Regulation of Superoxide Production by the Macula Densa during TGF

描述（由申请人提供）：致密斑中超氧化物（O2-）产生的机制尚不清楚。我们将以下列具体目标来检验我们的假设。目标1：假设：启动肾小管肾小球反馈的肾小管NaCl浓度的增加主要从致密斑NAD（P）H氧化酶（NOX）增强O2-产生。我们将在增加致密斑中的腔NaCl的同时测量O2产生。为了研究致密斑中表达的Nox亚型，我们将使用激光捕获显微切割（LCM）和真实的时间PCR分离致密斑细胞。目的2：假设：致密斑去极化激活O2-的产生。致密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产生，同时：a）使致密斑去极化;和B）使用显性负性Rac和组成型活性Rac表达的致密斑细胞。目标3：假设：通过增加致密斑细胞内pH来增强O2-产生，该过程涉及通过Na/H交换和/或H/K ATP酶将O2-产生期间产生的质子转运出致密斑。我们将在增加管腔NaCl的同时测量O2产生：a）在存在和不存在H/K ATP酶抑制剂的情况下;和B）在钳制细胞内pH的情况下。我们将在增加H/K ATP酶敲除小鼠中管腔NaCl的同时测量O2产生。目标4假设：SHR根尖Na/H交换体活性高于WKY，导致致密斑pH值升高，增强NAD（P）H氧化酶活性和超氧化物产生，从而增强肾小管-肾小球反馈。抑制致密斑顶端Na/H交换倾向于使SHR的肾小管肾小球反馈正常化。我们将研究SHR和WKY致密斑顶端和基底侧Na/H交换体的活性。我们将使用LCM分离致密斑的顶膜和基底外侧膜，并使用实时PCR研究Na/H交换剂的表达水平。我们将测量SHR中的管球反馈，同时将致密斑细胞内pH钳制到与WKY相同的程度。本研究旨在探讨超氧化物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.