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.

我们以前的研究表明，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）对饮食盐摄入量增加的反应， 抑制Na+-K+-2Cl-（NKCC 2）协同转运蛋白。因此，我们开发了两种小鼠模型，其中TNF 在以下基因中缺失：1）Henle袢（TAL）的粗升支，和2）远端肾单位 下游的近端小管（PT），这将被应用于确定TNF的作用和机制 由肾上皮细胞产生，作为新兴小管内TNF系统的一部分， 高盐（HS）摄入引起的血压。使用PT和TAL特异性TNF沉默的互补方法 慢病毒构建体将用于特异性抑制这些肾单位节段的TNF产生， 来源于肾上皮细胞的TNF对该细胞因子在肾中的调节作用的贡献。 遗传学和慢病毒方法也将用于确定TNF-α与细胞凋亡的分子机制。 调节NKCC 2磷酸化和亚型表达、肾功能和血压。初步数据显示 TNF通过激活TNF受体1（TNFR 1），通过抑制磷酸化NKCC 2（pNKCC 2）的表达， 涉及丝氨酸/苏氨酸磷酸酶、钙调磷酸酶（CN）的活化的机制。TNF对 尚未研究肾脏中的CN，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。基因和慢病毒 策略将适用于确定盐摄入对TNFR 1依赖性CN介导的抑制的影响， pNKCC 2表达、电解质排泄和血压对HS摄入的反应。NKCC 2的微调 功能依赖于NKCC 2A和NKCC 2B同种型，它们战略性地定位于沿着 哺乳动物TAL并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，表明这种细胞因子在mTAL和 和TAL的cTAL/MD片段。此外，TNF调节肾功能，涉及这些异构体， 限制NaCl再吸收的方式。然而，TNF抑制的分子机制 NKCC 2A和NKCC 2B mRNA的积累分别响应于高盐和低盐摄入， 确定了TAL的miRNA谱分析结合初步数据已经鉴定了 调节NKCC 2亚型mRNA丰度、磷酸化和BP，第一个显示NKCC 2功能的数据是 由miRNA依赖性机制调节。TAL特异性慢病毒操作将连接miRNA-195 由TAL衍生的TNF诱导的表达与NKCC 2A依赖性机制有关，该机制减弱了 小鼠摄食HS。总的来说，这些研究将揭示小管内调节系统， 其中肾小管上皮细胞响应盐摄入增加而产生的TNF调节 NKCC 2亚型表达，通过CN磷酸化，电解质排泄，并有助于BP稳态。