Regulation of Superoxide Production by the Macula Densa during TGF

描述(申请人提供)：致密黄斑中产生超氧化物(O2-)的机制尚不清楚。我们将用以下具体目标来检验我们的假设。目的1：假说：肾小管小球反馈引起的肾小管NaCI浓度升高主要增强致密黄斑NAD(P)H氧化酶(NOX)产生O2-的能力。我们将测量致密黄斑中O2-的产生，同时增加腔内NaCI。为了研究致密黄斑中NOx亚型的表达，我们将使用激光捕获显微解剖(LCM)和实时定量聚合酶链式反应(Real Time PCR)分离致密黄斑细胞。目的2：假设：致密黄斑去极化激活O2-产生。致密黄斑的去极化通过刺激GTP结合蛋白RAC激活NAD(P)H氧化酶。我们将测量O2-的产生，同时：a)去极化致密斑细胞；以及b)使用显性负性RAC和结构性活性RAC表达致密黄斑细胞。目的3：假说：致密斑细胞内pH升高可促进O2-的产生，这一过程涉及通过Na/H交换和/或H/K ATPase将产生O2-的质子从致密斑内转运出去。我们将在增加腔内NaCI的同时测量O2-的产生：a)在存在和不存在H/K ATPase抑制剂的情况下；以及b)在钳制细胞内pH的情况下。我们将测量H/K-ATPase基因敲除小鼠在增加腔内NaCI的同时产生O2-。目的4假说：自发性高血压大鼠心尖部Na/H交换器活性高于WKY组，导致致密黄斑pH值升高，从而增强NAD(P)H氧化酶活性和超氧化物歧化产物，从而增强肾小管球反馈。抑制致密斑尖Na/H交换使自发性高血压大鼠肾小管球反馈趋于正常化。我们将研究SHR和WKY的顶端和基底外侧致密斑区Na/H交换器的活性。我们将用激光共聚焦显微镜分离致密黄斑的顶膜和基侧膜，并用实时荧光定量聚合酶链式反应研究Na/H交换器的表达水平。我们将测量SHR的小管小球反馈，同时将致密黄斑的细胞内pH钳制到与WKY相同的程度。在本研究中，我们将研究超氧化物歧化对肾脏血流动力学和血压的影响及其机制。我们相信，本研究的结果将有助于我们更好地了解高血压的原因，并可能揭示新的治疗方法。

DESCRIPTION (provided by applicant): The mechanism of superoxide (O2-) production in the macula densa is not known. We will test our hypotheses with the following specific aims. Aim 1: Hypothesis: The increase in tubular NaCI concentration that initiates tubuloglomerular feedback enhances O2- production primarily from macula densa NAD(P)H oxidase (NOX). We will measure O2- production while increasing luminal NaCI in macula densa. To investigate the Nox isoform expressed at the macula densa, we will isolate macula densa cells using laser capture Microdissection (LCM) and real time PCR. Aim 2: Hypothesis: O2- production is activated by macula densa depolarization. Depolarization of the macula densa activates NAD(P)H oxidase by stimulating the GTP-binding protein Rac. We will measure O2- generation while: a) depolarizing the macula densa; and b) using dominant negative Rac and constitutively active Rac expressed macula densa cells. Aim 3: Hypothesis: O2- production is enhanced by increased macula densa intracellular pH, which process is involved in transporting protons generated during O2- production out of the macula densa by Na/H exchange and/or H/K ATPase. We will measure O2- production while increasing luminal NaCI: a) in the presence and absence of H/K ATPase inhibitors; and b) while clamping intracellular pH. We will measure O2- generation while increasing luminal NaCI in H/K ATPase knockout mice. Aim 4 Hypothesis: Activity of apical Na/H exchangers is higher in SHR than in WKY, resulting in higher macula densa pH, which enhances NAD(P)H oxidase activity and superoxide production and thus augments tubuloglomerular feedback. Inhibition of macula densa apical Na/H exchange tends to normalize tubuloglomerular feedback in SHR. We will study the activity of apical and basolateral Na/H exchangers at the macula densa in SHR and WKY. We will isolate the apical and basolateral membranes of the macula densa using LCM and study the Na/H exchanger expression level using real-time PCR. We will measure tubuloglomerular feedback in SHR while clamping macula densa intracellular pH to the same degree as WKY. In present research, we will study the mechanism and effect of superoxide on renal hemodynamic and blood pressure. We believe the results of the present study will help us better understand the causes of hypertension and might reveal new approaches for treatment.