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和肾脏液体潴留有关。激素血管紧张素II （Ang II）是通过调节肾近端小管（PT）的盐和液体重吸收来维持体内盐、液体和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引起盐和液体潴留，从而导致血压升高。Ang II通过激活PT细胞顶膜和基底膜上的细胞表面受体，对PT钠和液体的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AT1 （AT1a）受体介导的机制被培养的PT细胞或肾脏吸收；b)直接向细胞内微量注射Ang II可诱导细胞内钙反应；c)胞内Ang II可诱导离体大鼠肾皮质核中主要钠和氢反转运蛋白NHE-3的转录。在这个项目中，我们假设在体内和体外，PT细胞对Ang II的摄取主要是由顶端（AP）而不是基底侧（BL）膜AT1 （AT1a）受体介导的，这涉及微管依赖的内吞途径。细胞内Ang II蛋白在PT细胞中的体外表达或肾内腺病毒转移可刺激细胞内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中得到检验。在Specific Aim I中，我们将在体外和体内验证在极化PT细胞中，AP膜AT1a受体在介导细胞内摄取细胞外Ang II方面比BL膜AT1a受体发挥更大的作用。在顶端AT1a受体缺失的情况下，AT1b受体或内吞受体meggalin可能部分承担AT1a受体的作用。在Specific Aim II中，我们将验证在极化PT细胞中，AP膜AT1a受体介导的Ang II的细胞内摄取是由细胞质尾部AT1a受体的两个主要内吞基体介导的，并受到微管和脂质筏/ CAV-1 （CAV-1）依赖机制的调节。在Specific Aim III中，我们将验证在极化PT细胞中，细胞内Ang II蛋白ECFP/AII的体外表达，通过激活[Ca2+]i/PKC1/2II， MAP激酶ERK1/2和NF-:B信号通路，增加AP膜中NHE-3的表达和活性的假设。最后，Specific Aim IV将验证肾内腺病毒基因在PTs中选择性地转移细胞内Ang II蛋白，增加盐和液体的重吸收，促进盐和液体潴留，从而通过激活AT1 （AT1a）受体，增加尖端NHE-3的表达和活性，从而诱导高血压。这些研究将为细胞内的Ang II在肾近端小管盐和液体重吸收的生理调节和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。

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.