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

肿瘤坏死因子-α 对 NKCC2 亚型和血压的调节

• 批准号: 10801043
• 负责人: NICHOLAS R FERRERI
• 金额: $ 2.09万
• 依托单位: NEW YORK MEDICAL COLLEGE
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-07-01 至 2025-08-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10801043
• 关键词: Affect; African American population; Attenuated; Blood Pressure; Calcineurin; Catalytic Domain; Data; Development; Distal; Electrolytes; Epithelial Cells; Excretory function; Exhibits; Genetic; Heart Diseases; Hypertension; Individual; Ingestion; Ions; Kidney; Kidney Diseases; Lentivirus; Link; Mediating; Messenger RNA; MicroRNAs; Molecular; Mus; Nephrons; Patients; Phosphoric Monoester Hydrolases; Phosphorylation; Population; Production; Protein Isoforms; Regulation; Renal function; Risk Factors; Role; Serine; Sodium Chloride; Stroke; Structure of ascending limb of Henle' s loop; System; TNF gene; TNFRSF1A gene; Thick; Threonine; Tubular formation; Tumor Necrosis Factor Activation; United States; absorption; blood pressure elevation; blood pressure regulation; cytokine; defined contribution; dietary salt; experimental study; hypertensive; mouse model; renal epithelium; response; salt intake; symporter

We previously showed that tumor necrosis factor-alpha (TNF) produced within the kidney is part of a system that regulates renal function and the blood pressure (BP) response to increases in dietary salt intake via inhibition of the Na+-K+-2Cl- (NKCC2) cotransporter. Thus, we developed two mouse models in which TNF has been genetically deleted in the: 1) thick ascending limb of Henle’s loop (TAL), and 2) distal nephron downstream of the proximal tubule (PT), which will be applied to determine the role and mechanisms 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. A complementary approach, using PT- and TAL-specific TNF silencing lentivirus constructs, will be used to specifically inhibit TNF production by these nephron segments to define the contribution of TNF derived from renal epithelial cells to the regulatory effects of this cytokine in the kidney. The genetic and lentivirus approaches also will be used to determine the molecular mechanisms by which TNF regulates NKCC2 phosphorylation and isoform expression, renal function, and BP. Preliminary data indicate 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 in the kidney have not been studied, 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. Fine-tuning of NKCC2 function is dependent upon the NKCC2A and NKCC2B isoforms, which 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 these isoforms suggesting a role for this cytokine in both the mTAL and cTAL/MD segments of the TAL. Moreover, TNF regulates renal function involving these isoforms in a manner that limits reabsorption of NaCl. However, the molecular mechanism by which TNF suppresses NKCC2A and NKCC2B mRNA accumulation in response to high and low salt intake, respectively, has not been determined. miRNA profiling of the TAL in combination with preliminary data have identified miRNAs that regulate NKCC2 isoform mRNA abundance, phosphorylation and BP, the first data to show NKCC2 function is regulated by a miRNA-dependent mechanism. TAL-specific lentivirus manipulations will link miRNA-195 expression induced by TNF derived from the TAL to a NKCC2A-dependent mechanism that attenuates BP in mice ingesting HS. Collectively, the studies will reveal mechanisms by which an intratubular regulatory system, in which TNF produced by renal tubular epithelial cells in response to increases in salt intake, regulates NKCC2 isoform expression, phosphorylation via CN, electrolyte excretion, and contributes to BP homeostasis.

我们之前表明，肾脏内产生的肿瘤坏死因子-α (TNF) 是一个系统的一部分 调节肾功能和血压 (BP) 对膳食盐摄入量增加的反应 抑制 Na+-K+-2Cl- (NKCC2) 协同转运蛋白。因此，我们开发了两种小鼠模型，其中 TNF 已在以下部位进行基因删除：1) 厚厚的亨利氏袢升肢 (TAL)，以及 2) 远端肾单位 近端小管（PT）下游，将用于确定TNF的作用和机制 由肾上皮细胞产生，作为新兴肾小管内 TNF 系统的一部分，可减弱 高盐（HS）摄入引起的血压。一种补充方法，使用 PT 和 TAL 特异性 TNF 沉默 慢病毒构建体将用于特异性抑制这些肾单位片段产生 TNF，以定义 源自肾上皮细胞的 TNF 对肾脏中该细胞因子的调节作用的贡献。 遗传和慢病毒方法也将用于确定 TNF 的分子机制。 调节 NKCC2 磷酸化和亚型表达、肾功能和血压。初步数据表明 TNF 通过激活 TNF 受体 1 (TNFR1)，抑制磷酸化 NKCC2 (pNKCC2) 的表达 机制涉及丝氨酸/苏氨酸磷酸酶、钙调神经磷酸酶 (CN) 的激活。 TNF对人的影响 尚未研究肾脏中的 CN，因此实验将解决 TNF 依赖性 CN 活性的增加 以及催化亚基 CNAb 和调节亚基 CNB 的表达。遗传和慢病毒 将调整策略以确定盐摄入量对 TNFR1 依赖性 CN 介导的抑制的影响 pNKCC2 表达、电解质排泄以及血压对 HS 摄入的反应。 NKCC2 的微调 功能依赖于 NKCC2A 和 NKCC2B 同工型，它们战略性地定位于 哺乳动物 TAL 有助于响应高盐和低盐条件的调节功能， 分别。 TNF 抑制这些亚型的表达，表明该细胞因子在 mTAL 和 和 TAL 的 cTAL/MD 段。此外，TNF 通过以下方式调节肾功能： 限制 NaCl 重吸收的方式。然而，TNF 抑制的分子机制 NKCC2A 和 NKCC2B mRNA 的积累分别响应高盐摄入和低盐摄入，并没有 已确定。 TAL 的 miRNA 分析与初步数据相结合，确定了以下 miRNA： 调节 NKCC2 同工型 mRNA 丰度、磷酸化和 BP，第一个显示 NKCC2 功能的数据是 受 miRNA 依赖性机制的调节。 TAL 特异性慢病毒操作将连接 miRNA-195 TAL 衍生的 TNF 诱导的表达通过 NKCC2A 依赖性机制减弱血压 小鼠摄入HS。总的来说，这些研究将揭示管内调节系统的机制， 其中肾小管上皮细胞响应盐摄入量的增加而产生 TNF，调节 NKCC2 亚型表达、通过 CN 磷酸化、电解质排泄，并有助于血压稳态。