Novel Roles of Intracrine Angiotensin II in Proximal Tubule Cells

描述(由申请人提供)：高血压通常与过多的盐分和肾脏液体滞留有关。血管紧张素Ⅱ(Ang II)通过调节肾脏近端小管(PT)对盐分和液体的重吸收，是维持体内盐分、液体和血压动态平衡的重要因素之一。因此，血管紧张素转换酶II在PTS中的产生和作用增加会导致盐分和液体滞留，从而导致血压升高。血管紧张素Ⅱ通过激活甲状旁腺细胞顶膜和基底膜上的细胞表面受体，对甲状旁腺细胞的钠和液体转运起重要作用。然而，我们有证据表明：a)循环和旁分泌Ang II可通过AT1(AT1a)受体介导的机制被培养的PT细胞或肾脏摄取；b)直接显微注射Ang II可诱导细胞内钙反应；c)细胞内Ang II可诱导主要钠氢逆向转运体NHE-3在分离的大鼠肾皮质核中转录。在这个项目中，我们假设顶端(AP)而不是基底侧(BL)膜AT1a受体在体外和体内介导了PT细胞对Ang II的大部分细胞内摄取，这涉及微管依赖的内吞途径。体外表达或肾内选择性转导血管紧张素Ⅱ蛋白可刺激细胞内AT1(AT1a)受体增加NHE-3的表达和活性，促进PT钠和液体的重吸收，从而诱发高血压。这一假设将在四个具体目标上得到检验。为了明确目标I，我们将在体外和体内验证这一假设，即在极化的PT细胞中，AP膜AT1a受体在介导细胞外Ang II的摄取方面发挥着比BL膜AT1a受体更大的作用。在没有心尖AT1a受体的情况下，AT1b受体或内吞受体megalin可能部分承担AT1a受体的作用。在特定的目标II中，我们将检验这样的假设：在极化的PT细胞中，AP膜AT1a受体介导的细胞内摄取Ang II是由细胞质尾部AT1a受体的两个主要内吞基序介导的，并受微管和脂筏/小窝蛋白-1(CAV-1)依赖的机制调节。在特定的目标III中，我们将验证这样的假设：在极化的PT细胞中，细胞内Ang II蛋白ECFP/AII的体外表达增加了AP膜上NHE-3的表达和活性，这是通过激活[Ca2+]i/PKC1/2ii、MAP激酶ERK1/2和NF-：B信号通路来实现的。最后，特殊目的IV将验证这样的假设，即在PTS中选择性地将肾内Ang II蛋白的腺病毒基因转移增加盐和液体的重吸收，促进盐和液体保留，从而通过激活AT1(AT1a)受体增加心尖部NHE-3的表达和活性而诱导高血压。这些研究将对细胞内Ang II在肾脏近端小管盐和液体重吸收的生理调节以及在高血压发病机制中的重要作用和潜在的细胞和分子机制提供新的见解。 与公共卫生相关：大约三分之一的美国成年人会在一生中患上高血压或高血压相关并发症，治疗高血压疾病每年要花费美国经济数千亿美元。由于激素血管紧张素II的作用，盐和液体滞留仍然是高血压发生和发展的最重要的因素之一。该项目研究了调节肾脏近端小管细胞摄取血管紧张素II的信号机制，并研究了内化的血管紧张素II如何作用于增加近端小管对盐和液体的重吸收，促进体内盐分和液体滞留，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.