Role of MicroRNAs in Kidney Sodium Regulation

MicroRNA 在肾钠调节中的作用

• 批准号: 10756627
• 负责人: Michael B Butterworth
• 金额: $ 7.61万
• 依托单位: UNIVERSITY OF PITTSBURGH AT PITTSBURGH
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-05-04 至 2025-04-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10756627
• 关键词: Address; Adrenal Glands; Aldosterone; Angiotensin I; Angiotensin II; Angiotensinogen; Angiotensins; Applications Grants; Atrial Fibrillation; Blood Pressure; Blood Pressure Monitors; Body Fluids; Cardiovascular system; Cell Line; Cells; Cortical Nephron; Cytoprotection; Data; Disease; Distal; Duct (organ) structure; Electrolyte Balance; Epithelium; Equilibrium; Feedback; Female; Funding; Glucocorticoids; Goals; Grant; Health; Heart Hypertrophy; Homeostasis; Hormones; Hour; Hyperaldosteronism; Hypertension; Kidney; Knockout Mice; Link; Messenger RNA; Metabolic syndrome; MicroRNAs; Mineralocorticoid Receptor; Mineralocorticoids; Mus; Nature; Nephrons; Nucleotides; Phosphotransferases; Physiological; Premenopause; Production; Proteins; Regulation; Renin; Repression; Research; Research Personnel; Resistant Hypertension; Risk; Role; Serum; Sex Differences; Signal Pathway; Signal Transduction; Signaling Protein; Sodium; Sum; System; Testing; Time; Untranslated RNA; Up-Regulation; Woman; Women' s Health; absorption; arterial stiffness; blood pressure control; blood pressure elevation; blood pressure regulation; cardiovascular disorder risk; collecting tubule structure; coronary fibrosis; desensitization; dietary; dietary salt; in vivo; kidney cell; kidney fibrosis; male; mortality; mouse model; novel; osmotic minipump; parent grant; protein expression; response; salt intake; sex; steroid hormone; stroke risk; tool

