Regulation of Aldosterone Signaling by MicroRNAs

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Regulation of Aldosterone Signaling by MicroRNAs Michael B. Butterworth*,1, Diego Alvarez de la Rosa†,1 *Department of Cell Biology, University of Pittsburgh School of Medicine, Pittsburgh, PA, United States † Departamento de Ciencias Medicas Ba´sicas (Fisiologı´a), Instituto de Tecnologı´as Biomedicas, Universidad de La Laguna, Tenerife, Spain 1 Corresponding authors: e-mail address: [email protected]; [email protected]

Contents 1. 2. 3. 4. 5. 6. 7. 8.

Introduction to Non-Coding RNAs MiR Production Regulation of MiR Expression by Aldosterone MiR Regulation of Aldosterone Signaling Regulation of Related Steroid Hormone Signaling by MiRs MiR Role in Modulating Aldosterone Effects in the Kidney MiR Regulation of Other Kidney Transporters and Regulatory Proteins MiRs in Aldosterone-Related Disease 8.1 Implications of MiRs in Aldosterone Deleterious Effects in the Cardiovascular System 8.2 Aldosterone, MiRs and Kidney Injury 8.3 Other Diseases Acknowledgments References

2 4 6 8 10 11 14 15 16 22 24 25 25

Abstract The mineralocorticoid hormone aldosterone is released by the adrenal glands in a homeostatic mechanism to regulate blood volume. Several cues elicit aldosterone release, and the long-term action of the hormone is to restore blood pressure and/or increase the retrieval of sodium from filtered plasma in the kidney. While the signaling cascade that results in aldosterone release is well studied, the impact of this hormone on tissues and cells in various organ systems is pleotropic. Emerging evidence indicates aldosterone may alter non-coding RNAs (ncRNAs) to integrate the hormonal response, and these ncRNAs may contribute to the heterogeneity of signaling outcomes in aldosterone target tissues. The best studied of the ncRNAs in aldosterone action are the small ncRNAs, microRNAs. MicroRNA expression is regulated by aldosterone stimulation, and microRNAs are able to modulate protein expression at all steps in the reninangiotensin-aldosterone-signaling system. The discovery and synthesis of microRNAs

Vitamins and Hormones ISSN 0083-6729 https://doi.org/10.1016/bs.vh.2018.09.002

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2018 Elsevier Inc. All rights reserved.

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will be briefly covered followed by a discussion of the reciprocal role of aldosterone/ microRNA regulation, including misregulation of microRNA signaling in aldosteronelinked disease states.

ABBREVIATIONS AngII BNIP3L CamKII Cav1.2 HCN Kim-1 MR MuRF1 SATB2 Sox6 Wnt

angiotensin II BCL2/adenovirus E1B 19 kDa protein-interacting protein 3-like Ca2 +/calmodulin-dependent protein kinase II voltage-dependent L-type Ca2 + channel 1.2 hyperpolarization-activated cyclic nucleotide-gated ion channel kidney injury molecule 1 mineralocorticoid receptor muscle RING-finger protein-1 special AT-rich sequence-binding protein 2 Sry-related HMG box 6 wingless-related integration site

1. INTRODUCTION TO NON-CODING RNAs Elucidation of the DNA structure over 60 years ago established the genomic era of scientific discovery (Watson & Crick, 1953). An explosion of technical and scientific advances has made the sequencing of an organisms’ genetic code routine, and unlocked new studies into the genetic basis of life and disease (Hawkins, Hon, & Ren, 2010; Wang, Gerstein, & Snyder, 2009). Classic research linked the DNA code to messenger RNA and protein production as the linear decoding of genes. However, after sequencing of the human genome it became apparent that protein coding genes comprised only a fraction (
2%) of the total genetic code (Gardiner, 1995; Zuckerkandl, 1997). For many years the “junk” DNA went largely ignored, even though, in humans, the majority of this DNA was transcribed into RNA that was not translated into proteins. A second genomic revolution is underway as researchers seek to understand the role of the non-coding RNA (ncRNA) (Eddy, 2001). Research suggests that ncRNA and its interactions with DNA, RNA and proteins represent a unique mechanism that likely controls the majority of cellular functions (Engreitz, Ollikainen, & Guttman, 2016; Guttman & Rinn, 2012; He & Hannon, 2004). NcRNAs have been classed into small and long ncRNA at an arbitrary 200 base pair (bp) cutoff. The small inhibitory ncRNAs (<200 bp) have been further subcategorized into three classes, namely, small interfering

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RNAs (siRNAs), PIWI interacting RNAs (piRNAs) and the most extensively studied microRNAs (miRs) (Ha & Kim, 2014). These three classes of small inhibitory ncRNAs are between 20 and 30 nucleotides (nt) long and have similar functions to silence DNA transcripts. SiRNAs are similar to miRs in that they are derived from longer double-stranded RNA precursors that need to be processed into a mature form by the RNase III-protein Dicer (similar to miRs, see below) (Ha & Kim, 2014). These mature
21 nt siRNAs suppress transcripts and transposons and are best characterized for their antiviral roles. The piRNAs are single-stranded RNAs that do not need RNase-III enzymes to be cleaved. They are processed by an endonuclease from a precursor form before associating with a family of the Argonaute processing enzymes, PIWI. The piRNAs are best described in their role to silence germline transposons (Iwasaki, Siomi, & Siomi, 2015). The best characterized small ncRNAs are the miRs, and their importance has been established in growth, development, physiology and disease (Erson & Petty, 2008; Flynt & Lai, 2008). MicroRNAs are typically 22 or 23 nt in length and were first identified in Caenorhabditis elegans as a non-translated RNA species that interacted with developmental genes at a posttranscriptional level. These first identified miRs regulated the early developmental patterning by repressing protein expression (Lee, Feinbaum, & Ambros, 1993; Saal & Harvey, 2009; Wightman, Ha, & Ruvkun, 1993). Initially it was not appreciated that these small ncRNAs were present in other organisms. However, when let-7 was identified in higher organisms, including humans, an explosion of research was initiated (Axtell & Bartel, 2005; Lau, Lim, Weinstein, & Bartel, 2001; Pasquinelli et al., 2000). MiRs have now been described in a range of organisms. The latest release of the miRbase database lists over 38,000 potential hairpin precursor miRNAs, with >48,000 mature miRNA products in 271 species identified (Griffiths-Jones, Saini, van Dongen, & Enright, 2008; Kozomara & Griffiths-Jones, 2011, 2014). New miR species are continually being described and bona fide miRs are curated from these lists as experimental evidence aligns with the initial identification and description. While several miRs are eliminated during these rounds of validation, the list of confirmed miRs is expanding as researchers confirm functional roles for the identified miRs. MiRs act primarily to repress protein expression by binding to the untranslated region of mRNA to prevent protein translation and accelerate mRNA degradation (to be described below).

