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

项目总结 高血压是中风、心肌梗死、心脏病等疾病发病率和死亡率的主要原因 衰竭和慢性肾脏疾病。尽管控制血压很重要，但其发病机制 高血压病的病因仍知之甚少。在过去的几年里，情况变得很明显 钠可以积聚在间质中，特别是在皮肤和骨骼肌中，这些 适度升高的钠浓度可以促进免疫细胞的激活。我们的实验室有 最近描述了胞外钠激活树突状细胞中NADPH氧化酶的新途径 细胞，并表明这促进了异uglandin加合物，这些加合物被认为是非我的，并引起 一种免疫反应。我认为盐通过稳定NADPH氧化酶亚基，特别是p22Phox， 血清和树突状细胞(DC)中糖皮质激素调节的激酶1(SGK1)，从而导致 促进高血压的发展。在目标1中，我将测试假设p22Phox蛋白在 对盐的反应依赖于SGK1，并确定这是否促进DC激活和 高血压。在这个目的中，我将使用我们特别在DC中删除SGK1的小鼠。在第一个 作为这一目标的一部分，我将证明这种增加的钠是否真的增强了NADPH的稳定性 如果这依赖于SGK1。在第二系列实验中，我将 检测SGK1对DC表型的影响。将用流式细胞仪对DC进行分析， 电子自旋共振产生超氧化物。在其他实验中，我将检查体内的 SGK1在DC中的作用我将进行与甘露醇或HIGH共处理的树突状细胞过继转移 将盐注入幼鼠体内，并用无线电遥测法测量血压。我预测SGK1的缺失 防止NADPH氧化酶亚基稳定，产生超氧化物，并增加血液中 在目标2中，我将确定NADPH氧化酶亚基p22Phox， P47Phox和/或gp91Phox通过SGK1泛素化，以响应盐的反应，并确定这是否 促进DC激活和高血压。在这些研究中，我们将进行免疫沉淀 NADPH氧化酶亚基p22Phox、p47Phox和gp91Phox。我们将使用质谱学来鉴定 NADPH氧化酶亚基的泛素化赖氨酸。在其他实验中，我们将评估p22Phox， 利用盐敏感型高血压啮齿动物模型体内的p47Phox和gp91Phox泛素化。我 预测NADPH氧化酶亚基在高盐处理过程中会稳定下来，并且遗传 删除SGK1将在DC中阻止这一点。这将促进我们对高血压的理解，并将 为这种疾病提供新的治疗方向。

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.