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

The FASEB JournalVolume 34, Issue 9 p. 11714-11728 RESEARCH ARTICLEFree Access Aldosterone-induced microRNAs act as feedback regulators of mineralocorticoid receptor signaling in kidney epithelia Nejla Ozbaki-Yagan, Department of Cell Biology, University of Pittsburgh, Pittsburgh, PA, USASearch for more papers by this authorXiaoning Liu, Department of Cell Biology, University of Pittsburgh, Pittsburgh, PA, USASearch for more papers by this authorAndrew J. Bodnar, Division of Nephrology, Department of Pediatrics, University of Pittsburgh, Pittsburgh, PA, USASearch for more papers by this authorJacqueline Ho, Division of Nephrology, Department of Pediatrics, University of Pittsburgh, Pittsburgh, PA, USASearch for more papers by this authorMichael Bruce Butterworth, Corresponding Author michael7@pitt.edu Department of Cell Biology, University of Pittsburgh, Pittsburgh, PA, USA Correspondence Michael Bruce Butterworth, Department of Cell Biology, University of Pittsburgh School of Medicine, S314 BST, 200 Lothrop St., Pittsburgh, PA, 15261, USA. Email: michael7@pitt.eduSearch for more papers by this author Nejla Ozbaki-Yagan, Department of Cell Biology, University of Pittsburgh, Pittsburgh, PA, USASearch for more papers by this authorXiaoning Liu, Department of Cell Biology, University of Pittsburgh, Pittsburgh, PA, USASearch for more papers by this authorAndrew J. Bodnar, Division of Nephrology, Department of Pediatrics, University of Pittsburgh, Pittsburgh, PA, USASearch for more papers by this authorJacqueline Ho, Division of Nephrology, Department of Pediatrics, University of Pittsburgh, Pittsburgh, PA, USASearch for more papers by this authorMichael Bruce Butterworth, Corresponding Author michael7@pitt.edu Department of Cell Biology, University of Pittsburgh, Pittsburgh, PA, USA Correspondence Michael Bruce Butterworth, Department of Cell Biology, University of Pittsburgh School of Medicine, S314 BST, 200 Lothrop St., Pittsburgh, PA, 15261, USA. Email: michael7@pitt.eduSearch for more papers by this author First published: 11 July 2020 https://doi.org/10.1096/fj.201902254RRCitations: 5AboutSectionsPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onEmailFacebookTwitterLinked InRedditWechat Abstract The final steps in the Renin-Angiotensin-Aldosterone signaling System (RAAS) involve binding of the corticosteroid hormone, aldosterone to its mineralocorticoid receptor (MR). The bound MR interacts with response elements to induce or repress the transcription of aldosterone-regulated genes. A well characterized aldosterone-induced gene is the serum and glucocorticoid-induced kinase (SGK1), which acts downstream to increase sodium transport in distal kidney nephron epithelial cells. The role of microRNAs (miRs) induced by extended aldosterone stimulation in regulating MR and SGK1 has not been reported. In these studies, miRs predicted to bind to the 3ʹ-UTR of mouse MR were profiled by qRT-PCR after aldosterone stimulation. The miR-466a/b/c/e family was upregulated in mouse kidney cortical collecting duct epithelial cells. A luciferase reporter assay confirmed miR-466 binding to both MR and SGK1 3ʹ-UTRs. Inhibition of miR-466 increased MR and SGK1 mRNA and protein levels. Inhibiting miR-466b and preventing its upregulation after aldosterone stimulation increased amiloride-sensitive sodium transport and sensitivity to aldosterone stimulation. In vivo upregulation of miR-466 was confirmed in distal nephrons of mice on low Na+ diets. Repression of MR and SGK1 by aldosterone-induced miRs may represent a negative feedback loop that contributes