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2. MIR PRODUCTION A common feature of most miRs is a stem-loop secondary structure of the RNA sequence. The sequences that produce this stem-loop structure are encoded throughout the genome, both embedded within protein coding genes (introns and exons) or encoded as stand-alone sequences. While there is a diversity of strategies that transcribe miRs (detailed in numerous reviews, for example, Bhatt, Mi, & Dong, 2011; Chandrasekaran et al., 2012; Ha & Kim, 2014; Kim & Kim, 2007; Trionfini, Benigni, & Remuzzi, 2015; Wei, Mi, & Dong, 2013; Zhang et al., 2010), all stem-loop structures need to be processed to progress to a mature 23 nt miR. MiRs can be transcribed by a unique promoter (Breving & Esquela-Kerscher, 2010; Lee et al., 2008; Marson et al., 2008) or contained within genes to therefore share the promoters of the encoded gene (Hinske, Galante, Kuo, & Ohno-Machado, 2010; Liu et al., 2007). This either allows for independent regulation of miR expression or changes in miR expression linked to the gene in which the miR is embedded. MiRs can be produced as a single immature species, but can also be found with several stem-loop sequences encoded in close genomic proximity as a miR cluster (Chhabra, Dubey, & Saini, 2010; Janga & Vallabhaneni, 2011). MiRs production begins with transcription by RNA polymerase II into primary miRs (pri-miRs) (Bartel, 2004; Lee et al., 2003; Lee, Jeon, Lee, Kim, & Kim, 2002). Pri-miRs typically produce the characteristic stem-loop hairpin structures, but are longer than mature miRs (>70 nt) with a polyadenylated tail (Ha & Kim, 2014; Han et al., 2004; Lee & Kim, 2007). These long pri-miRs are first trimmed in the nucleus in a complex that contains the polymerase III enzyme Drosha, to produce a precursor miR (pre-miR) of approximately 70 nt in length (Kim, 2005b; Lee et al., 2003; Ying, Chang, Miller, & Lin, 2006). Pre-miRs are exported from the nucleus to cytoplasm by the action of Exportin-5 (Kim, 2004; Lund, Guttinger, Calado, Dahlberg, & Kutay, 2004). In the cytoplasm the pre-miRs are trimmed by the enzyme Dicer. The elongated tail is removed, and the loop clipped to produce a double-stranded RNA 22 or 23 nt long (Ha & Kim, 2014; Park et al., 2011). This mature miR is now the appropriate length to be loaded onto a member of the Argonaute protein family, Argonaute-2 (AGO2). AGO2 is itself an endonuclease and it forms the central component of a complex of proteins that together recognize and bind to target RNA sequences. The complex is guided by the miR, and

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inhibits protein translation or degrades the RNA. This protein assembly with AGO2 has been termed the RNA-induced silencing complex (RISC) (Choe, Cho, Lee, & Kim, 2010; Okamura, Ishizuka, Siomi, & Siomi, 2004; Perron & Provost, 2009; Siomi, Tsukumo, Ishizuka, Nagami, & Siomi, 2005; Sontheimer & Carthew, 2004; Vaucheret, Vazquez, Crete, & Bartel, 2004). While this is the most well characterized miR synthesis pathway, it is not the only mechanism to produce mature miRs, and numerous exceptions to this pathway have been identified. At almost every point in the transcription and processing of miRs, alternatives have been described. Therefore, transcription by RNA polymerase III as opposed to II has been detailed (Ha & Kim, 2014; Han et al., 2004; Lee et al., 2003). MiRs have been found in coding gene introns that can be directly excised by spliceosome action to generate pre-miR. These introns that generate mature miRs are termed mirtrons (Berezikov, Chung, Willis, Cuppen, & Lai, 2007; Flynt, Greimann, Chung, Lima, & Lai, 2010; Okamura, Chung, & Lai, 2008; Okamura, Hagen, Duan, Tyler, & Lai, 2007; Ruby, Jan, & Bartel, 2007). MiRs can be derived from a range of RNA species including small nucleolar RNAs, short hairpin RNAs (shRNAs), siRNAs or transfer RNAs (Breving & Esquela-Kerscher, 2010; Suzuki & Miyazono, 2011; Wahid, Shehzad, Khan, & Kim, 2010). MiRs produced from these sources do not require processing by Drosha, and both Drosha and Dicer independent processing of pri-miR/pre-miRs are known (Cheloufi, Dos Santos, Chong, & Hannon, 2010; Yang & Lai, 2010; Yang et al., 2010). This diversity of miR production allows for a range of regulatory strategies to produce the miRs for loading into the RISC (Guo, Ingolia, Weissman, & Bartel, 2010; Ha & Kim, 2014; Kim & Kim, 2007; Miyoshi, Miyoshi, Hartig, Siomi, & Siomi, 2010; Miyoshi, Miyoshi, & Siomi, 2010; Suzuki & Miyazono, 2011; Zhang et al., 2010). The predominant function of miRs is to decrease protein expression. This is achieved in several ways, but in the best described mechanism for protein downregulation, miRs bind to the 30 -untranslated region (UTR) of target mRNAs by base-pairing and this leads to the endonuclease degradation of the mRNA by the RISC, or delay in protein translation by hindering access of the translational machinery proteins to the target mRNA (Miyoshi, Miyoshi, & Siomi, 2010). Alternatively, miRs have been shown to bind to the 50 -UTR of mRNA or bind directly to coding sequences of proteins to interrupt their translation. MiRs may also alter the regulation of RNA synthesis itself (Chua, Armugam, & Jeyaseelan, 2009;

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Flynt et al., 2010; Ha & Kim, 2014; Kim, 2005a; Kim, Han, & Siomi, 2009; Lee et al., 2002; Miyoshi, Miyoshi, & Siomi, 2010; Okamura et al., 2007; Yang et al., 2010). As a rule, the major function of miRs is to downregulate or repress protein expression. There are exceptions to this rule; however, targeting of miRs to specific mRNAs is a mechanism that cells can employ to fine-tune protein levels. When this regulation is lifted it often results in disease states (see below).

3. REGULATION OF MIR EXPRESSION BY ALDOSTERONE Given that aldosterone receptor, the mineralocorticoid receptor (MR), is a member of the nuclear receptor family of transcription factors (Gomez-Sanchez & Gomez-Sanchez, 2014), it is reasonable to ask whether aldosterone, acting through its receptor, modulates specific pri-miRNA transcription, thus controlling the expression of their microRNA products, which would then contribute to the effects of the hormone in different cell types. Genome-wide binding of MR has been investigating using chromatin immunoprecipitation followed by deep sequencing in renal epithelial cells (Le Billan et al., 2015; Ueda et al., 2014) and hippocampus (van Weert et al., 2017). None of these studies specifically examined binding of MR to known or predicted pri-miRNA promoters, or to promoters of genes known to host pri-miRNAs. On the other hand, transcriptomic analysis demonstrated that aldosterone modulates miR expression in cultured mouse kidney cortical collecting duct (CCD) cells (Edinger et al., 2014). This finding was corroborated in the same study using CCD cells purified from mice treated with a low Na+ diet, a stimulus that increases aldosterone circulating levels. In addition to identifying miRs relevant for aldosterone function in the kidney, this study generally supports the idea that aldosterone, presumably acting through MR, is able to specifically modify miR expression. This notion is further supported by other studies in distal nephron cells (Liu et al., 2017) and in vascular smooth muscle cells (Bretschneider et al., 2016). In addition, DuPont et al. uncovered MR-dependent regulation of miR expression, directly demonstrating in the case of miR-155 that MR modulates the host-gene promoter in a ligand-independent way (DuPont et al., 2016). This last study uncovers the possibility that regulation of MR activity by pathways other than aldosterone signaling, which have been previously demonstrated to impact coronary vascular function and chronic kidney disease ( Jaffe & Mendelsohn, 2005; Shibata et al., 2008),

