证明，流动刺激NO。在没有L-精氨酸的情况下，FLOW也会增强O2-(因此也会增加NO)。相反，在L的存在下，精氨酸流动诱导的O2-减少了80%。到目前为止，人们认为NO只会通过清除产生剧毒的ONOO来减少O2。然而，我们发现NO引起的O2-的减少依赖于cGMP信号。这一新发现以前从未被报道过。与清除相比，cGMP诱导的O2-减少将是有益的，因为剧毒的ONOO-不会像没有清除O2时那样形成，NO也不会被破坏。NO诱导的O2-减少可能是由于：1)NADPH氧化酶(其主要来源为Thals)减少O2-的产生；或2)超氧化物歧化酶促进O2-的降解。产量的下降可能是由于抑制了p47Phox和p67Phox亚基与催化亚基的结合而导致的。这种组装通常是由蛋白激酶C(PKC)依赖的p47Phox的磷酸化刺激的。然而，我们不知道NO是如何调节O2-水平的，也不知道它对盐吸收的影响。我们推测，在Thals中，流动刺激的NO主要通过激活cGMP依赖的蛋白激酶(PKG)来减少流动诱导的O2-；这降低了PKC的活性，进而阻止了基于NOx 4的NADPH氧化酶的流动诱导激活，从而阻止了O2依赖的氯化钠吸收。NO减少O2-产生的能力缺陷导致了氯化钠滞留和盐敏性高血压。这一假设将在四个目标上得到检验。目的1将通过cGMP依赖的机制，测试在Thals中，流刺激的NO是否通过cGMP依赖机制减少流诱导的O2-，从而减少O2-依赖的氯化钠吸收。这种影响在盐敏感型高血压中会减弱。Aim 2将测试流动刺激的NO/cGMP是否通过激活PKG II来减少基于NOx 4的NADPH氧化酶产生O2-。Aim 3将测试流动刺激的NO/cGMP/PKG II是否会降低流动刺激的PKC活性，从而减少基于NOx 4的NADPH氧化酶产生O2-。目的4将测试非激活的PKG是否通过降低PKC活性从而钝化p47Phox的激活和易位来抑制流动诱导的基于NOx4的NADPH氧化酶组装。在盐敏感型高血压中，NO的作用减弱。我们将使用从整体动物生理学到分子生物学的广泛技术。这些技术中的许多都是高度创新的，是我们实验室为这些和类似的研究而开发的。这些研究的成功完成将进一步加深我们对NO和O2-在生理和病理生理条件下如何调节肾功能的理解。它们还可能为高血压的治疗带来新的靶点。 公共卫生相关性：叙述性氧化应激在多种病理条件下发挥重要作用，包括多种形式的高血压、糖尿病引起的肾功能改变、慢性肾功能衰竭和肾脏缺血损伤。然而，调控粗大上肢氧化应激的因素还没有得到广泛的研究。与氧化应激相反，一种名为一氧化氮的化合物促进利钠和利尿，其缺失已被认为与肾脏损伤和高血压有关。在这项提案中，我们将研究流动刺激的一氧化氮是否会抑制超氧化物的产生，这是一种导致最大氧化应激的化学物质。如果被证明是正确的，我们的假设可能解释为什么：1)一氧化氮的减少和超氧化物的增加都与盐敏感型高血压有关；2)在糖尿病的早期阶段，肾脏几乎没有损害。这项提案的数据将增加我们对钠滞留原因的理解，并可能导致开发新的利尿剂和治疗高血压引起的肾损伤和慢性肾功能衰竭的新疗法。

DESCRIPTION (provided by applicant): Abnormal thick ascending limb (THAL) NaCl reabsorption has been implicated in several forms of hypertension including salt-sensitive hypertension. We reported that THAL NaCl reabsorption is inhibited by NO and stimulated by O2- . Both reductions in NO and increases in O2- have been implicated in salt-sensitive as well as other forms of hypertension. A high-salt diet enhances luminal flow through THALs. We have shown that flow stimulates NO. Flow also enhances O2- in the absence of L-arginine (and therefore NO). In contrast, in the presence of L-arginine flow-induced O2- is reduced by >80%. Until now, it was thought that NO only reduces O2- by scavenging, producing ONOO- which is highly toxic. However, we found that the reduction in O2- caused by NO depends upon cGMP signaling. This novel finding has not been reported before. cGMP-induced reductions in O2- would be expected to be beneficial compared to scavenging because the highly toxic ONOO- is not formed as it is when NO scavenges O2- and NO is not destroyed. NO-induced reductions in O2- may result from either: 1) a decrease in O2- production by NADPH oxidase (its primary source in THALs); or 2) enhanced degradation by superoxide dismutase. A decrease in production may result from inhibiting the association of the p47phox and p67phox subunits with the catalytic subunit. This assembly is normally stimulated by protein kinase C (PKC)- dependent phosphorylation of p47phox. However, we do not know how NO regulates O2- levels, nor its effects on NaCl absorption. We hypothesize that in THALs flow-stimulated NO reduces flow-induced O2- primarily by activating cGMP-dependent protein kinase (PKG); this reduces PKC activity, which in turn prevents flow-induced activation of Nox 4-based NADPH oxidase and consequently O2--dependent NaCl absorption. Defects in the ability of NO to reduce O2- production contribute to NaCl retention and salt-sensitive hypertension. This hypothesis will be tested in four aims. Aim 1 will test whether in THALs flow-induced O2- and therefore O2--dependent NaCl absorption is reduced by flow-stimulated NO via a cGMP-dependent mechanism. This effect is blunted in salt- sensitive hypertension. Aim 2 will test whether flow-stimulated NO/cGMP reduces O2- production by Nox 4-based NADPH oxidase via activation of PKG II. Aim 3 will test whether flow-stimulated NO/cGMP/PKG II reduces flow- stimulated PKC activity and hence O2- production by Nox 4-based NADPH oxidase. Aim 4 will test whether NO- activated PKG inhibits flow-induced Nox 4-based NADPH oxidase assembly by reducing PKC activity and thereby blunting p47phox activation and translocation. NO's effect is reduced in salt-sensitive hypertension. We will use a wide range of techniques from whole animal physiology to molecular biology. Many of these techniques are highly innovative and were developed in our laboratory for these and similar studies. Successful completion of the proposed studies will further our understanding of how NO and O2- regulate renal function under physiological and pathophysiological conditions. They may also lead to new targets for the treatment of hypertension. PUBLIC HEALTH RELEVANCE: Narrative Oxidative stress plays important roles in several pathological conditions, including many forms of hypertension, diabetes-induced changes in renal function, chronic renal failure and renal ischemic injury. However, the factors that regulate oxidative stress in the thick ascending limb have not been extensively studied. In contrast to oxidative stress, a compound called nitric oxide promotes natriuresis and diuresis, and its loss has been implicated in renal damage and hypertension. In this proposal, we will study whether flow-stimulated nitric oxide inhibits superoxide production, a chemical that causes most oxidative stress. If proven correct, our hypothesis may explain why: 1) decreases in nitric oxide and increases in superoxide are both implicated in salt-sensitive hypertension; and 2) there is little renal damage in the early stages of diabetes. Data from this proposal will increase our understanding of the causes of sodium retention and may result in the development of new diuretics and new therapies for hypertension-induced renal damage and chronic renal failure.