Regulation of Superoxide Production by the Macula Densa during TGF

描述（由申请人提供）：致密黄斑中超氧化物（O2-）产生的机制尚不清楚。我们将通过以下具体目标来检验我们的假设。目标 1：假设：肾小管 NaCl 浓度的增加引发肾小管肾小球反馈，从而增强主要由致密斑 NAD(P)H 氧化酶 (NOX) 产生的 O2- 。我们将测量 O2- 的产生，同时增加致密斑中的腔内 NaCl。为了研究在致密斑处表达的 Nox 同种型，我们将使用激光捕获显微切割 (LCM) 和实时 PCR 分离致密斑细胞。目标 2：假设：O2- 的产生是由致密黄斑去极化激活的。致密黄斑的去极化通过刺激 GTP 结合蛋白 Rac 来激活 NAD(P)H 氧化酶。我们将在以下情况下测量 O2 生成： a) 对致密黄斑进行去极化； b)使用显性失活的Rac和组成型活性的Rac表达的致密斑细胞。目标 3：假设：致密黄斑细胞内 pH 值的增加可增强 O2- 的产生，该过程涉及通过 Na/H 交换和/或 H/K ATP 酶将 O2- 产生过程中产生的质子从致密斑转运出去。我们将在增加腔内 NaCl 的同时测量 O2- 产生： a) 在存在和不存在 H/K ATPase 抑制剂的情况下； b) 同时限制细胞内 pH 值。我们将在H/K ATP酶敲除小鼠中测量O 2 生成，同时增加腔内氯化钠。目标 4 假设：SHR 中顶端 Na/H 交换器的活性高于 WKY，导致致密斑 pH 值更高，从而增强 NAD(P)H 氧化酶活性和超氧化物产生，从而增强肾小球小球反馈。抑制黄斑顶端 Na/H 交换往往会使 SHR 中的肾小管球反馈正常化。我们将研究 SHR 和 WKY 中致密斑顶部和基底外侧 Na/H 交换器的活性。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 交换体的表达水平。我们将测量 SHR 中的肾小球反馈，同时将致密斑细胞内 pH 值钳制到与 WKY 相同的程度。在本研究中，我们将研究超氧化物对肾脏血流动力学和血压的作用机制和影响。我们相信本研究的结果将帮助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.