to a form of aldosterone escape in vivo. Abbreviations ACE2 angiotensin-converting enzyme 2 AGTR1B angiotensin II receptor, type 1b ATP1A2 ATPase, Na⁺/K⁺ transporting, alpha 2 polypeptide ATP1B4 ATPase Na+/K+ transporting family member beta 4 miRs microRNAs MR mineralocorticoid receptor NCC sodium-chloride co-transporter (SLC12A3) SGK1 serum and glucocorticoid-induced kinase 1 WNK1 WNK lysine deficient protein kinase 1 1 INTRODUCTION Inappropriately elevated levels of the mineralocorticoid steroid hormone aldosterone lead to the development of hypertension.1-3 Aldosterone is the final constituent of the renin-angiotensin-aldosterone (RAAS) signaling cascade.4-7 Aldosterone is produced in the zona glomerulosa cells of the adrenal gland in response to renin, which is released when plasma sodium (Na+) or blood volume is low.8-11 By binding to the mineralocorticoid receptor (MR) aldosterone induces the transcription of proteins that function together to establish a coordinated cellular genomic response.12, 13 The purpose of the final signaling step is to increase Na+ reabsorption from glomerular filtrate. Homeostatic feedback following aldosterone stimulation reverses the signaling cascade as the cues to release aldosterone (reduced plasma volume or lower Na+ levels) are diminished when plasma Na+ and volume are restored.14-16 There are known cellular mechanisms that reverse aldosterone's action and keep the signaling cascade in check. Studies have demonstrated that aldosterone stimulation results in posttranslational modifications to the MR that cause its degradation.17, 18 A recent study demonstrated that mRNA expression of MR was reduced in response to long-term aldosterone stimulation.19 A decrease in MR would diminish the ability to elicit a full aldosterone stimulation and protect the cells from aldosterone excess. A major aldosterone-induced protein is the serum and glucocorticoid kinase (SGK1). SGK1 is responsible for transducing many of the cellular responses to aldosterone stimulation and coordinates the upregulation of Na+ transport via a number of channels and transporters.20-23 The rapid induction of SGK1 mRNA is not accompanied by an equivalent change in protein expression.24 This moderated cellular response protects the cells from dramatic swings in SGK1 action. The actions of aldosterone are considered pleiotropic with different cellular responses noted in different cell and tissue types, given the same hormonal input.25-27 The posttranscriptional regulation by microRNAs could account for both the restriction of aldosterone signaling (feedback regulation), and the heterogeneous actions of the hormone noted in prior studies. MicroRNAs (miRs) are noncoding RNAs between 18-23 nucleotides long and function primarily to degrade target mRNA and prevent protein synthesis.28-32 Precursor miRs are synthesized in the nucleus, either embedded in the introns (and occasionally the exons) of protein coding genes or encoded as stand-alone and cluster miRs driven by promotors and enhancers specifically regulating the production of the miRs.33-36 Aldosterone can induce or repress miRs, and mineralocorticoid response elements have been noted upstream of aldosterone-induced miRs that would account for their upregulation.37-39 Precursor miRs are exported to the cytoplasm for processing by the endonuclease Dicer.31, 40-44 Dicer processes the stem-loop RNA structure to produce a mature miR strand that is loaded into an inhibitory RNA-Induced Silencing Complex (RISC) with a second endonuclease, Argonaute.45-47 RISC is responsible for target recognition by sequence complementarity of the miRs with