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.

我们之前已经证明，在肾脏中产生的肿瘤坏死因子-α是系统的一部分。 它通过调节肾功能和血压(BP)对食盐摄入量增加的反应 抑制Na+-K+-2Cl-(NKCC2)共转运体。因此，我们开发了两种小鼠模型，在其中，肿瘤坏死因子 基因缺失：1)粗大的Henle氏环(TAL)升支，2)远端肾单位 近端小管(PT)下游，用于确定肿瘤坏死因子的作用和机制 由肾上皮细胞产生，作为新出现的肾小管内肿瘤坏死因子系统的一部分，该系统可减缓 高盐(HS)摄入引起的BP。一种互补的方法，使用PT和TAL特异性的肿瘤坏死因子沉默 慢病毒构建，将被用来特异性地抑制这些肾单位节段产生肿瘤坏死因子，以定义 肾上皮细胞来源的肿瘤坏死因子对该细胞因子在肾脏中的调节作用的贡献。 遗传和慢病毒方法也将被用来确定肿瘤坏死因子 调节NKCC2的磷酸化和异构体表达、肾功能和血压。初步数据显示 肿瘤坏死因子通过激活肿瘤坏死因子受体1(TNFR1)抑制磷酸化NKCC2(PNKCC2)的表达。 涉及丝氨酸/苏氨酸磷酸酶，钙调神经磷酸酶(CN)的激活机制。肿瘤坏死因子对血管内皮细胞生长的影响 CN在肾脏中的作用尚未得到研究，因此实验将解决肿瘤坏死因子依赖的CN活性增加问题 以及催化亚单位CNAb和调节亚基CNB的表达。基因和慢病毒 将采用策略来确定盐摄入量对依赖于TNFR1的CN介导的抑制的影响 PNKCC2表达、电解质排泄和血压对HS摄入量的反应。NKCC2的微调 功能依赖于NKCC2A和NKCC2B亚型，这两种亚型在战略上沿着 哺乳动物的TAL，并有助于对高盐和低盐条件的调节功能， 分别进行了分析。肿瘤坏死因子抑制这些异构体的表达，提示该细胞因子在mTAL和mTAL中的作用 和TAL的cTAL/MD节段。此外，肿瘤坏死因子调节肾功能，涉及这些异构体。 限制重吸收氯化钠的方式。然而，肿瘤坏死因子抑制的分子机制 NKCC2A和NKCC2B对高盐摄入量和低盐摄入量的反应分别没有 已经确定了。结合初步数据，TAL的miRNA图谱已经确定了miRNAs 调节NKCC2亚型的mRNA丰度、磷酸化和BP，首次数据显示NKCC2的功能是 受依赖于miRNA的机制调节。针对TAL的慢病毒操作将把miRNA-195 肿瘤坏死因子诱导的TAL表达可能是一种NKCC2A依赖机制，可减轻血压 摄入HS的小鼠。总而言之，这些研究将揭示肾小管内调控系统， 其中，肾小管上皮细胞产生的肿瘤坏死因子对盐摄入量的增加做出反应，调节 NKCC2的异构体表达、通过CN的磷酸化、电解质排泄，并有助于BP的动态平衡。