The role of salt and SGK1 on NADPH oxidase stabilization in dendritic cells in hypertension

盐和 SGK1 对高血压树突状细胞 NADPH 氧化酶稳定的作用

• 批准号: 10063425
• 负责人: Justin Pieter Van Beusecum
• 金额: $ 5.92万
• 依托单位: VANDERBILT UNIVERSITY MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-05-01 至 2021-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10063425
• 关键词: Adoptive Transfer; Adult; Age; American; Angiotensin II; Blood Pressure; Blood Vessels; Cardiovascular Diseases; Cardiovascular system; Cells; Chronic Kidney Failure; Coronary heart disease; Data; Dendritic Cells; Dendritic cell activation; Disease; Docking; Dose; Electron Spin Resonance Spectroscopy; Environment; Essential Hypertension; Event; Excess Dietary Salt; Flow Cytometry; General Population; Genetic; Guidelines; Half-Life; Heart failure; Hypertension; Immune; Immune response; Immune system; Immunoprecipitation; Inflammation; Kidney; Laboratories; Lysine; Mannitol; Mass Spectrum Analysis; Measures; Mentorship; Morbidity - disease rate; Mus; Myocardial Infarction; NADPH Oxidase; Pathogenesis; Pathway interactions; Pharmacology; Phenotype; Phosphotransferases; Physiology; Positioning Attribute; Postdoctoral Fellow; Production; Protein Subunits; Proteins; Research; Rodent Model; Role; Series; Sgk protein; Site; Skeletal Muscle; Skin; Societies; Sodium; Sodium Chloride; Stroke; Superoxides; Testing; Training; Ubiquitination; Up-Regulation; Work; adduct; base; blood pressure regulation; career; epithelial Na+ channel; experience; experimental study; extracellular; immune activation; in vivo; mortality; novel; novel therapeutics; prevent; renal damage; response; salt intake; salt sensitive hypertension

PROJECT SUMMARY Hypertension is the leading cause of morbidity and mortality from stroke, myocardial infarction, heart failure, and chronic kidney disease. Despite the importance of blood pressure control, the pathogenesis of essential hypertension remains poorly understood. In the past several years it has become clear that sodium can accumulate in the interstitium, particularly in the skin and skeletal muscle and that these modestly elevated concentrations of sodium can drive immune cell activation. Our laboratory has recently described a new pathway by which extracellular sodium activates NADPH oxidase in dendritic cells and showed that this promotes isolevuglandin-adducts that are recognized as non-self and evoke an immune response. I propose that salt stabilizes NADPH oxidase subunits, specifically p22phox, via serum and glucocorticoid-regulated kinase 1 (SGK1) in dendritic cells (DCs), which leads to the promotion of hypertension. In Aim 1, I will test the hypothesis that stabilization of p22phox protein in response to salt is dependent on SGK1 and to determine if this promotes DC activation and hypertension. In this aim I will use mice in which we have deleted SGK1 specifically in DCs. In the first part of this aim, I will demonstrate if this increased sodium indeed enhances stability of the NADPH oxidase protein subunits and if this is dependent on SGK1. In a second series of experiments, I will examine the effect of SGK1 on the phenotype of DCs. DCs will be analyzed by flow cytometry and for superoxide production by electron spin resonance. In additional experiments, I will examine the in vivo role of SGK1 in DCs. I will perform adoptive transfer of dendritic cells co-treated with mannitol or high salt into naïve mice and measure blood pressure by radiotelemetry. I predict that deletion of SGK1 prevents NADPH oxidase subunit stabilization, production of superoxide, and increase is blood pressure with low dose angiotensin II. In aim 2, I will determine if NADPH oxidase subunits p22phox, p47phox, and/or gp91phox are ubiquitinated in response to salt via SGK1, and to determine if this promotes DC activation and hypertension. In these studies, we will perform immunoprecipitation of the NADPH oxidase subunits p22phox, p47phox and gp91phox. We will use mass spectrometry to identify ubiquitinated lysines of the NADPH oxidase subunits. In additional experiments, we will assess p22phox, p47phox, and gp91phox ubiquitination in vivo utilizing a rodent model of salt-sensitive hypertension. I predict that NADPH oxidase subunits will be stabilized during high salt treatment, and that genetic deletion of SGK1 will prevent this in DCs. This will advance our understanding of hypertension and will provide new therapeutic directions for this disease.

项目摘要 高血压是中风，心肌梗塞，心脏的发病率和死亡率的主要原因 失败和慢性肾脏疾病。尽管血压控制很重要，但发病机理 基本的高血压仍然知之甚少。在过去的几年中，很明显 钠可以在间质中积聚，尤其是在皮肤和骨骼肌中，这些 钠的浓度适度升高会驱动免疫细胞激活。我们的实验室有 最近描述了一种新途径，细胞外钠激活树突状的氧化物 细胞并表明这促进了被认为是非自我并引起唤起的异甲列列扬蛋白添加剂 免疫响应。我建议盐稳定纳德氧化物亚基，特别是p22phox， 树突状细胞（DCS）中的血清和糖皮质激素调节的激酶1（SGK1），这导致导致 促进高血压。在AIM 1中，我将检验以下假设：p22phox蛋白在 对盐的反应取决于SGK1，并确定这是否促进了直流激活和 高血压。在此目标中，我将使用在DC中专门删除SGK1的小鼠。在第一个 该目标的一部分，我将证明这种增加的钠是否确实提高了NADPH的稳定性 氧化酶蛋白亚基，如果这取决于SGK1。在第二个实验中，我将 检查SGK1对DC表型的影响。 DC将通过流式细胞仪和用于分析 通过电子自旋共振产生超氧化物。在其他实验中，我将检查体内 SGK1在DC中的作用。我将执行与甘露醇或高的树突状细胞的自适应转移 盐分为幼稚的小鼠，并通过放射性骨化测量血压。我预测SGK1的删除 防止NADPH氧化亚基稳定，超氧化物的产生和增加是血液 低剂量血管紧张素II的压力。在AIM 2中，我将确定NADPH氧化物亚基是否是P22Phox， p47phox和/或gp91phox因通过sgk1响应盐而泛素化，并确定这是否是 促进直流激活和高血压。在这些研究中，我们将对 NADPH氧化酶亚基P22Phox，P47Phox和GP91Phox。我们将使用质谱法来识别 NADPH氧化酶亚基的泛素化歌词。在其他实验中，我们将评估P22Phox， p47phox和GP91PHOX在体内使用啮齿动物敏感高血压模型。我 预测NADPH氧化亚基将在高盐处理期间稳定，并且该遗传 SGK1的删除将在DCS中阻止这种情况。这将提高我们对高血压的理解，并将 为该疾病提供新的治疗方向。