Salt-sensitive hypertension: Role of renal superoxide

描述（由申请人提供）：异常增厚的升支（塔尔）NaCl重吸收与多种形式的高血压有关，包括盐敏感性高血压。我们报道了NO抑制塔尔对NaCl的重吸收，而O ~（2-）则促进其重吸收。NO的减少和O2-的增加都与盐敏感性以及其他形式的高血压有关。高盐饮食增强通过THAL的管腔流动。我们已经表明，流量刺激NO。流量也增强O2-在L-精氨酸（因此NO）的情况下。相比之下，在L-精氨酸的存在下，流动诱导的O2-减少> 80%。到目前为止，人们认为NO只是通过清除来减少O2-，产生毒性很强的ONOO-。然而，我们发现NO引起的O2-减少依赖于cGMP信号。这一新发现以前从未报道过。cGMP诱导的O2-减少预期与清除相比是有益的，因为当NO清除O2-时不会形成高毒性的ONOO-，并且NO不会被破坏。NO诱导的O2-减少可能是由于：1）NADPH氧化酶（其在THAL中的主要来源）产生的O2-减少;或2）超氧化物歧化酶的降解增强。生产的减少可能是由于抑制p47 phox和p67 phox亚基与催化亚基的结合。这种组装通常由p47 phox的蛋白激酶C（PKC）依赖性磷酸化刺激。然而，我们不知道NO如何调节O2-水平，也不知道它对NaCl吸收的影响。我们假设，在THAL流量刺激NO减少流量诱导O2-主要通过激活cGMP依赖性蛋白激酶（PKG），这降低了PKC活性，这反过来又阻止了流量诱导的激活Nox 4-NADPH氧化酶，因此O2-依赖性NaCl吸收。NO减少O2-产生能力的缺陷有助于NaCl潴留和盐敏感性高血压。这一假设将在四个目标进行检验。目的1将测试是否在THALs流量诱导的O2-，因此O2-依赖的NaCl吸收减少流量刺激NO通过cGMP依赖的机制。这种效应在盐敏感性高血压中减弱.目的2将测试流量刺激的NO/cGMP是否通过PKG II的活化减少基于Nox 4的NADPH氧化酶的O2产生。目的3将测试流动刺激的NO/cGMP/PKG II是否降低流动刺激的PKC活性，并因此降低基于Nox 4的NADPH氧化酶的O2产生。目的4将测试NO激活的PKG是否通过降低PKC活性抑制流动诱导的基于Nox 4的NADPH氧化酶组装，从而钝化p47 phox激活和易位。NO的作用在盐敏感性高血压中降低。我们将使用从整体动物生理学到分子生物学的广泛技术。其中许多技术是高度创新的，是在我们的实验室为这些和类似的研究开发的。成功完成拟议的研究将进一步了解NO和O2-如何在生理和病理生理条件下调节肾功能。它们还可能为高血压的治疗带来新的靶点。

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.