Role of MicroRNAs in Kidney Sodium Regulation

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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.