mRNAs, mainly in the mRNA 3ʹ-untranslated regions (3ʹ-UTRs).48-50 The targeted mRNA is degraded by the RISC or protein translation is sterically hindered by binding of the RISC to the mRNA so that efficient protein synthesis is interrupted. The net result is a decrease in the steady-state protein level due to the binding of miRs. It is considered that the majority of protein coding mRNAs are targeted by miRs, and that noncoding RNAs (including miRs) regulate up to 80% of all protein coding genes.51-53 MiRs have been shown to target constituents of the RAAS cascade. Production of signaling hormones, expression of receptors, and signaling proteins are all impacted by changes in miR expression.39, 54-59 However, the ability for aldosterone-induced miRs to feedback and alter the effectors of RAAS signaling has not been systematically investigated. In this study, we report on a family of miRs, mmu-miR-466, that are coordinately upregulated in principal epithelial cortical collecting duct (CCD) cells of the distal nephron in response to extended aldosterone stimulation. The miRs’ targets, as demonstrated using a dual luciferase reporter assay, include MR and SGK1. Binding of miR-466 reduces the production of both proteins. By inhibiting miR-466 using antagomirs, this repression is relieved and the protein levels of both MR and SGK1 are elevated after aldosterone stimulation. Sensitivity to extended aldosterone exposure, as measured by the amiloride-sensitive short circuit current activity in cultured mCCD-cl1 cells, is greater when the miR upregulation is prevented. A similar increase in miR-466 expression was observed in vivo in CCD cells isolated from mice placed on low Na+ diets to stimulate aldosterone release. These miRs contribute to a negative feedback loop that dampens long-term aldosterone signaling. This would constitute a form of aldosterone escape that reduces cellular sensitivity to aldosterone to protect aldosterone-sensitive tissues from excessive aldosterone exposure. 2 MATERIALS AND METHODS 2.1 Antibodies and reagents All chemicals were purchased from Sigma-Aldrich (St. Louis, MO) or Thermo Fisher (Pittsburgh, PA) unless noted otherwise. Antibodies used are as follows: anti-mineralocorticoid receptor (mouse monoclonal, anti-rat, isotype MIgG1, kappa light chain from the Developmental Studies Hybridoma Bank (DSHB) at the University of Iowa. The hybridoma was deposited to the DSHB by Gomez-Sanchez, C. (DSHB Hybridoma Product rMR1-18 1D5). Anti-tubulin (mouse monoclonal, anti-Tetrahymena thermophila, and Tetrahymena pyriformis (mixture)), isotype MIgG1 from the DSHB at the University of Iowa. The hybridoma was deposited to the DSHB by Frankel, J. Nelsen, EM (DSHB Hybridoma Product 12G10 anti-alpha-tubulin). Anti-SGK1 Rabbit IgG (Cell Signaling Technology, Cat # D27C11, #12103, Davers, MA). 2.2 Mice and metabolic experiments C57Bl/6 two month old male mice (obtained from Charles River Laboratories, Wilmington, MA) were fed in their home cage with standard diet (0.25% of sodium). Half the mice were then switched to a low salt diet (0.01%-0.02% sodium, Sodium Deficient Diet, Harlan, Frederick, MD) for 3 days as described before.60 All animals were housed in the vivarium at Rangos Research Center at UPMC Children's Hospital of Pittsburgh, Pittsburgh, Pennsylvania, and all animal experiments were carried out in accordance with the policies of the Institutional Animal Care and Use Committee at the University of Pittsburgh. 