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

基本信息

批准号：
10684910
负责人：
NICHOLAS R FERRERI
金额：
$ 41万
依托单位：
NEW YORK MEDICAL COLLEGE
依托单位国家：
美国
项目类别：
财政年份：
2021
资助国家：
美国
起止时间：
2021-09-01 至 2025-08-31
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10684910
关键词：
Address Affect African American population Angiotensinogen Attenuated Binding Blood Pressure Calcineurin Calcineurin inhibitor Cardiovascular Diseases Catalytic Domain Chronic Cyclosporine Data Development Distal Electrolytes Epithelial Cells Excretory function Exhibits Framingham Heart Study Functional disorder Genes Genetic Heart Diseases Homeostasis Human Hypertension Individual Inflammatory Ingestion Intake Ions Kidney Kidney Diseases Lentivirus Limb structure Macula densa Mediating Messenger RNA MicroRNAs Molecular Mus Mutation Nephrons Nucleotides PPP3CA gene Patients Phosphoric Monoester Hydrolases Phosphorylation Play Population Production Protein Isoforms Regulation Renal function Renal tubule structure Renin-Angiotensin System Risk Factors Role Serine Sodium Chloride Source Stroke Structure of ascending limb of Henle&apos s loop System TNF gene TNFRSF1A gene Tacrolimus Thick Threonine Tubular formation Tumor Necrosis Factor Activation United States Untranslated RNA Untranslated Regions Up-Regulation Water absorption blood pressure control blood pressure elevation blood pressure reduction blood pressure regulation cardiovascular risk factor cell type cytokine dietary salt experimental study hypertensive kidney cell mRNA Transcript Degradation member miRNA expression profiling mortality risk mouse model novel posttranscriptional receptor renal epithelium response salt intake saluretic screening side effect symporter

项目摘要

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.

我们之前表明，肾脏内产生的肿瘤坏死因子-α (TNF) 是调节肾功能和血压 (BP) 对膳食盐增加的反应的机制摄入量。我们最近的研究表明，TNF 对肾脏的影响在髓质 (m) 厚度中很明显肾单位的升肢 (TAL)、近端小管 (PT) 和皮质 (c) TAL/致密斑 (MD) 区域。然而，产生这些效应的 TNF 的肾脏内细胞来源尚未发现。尚未确定，也尚未确定其分子机制。为此，我们开发了两款鼠标 TNF 已在以下部位进行基因删除的模型：1) TAL，2) PT 下游的远端肾单位，它将用于了解肾上皮细胞产生的 TNF 作为新兴疾病的一部分的作用管内 TNF 系统可减弱高盐 (HS) 摄入引起的血压升高。我们还有使用 PT 和 TAL 特异性 TNF 沉默慢病毒构建体定制了一种补充方法，特异性抑制这些细胞类型的 TNF 产生。遗传和慢病毒方法将用于串联以确定 TNF 调节 Na+-K+-2Cl- (NKCC2) 磷酸化的机制和同种型表达、肾功能和血压。初步数据表明，TNF 通过激活 TNF 受体 1 (TNFR1)，通过涉及激活 NKCC2 的机制抑制磷酸化 NKCC2 (pNKCC2) 表达丝氨酸/苏氨酸磷酸酶、钙调神经磷酸酶 (CN)。 TNF 对 CN 的影响尚未在肾，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。遗传和慢病毒策略将适应确定盐摄入量对 TNFR1 依赖性 CN 介导的 pNKCC2 表达抑制的影响，电解质排泄，以及血压对 HS 摄入的反应。 NKCC2A 和 NKCC2B 亚型具有战略意义位于哺乳动物 TAL 上，有助于响应高盐和低盐的调节功能条件分别。 TNF 抑制两种亚型的表达，表明该细胞因子在两种亚型中均发挥作用 TAL 的 mTAL 和 cTAL/MD 段。我们之前表明，在每种情况下，TNF 都会调节肾功能以限制 NaCl 重吸收的方式涉及这些亚型。然而，分子 TNF 响应高盐和低盐抑制 NKCC2A 和 NKCC2B mRNA 的机制摄入量分别尚未确定。 TAL 先前的 miRNA 分析与新的 miRNA 分析相结合初步数据已鉴定出 3 个调节 NKCC2 同工型 mRNA 丰度的候选 miRNA。为了例如，miRNA-195 表达由 TAL 衍生的 TNF 诱导，并抑制 NKCC2A mRNA 摄入 HS 的小鼠体内的积累和 pNKCC2 表达。总的来说，这些研究将定义一部小说肾小管内调节系统，其中肾小管上皮细胞产生 TNF，响应增加盐摄入量，调节 NKCC2 亚型表达和功能，并有助于血压稳态。