The Effect of Salt on T Cell Function in Hypertension

 描述(申请人提供)：高血压是心血管疾病发病率和死亡率的主要原因，也是心肌梗死、中风、心力衰竭和慢性肾脏疾病的关键因素。仅在美国，高血压患病率就达到了30%。在过去的几十年里，我们已经提出证据表明，高血压是一种由先天性和获得性免疫系统的细胞介导的炎症性疾病，特别是由产生IL17的T细胞介导的。此外，许多研究已经将饮食中的食盐摄入量与高血压联系起来。在初步研究中，我们和其他人已经表明，过量的盐促进产生IL17A的CD4+(Th17)和CD8+(Tc17)细胞的分化。作为对盐的响应，这些细胞上调渗透敏感转录因子TONEBP(NFAT5)及其下游靶标之一盐敏感激酶1(SGK1)。SGK1在调节T细胞功能中的潜在重要性是有趣的，因为它直接将盐与炎症联系起来。然而，盐影响高血压进展的机制尚不清楚。此外，T细胞的一部分具有电压门控的钠通道。我们有初步数据显示，当CD4+和CD8+T细胞暴露在Th17极化细胞因子和盐中时，这些细胞的Na+通道SCN5A上调。因此，我假设SGK1对T细胞通过调节SCN5A调节高血压的能力是必不可少的，并增加由此产生的Th17/Tc17细胞的致病性。为了检验这一假设，我首先计划 通过基因删除小鼠所有T细胞中的SGK1，以确定T细胞SGK1是否是血管紧张素II和DOCA盐引起的高血压和炎症反应所必需的。为了做到这一点，我们创造了一个CD4cre/SGK1fl/fl小鼠群体。我们将使用这些小鼠来确定T细胞中SGK1的缺失是否会导致与对照组小鼠相比，在暴露于高血压刺激时，血压降低，肾脏/血管功能增强，肾脏/血管炎症减轻。接下来，我将确定SCN5A是否受SGK的调控。为此，我将对野生型小鼠(C57BL/6J)或CD4cre/SGK1fl/fl小鼠的极化T细胞进行脉冲追逐分析、RT-PCR和磷酸化流动分析。最后，我将确定SCN5A活性是否是Th17/Tc17细胞致病所必需的。我将用siRNA敲除SCN5A，并用盐和Th17细胞因子极化细胞后，用RT-PCR检测IL17A的含量。我还将比较高血压小鼠、正常血压小鼠和人类T细胞全细胞膜片钳测得的钠电流。此外，我将量化膜电位和进入T细胞的钠的量，这些T细胞已经被Th17细胞因子极化，然后被过量的盐电击。这些研究的完成将理想地导致确定治疗高血压的新的治疗靶点。

 DESCRIPTION (provided by applicant): Hypertension is a leading cause of cardiovascular disease morbidity and mortality and is a key contributor to myocardial infarction, stroke, heart failure and chronic kidney disease. The prevalence of hypertension reaches 30% of adults in the United States alone. We have put forth evidence in the past few decades suggesting that hypertension is an inflammatory disease mediated by cells of the innate and adaptive immune systems, specifically by IL17-producing T cells. In addition, many studies have linked dietary salt intake to hypertension. In preliminary studies, we and others have shown that excess salt promotes differentiation of IL17A-producing CD4+ (Th17) and CD8+ (Tc17) cells. In response to salt, these cells upregulate the osmosensitive transcription factor, TonEBP (NFAT5) and one of its downstream targets, the salt-sensing kinase 1 (SGK1). The potential importance of SGK1 in modulating T cell function is interesting as it directly connects salt to inflammation. However, th mechanism by which salt acts to affect the progression of hypertension is unknown. Additionally, a subset of T cells possesses voltage-gated sodium channels. We have preliminary data showing that the Na+ channel SCN5A is upregulated in CD4+ and CD8+ T cells when these cells are exposed to Th17 polarizing cytokines and salt. Therefore, I hypothesize that SGK1 is essential to the ability of T cells to mediate hypertension by regulating SCN5A; and increasing the pathogenicity of the resultant Th17/Tc17 cells. To test this hypothesis I first plan to determine whether T cell SGK1 is necessary for the hypertensive and inflammatory response to angiotensin II and DOCA-salt by genetically deleting SGK1 in all T cells in mice. To do this, we have generated a colony of mice that are CD4cre/SGK1fl/fl. We will use these mice to determine if loss of SGK1 in T cells results in lowered blood pressure, increased renal/vascular function and decreased renal/vascular inflammation compared to control mice when exposed to hypertensive stimuli. Next I will determine if SCN5A is regulated by SGK. To do this, I will conduct pulse chase analyses, RT-PCR and phospho-flow on polarized T cells from wild type mice (C57BL/6J) or from CD4cre/SGK1fl/fl mice. Finally, I will determine if SCN5A activity is required for Th17/Tc17 cell pathogenicity. I will knock down SCN5A with siRNA and measure the amount of IL17A by RT-PCR after the cells are polarized with salt and Th17 cytokines. I will also compare sodium currents measured by whole cell patch clamping in T cells from hypertensive and normotensive mice and humans. In addition, I will quantify the membrane potential and the amount of sodium entering T cells that have been polarized with Th17 cytokines and then shocked with excess salt. Completion of these studies will ideally lead to the identification of novel therapeutic targets to treat hypertension.