RESEARCH PLAN Summary and Aims of Funded Parent Grant Summary The funded grant (DK102843) is investigating the role of small non‐coding RNAs, termed microRNAs (miRs), in the regulation of sodium (Na+) transport in the distal kidney nephron. We are testing the hypothesis that the mineralocorticoid hormone, aldosterone regulates the expression of specific miR clusters in the distal kidney nephron cortical collecting duct (CCD). These miRs target mRNAs to alter Na+ transport. Extended up- regulation of the aldosterone-induced miRs then feedback to reduce the aldosterone response (negative feedback regulation). We propose to test this novel role of miRs in Na+ homeostasis by using a miR-17~92 cluster KO mouse line. The miR-17~92 cluster will be deleted from the kidney nephron using a conditional, inducible KO mouse model (already generated) and the impact on long-term aldosterone signaling will be investigated. In the second part of the proposal, the mechanisms miRs use to alter Na+ transport will be investigated in primary kidney cells. We will then determine how this regulation impacts blood pressure homeostasis. The proposed supplement represents a “carve-out” of the parent grant to look at sex differences in miR-17~92 regulation of aldosterone signaling and blood pressure homeostasis. This was proposed as sum- aims in all three specific aims, and aligns well with the applicant’s stated goals of conducting health relevant research, that can be specifically applied to women’s health. The role of miR in blood pressure regulation has not been extensively explored, and sex differences in miR expression in the kidney will be detailed for the first time in these studies. Parent grant specific aims Hypertension is associated with an increased risk of cardiovascular disease and is the largest contributor to cardiovascular mortality worldwide. The steroid hormone aldosterone (aldo) helps to maintain body fluid volume and sodium (Na+) balance within narrow limits. Primary aldosteronism is a disorder caused by excessive aldo production and accounts for almost 20% of resistant hypertension. Elevated aldo levels or defective signaling in the renin-angiotensin-aldosterone signaling (RAAS) cascade are linked to increased blood pressure, risk of stroke, atrial fibrillation, cardiac hypertrophy and fibrosis and arterial stiffening. These risks are lowered in premenopausal women due, in part, to reduced RAAS signaling and lower aldo levels. Understanding aldo signaling and the systems that keep the RAAS pathway in check are vital to address disorders associated with aldo excess, like metabolic syndrome, resistant hypertension and renal and cardiac fibrosis. MicroRNAs (miRs) are small (~23 nucleotide) non-coding RNAs that are negative regulators of protein expression. MiRs act as dampers in signaling cascades making them ideally suited for the regulation of RAAS proteins. Angiotensinogen Adrenal Aldo Angiotensin I ACE Kidney Angiotensin II Aldosterone MR miR-17~92 SGK1 miR-466a-e Na+ Transporters ↑Na+ Reabsorption Fig.1.Schematic depiction of the hypothesis. MiR-17~92 is induced by aldo to act as a negative regulator of RAAS signaling. (green/blue = stimulation, red = inhibition) We identified a role for miRs in aldo signaling, linking aldo-induced changes in miR expression with alterations in Na+ transport in a kidney collecting duct cell line (mCCD). We recently showed that miRs act as negative feedback regulators of aldo signaling in mCCD cells. The role of miR feedback in maintaining Na+ balance in vivo is unknown. This study will demonstrate that the miR-17~92 cluster is elevated during extended aldo exposure, in female mice, to reduce the cellular aldo response (see schematic Fig.1). We will investigate the overarching hypothesis that extended aldosterone signaling upregulates miRs that target RAAS mRNAs to reduce protein expression, acting as a negative feedback regulator in Na+ homeostasis. These studies will establish a role for miR-17~92 in the RAAS pathway and provide the genesis for identifying sex differences in aldo signaling and blood pressure control. We have three aims. Aim #1: Determine the time-course and sex-specific regulation of miRs after aldo stimulation. Based on our preliminary data we hypothesize that miR-17~92 is upregulated in the distal kidney nephron epithelia of female mice following prolonged aldo exposure. We will: 1.1) Determine the changes in miR expression in mice of both sexes following extended aldo stimulation. 1.2) Track the time-course of miR regulation by aldo from short (hours) to long exposures (days-weeks) induced by changes to dietary Na+ or by using aldo osmotic minipumps. Aim #2: Use a miR-17~92 KO mouse to establish the miRs’ role in maintaining Na+ homeostasis. We have data to show that altering the expression of miR-17~92 changes target RAAS expression. We hypothesize that deleting miR-17~92 will increase regulated Na+ reabsorption and blood pressure in response to aldo and/or dietary Na+ changes. We will: 2.1) Use a nephron-specific, inducible, miR-17~92 KO mouse to define the impact on Na+ reabsorption, Na+-transporter activation and electrolyte balance during short and long-term aldo stimulation. 2.2) Monitor blood pressure changes in KO mice with physiological and exogenous changes to aldo. Aim #3: Define the miR-RAAS interactome and mechanisms of aldo desensitization by miR-17~92. MiR-17~92 targets include the serum and glucocorticoid kinase and mineralocorticoid receptor. Inhibition of these aldo-regulated proteins would facilitate negative feedback regulation of aldo signaling. We hypothesize that release of miR-17~92 repression would sensitize cells to aldo. To test this we will: 3.1) Use ex-vivo primary cultures to compare aldo sensitivity in male vs female, control and KO mice. 3.2) Employ established miR tools to override miR expression and link miRs to changes in aldo sensitivity. The PI and co-investigators have been studying miR regulation in the kidney and the role of dietary salt intake in blood pressure regulation for years. This proposal expands on our studies that identified aldo- sensitive miRs in the kidney. The role of miRs to act as a rheostat in feedback regulation of Na+ homeostasis will be examined and the sex-specific nature of miR regulation demonstrated. These studies will place miRs as integral components in RAAS signaling, protecting cells from extended aldo exposure.