2.3 Cell culture The mCCD-cl1 cells (kindly provided by B. Rossier and L. Schild, Université de Lausanne, Lausanne, Switzerland) were grown in flasks (passages 30-40) in defined (supplemented) medium at 37°C in 5% of CO2 as described previously.60, 61 The medium was changed every second day. For electrophysiological experiments, the mCCD cells were subcultured onto permeable filter supports (0.4 µm pore size, 0.33 cm2 or 4 cm2 surface area; Transwell, Corning, Lowell, MA). Typically, 24 hours before use in any investigation, medium incubating filter-grown cells were replaced with a minimal medium (without drugs or hormones) that contained DMEM and Ham F12 only. HEK-293 cells (from ATCC) were grown in flasks in DMEM (Invitrogen) supplemented with 10% of FCS. 2.4 Plasmid construction The 3ʹ UTRs of mouse MR and SGK1 were constructed using Gibson assembly of synthesized gene fragments (Integrated DNA Technologies, Coralville, IA) from the known sequences downloaded from the National Center for Biotechnology Information (NCBI) databases. For SGK1, we used GenBank Accession # NM_001161850 and MR (NR3C2) GenBank Accession # NM_001083906. The MR-UTR construct was engineered with AccI/SbfI and the SGK1-UTR with XhoI/XbaI restriction sequences for incorporation into a digested pMir-Glo dual luciferase reporter plasmid (Promega, Madison, WI). The putative miR-466 binding sites, located at positions 456 and 699 bp away from the termination codon in the MR-UTR and, 868 bp in the SGK1-UTR (see Figure 2) were eliminated and gene fragments assembled (as above) to produce UTRs, in which the predicted miR-466 sites were absent. All constructs were sequenced (GeneWiz, South Plainfield, NJ) to verify the correct sequence and desired deletions were incorporated. 2.5 Short-circuit current recordings and equivalent current measurements Inserts were mounted in modified Ussing chambers (P2300; Physiologic Instruments, San Diego, CA) and continuously short circuited with an automatic voltage clamp (VCC MC8; Physiologic Instruments) as described previously.60-62 The apical and basolateral chambers each contained 4 mL of Ringer solution (120 mM of NaCl, 25 mM of NaHCO3, 3.3 mM of KH2PO4, 0.8 mM of K2HPO4, 1.2 mM of MgCl2, 1.2 mM of CaCl2, and 10 mM of glucose). Chambers were constantly gassed with a mixture of 95% of O2, 5% of CO2 at 37°C, which maintained the pH at 7.4 and established a circulating perfusion bath within the Ussing chamber. Simultaneous transepithelial resistance was recorded by applying a 2-mV pulse per minute via an automated pulse generator. Recordings were digitized and analyzed using PowerLab (AD Instruments, Colorado Springs, CO). To screen for aldosterone-regulated miRs from mCCD-cl1 cells in culture, equivalent open circuit currents were obtained from cells using chopstick electrodes and an Epithelial Volt/Ohm Meter (EVOM) (World Precision Instruments, Sarasota, FL). Currents were calculated from cells stimulated with or without 50 nM aldosterone for 24 hours using Ohm's Law from the EVOM-measured open circuit voltages and transepithelial resistance. 2.6 Transfections: RNA interference, miRNA overexpression, and depletion DNA plasmids and RNA oligonucleotide constructs were transiently transfected into the mCCD and HEK293 cells using Lipofectamine 2000 (Invitrogen) according to the manufacturer's instructions and as described previously.60-62 The sequences for siRNA and all primers are listed in Table 1. Double-stranded RNA miR mimics (miRIDIAN microRNA Mimics) were obtained from Thermo Fisher Scientific. To inhibit processing to mature miRs and reduce endogenous miR expression, miRNA inhibitors, antisense modified RNA oligonucleotides (antagomirs), along with nontargeting miRNA inhibitor controls were obtained from IDT (Woburn, MA). Quantitative RT-PCR (below) was used to confirm miR overexpression or inhibition. For all transfections, mCCD cells were seeded onto 6-well culture dishes at low density (~40%) and transfected with the chosen construct using Lipofectamine 2000. The cells remained in OptiMEM with the Lipofectamine 2000 overnight. The next day, cells were washed, lifted from the 6-well plates and seeded at super-density onto Transwell filters in fully supplemented medium. After 24 hours, during which time the cells attached to the filter supports, the cells were washed and placed in a minimal medium that contained only DMEM/F12 without additional supplementation (24 hours) in preparation for aldosterone stimulation. Cells were then stimulated with aldosterone for the indicated times. Therefore, at the earliest stimulation timepoint (24-hours aldosterone) cells had been transfected for 96 hours, and were on filters for 72 hours. TABLE 1. Table listing sequences of primers, oligonucleotides, and summary of the qPCR protocol used in the study qPCR primers Gene Forward primer sequence Reverse primer sequence mouse Actin 5ʹ-GCAGCTCCTTCGTTGCCGGT-3ʹ 5ʹ-GGGGCCACACGCAGCTCATT-3ʹ GAPDH 5ʹ-CATCACCATCTTCCAGGAGCG-3ʹ 5ʹ-GAGGGGCCATCCACAGTCTTC-3ʹ SGK1 5ʹ-CTGCTCGAAGCACCCTTACC-3ʹ 5ʹ-TCCTGAGGATGGGACATTTTCA-3ʹ αENaC 5ʹ-GCTCAACCTTGACCTAGACCT-3ʹ 5ʹ-GGTGGAACTCGATCAGTGCC-3ʹ Nedd4-2 5ʹ-TTGGTGATGTCGACGTGAACGACT-3ʹ 5ʹ-TGGAGGTGCCTGTGACAAACTGTA-3ʹ NR3C2 5ʹ-CAACTATCTGTGTGCTGGAAGA-3ʹ 5ʹ-CCTTGGTAGGAGCAATGTATGT-3ʹ NR3C1 5ʹ-CCTTCGGGAGCTTTAGGTTT-3ʹ 5ʹ-GCAGGGTATTTAGGAGGGTATTT-3ʹ microRNA Primer sequence mmu-let-7a-1-3p CTATACAATCTACTGTCTTTCC mmu-let-7b-3p CTATACAACCTACTGCCTTCCC mmu-let-7c-2-3p CTATACAATCTACTGTCTTTCC mmu-let-7f-1-3p CTATACAATCTATTGCCTTCCC mmu-miR 10a TACCCTGTAGATCCGAATTTGT mmu-miR-101b-3p GTACAGTACTGTGATAGCT mmu-miR-127-5p CTGAAGCTCAGAGGGCTCTGAT mmu-miR-135a-5p TATGGCTTTTTATTCCTATGTGA mmu-miR-186-5p CAAAGAATTCTCCTTTTGGGCT mmu-miR-19a-3p TGTGCAAATCTATGCAAAACTGA mmu-miR-204-5p TTCCCTTTGTCATCCTATGCCT mmu-miR-211-5p TTCCCTTTGTCATCCTTTGCCT mmu-miR-216b-3p ACACTTACCTGTAGAGATTCTT mmu-miR-28a-3p CACTAGATTGTGAGCTGCTGGA mmu-miR-365-3p TAATGCCCCTAAAAATCCTTAT mmu-miR-466a-3p TATACATACACGCACACATAAGA mmu-miR-466b-3p ATACATACACGCACACATAAGA mmu-miR-466c-3p ATACATACACGCACACATAAGA mmu-miR-466e-3p TATACATACACGCACACATAAGA mmu-miR-466p-3p ATACATACACGCACACATAAGA mmu-miR-467c-3p ATATACATACACACACCTATAC mmu-miR-467d-3p ATATACATACACACACCTACAC mmu-miR-467e-3p ATATACATACACACACCTATAT mmu-miR-669d-2-3p ATATACATACACACCCATATAC mmu-miR-669d-3p TATACATACACACCCATATAC mmu-miR-669l-3p ATATACATACACACCCATATAT mmu-miR-669m-3p ATATACATCCACACAAACATAT mmu-miR-669n ATTTGTGTGTGGATGTGTGT mmu-miR-6715-3p CCAAACCAGGCGTGCCTGTGG MicroRNA mimic and inhibitor miRNA Sequence miR 466b-3p 5ʹ-AUACAUACACGCACACAUAAGA-3ʹ miR 466b-3p Inhibitor 5ʹ-CUUAUGUGUGCGUGUAUGUA-3ʹ miR negative control 5ʹ-ACCAUAUUGCGCGUAUAGUCGC-3ʹ qPCR protocol 50°C–2 min 95°C–2 min 40 cycles 95°C 15 s, 60°C 1 min 2.7 Dual luciferase assays HEK cells were transfected with pMIR-Glo containing MR and SGK1 wt or mutant 3ʹ UTRs with or without miR-466 mimics. The following day the cells were sub-cultured to a white 96-well plate (Falcon, Thermo Fisher) and returned to the incubator. After 24 hours, luciferase activity was determined using the Dual-Glo Luciferase Assay System (Promega, Madison, WI) according to the manufacturer's protocol. Bioluminescence activity was recorded as an endpoint assay on a Synergy 1H plate reader (BioTek Instruments, Winooski, VT), with an integration time of 100ms at a sensitivity of 200. 