Novel Roles of Intracrine Angiotensin II in Proximal Tubule Cells

描述（由申请人提供）：高血压通常与肾脏的过量盐和液体潴留有关。激素血管紧张素II（Ang II）是通过调节肾脏近端小管（PT）的盐和液体重吸收来维持体内盐和液体以及血压稳态的最重要因素之一。因此，PT中Ang II的产生和作用增加可导致盐和液体潴留，从而增加血压。血管紧张素Ⅱ通过激活PT细胞顶膜和基底膜上的细胞表面受体，对PT钠和液体转运产生强大的影响。然而，我们有证据表明：a）循环和旁分泌的Ang II通过AT 1（AT 1a）受体介导的机制被培养中的PT细胞或肾脏吸收; B）将Ang II直接显微注射到细胞中可以诱导细胞内钙反应; c）细胞内Ang II可以诱导分离的大鼠肾皮质核中主要钠和氢逆向转运蛋白NHE-3的转录。在这个项目中，我们假设，顶端（AP），而不是基底外侧（BL），膜AT 1（AT 1a）受体介导的PT细胞在体外和体内，这涉及微管依赖性内吞途径的血管紧张素II的细胞内摄取的大部分。在PT细胞中选择性地在体外表达或肾内腺病毒转移细胞内Ang II蛋白刺激细胞内AT 1（AT 1a）受体以增加NHE-3的表达和活性，促进PT钠和液体重吸收，并因此诱导高血压。这一假设将在四个具体目标中得到检验。是具体的目标我，我们将测试的假设，在极化PT细胞，AP膜AT 1a受体发挥更大的作用，在介导细胞内摄取的细胞外血管紧张素II比BL膜AT 1a受体在体外和体内。在缺乏顶端AT 1a受体的情况下，AT 1b受体或内吞受体巨蛋白可能部分承担AT 1a受体的作用。在Specific Aim II中，我们将检验以下假设：在极化PT细胞中，AP膜AT 1a受体介导的Ang II细胞内摄取由胞质尾区AT 1a受体的两个主要内吞基序介导，并受微管和脂筏/小窝蛋白-1（CAV-1）依赖性机制调节。在特定目标III中，我们将检验以下假设：在极化PT细胞中，细胞内Ang II蛋白ECFP/AII的体外表达通过激活[Ca 2 +]i/PKC 1/2 II、MAP激酶ERK 1/2和NF-：B信号通路增加AP膜中NHE-3的表达和活性。最后，特定目的IV将测试以下假设：肾内腺病毒基因转移细胞内Ang II蛋白选择性地在PT中增加盐和液体重吸收，促进盐和液体潴留，从而通过AT 1（AT 1a）受体的激活增加顶端NHE-3的表达和活性来诱导高血压。这些研究将提供新的见解的重要作用和潜在的细胞和分子机制的细胞内血管紧张素II的生理调节盐和液体重吸收的近端肾小管和高血压的发病机制。 公共卫生相关性：大约三分之一的美国成年人在他们的一生中会患上高血压或高血压相关的并发症，治疗高血压疾病每年花费美国经济数千亿美元。由于激素血管紧张素II的作用导致的盐和液体潴留仍然是高血压发展和进展中最重要的因素之一。本项目研究调节肾脏近端小管细胞摄取血管紧张素II的信号机制，并研究内化的血管紧张素II如何增加近端小管的盐和液体重吸收，促进体内盐和液体潴留，从而导致高血压。该项目产生的新信息将有助于我们更好地了解高血压的肾脏机制，并开发治疗高血压的新药。

DESCRIPTION (provided by applicant): High blood pressure is often associated with excessive salt and fluid retention from the kidney. The hormone angiotensin II (Ang II) is one of the most important factors in maintaining body salt and fluid and blood pressure homeostasis by regulating salt and fluid reabsorption from proximal tubules (PT) of the kidney. Thus increased production and actions of Ang II in PTs can cause salt and fluid retention and consequently increase blood pressure. Ang II exerts powerful effects on PT sodium and fluid transport by activating cell surface receptors on apical and basolateral membranes of PT cells. However, we have evidence that a) circulating and paracrine Ang II is taken up by PT cells in culture or by the kidney via an AT1 (AT1a) receptor-mediated mechanism; b) microinjection of Ang II directly into the cells can induce intracellular calcium responses; and c) intracellular Ang II can induce transcription of the major sodium and hydrogen antiporter, NHE-3, in isolated rat renal cortical nuclei. In this project, we hypothesize that apical (AP), rather than basolateral (BL), membrane AT1 (AT1a) receptors mediate the majority of intracellular uptake of Ang II by PT cells in vitro and in vivo, which involves the microtubule-dependent endocytic pathway. In vitro expression or intrarenal adenoviral transfer of an intracellular Ang II protein selectively in PT cells stimulates intracellular AT1 (AT1a) receptors to increase the expression and activity of NHE-3, promotes PT sodium and fluid reabsorption, and therefore induce hypertension. This hypothesis will be tested in four specific aims. Is Specific Aim I, we will test the hypothesis that in polarized PT cells, AP membrane AT1a receptors play a greater role in mediating intracellular uptake of extracellular Ang II than BL membrane AT1a receptors in vitro and in vivo. In the absence of apical AT1a receptors, AT1b receptors or the endocytic receptor megalin may partially assume the role of AT1a receptors. In Specific Aim II, we will test the hypothesis that in polarized PT cells, AP membrane AT1a receptor-mediated intracellular uptake of Ang II is mediated by two major endocytic motifs of AT1a receptors in the cytoplasmic tail, and is regulated by the microtubules- and lipid rafts/caveolin-1 (CAV-1)-dependent mechanisms.. In Specific Aim III, we will test the hypothesis that in polarized PT cells, in vitro expression of an intracellular Ang II protein, ECFP/AII, increases the expression and the activity of NHE-3 in AP membranes via the activation of [Ca2+]i/PKC1/2II, MAP kinases ERK1/2, and NF-:B signaling pathways. Finally, Specific Aim IV will test the hypothesis that intrarenal adenoviral gene transfer of an intracellular Ang II protein selectively in PTs increases salt and fluid reabsorption, promotes salt and fluid retention, and thereby induce hypertension by increasing the expression and activity of apical NHE-3 via activation of AT1 (AT1a) receptors. These studies will provide novel insights into the important roles and underlying cellular and molecular mechanisms of intracellular Ang II in the physiological regulation of salt and fluid reabsorption in proximal tubules of the kidney and in the pathogenesis of hypertension. PUBLIC HEALTH RELEVANCE: About one in three U.S. adults will develop hypertension or hypertension-related complications in their life time, and treating hypertensive diseases costs the U.S. economy several hundreds of billion dollars a year. Salt and fluid retention due to the actions of the hormone angiotensin II remains one of the most important factors in the development and progression of hypertension. This project investigates the signaling mechanisms that regulate the uptake of angiotensin II by proximal tubule cells of the kidney and studies how internalized angiotensin II acts to increase salt and fluid reabsorption from proximal tubules, promote body salt and fluid retention, and therefore cause hypertension. The new information generated by this project will help us better understand renal mechanisms of hypertension and develop new drugs to treat hypertension.