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

我们之前已经证明，在肾脏中产生的肿瘤坏死因子-α(TNF)是 调节肾功能和血压(BP)对食盐增加的反应的机制 入口处。我们最近的研究表明，肿瘤坏死因子在肾脏中的作用在髓质(M)厚度上是明显的。 肾单位的上肢(TAL)、近端小管(PT)和皮质(C)TAL/致密斑区(MD)。 然而，肾脏内产生肿瘤坏死因子的细胞来源并没有 目前还没有确定其分子机制。因此，我们开发了两只小鼠 肿瘤坏死因子在TAL中基因缺失的模型：1)TAL，2)PT下游的远端肾单位， 这将被用来理解由肾上皮细胞产生的肿瘤坏死因子作为新出现的 肾小管内肿瘤坏死因子系统，可减轻高盐(HS)摄入引起的血压升高。我们还有 定制了一种补充方法，使用PT和TAL特异性的肿瘤坏死因子沉默慢病毒构建物，以 特异性地抑制这些细胞类型产生的肿瘤坏死因子。基因和慢病毒方法将用于 确定肿瘤坏死因子调节Na+-K+-2Cl-(NKCC2)磷酸化和 异构体表达、肾功能和血压。初步数据提示，通过激活肿瘤坏死因子受体， 1(TNFR1)，抑制磷酸化NKCC2(PNKCC2)的表达，机制涉及激活 丝氨酸/苏氨酸磷酸酶，钙调神经磷酸酶(CN)。肿瘤坏死因子对CN的影响尚未在 肾脏，因此实验将解决肿瘤坏死因子依赖的CN活性的增加以及 催化亚基CNAb和调节亚基CNB。基因和慢病毒策略将被调整为 确定盐摄入量对TNFR1依赖的CN介导的pNKCC2表达抑制的影响， 电解质排泄和血压对HS摄入量的反应。NKCC2A和NKCC2B亚型在战略上是 定位于哺乳动物的TAL，有助于调节对高盐和低盐的反应 条件分别为。肿瘤坏死因子抑制这两种异构体的表达，提示该细胞因子在 TAL的mTAL和cTAL/MD段。我们之前已经证明，在每一种情况下，肿瘤坏死因子都能调节 与这些异构体有关的肾功能，限制了对氯化钠的重吸收。然而，分子 肿瘤坏死因子同时抑制NKCC2A和NKCC2B对高盐和低盐反应的机制 这两种药物的摄入量尚未确定。TAL以前的miRNA分析结合新的 初步数据已鉴定出3个调控NKCC2亚型mRNA丰度的候选miRNAs。为 例如，miRNA-195的表达是由来自TAL的肿瘤坏死因子诱导的，并抑制NKCC2AmRNA 摄入HS小鼠体内蓄积和pNKCC2的表达总的来说，这些研究将定义一部小说 肾小管上皮细胞产生肿瘤坏死因子的肾小管内调控系统 增加盐的摄入量，调节NKCC2亚型的表达和功能，有助于血压的动态平衡。

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.