2.8 RNA isolation and qRT-PCR RNA from cultured or primary CCD cells was isolated using the miRNeasy RNA isolation kit (Qiagen, Germantown, MD) according to the manufacturer's protocol. The kit facilitated isolation of both miRNA and total RNA from each sample for use in qRT-PCR and RT-PCR. Total RNA (containing miRNAs) concentration and quality were evaluated for inclusion in subsequent in vitro transcription assays based on a spectrophotometric absorption ratio of 260/280 > 1.8 (NanoDrop, Wilmington, DE). Primers and primer pairs used for all PCRs are listed in Table 1. For qRT-PCR of miRNA, EvaGreen with ROX (MidSci, St Louis, MO) master mix was used. For all miRNA qPCRs, the miRNA-specific forward primers were paired to a universal reverse primer as described before.60, 62 Real-time PCR was carried out using an Applied Biosystems 7900HT Fast Real-Time PCR System (Applied Biosystems, Life Technologies, Grand Island, NY). Detected signals from miR amplifications were normalized to the relative expression of small nucleolar RNA (SNO-202 and SNO-135) with each reaction/sample run in triplicate. Negative controls included no template and no primer omissions. The standard qPCR protocol is provided in Table 1. For qPCR of mRNA, primer pairs were used as listed (Table 1). Relative mRNA was normalized to the glyceraldehyde 3-phosphate dehydrogenase or actin message from each sample, and expression is presented as a fold change from control untreated samples (ΔΔCT). 2.9 Ex vivo kidney CCD cell isolation Distal kidney nephron principal epithelial cells were isolated from a crude kidney tubule preparation (1 kidney from each animal) using a lectin binding and magnetic bead isolation technique as described before.60, 62 The isolated cells were immediately processed for RNA isolation; isolated RNA was used immediately or stored at −80°C until needed. 2.10 Western blot Lysates were prepared in cell lysis buffer (0.4% of deoxycholic acid, 1% of Nonidet P-40, 50 mM of EGTA, 10 mM of Tris-Cl, pH 7.4) plus protease inhibitors at 4°C for 10 min. The lysates were heated to 70°C for 5 min, separated by SDS-PAGE, transferred to Immobilon-P (EMD Millipore) and subjected to Western blot analysis using antibodies as indicated. 2.11 Blot quantification and statistical analyses Densitometric quantification of protein band intensities was carried out in Adobe Photoshop CS5.1 (Adobe Systems, Inc, San Jose, CA), and values were expressed as a percentage or fold change of control (unstimulated) signal, following background subtraction, and normalization to total protein expression (tubulin). Statistical analyses were performed using GraphPad Prism (Systat, La Jolla, CA). All data are presented as mean ± SEM. Repeats are defined as “N” for biological replicate and “n” for individual observations/samples. Data sets to be compared were tested for equal variance, and comparisons were performed using Mann-Whitney rank-sum and one-way ANOVA (multiple comparison) tests. Groups were considered statistically significant different at P < .05. Significance is denoted by *** or ###P < .001, ** or ##P < .01, * or #P < .05. 3 RESULTS 3.1 Aldosterone regulates miRs that target MR To determine if the mouse MR 3ʹ-UTR was targeted by aldosterone-regulated miRs, target prediction algorithms (miRDB and Diana Tools) were used to rank potential MR binding miRs (Table 2).63 Approximately 30 of the top ranked miRs were tested by qRT-PCR to determine if their expression was increased by aldosterone (50 nM, 24 hours) in a mouse cortical collecting duct (mCCD) cell line. Total RNA from mCCD cells grown on filters was collected after a >50% increase in equivalent current aldosterone stimulation was observed (as measured using chopstick electrodes, data not shown). Of the