The Mineralocorticoid Receptor (MR), a hormone-activated transcription factor that mediates sodium-retaining action of aldosterone, is highly expressed in the distal nephron in which large variations in extracellular fluid tonicity are generated by the cortico-papillary gradient. However, mechanisms regulating MR expression remain sparse. We recently showed that extracellular tonicity modulates renal MR expression through posttranscriptional mechanisms (Viengchareun, Mol Endocrinol, 2009) involving recruitment of RNA-binding proteins, which modulate the half-life time of MR transcript. Indeed, hypertonicity compromises MR signaling through Tis11b-mediated MR mRNA degradation (Viengchareun, J Am Soc Nephrol, 2014), while HuR favors MR mRNA stability (Lema, Cell Mol Life Sci, 2017) and edits MR transcript (Lema, Sci Rep, 2017), thus enhancing renal MR signaling under hypotonicity. Herein, we showed that MR transcript is also targeted by microRNAs (miRNAs), which represent another class of posttranscriptional regulators. We first demonstrated that expression of miR-324-5p increases under hypertonicity (×1.5) in the differentiated cortical collecting duct KC3AC1 cells, but also in mouse kidneys (×4) under conditions mimicking extracellular distal hypertonicity (furosemide exposure). Next, we showed that miR-324-5p functionally interacts with MR 3-untranslated region (3-UTR). Indeed, miR-324-5p mimics, leading to miRNA overexpression in transfected HEK293T cells, significantly decrease in a dose-dependent manner the luciferase activity of a reporter plasmid in which the MR 3-UTR was cloned downstream of the luciferase reporter gene. Stable clones of KC3AC1 cells, overexpressing inducible miR-324-5p, are currently generated using lentiviral strategy to further investigate functional consequences on aldosterone signaling and sodium transport. Moreover, we showed that miR-324-5p is secreted into the apical compartment of KC3AC1 cells, suggesting that this miRNA may also act in a paracrine and/or endocrine manner on other cell types. We have also performed pilot experiments that conclusively demonstrate the feasibility to quantify this miRNA in urine samples of humans and mice subjected to osmotic stress (hypertonicity or hypotonicity). As a result, we set up a collaboration with the Clinical Investigation Center of Poitiers Hospital to get plasma and urine samples of well-characterized patients presenting with cardio-renal pathologies such as diabetic nephropathy. Thus, miR-324-5p might represent a promising new biomarker or a novel therapeutic target for renal dysfunction. Collectively, we demonstrate for the first time that renal MR expression is post-transcriptionally regulated by both RNA-Binding Proteins and miRNAs. Such regulatory mechanisms could lead to new pharmacological perspectives of modulating aldosterone signaling during the management and follow-up of cardio-renal pathologies.
18 - 21 May 2019
European Society of Endocrinology