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may impact miR regulation. Further genome-wide screenings combining transcriptomics with MR DNA-binding profile should further clarify whether direct regulation of miR transcription is a prevalent feature in MR aldosterone-dependent and -independent effects. The renin-angiotensin-aldosterone-signaling system (RAAS) is the wellestablished signaling cascade that leads to the release of aldosterone in response to deceased effective circulating volume. MiRs are implicated at every step in the cascade. Experimental evidence has demonstrated both regulation of miRs by RAAS members and regulation of RAAS components by miRs. Examples of this reciprocal expression will be described, with a particular emphasis on the kidney. Downregulation of renin expression (encoded by REN) has been demonstrated after increases in miR-181a and miR-663 levels. These miRs were shown experimentally to bind to the 30 -UTR of REN (Marques et al., 2011). As an additional confirmation of miR-181a’s role in renin regulation, transfection of a miR-181a mimic into the kidney resulted in a reduction in renal renin levels (Morris, 2015). MiR-483 was downregulated by angiotensin II (AngII) to feedback and alter the expression of several RAAS components, including angiotensinogen (Kemp et al., 2014). MiR-483 also targeted the angiotensin converting enzyme (ACE) 1 and 2, and the angiotensin receptor 2. ACE1 has been identified as a target of miR-145 (Boettger et al., 2009) and miR-27a/b, whereas ACE2 is targeted by miR-421 and miR-143 (Chen, Xu, Yu, Chang, & Zhong, 2015). On the other hand, AngII decreased the expression of miR-29b in the renal cortex of spontaneously hypertensive rats (Pan et al., 2014). In a separate study AngII increased the expression of miR-132 and miR-212 in cardiac fibroblasts and myocytes ( Jeppesen et al., 2011). In cardiac fibroblasts, AngII stimulation upregulated miRs-132, -125b and -146b, and decreased expression of miR-300-5p, -204-3p and -181b ( Jiang, Ning, & Wang, 2013). Aldosterone production via aldosterone synthase (AS) is mainly stimulated by AngII or increased plasma K+ and is encoded by the gene CYP11B2 in the adrenal cortex (Hattangady, Olala, Bollag, & Rainey, 2012). Overexpression of miR-24 reduced the expression of both CYP11B1 (11β-hydroxylase, also essential for aldosterone synthesis) and CYP11B2 genes. Conversely, depletion of miR-24 increased mRNA levels of both (Robertson et al., 2013). MiR-24 has been demonstrated to be upregulated by aldosterone (Lin et al., 2009). It is possible then that miR-24 could constitute a feedback signaling loop, repressing the expression of AS when aldosterone levels are elevated.

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4. MIR REGULATION OF ALDOSTERONE SIGNALING Release of aldosterone is the termination of the RAAS pathway that produces the hormone responsible for changes in Na+ and blood volume regulation. Aldosterone binds to the MR (encoded by NR3C2 gene) in the cytoplasm of target cells, and together they translocate to the nucleus. Here the bound receptor targets chromosomal mineralocorticoid response elements (MREs) to modulate the transcription of aldosterone-regulated genes. At this final step miRs have been shown to be involved in the regulation of the RAAS signaling pathway. Sober et al. used bioinformatic analysis to study whether a group of 160 different genes related to blood pressure control displayed overrepresented miR target sites in their 30 -UTRs (Sober, Laan, & Annilo, 2010). Even though the authors found no evidence for selected miR sites in the selected group of genes, they did detect a high number of miR binding sites in the 30 -UTR of a handful of genes related to the RAAS, most notably MR. This, together with the unusually long 30 -UTR of this receptor and the sequence conservation of this region in different species, prompted the authors to focus on experimental validation of miR binding to MR. Based on the robustness of miR binding prediction using different algorithms, miR-124, miR-135a, miR-30e, miR-19b, and miR-130a were considered for further analysis (Sober et al., 2010). Of those miRs, miR-124 and miR-135a downregulated the expression of a MR 30 -UTR luciferase reporter construct in a non-additive manner. The effect of miR-124 and miR-135a did not correlate with decreased reporter mRNA expression, nor with decreased endogenous expression of MR mRNA, suggesting that these two miRs may be controlling protein translation (Sober et al., 2010). An independent study found both decreased MR 30 -UTR luciferase reporter activity and decrease MR protein expression by miR-124 and miR-135a (Mannironi et al., 2013). Given that the same study demonstrated negative regulation of both miRs in the amygdala of mice subjected to acute stress, and that this correlated with increased MR protein expression, the authors proposed that miR-124 and miR-135a may be important in regulating the role of MR as an effector of early stress responses (Mannironi et al., 2013). An additional search for miR binding sites in the 30 -UTR of RAAS genes focused on single-nucleotide polymorphisms (SNPs) affecting those sites (Nossent et al., 2011). A rare allele of SNP rs5534 in the 30 -UTR of MR was associated to increased risk of myocardial infarction. This SNP is located in a miR-383 binding site in the 30 -UTR of MR. This miR

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inhibited expression of a MR 30 -UTR luciferase reporter construct and the variation in the binding site corresponding to the different alleles of the SNP altered the extent of this inhibition (Nossent et al., 2011). Further information about the role of miRs on regulating MR expression is scarce. It has recently been demonstrated that miR-301b suppresses the expression of MR in pancreatic ductal adenocarcinoma tumors expressing high levels of macrophage migration inhibitory factor, a pathway that contributed to enhance aggressiveness of the tumor (Yang et al., 2016). However, so far there is no additional information supporting a role for miR-301b in controlling the physiological actions of aldosterone. Regulation of aldosterone signaling through MR is complicated by the fact that MR displays poor specificity toward corticosteroids, binding aldosterone with the same high affinity as glucocorticoids such as cortisol. Both types of hormones potently activate MR (Arriza et al., 1987). In fact, each hormone triggers specific responses, but an excess of glucocorticoid signaling produces mineralocorticoid-like effects, particularly hypertension (Fuller & Young, 2005). Given that glucocorticoids circulate at much higher concentrations than aldosterone, how can mineralocorticoids generate specific signals through MR? This essential question was apparently solved by the discovery of a pre-receptor specificity mechanism conferred by the enzyme 11β-hydroxysteroid-dehydrogenase type 2 (11β-HSD2), which metabolizes glucocorticoids to produce the biologically inactive 11-ketosteroids (Chapman, Holmes, & Seckl, 2013). Aldosterone-target cells express 11β-HSD2, which creates a low glucocorticoid environment, allowing aldosterone to have specific access to its receptor (Funder, Pearce, Smith, & Smith, 1988; Odermatt & Kratschmar, 2012). The importance of 11β-HSD2 in aldosterone signaling is firmly established by multiple lines of evidence, including genetic linkage in humans between mutations in this enzyme and the Syndrome of Apparent Mineralocorticoid Excess (SAME). SAME patients present extremely high blood pressure and other symptoms compatible with aldosterone excess, but circulating levels of the hormone are suppressed. Thus, it has long been proposed that the mechanism underlying SAME is the lack of protection against glucocorticoidmediated MR occupancy (Funder, 2017; White, Mune, & Agarwal, 1997). Characterization of SAME-causing mutations shows a good correlation between 11β-HSD2 activity and the severity of symptoms (White et al., 2000). In addition, it has recently been shown that MR nuclear translocation and activity are modulated by 11β-HSD2 SUMOylation, a modification that does not grossly alter enzyme activity ( Jimenez-Canino et al., 2017). Therefore, even small changes in the amount/activity of the enzyme may