研究计划 资助家长补助金的摘要和目标 概括 资助项目 (DK102843) 正在研究小型非编码 RNA（称为 microRNA (miR)）的作用， 我们正在验证以下假设： 盐皮质激素、醛固酮调节远端肾脏中特定 miR 簇的表达 这些 miR 靶向 mRNA 来改变 Na+ 转运。 醛固酮诱导的 miR 的调节然后反馈以减少醛固酮反应（负 我们建议使用 miR-17~92 来测试 miR 在 Na+ 稳态中的新作用。 簇 KO 小鼠系将使用条件从肾单位中删除 miR-17~92 簇。 可诱导的 KO 小鼠模型（已生成）以及对长期醛固酮信号传导的影响将是 在该提案的第二部分中，将研究 miR 用于改变 Na+ 转运的机制。 然后我们将在原代肾细胞中进行研究，以确定这种调节如何影响血压。 拟议的补充代表了对家长补助金的“剔除”，以考虑性别差异。 miR-17~92 对醛固酮信号传导和血压稳态的调节 旨在实现所有三个具体目标，并且与申请人进行健康相关的既定目标非常一致 miR 在血压调节中的作用可以专门应用于女性健康。 尚未得到广泛探索，肾脏中 miR 表达的性别差异将首次详细说明 花时间在这些研究上。 家长补助金的具体目标 高血压与心血管疾病风险增加有关 疾病，是导致心血管死亡的最大因素 在世界范围内，类固醇激素醛固酮 (aldo) 有助于维持身体健康。 液体量和钠 (Na+) 平衡在狭窄范围内。 醛固酮增多症是一种由醛固酮生成过多引起的疾病 约占难治性高血压的 20%。 肾素-血管紧张素-醛固酮信号传导 (RAAS) 信号传导缺陷 级联与血压升高、中风风险、心房 颤动、心脏肥大和纤维化以及动脉硬化。 绝经前女性的风险降低，部分原因是 RAAS 减少 了解 ALDO 信号传导和降低 ALDO 水平。 控制 RAAS 路径的系统对于解决问题至关重要 与醛过多相关的疾病，如代谢综合征、耐药性 高血压以及肾脏和心脏纤维化。 MicroRNA (miR) 是小型（约 23 个核苷酸）非编码 RNA， 蛋白质表达的负调节因子在信号传导中起到阻尼器的作用。 级联使它们非常适合 RAAS 蛋白的调节。 肾上腺血管紧张素原 奥尔多 血管紧张素I ACE肾 血管紧张素II 醛固酮 先生 miR-17~92 SGK1 miR-466a-e Na+转运蛋白 ↑Na+重吸收 图1.示意图 假设是MiR-17~92。 由 aldo 诱导充当 RAAS负调节因子 信号（绿色/蓝色 = 刺激，红色=抑制） 我们确定了 miR 在 aldo 信号传导中的作用，将 aldo 诱导的 miR 表达变化与 肾集合管细胞系 (mCCD) 中 Na+ 转运的改变 我们最近表明 miR 起到了作用。 mCCD 细胞中 aldo 信号的负反馈调节器 miR 反馈在维持 Na+ 中的作用。 体内平衡尚不清楚，本研究将证明 miR-17~92 簇在过程中升高。 延长雌性小鼠的醛酮暴露量，以减少细胞醛酮反应（见图 1）。 我们将研究扩展醛固酮信号传导上调 miR 的总体假设 靶向 RAAS mRNA 来减少蛋白质表达，充当 Na+ 的负反馈调节剂 这些研究将确定 miR-17~92 在 RAAS 通路中的作用并提供 识别 ALDO 信号传导和血压控制中的性别差异的起源 我们有三个目标。 目标#1：确定 ALDO 刺激后 miR 的时程和性别特异性调节。 根据我们的初步数据，我们认为 miR-17~92 在远端肾单位中表达上调 长期暴露于醛醇后雌性小鼠的上皮细胞我们将： 1.1)确定两种性别小鼠在延长aldo刺激后miR表达的变化。 1.2) 跟踪 aldo 从短（小时）到长暴露（天-周）调节 miR 的时间过程 由饮食 Na+ 的变化或使用醛渗透微型泵引起。 目标#2：使用 miR-17~92 KO 小鼠确定 miR 在维持 Na+ 稳态中的作用。 我们有数据表明，改变 miR-17~92 的表达会改变目标 RAAS 的表达。 删除 miR-17~92 将增加调节的 Na+ 重吸收和血压 对醛醇和/或饮食 Na+ 变化的反应我们将： 2.1) 使用肾单位特异性、可诱导的 miR-17~92 KO 小鼠来定义对 Na+ 重吸收的影响， 短期和长期 aldo 刺激期间 Na+ 转运蛋白激活和电解质平衡。 2.2) 监测 KO 小鼠的血压变化，以及 ALDO 的生理和外源性变化。 目标#3：定义 miR-RAAS 相互作用组和 miR-17~92 的 ALDO 脱敏机制。 MiR-17~92 靶点包括血清和糖皮质激素激酶以及盐皮质激素受体抑制。 这些 ALDO 调节蛋白的表达将促进 ALDO 信号传导的负反馈调节。 坚持认为 miR-17~92 抑制的释放会使细胞对 ALDO 敏感。为了测试这一点，我们将： 3.1) 使用离体原代培养物比较雄性与雌性、对照和 KO 小鼠的 aldo 敏感性。 3.2) 使用已建立的 miR 工具来覆盖 miR 表达并将 miR 与 aldo 敏感性的变化联系起来。 PI 和共同研究人员一直在研究肾脏中的 miR 调节以及膳食盐的作用 该提案扩展了我们多年来确定的醛固酮摄入量的研究。 肾脏中敏感的 miR 充当变阻器在 Na+ 反馈调节中的作用。 这些研究将检查体内平衡并证明 miR 调节的性别特异性。 将 miR 作为 RAAS 信号传导的组成部分，保护细胞免受长时间的 ALDO 暴露。

