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.

我们先前表明肾脏内产生的肿瘤坏死因子-Alpha（TNF）是系统的一部分 调节肾功能和血压（BP）对饮食盐摄入量增加的反应 抑制Na+-k+-2Cl-（NKCC2）共转运蛋白。那，我们开发了两个鼠标模型，其中TNF具有 在遗传上删除了：1）亨尔环的较厚的升节（tal）和2）远端肾单位 近端管（PT）的下游，将应用于确定TNF的作用和机制 由肾上皮细胞产生，作为新出现的管内TNF系统的一部分，该系统减弱了 由高盐（HS）摄入诱导的BP。使用PT和TAL特异性TNF沉默的完整方法 慢病毒构建体将用于专门抑制这些肾单位段的TNF生产以定义 源自肾上皮细胞对该细胞因子在肾脏中调节作用的TNF的贡献。 遗传和慢病毒方法还将用于确定TNF的分子机制 调节NKCC2磷酸化和同工型表达，肾功能和BP。初步数据指示 通过激活TNF受体1（TNFR1），TNF抑制A磷酸-NKCC2（PNKCC2）的表达 涉及丝氨酸/苏氨酸磷酸酶钙调神经酶（CN）激活的机制。 TNF对 肾脏中的CN尚未研究，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的表达。遗传和慢病毒 将适应策略以确定盐摄入对TNFR1依赖性CN介导的抑制的影响 PNKCC2表达，电解质排泄和对HS摄入的BP响应。 NKCC2的微调 功能取决于NKCC2A和NKCC2B同工型，它们在战略上沿着策略性定位 哺乳动物TAL，并为高盐和低盐条件响应调节功能做出贡献， TNF抑制这些同工型的表达，这表明该细胞因子在MTAL中起作用 和TAL的CTAL/MD段。此外，TNF调节涉及这些同工型的肾功能 限制NaCl的重吸收的方式。但是，TNF抑制的分子机制 NKCC2A和NKCC2B mRNA积累分别响应高盐和低盐的摄入量尚未 TAL与初步数据结合的miRNA分析已确定miRNA 调节NKCC2同工型mRNA抽象，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特异性慢病毒操纵将链接miRNA-195 TNF诱导的表达来自TAL到NKCC2A依赖性机制，该机制减弱了BP 摄入HS的老鼠。总的来说，研究将揭示一个机制 其中肾小管上皮细胞产生的TNF响应盐摄入量增加，调节 NKCC2同工型表达，通过CN磷酸化，电解质排泄，并导致BP稳态。