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have an impact on aldosterone signaling and MR biology. The pathophysiological importance of 11β-HSD2 has generated interest on mechanisms controlling its expression (Chapman et al., 2013). A role for miRs in the control of 11β-HSD2 expression was suggested by Shang, Yang, Zhang, Zou, and Zhao (2012), who reported a decrease in protein abundance without changes in mRNA, correlating with increased expression of miR-498. However, no direct evidence of miR-498 role on controlling 11β-HSD2 translation was provided. The first systematic search for miRs regulating 11β-HSD2 was performed by Rezaei et al. (2014), who found multiple putative miR binding sites in human and rat 11β-HSD2 mRNA 30 -UTR by in silico analysis. This study demonstrated that both human and rat 11β-HSD2 30 -UTRs are relevant to control protein translation. Most importantly, Rezaei et al. compared miR expression profiles between two different rat kidney tubule segments, the proximal convoluted tubule (PCT), which lacks 11β-HSD2 expression, and the cortical collecting duct (CCD), which displays prominent expression of the enzyme. Of the 53 differentially expressed miRs, 13 were predicted to bind to rat 11βHSD2 30 -UTR (Rezaei et al., 2014). MiR expression profile was altered in two different models that display differential 11β-HSD2 expression: uninephrectomized vs. control rats and Sprague-Dawley vs. Wistar rat strains. In both cases, differences in 11β-HSD2 expression correlated with differential expression of miRs. Comparison between Sprague-Dawley and Wistar rats revealed three differentially expressed miRs, including miR-20a, which was predicted to bind to 11β-HSD2 30 -UTR. Expression of pri-miR-20a in HT29 and SW620 cell lines, which endogenously express 11β-HSD2, significantly decreased the activity of the enzyme, although whether this was a direct or indirect effect and also whether the reduced enzyme activity was due to reduced protein expression remains to be investigated (Rezaei et al., 2014).

5. REGULATION OF RELATED STEROID HORMONE SIGNALING BY MIRS The functional interactions between glucocorticoid and mineralocorticoid signaling pathways, which are mainly due to the promiscuity shown by MR toward both types of hormones, implicate that regulation of glucocorticoid abundance interferes with aldosterone signaling. As mentioned above, regulation of 11β-HSD2 expression, including the role of miRs in the process, has the potential to directly impact aldosterone access to its

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receptor. In addition, the activity of isozyme 1 of 11β-dehydrogenase (11βHSD1) may have important implications in aldosterone signaling. 11βHSD1 acts as an NADP(H)-dependent reductase that catalyzes the conversion of inactive cortisone to the active glucocorticoid cortisol, thus increasing local glucocorticoid concentrations in cells that express the enzyme (Chapman et al., 2013). Increased expression of 11β-HSD1 has been linked to several disorders, including obesity or glucocorticoidassociated cognitive decline (Chapman et al., 2013) and has the potential to induce inappropriate activation of MR and displacement of aldosterone binding to its receptor. The role of miRs in 11β-HSD1 expression was explored by Han et al., which identified using a bioinformatics analysis tool potential binding sites to three miRs, hsa-miR-340, -561 and -579 (Han, Staab-Weijnitz, Xiong, & Maser, 2013). The role of two of the candidates, hsa-miR-561 and -579, on regulating 11β-HSD1 expression was confirmed using a luciferase reporter construct. Both miRs were expressed in the liver and the authors suggested that they may constitute a tissue-specific mechanism for regulating 11β-HSD1 expression (Han et al., 2013). In addition to the competing effects of glucocorticoid levels on aldosterone action, it is important to take into account that relative levels of GR and MR in any given cell may affect the outcome of corticosteroid signaling. Even though GR has low affinity for aldosterone, it has been demonstrated that both receptors can heterodimerize and regulate gene expression, with specific effects depending on the gene context (Farman & Rafestin-Oblin, 2001; Liu, Wang, Sauter, & Pearce, 1995; Trapp & Holsboer, 1996). Therefore, control of GR expression by different mechanisms, including miRs directly interacting with its 30 -UTR, could potentially impact aldosterone function. A recently published review summarizes the various roles of miRs in regulating glucocorticoid signaling, including GR expression and glucocorticoid production in the adrenal gland (Clayton, Jones, KurowskaStolarska, & Clark, 2018). The possible interactions between aldosterone/MR and glucocorticoid/GR regulation by miRs remain to be studied.

6. MIR ROLE IN MODULATING ALDOSTERONE EFFECTS IN THE KIDNEY The major target of aldosterone signaling in the kidney is the regulation of tubular ion transport, where it alters the regulation of Na+ and K+ transport. A consequence of increased Na+ transport is the osmotic

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reabsorption of water to augment blood plasma volume. In investigating the role of miR in aldosterone signaling in the kidney, studies have focused on Na+ and K+ reabsorption. In one of the first studies, mouse miR-192 was shown to be inhibited when circulating aldosterone levels was increased (Elvira-Matelot et al., 2010). This change in aldosterone was induced by K+ load, salt depletion or chronic aldosterone infusion, all maneuvers that increase circulating aldosterone levels. One of the confirmed targets of miR-192 was the serine-threonine kinase, with no lysine (WNK1), specifically the long form (L-WNK1). Therefore, when miR-192 was inhibited, a de-repression of L-WNK1 protein expression could occur (Elvira-Matelot et al., 2010). The role of L-WNK and the kidney specific form, KS-WNK have been elucidated and it is known that L-WNK1 is an important regulator of both K+ and Na+ transport (Huang & Kuo, 2007; Lang, Capasso, Schwab, & Waldegger, 2005; Subramanya, Yang, McCormick, & Ellison, 2006; Wade et al., 2006; Yang, Zhu, Wang, Subramanya, & Ellison, 2005). Regulation of renal outer medullary potassium (ROMK) channels by miRs has been reported. By changing the K+ diet in mouse models, an increased miR-194 expression was reported, while a reduction in K+ causes a decrease in this miR (Lin, Yue, Zhang, & Wang, 2014). Looking for a target for this miR that could account for the reciprocal changes linked to altered K+ diet, the authors describe a scaffold and regulatory protein, intersectin 1, that was regulated by miR-194. Decreasing intersectin 1 expression prevented the internalization of ROMK which then increased K+ transport. An earlier study by the same group identified miR-802 similarly regulated by altered K+ diet. In this case the target protein was caveolin-1 (Lin, Yue, Pan, Sun, & Wang, 2011). A high K+ diet, which would result in an increase in circulating aldosterone levels, induced miR-802 production which inhibited caveolin-1 expression and reduced ROMK internalization. This then provides two separate mechanisms to regulate K+ transport, but with similar outcomes. Na+ regulation by miRs has been reported mainly for the epithelial sodium channel (ENaC). Direct regulation of ENaC function by miRs has been demonstrated (Edinger et al., 2014). In these studies, both a cortical collecting duct (mCCD) cell line treated with aldosterone and mice placed on a low Na+ diet to induce aldosterone release, produced parallel changes in miR expression. A cohort of miRs was described to be both upregulated and repressed after 24 h aldosterone stimulation (in the mCCD cells) or on

