Role of the kidneys in hypertension: paracrine actions of NO in the renal medulla

描述(由申请人提供)：高血压困扰着大约三分之一的美国人口。肾脏通过调节细胞外体积在调节血压中起着重要作用。肾脏一氧化氮(NO)在调节细胞外容量和血压方面起着重要作用。一般情况下，抑制肾脏和肾髓质中NO的产生会导致高血压。后者是由于髓质血流和肾单位运输的改变所致。在肾髓质中，NO扩张直血管周细胞以增加髓质血流量。其中至少有一部分来自邻近粗大的亨勒环(Thal)升支。自由扩散被认为是NO离开Thal进入周细胞的主要机制。然而，我们最近发现，水通道蛋白-1(AQP-1)跨细胞膜转运NO的速度是自由扩散的4倍，而且AQP-1依赖的NO转运是内皮诱导的胸主动脉松弛所必需的。尽管Thals不会再吸收水分，但它们的基侧膜是透水的。我们的初步数据显示，这在一定程度上是由于AQP-1，它也在直血管中表达。我们假设AQP-1将NO转运出THAL并进入直血管周细胞。首先，我们将通过使用非选择性电极测量从野生型和AQP-1基因敲除(-/-)小鼠分离的单个微灌流Thal的NO外流，来研究AQP-1是否将NO转运出Thal。其次，我们将使用荧光染料和荧光共聚焦显微镜，通过测量从野生型和AQP-1-/-小鼠分离的单个微灌流直肠血管，研究AQP-1是否将NO转运到直血管周细胞。第三，我们将通过测量THAL刺激NO的产生对野生型和AQP-1-/-小鼠的NO流入AQP-1-/-小鼠下行直血管的影响，来研究AQP-1依赖的NO转运是否参与了THAL和直血管周细胞之间的肾小管血管串扰。最后，我们将检测通过基因转移技术恢复AQP-1在Thal和/或Vasa Recta周细胞中的表达的效果：1.THAL的NO外流，2.NO内流到Vasa recta，3)Thal产生的NO依赖的直血管松弛。这项建议的数据将有助于我们理解肾血流的调节。AQP-1依赖的NO从Thal转运到DVR的缺陷可能在高血压的发生发展中起作用。这一建议的结果可能为开发治疗高血压的药理工具提供新的靶点。

DESCRIPTION (provided by applicant): Hypertension afflicts approximately 1/3 of the U.S. population. The kidney plays an important role in the regulation of blood pressure through the regulation of extracellular volume. Renal nitric oxide (NO) plays an important role in the regulation of extracellular volume and thus blood pressure. Inhibition of renal NO production in general and in the renal medulla specifically causes hypertension. The latter is due to changes in medullary blood flow and nephron transport. In the renal medulla, NO dilates the vasa recta pericytes to increase medullary blood flow. At least some of this NO comes from the adjacent thick ascending limb of the loop of Henle (THAL). Free diffusion is assumed to be the primary mechanism whereby NO leaves the THAL and enters the pericyte. However we have recently shown that aquaporin-1 (AQP-1) transports NO across cell membranes 4 times faster than free diffusion and that AQP-1-dependent NO transport is required for endothelium-induced relaxation of thoracic aortas. Although THALs reabsorb no water, their basolateral membranes are water permeable. Our preliminary data show that this is in part due to AQP-1, which is also expressed in the vasa recta. We hypothesize that AQP-1 transports NO out of the THAL and into the vasa recta pericytes. First, we will investigate whether AQP-1 transports NO out of the THAL by measuring NO efflux from single, microperfused THALs isolated from wild-type and AQP-1 knockout (-/-) mice using a NO-selective electrode. Second, we will investigate whether AQP-1 transports NO into vasa recta pericytes by measuring NO influx into single, microperfused vasa recta isolated from wild-type and AQP-1 -/- mice using fluorescent dye and fluorescent confocal microscopy. Third, we will investigate whether AQP-1-dependent NO transport is involved in tubular vascular crosstalk between the THAL and vasa recta pericytes by measuring the effect of stimulating NO production by the THAL on NO influx into descending vasa recta from wild-type and AQP-1 -/- mice using a single, isolated THAL with an adjacent descending vasa recta attached. Finally, we will measure the effect of restoring AQP-1 expression by gene transfer technology specifically in the THAL, vasa recta pericytes, or both on: 1. Efflux of NO out of the THAL, 2. Influx of NO into the Vasa recta and 3) THAL-derived NO-dependent relaxation of vasa recta. Data from this proposal will contribute to our understanding of regulation of renal blood flow. Defects in AQP-1 - dependent NO transport from the THAL to the DVR may play a role in the development of hypertension. Results from this proposal may offer new targets for the development of pharmacological tools for the treatment of hypertension.