Regulation of NKCC2 isoforms and blood pressure by tumor necrosis factor-alpha

我们以前的研究表明，肾脏内产生的肿瘤坏死因子-α（TNF）是一种肿瘤坏死因子的一部分。 调节肾功能和血压（BP）对膳食盐增加的反应的机制 摄入我们最近的研究表明，TNF在肾脏中的作用在髓质（m）厚度中是明显的。 肾单位的升支（TAL）、近端小管（PT）和皮质（c）TAL/致密斑（MD）区域。 然而，肾脏内产生TNF的细胞来源并没有引起这些效应。 目前还没有确定，也没有确定分子机制。因此，我们开发了两种小鼠 模型中TNF已在以下基因中缺失：1）TAL，和2）PT下游的远端肾单位， 这将被用来了解肾上皮细胞产生的TNF的作用， 小管内TNF系统，其减弱由高盐（HS）摄入诱导的BP升高。我们也有 定制了一种互补的方法，使用PT和TAL特异性TNF沉默慢病毒构建体， 特异性抑制这些细胞类型的TNF产生。基因和慢病毒方法将用于 串联以确定TNF调节Na+-K+-2Cl-（NKCC 2）磷酸化的机制， 同种型表达、肾功能和血压。初步数据表明，TNF，通过激活TNF受体， 1（TNFR 1），通过激活磷酸化NKCC 2（pNKCC 2）的机制抑制磷酸化NKCC 2的表达。 丝氨酸/苏氨酸磷酸酶，钙调神经磷酸酶（CN）。TNF对CN的影响尚未在研究中探讨。 因此，实验将解决CN活性的TNF-依赖性增加以及 催化亚基CNAb和调节亚基CNB。遗传和慢病毒策略将被调整， 确定盐摄入对TNFR 1依赖性CN介导的pNKCC 2表达抑制的影响， 电解质排泄和血压对HS摄入的反应。NKCC 2A和NKCC 2B亚型在战略上是 位于沿着哺乳动物TAL，并有助于响应高盐和低盐的调节功能 条件分别。TNF抑制这两种亚型的表达，表明这种细胞因子在两种疾病中的作用。 TAL的mTAL和cTAL/MD片段。我们先前表明，在每种情况下，TNF调节 以限制NaCl重吸收的方式涉及这些亚型的肾功能。然而，分子 TNF抑制NKCC 2A和NKCC 2B mRNA对高盐和低盐反应的机制 分别的摄入量尚未确定。TAL的先前miRNA谱分析与新的 初步数据已经鉴定了3种调节NKCC 2同种型mRNA丰度的候选miRNA。为 例如，miRNA-195的表达被来自TAL的TNF诱导并抑制NKCC 2A mRNA 在小鼠摄入HS中的蓄积和pNKCC 2表达。总的来说，这些研究将定义一部小说 肾小管内调节系统，其中肾小管上皮细胞响应于 增加盐摄入，调节NKCC 2亚型表达和功能，并有助于BP稳态。

We previously showed that tumor necrosis factor-alpha (TNF) produced within the kidney is part of a mechanism that regulates renal function and the blood pressure (BP) response to increases in dietary salt intake. Our recent studies suggest that TNF effects in the kidney are evident in the medullary (m) thick ascending limb (TAL), proximal tubule (PT), and cortical (c) TAL/macula densa (MD) regions of the nephron. However, the cellular sources within the kidney that produce the TNF that accounts for these effects have not been determined, nor have the molecular mechanisms been identified. Thus, we developed two mouse models in which TNF has been genetically deleted in the: 1) TAL, and 2) distal nephron downstream of the PT, which will be used to understand the role of TNF produced by renal epithelial cells as part of an emerging intratubular TNF system that attenuates increases in BP induced by high salt (HS) intake. We also have tailored a complementary approach, using PT- and TAL-specific TNF silencing lentivirus constructs, to specifically inhibit TNF production by these cell types. The genetic and lentivirus approaches will be used in tandem to determine the mechanism by which TNF regulates Na+-K+-2Cl- (NKCC2) phosphorylation and isoform expression, renal function, and BP. Preliminary data suggest that TNF, via activation of TNF receptor 1 (TNFR1), inhibits phospho-NKCC2 (pNKCC2) expression by a mechanism involving activation of the serine/threonine phosphatase, calcineurin (CN). The effects of TNF on CN have not been explored in the kidney, thus experiments will address TNF-dependent increases in CN activity as well as expression of the catalytic subunit CNAb and regulatory subunit CNB. The genetic and lentivirus strategies will be adapted to determine the effects of salt intake on TNFR1-dependent CN-mediated inhibition of pNKCC2 expression, electrolyte excretion, and the BP response to HS intake. The NKCC2A and NKCC2B isoforms are strategically localized along the mammalian TAL and contribute to regulatory functions in response to high and low salt conditions, respectively. TNF inhibits the expression of both isoforms suggesting a role for this cytokine in both the mTAL and cTAL/MD segments of the TAL. We previously showed that in each instance, TNF regulates renal function involving these isoforms in a manner that limits reabsorption of NaCl. However, the molecular mechanism by which TNF suppresses both NKCC2A and NKCC2B mRNA in response to high and low salt intake, respectively, has not been determined. Previous miRNA profiling of the TAL in combination with new preliminary data have identified 3 candidate miRNAs that regulate NKCC2 isoform mRNA abundance. For instance, miRNA-195 expression is induced by TNF derived from the TAL and inhibits NKCC2A mRNA accumulation and pNKCC2 expression in mice ingesting HS. Collectively, the studies will define a novel intratubular regulatory system in which TNF production by renal tubular epithelial cells, in response to increases in salt intake, regulates NKCC2 isoform expression and function and contributes to BP homeostasis.