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extended Na+-deficient diets in the CCD cells isolated from mouse kidney (Edinger et al., 2014). The downregulated miRs (mmu-miR-290-5p, mmu, miR-335-3p, mmu-miR-1983) were predicted not to interact directly with ENaC or any of the well characterized proteins that are known to regulate Na+ transport (for example, SGK1 or Nedd4l). By using prediction algorithms, the authors were able to identify a membrane regulatory and scaffold protein, ankyrin 3 (Ank3), as a target for these miRs. Direct binding of the regulated miRs to the 30 -UTR was demonstrated using a luciferase assay. The protein expression of Ank3 was increased when the miRs were inhibited and decreased when the miRs were overexpressed, demonstrating direct regulation of the protein by the aldosterone-altered miRs. The mechanism of ENaC regulation by Ank3 was elucidated in a followup study (Klemens, Edinger, Kightlinger, Liu, & Butterworth, 2017). It was demonstrated that increasing Ank3 expression accelerated the delivery of ENaC to the apical surface of mCCD cells. The result of increasing ENaC abundance at the membrane surface was a net increase in Na+ reabsorption across the epithelial cells, a reported action of aldosterone in these cells. In addition to the downregulation of miRs, aldosterone has been shown to increase miR expression in response to aldosterone stimulation as described for the regulation of K+ transport. For ENaC regulation, the miR cluster mmu-miR-23
24
27, was significantly upregulated by aldosterone, along with several other miRs reported in the same study (Liu et al., 2017). These miR clusters are mmu-miR-23a
27a
24-2, encoded in a
400 bp stretch on mouse chromosome 8 and mmu-miR-23b
27b
24-1 found within
800 bps on chromosome 13. These clusters are evolutionarily conserved among vertebrates and retained in humans. In most studies investigating these clusters, miR family members have been co-regulated, likely as a result of their chromosomal proximity (Chhabra et al., 2010). While these two clusters are located on different chromosomes, pri-miRs of the miR-23 a/b family members are highly homologous and the mature miR forms of miR-23
24
27 are identical, except for a single nucleotide. It is therefore likely that the two clusters target similar UTRs, even if each cluster is regulated independently. These clusters were previously reported to be enriched in the kidney (Tian, Greene, Pietrusz, Matus, & Liang, 2008). Alteration in the expression of these miRs has been reported in numerous disease states (see below), such as cardiac hypertrophy (Lin et al., 2009) or interstitial lung disease (ILD) (Hunter, Russo, & O’Bryan, 2013). Nedd4l was a target for the cluster and with a downregulation in ILD.

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The induction of these miRs by aldosterone in the kidney CCD was confirmed in both an mCCD cell line, and in isolated CCD cells from mice placed on low Na+ diets (Liu et al., 2017). This increase in all family members was likely due to an MRE upstream of the clusters, although this has yet to be validated. The predicted targets of these induced miRs were again carried out by in silico analysis. The top candidate was another member of the intersectin family of proteins, intersectin-2 (Itsn2). Similar to the reports for ROMK and the regulation of K+ transport, when Itsn2 protein was decreased, an increase in ENaC-mediated Na+ transport was observed. The 30 -UTR of Itsn2 was shown to be a target of these upregulated miRs, and a reduction in Itsn2 protein levels increased Na+ transport via ENaC with a reciprocal finding when Itsn2 was upregulated. The mechanism of action may well be similar to that reported for ROMK, but this has yet to be experimentally validated. Regulation of ENaC by other miRs has been reported, for example, miR-16 in alveolar epithelial cells (Tamarapu Parthasarathy et al., 2012) and regulation of miR-101 and miR-199 by ENaC in endometrial cells (Sun et al., 2014). MiR-263a downregulates the expression of epithelial sodium channel (ENaC) subunits in enterocytes to maintain osmotic homeostasis. None of these reports focused on miR regulation by hormones like aldosterone.

7. MiR REGULATION OF OTHER KIDNEY TRANSPORTERS AND REGULATORY PROTEINS MiR expression down the length of the kidney nephron has been examined under normal and low Na+ diets. Mladinov et al. examined miR expression in epithelial cells in proximal vs. distal kidney nephron segments in rodents. Enrichment of several miRs was established for each segment (Mladinov, Liu, Mattson, & Liang, 2013). In this study, miR192’s expression decreased in the proximal tubule when animals were placed on a low Na+ diet. The target of miR-192 investigated and characterized in this study was the β-1 subunit of the Na/K-ATPase. Knockdown of miR192 increased the β-1 subunit expression, and regulation was suggested to occur through the 50 -UTR rather than the 30 -UTR. Additional regulated miRs in this study included miRs-16, -195, -382 thought to interact with ROMK and the Na/K/Cl-co-transporter.

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In a study investigating miRs induced by osmotic stress, a transgenic mouse model was generated overexpressing miR-466a which had previously been linked to osmotic stress pathways (Luo et al., 2014). These mice demonstrated an impaired ability to concentrate urine. Mice with this miR overexpressed had reduced expression for a range of transporters and proteins in the kidney linked to osmoregulation, including aquaporins 1, 2 and 3, urea transporter 1, 2 and 3, the serum and glucocorticoid kinase1 (SGK1) and osmotic response element binding proteins. SGK1 is a known regulator of ENaC (Boini et al., 2008; Pearce & Kleyman, 2007; Rotin, 2000; Verrey, Loffing, Zecevic, Heitzmann, & Staub, 2003). SGK1 was found to be regulated by another miR-466 family member, miR-466g in cultured CCD cells ( Jacobs et al., 2016). A CCD epithelial cell line, mpkCCDc14, was treated with aldosterone for 1 h to observe the rapid regulation of miRs and targets. Expression of miR-466g was significantly decreased after 1 h of aldosterone stimulation, and a target of miR-466g was confirmed to be the 30 -UTR of SGK1. Altering the steady-state levels of miR-466g in these cells chronically shifted the response to aldosterone. The mRNA levels of SGK1 were significantly reduced when miR-466g was kept artificially high, and a response to aldosterone stimulation, as determined by the activity of Na+-transport via ENaC, was blunted. The in vitro studies described here point to a network of miRs that are regulated by aldosterone to augment well established signaling pathways. MiR expression is both increased and decreased following aldosterone stimulation, and this is seen in a rapid time course (1 h) and over an extended aldosterone stimulation. Much of the in vitro alteration in response to aldosterone was validated in in vivo models. It is therefore likely that miRs underpin the aldosterone signaling cascade and act as a rheostat to keep homeostatic balance, in particular of ion transport regulation. The role of miRs in the RAAS signaling pathway is depicted schematically in Fig. 1.