项目成果

MicroRNAs in kidney development and disease.

MicroRNA 在肾脏发育和疾病中的作用。

• 发表时间: 2022-05-09
• 影响因子: 8
• 作者: Cerqueira, Débora Malta;Tayeb, Maliha;Ho, Jacqueline
• 通讯作者: Ho, Jacqueline

Non-coding RNAs and the mineralocorticoid receptor in the kidney.

肾脏中的非编码 RNA 和盐皮质激素受体。

• 发表时间: 2021
• 影响因子: 4.1
• 作者: Butterworth; Michael B
• 通讯作者: Michael B

The tale of two (distal nephron) cell types.

两种（远端肾单位）细胞类型的故事。

• 发表时间: 2018-05-01
• 作者: Butterworth; Michael B
• 通讯作者: Michael B

Aldosterone-induced microRNAs act as feedback regulators of mineralocorticoid receptor signaling in kidney epithelia.

醛固酮诱导的 microRNA 作为肾上皮中盐皮质激素受体信号传导的反馈调节剂。

• 发表时间: 2020
• 作者: Ozbaki;Liu, Xiaoning;Bodnar, Andrew J;Ho, Jacqueline;Butterworth, Michael Bruce
• 通讯作者: Butterworth, Michael Bruce

Expression of a Diverse Array of Ca2+-Activated K+ Channels (SK1/3, IK1, BK) that Functionally Couple to the Mechanosensitive TRPV4 Channel in the Collecting Duct System of Kidney.

多种 Ca2 激活 K 通道（SK1/3、IK1、BK）的表达，这些通道在功能上与肾集合管系统中的机械敏感 TRPV4 通道耦合。

• 发表时间: 2016
• 影响因子: 3.7
• 作者: Li, Yue;Hu, Hongxiang;Butterworth, Michael B;Tian, Jin;Zhu, Michael X;O'Neil, Roger G
• 通讯作者: O'Neil, Roger G