*3.1. miRNAs in the Regulation of the Renin-Angiotensin Aldosterone System*

The renin-angiotensin aldosterone system (RAAS) is a hormonal system that is paramount in the regulation of blood pressure by its influence on cardiac contractility, blood volume and resistance in the vasculature. The RAAS is a collaboration of the physiological workings of various organs and systems: the renal system, the cardiovascular system, the central nervous system and adrenal glands [38]. The RAAS involves a number of molecular players: peptides (angiotensin II), substrate (angiotensinogen (AGT), enzymes (angiotensin converting enzymes 1 and 2 (ACE1 and ACE2, respectively), aldosterone and vasopressin (known as anti-diuretic hormone (ADH)) and receptors (angiotensin II receptor type 1 and type 2 (AT1R and AT2R encoded by AGTR1 and AGTR2 mRNAs, respectively), bradykinin receptor 2 (B2R) and thromboxane A2 receptor (TBXA2R)). Hypertension develops when this well-balanced system of RAAS is over-activated. A number of microRNAs interact with the major players of RAAS in the hypertensive human cases and animal and in vitro experiments, as shown in Table 1.



*Int. J. Mol. Sci.* **2018**, *19*, 927

Wky: Wistar-Kyoto

normotensive

 blood pressure mice.

 rats; SHR: spontaneous

 hypertensive

 rats; n/iCMIT:

normal/increased

 carotid intima-media

 thickness; BPH/2J: hypertensive

 blood pressure mice; BPN/3J:

It has been shown that many major players of RAAS are regulated by miRNAs. From single-nucleotide polymorphism (SNP) datasets, the *AGTR1* gene, which encodes for angiotensin II receptor type 1, is regulated by miR-155 by its preferential binding to the A allele at position +1166 of the 3 UTR of *AGTR1* [40]. Interestingly, in hypertension cases, there is a higher prevalence for the C allele than the A allele, which reduces the ability of miR-155 to bind to *AGTR1* [40], and individuals who were homozygous for the C allele showed lower miR-155 expression and higher *AGTR1* expression, which resulted in elevated blood pressure [39]. The role of miR-155 in repressing *AGTR1* was tested and confirmed in rat cardiomyocytes, which also reduced cardiac hypertrophy [74]. Furthermore, within a hypertensive cohort, SNPs found in the miRNA binding sites of the RAAS protein genes were associated with elevated blood pressure (in *AVPR1A*) or lower blood pressure (in *BDKRB2* and *TBXA2R*). This finding suggests the role of miRNAs in blood pressure regulation via the genes of RAAS [41]. There has been a collection of reports of SNPs identified in miRNA binding sites, as well the miRNA promoter, and these have modified the binding proficiency of miRNAs to the corresponding target gene and are associated with elevated blood pressure [75–77]. The most recent SNP in the 3 UTR gene is found in the Chinese Han population, within the miRNA miR-495 binding site. The mutant C allele has increased the hypertension susceptibility, but further tests are required to determine if this SNP has altered the miRNA binding efficiency [78]. Another report of an association study of hypertensives (156) and normotensives (187) has discovered SNP rs4705342 in the miRNA promoter with a lower frequency of minor C allele among the hypertensive group. However, no further tests have been done to verify the effect of the SNP on miR-143 expression or binding [79]. A list of more of these SNPs that alter miRNA binding sites is provided in Table 2. In addition, microarray data from human, rat and mice showed that miR-483-3p can downregulate AT2R, AGT, ACE1 and ACE2 [42]. Moreover, the cluster of miR-143/145 has been shown to increase under shear stress by activation of the AMPK-p53 pathway, which in turn downregulates the ACE expression [43]. Interestingly, an earlier study has found that expression of miR-143/145 is vital for the maintenance of the VSMC contractile phenotype [80]. A recent study showed that knockout of miR-143/145 in mice resulted in them developing the loss of myogenic tone, and under induction of AngII for increased blood pressure, these knockout mice developed severe vascular inflammation and fibrosis, compared to their wildtype littermates [44].


**Table 2.** SNPs associated with hypertension.

On the other side, the increase of AngII as a vasoconstrictor that induces the release of aldosterone and vasopressin has also altered miRNA expression. A study on Sprague-Dawley rats showed that 10 days of AngII intravenous infusion induces cardiac hypertrophy and fibrosis and increases miR-132 and miR-212 expression in rat hearts, aortas and kidneys [45]. When applied to the human setting, blocking AngII activity by AGTR1 blocker treatment in hypertensive patients reduces the expression

of miR-132 and miR-212 in the internal mammalian artery compared to the control group [45]. The β-blocker drug was also trialed, but the AGTR1 blocker was more potent in its attenuating effect. These studies suggest that miR-132 and miR-212 assist AngII-induced hypertension. Within the in vitro setting, AngII treatment in human adrenocortical cell lines increases miR-21 expression. This releases aldosterone secretion, but not cortisol. This result suggests the possibility that miR-21 could influence the abnormal aldosterone secretion in the hypertension setting and contribute to primary aldosteronism [46].

A number of studies has reported changes in the expressions of miRNAs and its possible use as a biomarker in connection with sodium homeostasis, blood pressure and renin expression. In a small cohort study (*n* = 10) that evaluated, using a microarray, the correlation of salt intake and blood pressure, 45 differentially-expressed miRNAs were found with miR-4516 displaying the highest expression change across salt intake variation [47]. Interestingly, the exosomes from the urine samples showed a reduction of miR-4516 expression in the inverse salt-sensitive (mean arterial pressure (MAP) decreases ≥7 mmHg with high salt intake) vs. the salt-resistant subjects (control; <7 mmHg MAP change with high salt intake) and, conversely, an increase in the expression in the salt-sensitive (≥7 mmHg increase in MAP) vs. the salt-resistant group [47]. Similarly, a recent report showed other miRNAs, miR-361-5p and miR-362-5p, being associated with salt sensitivity. Both of these miRNAs were downregulated in the salt-sensitive hypertensive group, compared to the salt-resistant essential hypertensive group [48]. Within another cohort of white hypertensive European subjects, microarray profiling on the medulla and cortex of kidney tissues along with qPCR validation showed: downregulation of miR-638 and let-7c, in the medulla, and in the renal cortex, downregulation of miR-181a, miR-638 and miR-663 and upregulation of miR-21, miR-126, miR-196 and miR-451 [49].

The above findings of the downregulation of miR-181a and miR-663 in the hypertensive human cohort can be correlated further to some in vitro and animal studies. In the human embryonic kidney cell cline (HEK293), miR-181a and miR-663 regulate the endogenous renin expression by targeting the 3 UTR of the human renin (REN) mRNA, and specifically also apoptosis-inducing factor mitochondrion-associated 1 (AIFM1) and apolipoprotein (APO E), respectively [49]. In the BHP/2J mouse circadian hypertension model [50], they found downregulation of miR-181a and increased renin expression in the active period [50].