tested miRs an increase in expression was observed for several miRs including mmu-miR-19a and mmu-miRs-466a/b/c/e-3p. The miR-466 family members represent a larger cluster of miRs in genomic proximity, which include the miR-466, miR-467 and miR-669 family members. However, the miR-467 and -669 members that were tested did not exhibit elevated expression after 24 hours aldosterone stimulation and were not further evaluated. The mature miRs-466a/b/c/d/e are nearly identical in mature sequence, with either the deletion of a single nucleotide at the beginning of the sequence (miRs-466b/c) or the omission of two nucleotides at the end of the sequence (miR-466d, Figure 1A). While the precursor miR sequences differ, mature miR-466a&e and miR-466b&c sequences are identical. Consequently, results for these miRs were grouped together as miR-466a/e and miR-466b/c, as primers used in qRT-PCR analysis would be unable to distinguish between these mature forms. Target site predictions based on the seed sequence, identify the same targets for miR-466a, b, c & e family members due to the identical seed sequences. We, therefore, chose to base these studies on the miR-466b sequence (for mimic and antagomir). A time-course of miR-466 expression following extended aldosterone stimulation was performed to verify the initial screen and determine if these miRs remained elevated with extended aldosterone stimulation. The relative expression of miRs-466a/e and miR-466b/c increased following aldosterone stimulation and were significantly higher at 72 hours of aldosterone stimulation (Figure 1B). Levels of miR-466d and control miR-10a were not significantly changed. To confirm that a similar upregulation of the miR-466 family members occurred in vivo, wt male mice were placed on a sodium deficient diet for 3 days. Distal nephron epithelial cells were isolated from kidneys of mice on control and sodium-deficient diets following enzymatic digestion and magnetic bead isolation as we have described previously.60, 62 The abundance of miRs-466a/e and miR-466b/c from isolated CCD cells in mice on a 72-hours low sodium diet was significantly greater relative to mice on normal sodium diets (Figure 1C). Expression of control miR-10a which is abundant in the CCD epithelial cells, and not regulated by aldosterone, related family member miR-466d, and unrelated miR-466g were not significantly altered in response to the low Na+ diet. To verify aldosterone stimulation in the ex vivo isolated CCD cells, relative mRNA levels of SGK1, MR, and αENaC were determined using qRT-PCR from mRNA isolated from in mice on low Na+ diets compared to CCD cells isolated from mice on normal Na+ diets (Figure 1D). SGK1 and αENaC mRNA levels were significantly greater in CCD cells isolated from the mice on low Na+ diets compared to control diets. TABLE 2. Table listing tested miRNAs predicted to bind to the mouse Nr3c2 3ʹ-UTR, ranked from highest to lowest (by miRDB.org) miRBD target score miTG score (Diana tools) miRNA name Fold change (24 hours Aldo) SEM 99 94 mmu-miR-669n 0.86 0.02 95 82 mmu-miR-466b/c-3p 1.89 0.60 95 82 mmu-miR-466p-3p 1.13 0.26 95 93 mmu-miR-124-3p 1.06 0.08 93 85 mmu-miR-216b-3p 1.02 0.01 92 83 mmu-miR-669d-2-3p 0.94 0.15 92 85 mmu-miR-467c-3p 0.81 0.21 92 85 mmu-miR-467d-3p 0.80 0.14 92 95 mmu-miR-204-5p 0.78 0.19 92 95 mmu-miR-211-5p 0.83 0.16 92 85 mmu-miR-467e-3p 0.54 0.00 92 71 mmu-miR-669m-3p 0.92 0.05 92 83 mmu-miR-669l-3p 0.92 0.27 92 99 mmu-miR-365-3p 0.96 0.12 89 ND mmu-miR-127-5p 0.80 0.32 85 98 mmu-miR-19a-3p 1.39 0.15 85 99 mmu-miR-135a-5p 0.84 0.05 83 98 mmu-miR-466a/e-3p 1.95 0.58 83 97 mmu-miR-669d-3p 0.94 0.15 78 