8. MiRS IN ALDOSTERONE-RELATED DISEASE It has long been known that miRs can play a prominent role in the development of human disease. Loss-of-function studies performed in mice demonstrate defects in the development of different organs and cell types and promote a wide range of disease phenotypes (Bartel, 2018). Accordingly, there has been increased interest in studying the possible role of miRs in diseases linked to inappropriate aldosterone signaling, either due to altered

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Fig. 1 A schematic representation of the renin-angiotensin-aldosterone-signaling cascade (RAAS, blue arrows). Points at which microRNAs are upregulated (green text and green arrow) or downregulated (red text and red line) are indicated. The regulation of microRNAs by RAAS components or proteins targeted by microRNAs is described in the text.

levels of the hormone or abnormalities in MR (summarized in Table 1). Excess MR activity is mostly deleterious for a variety of organs, both in the short and the long term (Gomez-Sanchez & Gomez-Sanchez, 2014; Jaisser & Farman, 2016). In addition, the similarity between MR and GR presents an additional problem when there is high corticosteroid circulating levels or when selectivity mechanisms conferring MR-specific activation by aldosterone are dysfunctional. In such situations, inappropriate MR activation can occur either by aldosterone or glucocorticoids, deregulating certain genes and modulating others that normally would be preferential GR targets (Gomez-Sanchez & Gomez-Sanchez, 2014; Jaisser & Farman, 2016).

8.1 Implications of MiRs in Aldosterone Deleterious Effects in the Cardiovascular System Primary aldosteronism, an excess secretion of aldosterone independently of physiological regulatory mechanisms, leads to hypertension and accounts for approximately 10% of treatment-resistant patients (Gomez-Sanchez, Rossi, Fallo, & Mannelli, 2010). Moreover, it is now well established that aldosterone/MR signaling associated to high NaCl intake produces

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MicroRNAs and Aldosterone

Table 1 MicroRNAs in Aldosterone-Related Disease MicroRNA Proposed Disease/ Stimulus Alteration Target Phenotype

Cardiac hypertrophy

Reference

Isoproterenol and aldosterone

miR-23a (up)

MuRF1

Isoproterenol and aldosterone

miR-9 (down)

Myocardin Cardiac hypertrophy

Wang, Long, Zhou, and Li (2010)

Local aldosterone production (heart)

miR-208a (down)

Sox6

Azibani et al. (2012)

Aldosterone

miR-21 (up)

Sprouty-1 Cardiac injury and dysfunction

Aldosterone

miR-21 (up)

Sprouty-1 Atrial fibrillation Adam et al. (2012)

Cardiac hypertrophy

Lin et al. (2009)

Ball et al. (2017)

Spironolactone miR-1 (up)

HCN ion channels

Ventricular arrhythmia

Yu et al. (2015)

Myocardial infarction

miR-31 (up)

MR

Myocardial infarction

Martinez et al. (2017)

MR expression/ aging

miR-155 (down) Cav1.2; Hypertension/ AngII aging receptor-1

DuPont et al. (2016)

Aldosterone

miR-29b (down) Multiple

VSMC phenotype switch

Bretschneider et al. (2016)

Aldosterone

miR-34b/c (down)

SATB2

VSMC calcification

Hao, Zhang, Cong, Ren, and Hao (2016)

MR

rno-miR-203 (down)

Kim-1

Acute kidney injury

Xiao, Tang, Zhou, Peng, and Yu (2016)

Aldosterone

miR-34c-5p (up) CamKII; Wnt signaling

Renal Park et al. tubulointerstitial (2018) fibrosis Continued

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Table 1 MicroRNAs in Aldosterone-Related Disease—cont’d MicroRNA Proposed Disease/ Stimulus Alteration Target Phenotype

Spironolactone miR-124, miR190, miR-217 and miR-188 (down)

Integrins Podocyte injury α3 and β1

Reference

Li, Lu, Jia, Zheng, and Lin (2013)

Aldosterone

miR-30e (down) BNIP3L

Podocyte apoptosis

Guo et al. (2017)

MR

miR-338-3p (up) Pyruvate kinase

Hepatocellular carcinoma

Nie et al. (2015)

Macrophage inhibitory factor

miR-301b (up)

Pancreatic ductal Yang et al. adenocarcinoma (2016)

MR

MiR change in expression is characterized as upregulated (up) or downregulated (down). Abbreviations: MuRF1, muscle RING-finger protein-1; Sox6, Sry-related HMG box 6; HCN, hyperpolarization-activated cyclic nucleotide-gated ion channel; MR, mineralocorticoid receptor; Cav1.2, voltage-dependent L-type Ca2+ channel 1.2; AngII, angiotensin II; SATB2, special AT-rich sequence-binding protein 2; Kim-1, kidney injury molecule 1; CamKII, Ca2+/calmodulin-dependent protein kinase II; Wnt, wingless-related integration site; BNIP3L, BCL2/adenovirus E1B 19 kDa protein-interacting protein 3-like.

cardiovascular and renal injury independently of changes in blood pressure. Clinical trials conclusively proved that MR antagonism is highly beneficial for patients with systolic heart failure (Pitt, Remme, et al., 2003; Pitt et al., 1999; Zannad et al., 2011), although it may lead to complications related to K+ homeostasis (Parviz et al., 2015). Knowledge about the molecular basis of aldosterone/MR deleterious effects on cardiac and vascular function has greatly expanded in the past few years (Bauersachs, Jaisser, & Toto, 2015; Davel, Anwar, & Jaffe, 2017; Tarjus, Amador, Michea, & Jaisser, 2015), including several studies addressing the role of miRs in the process. It is well established that excess aldosterone signaling leads to cardiac hypertrophy (Pitt, Reichek, et al., 2003; Sechi, Colussi, & Catena, 2010). A study designed to evaluate the role of miRs known to participate in cardiac hypertrophy proposed that miR-23a, a target of the pro-hypertrophic transcription factor in Nuclear factor of activated T cells isoform c3 (NFATc3), participates in aldosterone-mediated increase in cell surface area (Lin et al., 2009). However, it should be noted that this study showed mR-23a induction by aldosterone only at doses that are two to three orders of magnitude above physiological levels (500 nM), while 100 nM aldosterone did not produce any effect. This result strongly suggests that miR-

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23a induction is not an MR-mediated effect. The same group also reported that under the same model of cardiac hypertrophy aldosterone downregulates miR-9 expression (Wang et al., 2010). This effect was observed at 1 μM aldosterone and no other concentration was tested, again raising doubts about the participation of MR in the process and the possible relevance of miR-9 as a mediator of aldosterone-induced cardiac hypertrophy. Cardiac wall remodeling secondary to essential hypertension initially allows adaptation to pressure overload. This response involves re-expression of fetal genes such as the β-myosin heavy chain (β-MyHC). Azibani et al. (2012) using a mouse model with cardiac-specific expression of aldosterone synthase found that aldosterone inhibits expression of β-MyHC, worsening cardiac hypertrophy. The expression of β-MyHC is controlled by the transcription factor Sox6 and miR-208a. Expression of miR-208a, an intronic miR codified by the α-myosin heavy chain and thus named MyomiR (van Rooij et al., 2009), is repressed by aldosterone in the hypertrophic myocardium, as evidenced by the effect of MR antagonist eplerenone. This effect allows for increased Sox6 production, which in turn represses β-MyHC (Azibani et al., 2012). A more recent study examined left ventricular miR expression in a rat model of primary hyperaldosteronism, the aldosterone/salt model (Ball et al., 2017). The screening identified potent upregulation of miR-21. Paradoxically, downregulation of miR-21 using antagomirs exacerbated cardiac dysfunction, increasing hypertrophy and fibrosis, suggesting that miR-21 induction in the context of hyperaldosteronism may mediate compensatory mechanisms to limit cardiac damage. Using a similar model of hyperaldosteronism based on treatment with the mineralocorticoid deoxycorticosterone (DOCA) and high salt intake, Dickinson et al. performed a screening of circulating miRs with the goal of identifying new biomarkers of hypertension-related heart disease (Dickinson et al., 2013). There is only partial correlation between miRs with altered expression in the left ventricle and those with changed circulating levels, with three coincidences (miR-16, miR-19b, miR-223) and another three showing opposing patterns (miR-125a, mir-143, miR199a-3p) (Ball et al., 2017; Dickinson et al., 2013). Lack of correlation between circulating levels and tissue expression of miRs is not surprising, since the circulating pool is affected by the contribution of many different tissues. In addition to its effects on left ventricular remodeling, activation of MR has direct effects on cardiomyocyte electrical activity, supporting its role on promoting both atrial and ventricular fibrillation (Gomez-Sanchez, 2016;

