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：
10887848
负责人：
NICHOLAS R FERRERI
金额：
$ 8.47万
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/10887848
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.

我们先前表明，肾脏内产生的肿瘤坏死因子-Alpha（TNF）是A的一部分调节肾功能和血压（BP）对饮食盐增加的反应的机制进气。我们最近的研究表明，肾脏中的TNF效应在髓质（M）厚中很明显上升的肢体（TAL），近端小管（PT）和肾单位的皮质（C）TAL/MACULA DENSA（MD）区域。但是，产生有关这些影响的TNF的肾脏中的细胞来源尚未已经确定，也没有鉴定出分子机制。因此，我们开发了两只鼠标 TNF在以下遗传上删除的模型：1）TAL和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的作用，作为新兴的一部分高盐（HS）摄入诱导的BP增加的肿瘤内TNF系统。我们也有使用PT和TAL特异性TNF沉默的慢病毒构建体量身定制互补方法特别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确定TNF调节Na+-k+-2Cl-（NKCC2）磷酸化和磷酸化和同工型表达，肾功能和BP。初步数据表明，TNF通过激活TNF受体 1（TNFR1），通过涉及激活的机制抑制磷酸-NKCC2（PNKCC2）表达丝氨酸/苏氨酸磷酸酶，钙调神经酶（CN）。尚未探讨TNF对CN的影响肾脏，因此实验将解决CN活性的TNF依赖性增加以及表达催化亚基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表达的抑制作用，电解质排泄和BP对HS摄入的反应。 NKCC2A和NKCC2B同工型在战略上是沿哺乳动物TAL局部，并促进高盐和低盐的调节功能条件分别。 TNF抑制两种同工型的表达，表明该细胞因子在两者中都起作用 TAL的MTAL和CTAL/MD段。我们先前表明，在每种情况下，TNF都调节涉及这些同工型的肾功能以限制重吸收NaCl的方式。但是，分子 TNF抑制NKCC2A和NKCC2B mRNA的机制，响应高盐和低盐摄入量分别尚未确定。 TAL的先前miRNA分析与新组合初步数据已经确定了调节NKCC2同工型mRNA丰度的3个候选miRNA。为了实例，miRNA-195表达是由源自TAL的TNF诱导的，并抑制了NKCC2A mRNA 摄入HS的小鼠中的积累和PNKCC2表达。总体而言，研究将定义一个小说肾脏内调节系统，其中肾小管上皮细胞产生TNF，以响应于盐摄入量的增加，调节NKCC2同工型表达和功能，并有助于BP稳态。