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

项目摘要 高血压是脑卒中、心肌梗死、心脏病、高血压病等疾病发病率和死亡率的主要原因。 衰竭和慢性肾病。尽管血压控制的重要性， 对原发性高血压的认识仍然很少。在过去的几年里，很明显， 钠可以在骨骼肌中积累，特别是在皮肤和骨骼肌中， 适度升高的钠浓度可以驱动免疫细胞活化。本实验室 最近描述了一种新的途径，通过这种途径，细胞外钠激活树突状细胞中的NADPH氧化酶。 细胞，并表明这促进了isolevuglandin加合物，被认为是非自我的， 免疫反应我认为盐通过以下途径稳定NADPH氧化酶亚基，特别是p22 phox， 血清和糖皮质激素调节激酶1（SGK 1）在树突状细胞（DC），这导致了 促进高血压。在目的1中，我将检验p22 phox蛋白在细胞中的稳定性的假设。 对盐的反应依赖于SGK 1，并确定这是否促进DC活化， 高血压在这个目标中，我将使用我们在DC中特异性删除SGK 1的小鼠。上 作为这一目标的一部分，我将证明这种增加的钠是否确实增强了NADPH的稳定性 氧化酶蛋白亚基，如果这是依赖于SGK 1。在第二个系列的实验中，我将 检测SGK 1对DC表型的影响。将通过流式细胞术分析DC， 超氧化物生成电子自旋共振。在另外的实验中，我将检查体内 SGK 1在DC中的作用我将进行过继转移的树突状细胞共处理甘露醇或高 将盐注入未实验过的小鼠体内，并通过无线电遥测术测量血压。我预测SGK 1的缺失 防止NADPH氧化酶亚单位稳定，生产超氧化物，并增加是血液 低剂量血管紧张素II。在目标2中，我将确定NADPH氧化酶亚基p22 phox， p47 phox和/或gp 91 phox通过SGK 1响应于盐而被泛素化，并确定这是否 促进DC活化和高血压。在这些研究中，我们将进行免疫沉淀， NADPH氧化酶亚基p22 phox、p47 phox和gp 91 phox。我们会用质谱仪来鉴定 NADPH氧化酶亚基的泛素化赖氨酸。在另外的实验中，我们将评估p22 phox， p47 phox和gp 91 phox泛素化在体内利用盐敏感性高血压的啮齿动物模型。我 预测NADPH氧化酶亚基将在高盐处理期间稳定，并且遗传 删除SGK 1将防止DC中出现这种情况。这将促进我们对高血压的了解， 为该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.