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Gravez, Tarjus, & Jaisser, 2013). Aldosterone induces atrial fibrillation (AF) and remodeling (Reil et al., 2012) and MR antagonists reduce the incidence of AF in patients with systolic heart failure (Swedberg et al., 2012). AF-associated structural remodeling correlates with overexpression of miR-21 (Adam et al., 2012) in an MR-dependent way (Lavall et al., 2014). The authors of these studies also showed that aldosterone-mediated atrial remodeling is potentiated by 11β-HSD2 overexpression (Lavall et al., 2014). Interestingly, the loop diuretic torasemide, which improves cardiovascular morbidity and mortality (Cosin, Diez, & investigators, 2002), prevented this effect by directly inhibiting aldosterone synthesis and preventing overexpression of miR-21 expression (Adam et al., 2015). The cardiovascular effect of torasemide was originally thought to be due at least in part to MR inhibition, although this hypothesis was later disproved (Gravez, Tarjus, Jimenez-Canino, et al., 2013; Lavall et al., 2014). The effects of aldosterone/MR on cardiomyocyte excitability are mediated at least in part by direct or indirect regulation of multiple types of ion channels (see for instance Benitah & Vassort, 1999; Mesquita et al., 2018; Ouvrard-Pascaud et al., 2005; Sabourin, Bartoli, Antigny, Gomez, & Benitah, 2016). MR blocker spironolactone reduces ventricular arrhythmias decreasing expression of the hyperpolarization-activated cyclic nucleotidegated channel (HCN) (Song et al., 2011). Spironolactone was later found to increase miR-1 (one of the “MyomiRs”) expression in rat left ventricle in a model of myocardial infarction, contributing to repression of HCN protein expression, which may be related to decreased ventricular arrhythmia (Yu et al., 2015). A recent study studying miR expression post-myocardial infarction identified upregulation of miR-31, which in turn modulates the expression of several proteins including MR (Martinez et al., 2017). Paradoxically, miR-31 inhibition produced de-repression of target genes, improving the outcomes of myocardial infarction in rats. Vascular smooth muscle and endothelial cells express MR, which has a direct role in regulating vascular function and contributes to vascular dysfunction and remodeling in different contexts, including vascular injury, hypertension and obesity (DuPont & Jaffe, 2017; McCurley et al., 2012; Tarjus et al., 2015). Vascular smooth muscle cell (VSMC) MR directly contributes to increase vascular tone and blood pressure and mouse with VSMC-specific MR deletion are protected from age-related rise in blood pressure. Recently, DuPont et al. performed miR expression profiling in mouse aorta from young and aged mice expressing or not MR in VSMC

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(DuPont et al., 2016). From all miRs with twofold or more variation in expression 90% were downregulated and 10% upregulated. Most importantly, of all miRs showing altered expression with age (108 in total), approximately 70% were specific of MR-intact animals, indicating that MR plays a very prominent role in modulating miR expression profile remodeling with age (DuPont et al., 2016). miR remodeling correlated with increased MR expression with age. Detailed characterization of miR-155, the most potent downregulated miR with ageing in MR-intact mice, revealed that MR represses its host-gene promoter in a ligand-independent manner. MR ligand-independent activation by the GTPase Rac1 (Shibata et al., 2008) or by angiotensin II receptor 1 (AT1) signaling ( Jaffe & Mendelsohn, 2005) has been previously described, although it is still unclear whether any of those two mechanisms participate in MR-dependent miR expression remodeling with age. Interestingly, miR-155 restoration in MR-intact aged mice improved vascular function and decreased oxidative stress, decreasing expression of voltage-gated Ca2+ channels Cav1.2 and AT1 expression (DuPont et al., 2016). The role of aldosterone-dependent regulation of miR expression in the vasculature was investigated by Bretschneider et al. using aorta samples from mice and cultured human VSMC (Bretschneider et al., 2016). This study found aldosterone acting through MR decreased miR-29b expression both in aorta and in cultured VSMC, but not in endothelial cells. Reduced miR-29b produced increased production of extracellular matrix and promoted VSMC migration, also modulating cell proliferation and the balance between apoptosis and necrosis. This led the authors to propose that MR-regulated miR-29b expression as a pathologically relevant event in VSMC. Interestingly, MR-dependent regulation of miR-29b abundance occurred by increasing its rate of decay, rather than enhancing transcription, uncovering a new potential role of MR in the regulation of cellular miR profile (Bretschneider et al., 2016). The role of aldosterone-regulated miRs in VSMC has also been explored in the context of vascular calcification secondary to chronic kidney disease. Increased aldosterone circulating levels have been proposed to contribute to vascular calcification in uremic patients and in animal models of uremia by enhancing osteoblastic transdifferentiation of VSMC (Lang, Ritz, Alesutan, & Voelkl, 2014; Voelkl et al., 2013). Based on this idea and the findings that miR-34b/c regulates osteoblast differentiation and bone formation (Bae et al., 2012; Wei et al., 2012), Hao et al. investigated the role of this miR cluster in VSMC calcification both in vivo and in vitro

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(Hao et al., 2016). Their results using a rat model of uremia showed that miR-34b/c expression is downregulated during calcification, coinciding with increased aldosterone levels and upregulation of the aldosterone signaling pathway, with increased expression of MR, 11β-HSD2, aldosterone synthase, and also the osteogenic differentiation pathway mediated by SATB2/RUNX2. The effects on uremia on calcification and pathway upregulation were partially prevented by MR blocker eplerenone, as well as by a miR-34b/c agonist (Hao et al., 2016). Further experiments performed in a cell culture model of VSMC calcification showed that mi-34b/c repression directly enhanced SATB2 expression and promoted calcification, while the opposing effects were obtained by miR-34b/c overexpression (Hao et al., 2016).