83 mmu-miR-6715-3p 1.12 0.17 69 82 mmu-miR-186-5p 1.19 0.11 64 ND mmu-let-7a-1-3p 0.88 0.14 64 ND mmu-let-7b-3p 0.78 0.09 64 ND mmu-let-7c-2-3p 0.85 0.09 64 ND mmu-let-7f-1-3p 0.76 0.12 61 80 mmu-miR-28a-3p 1.15 0.15 60 ND mmu-miR-101b-3p 0.89 0.16 Note The equivalent ranking from Diana Tools is presented (where available, ND = not determined). The change in miRNA expression from mCCD cells cultured on filter supports and stimulated with aldosterone (50 nM for 24 hours) compared to unstimulated levels (n = 3) is expressed as a relative fold change (SEM = standard error of the mean) FIGURE 1Open in figure viewer A, The precursor and mature sequences of mouse miR-466 family members, with the genomic location of each are listed. The position of the mature miRs embedded in each precursor sequence is highlighted in blue text. Variations from the miR-466a sequence are highlighted with indicating deletions at the beginning of the sequence, or representing insertions/deletions within the sequence. Mature sequences of miRs-466 a & e and miRs-466 b & c are identical even though their precursor sequences differ. B, qRT-PCR determination of the relative expression of miRs-466 a/e, b/c, d and control miR-10a after aldosterone stimulation (50 nM, 24-72 hours) compared to unstimulated mCCD cells (N ≥ 5, n = 5-17 for each bar, ***P < .001 compared to unstimulated, one-way ANOVA). C, Relative expression of miR-466 a/e, b/c, d, miR-10a (control) and a nonfamily member miR-466g in CCD cells isolated from mice on low Na+ diets compared to miR expression on normal Na+ diets (N = 6, *** P < .001, one-way ANOVA). D, Relative expression of SGK1, MR and αENaC mRNA from the same samples as in Figure 1C. Both SGK1 and aENaC expression was significantly greater in CCD cells isolated from mice on low Na+ diet compared to control diet. (N = 6, *** P < .001, * P < .05 one-way ANOVA) 3.2 miR-466 binds to the MR 3ʹ-UTR To confirm that miR-466 could bind the predicted target sequence on the 3ʹ-UTR of MR (Figure 2A), a wt-UTR mouse MR or a MR-UTR with the miR-466 binding sites eliminated (MR-mutant) was inserted into a dual luciferase reporter construct. The reporter construct was co-transfected into HEK293 cells with a scrambled control or miR-466b mimic (50 nM) and the luciferase signal measured (Figure 2B). Compared to the nontargeting control RNA, miR-466 mimics decreased the relative (normalized) luciferase signal by ~50%. The repression of the luciferase signal was not observed in the MR-mutant reporter where the predicted miR-466 binding sites were eliminated (Figure 2B). FIGURE 2Open in figure viewer A, Predicted miR-466 binding sites on the MR-3ʹUTR (mouse NR3C2) that were eliminated in the MR-mutant construct (MR-Mut). B, Dual luciferase assay for MR luciferase constructs transfected into HEK cells with control or miR-466 mimic (50 nM). Luciferase signal was normalized to the second (nontargeted) luciferase reading and expressed as a % of the blank (pmiR-Glo) luciferase signal (N = 4 replicates,***P < .001, one-way ANOVA). C, Predicted miR-466 binding site on the SGK1-3ʹUTR (mouse SGK1) that was eliminated in the Sgk1-mutant construct (Sgk1-Mutant). D, Dual luciferase assay for SGK1 luciferase constructs transfected into HEK cells with control or miR-466 mimic (50 nM). Luciferase signal was normalized to the second (nontargeted) luciferase reading and expressed as a % of the blank (pmiR-Glo) luciferase signal (N = 3 replicates, ***P < .001, one-way ANOVA) 3.3 SGK1 is targeted by miR-466 We examined the predicted miR binding sites of other known aldosterone-regulated proteins or components of the RAAS pathway. In mo