8.2 Aldosterone, MiRs and Kidney Injury Acute kidney injury (AKI) is characterized by a sudden decrease in kidney function, reduced urine output and increased serum creatinine levels and can lead to chronic kidney disease (CKD) (Lameire et al., 2013). Excessive aldosterone/MR signaling also plays a prominent role in the development of acute kidney injury (AKI) and MR antagonists have been proposed as therapeutic tools to limit kidney damage associated to this condition (Barrera-Chimal, Bobadilla, & Jaisser, 2016; Ramirez et al., 2009). The etiology of AKI is very broad and its pathogenesis mechanisms complex. Several studies support the role of non-coding RNAs, including miRs, in the pathogenesis of AKI (Xue, Teng, Zhou, & Rui, 2016). Kidney injury molecule-1 (Kim-1) is a biomarker of AKI that associates with tubular injury during ischemia and proteinuria. Xiao et al. found that MR blocker spironolactone partially attenuates increased Kim-1 expression in a ischemiareperfusion (I/R) mouse model and in NRK-52E renal cells treated with antimycin-A to induce fibrosis or with a hypoxia/reoxygenation (H/R) protocol (Xiao et al., 2016). Since bioinformatics analysis predicts that Kim-1 is a target of rno-miR-203, the authors tested the expression of this miR in their models. Rno-miR-203 was downregulated by I/R in vivo or by antimycin-A or H/R treatment in cultured cells, and spironolactone was able to block these effects. Furthermore, rno-miR-203 was shown to repress Kim-1 through its 30 -UTR. Aldosterone-induced apoptosis of NRK-52E cells was associated with rno-miR-203 promoter hypermethylation, which represses its expression, triggering increased levels of Kim-1. Consistently with this model, pri-miR-203 partially prevented aldosterone-induced

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apoptosis, in an effect that was additive with spironolactone, suggesting the presence of additional signaling pathways (Xiao et al., 2016). Long-term upregulation of aldosterone/MR signaling associates with kidney inflammation, tubulointerstitial fibrosis, and glomerulosclerosis (Bertocchio, Warnock, & Jaisser, 2011; Remuzzi, Cattaneo, & Perico, 2008; Tesch & Young, 2017; Thomas, Dooley, & Harvey, 2010). In order to investigate whether aldosterone treatment alters miR expression during renal cell fibrosis, Park et al. treated mpkCCDc14 distal nephron cell line with 1 μM aldosterone and performed microarray analysis of mature and pre-mature miR expression (Park et al., 2018). The study identified approximately 50 miRs or pri-miRs with >30% change in expression, with 50% of them upregulated and the other half downregulated. Bioinformatic analysis of altered miR expression suggested several pathways that may be affected by the treatment, particularly the wingless-related integration site (Wnt) signaling pathway (Park et al., 2018). Three of the miRs predicted to participate in this pathway were analyzed and miR-34c-5p was predicted to affect Ca2+/ calmodulin-dependent protein kinase II (CamKII) β-chain expression. The study identified Wnt signaling and CamKII as possible mediators of aldosterone-induced tubular fibrosis through miR-34c-5p. Unfortunately, the relevance of this analysis for MR-regulated miR expression is unclear due to the high dose of aldosterone used. Podocyte injury is a key step on developing proteinuria and glomerulosclerosis. Aldosterone targets podocytes through MR activation, increasing oxidative stress and proteinuria (Shibata, Nagase, Yoshida, Kawachi, & Fujita, 2007). Glomerular capillary hypertension during diabetic nephropathy causes deleterious effects on podocytes, reducing their adhesion capacity in a process that can be ameliorated by spironolactone treatment (Lin et al., 2010). A subsequent study investigated the role of miRs on podocyte adhesion damage due to mechanical stress using an immortalized human podocyte cell line (Li et al., 2013). Cells seeded on flexible wells were placed under mechanical stress in the presence or absence of spironolactone. Mechanical stress increased aldosterone production by podocytes, which correlated with expression of MR and its target SGK1 and decreased integrin α3/β1 abundance in a spironolactone-dependent way. Microarray analysis under both conditions (mechanical stress or spironolactone treatment) revealed four miRs (miR-124, miR-190, miR-217 and miR-188) that were upregulated by mechanical stress and downregulated by spironolactone treatment (Li et al., 2013). Of those, miR-124 has predicted targets on integrins α3 and β1 and therefore the authors proposed that this miR may be a mediator of mechanical stress-induced,

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aldosterone-dependent podocyte injury, although it remains necessary to obtain functional confirmation of this prediction. The role of miRs (specifically the miR-30 family) on aldosteroneinduced podocyte injury was further studied by Guo et al. using cultured podocytes (Guo et al., 2017). The underlying hypothesis in this study was that this miR family is highly expressed in podocytes and one of them, miR-30a, has been shown to protect podocytes from injury (Wu et al., 2014). Aldosterone was found to downregulated miR-30e in cultured podocytes. However, this effect was seen only above 100 nM, which brings into question the involvement of MR in this observation (Guo et al., 2017). Interestingly, glucocorticoid treatment prevents podocyte injury in the same model (Wu et al., 2014), suggesting that the effects of aldosterone are not mediated by GR either or, alternatively, that aldosterone/GR signaling differs from that triggered by glucocorticoids/GR. Overexpression of miR30e protected podocytes from aldosterone-dependent apoptosis, while miR-30e inhibition directly induced it. The effects of miR-30e can be ascribed to targeting of BNIP3L and protection of mitochondria function (Guo et al., 2017).

8.3 Other Diseases Even though the importance of aldosterone/MR signaling in pathophysiological conditions outside the kidney and the cardiovascular system is well recognized and the importance of MR antagonists is becoming increasingly apparent in different settings ( Jaisser & Farman, 2016), the role of miRs in these very diverse conditions is only beginning to be explored. Moreover, the few available studies mostly focus on MR expression/function, and not in aldosterone signaling. For instance, Jung et al. explored the role of miRs on glucocorticoid resistance in macrophages caused by repeated social defeat ( Jung et al., 2015), which in turn is related to decreased expression of glucocorticoid and mineralocorticoid receptors (Quan et al., 2003). The study found specific alteration of miR expression in splenic macrophages due to repeated social defeat stress. Some of these miRs were predicted to interact with GR and MR mRNAs, although their specific roles on controlling MR expression and signaling remained to be analyzed ( Jung et al., 2015). In a different context, miR regulation of MR expression was found to be important in the progression of hepatocellular carcinoma (HCC) (Nie et al., 2015) or pancreatic ductal adenocarcinoma (Yang et al., 2016). In the first case, the authors found that MR suppresses HCC growth by repressing aerobic

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glycolysis (i.e., the Warburg effect) by upregulation of miR-338-3p, which in turn represses pyruvate kinase expression (Nie et al., 2015). Moreover, 80% of HCC tumors shown downregulation of MR expression, which closely associates to poor prognosis and correlates with miR-338-3p expression, strongly suggesting an important role of MR and miR regulation in this type of tumor (Nie et al., 2015). The possible role of MR as a tumor suppressor is further supported by the study of Yang et al., who identified that macrophage inhibitory factor (MIF), which is altered in highly aggressive pancreatic tumors, represses MR expression (Yang et al., 2016). The mechanism underlying MIF-promoted MR repression was found to be the upregulation of miR-301b, which targets MR 30 -UTR. MIF knockout abolished mir-301b effects on MR, which correlated with reduced metastasis and increased survival in a mouse model of reducing metastasis and prolonging survival in a genetically engineered mouse model of pancreatic ductal adenocarcinoma (Yang et al., 2016). The role of miRs on aldosterone/MR signaling in other conditions, including but not limited to metabolic, ocular or skin diseases, where MR antagonism has been proposed as an important therapeutic target, remains to be explored.

ACKNOWLEDGMENTS Work in the author’s laboratories is funded by the US National Institutes of Health (NIH NIDDK DK102843) to M.B.B. and Ministerio de Economı´a y Competitividad (MINECO, Spain, Grant BFU2016-78374-R) to D.A.d.l.R.

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