key: cord-0006595-q7m4srvp
authors: Nakagawa, Pablo; Gomez, Javier; Grobe, Justin L.; Sigmund, Curt D.
title: The Renin-Angiotensin System in the Central Nervous System and Its Role in Blood Pressure Regulation
date: 2020-01-10
journal: Curr Hypertens Rep
DOI: 10.1007/s11906-019-1011-2
sha: 0fcc3896445a8ac9bab74697da1081e8fa1c6279
doc_id: 6595
cord_uid: q7m4srvp

PURPOSE OF THE REVIEW: The main goal of this article is to discuss how the development of state-of-the-art technology has made it possible to address fundamental questions related to how the renin-angiotensin system (RAS) operates within the brain from the neurophysiological and molecular perspective. RECENT FINDINGS: The existence of the brain RAS remains surprisingly controversial. New sensitive in situ hybridization techniques and novel transgenic animals expressing reporter genes have provided pivotal information of the expression of RAS genes within the brain. We discuss studies using genetically engineered animals combined with targeted viral microinjections to study molecular mechanisms implicated in the regulation of the brain RAS. We also discuss novel drugs targeting the brain RAS that have shown promising results in clinical studies and trials. SUMMARY: Over the last 50 years, several new physiological roles of the brain RAS have been identified. In the coming years, efforts to incorporate cutting-edge technologies such as optogenetics, chemogenetics, and single-cell RNA sequencing will lead to dramatic advances in our full understanding of how the brain RAS operates at molecular and neurophysiological levels.

The physiological relevance of the renin angiotensin system (RAS) in blood pressure regulation and electrolyte homeostasis is well established and undisputable. The RAS is traditionally described as a hormone system, which promotes arterial blood pressure elevation primarily by inducing vasoconstriction, sodium retention, and aldosterone release. The sustained overactivation of the RAS could lead to hypertension, a disease affecting almost half of US American adults [1] . The activation of the endocrine RAS is initiated upon the release of renin from juxtaglomerular cell granules into the circulation. By catalyzing the cleavage of angiotensinogen to release angiotensin I peptide, renin acts as the rate limiting enzyme of the RAS, at least in humans. Thus, it is not surprising that there are a number of complex mechanisms regulating renin expression and secretion [2] . The subsequent conversion of angiotensin (ANG)-I to ANG-II is catalyzed by angiotensin converting enzyme (ACE) which is localized to endothelial cells and is abundant in the lungs. Most of the functions inducing blood pressure elevation are mediated through binding of ANG-II to angiotensin type 1 receptor (AT 1 R), whereas, binding of ANG-II to angiotensin type II receptors (AT 2 R) has been reported to generally oppose the actions of AT 1 R. Other peptides of the RAS, such as ANG-(1-7) and alamandine, also act to counter regulate the action of ANG-II at AT 1 R [3, 4] . Drugs targeting the RAS are effective as treatments for hypertension and other diseases including heart failure, chronic kidney disease, diabetic nephropathy, Marfan's syndrome, and some autoimmune diseases [5] [6] [7] [8] [9] [10] . However, it is unclear why these drugs are effective even in patients exhibiting low or normal circulating renin activity [11, 12] . The answer to this observation may lie in the existence of an independent autocrine/paracrine RAS acting locally within several tissues, including the brain.

The existence of the brain RAS, which was initially proposed by Bickerton and Buckley in 1961 , has changed the traditional view of the RAS [13] . Since the discovery that central ANG-II induces a potent pressor response, several new functions of the brain RAS have been identified. Central administration of ANG-II elicits potent dipsogenic responses, induces sodium intake, triggers sympathetic outflow to the kidney and other organs, and recently, evidence has established that the brain RAS modulates metabolic function primarily through distinct nuclei within the hypothalamus [14] [15] [16] [17] . Most of these effects can be attenuated by administration of RAS blockers or by genetically ablating AT 1 R in specific brain regions or cell types [18] [19] [20] .

Resistant hypertension, in which high blood pressure remains above 140/90 mmHg despite use of 3 or more antihypertensive drugs (including a diuretic), accounts for approximately 10% of patients with essential hypertension [21] . Resistant hypertension and sympathetic overactivity have been linked to brain RAS overactivation [22] . Thus, novel drugs targeting the brain RAS might be useful to treat resistant hypertension and/or diseases associated with elevated sympathetic outflow such as heart failure [23] . This article aims to bring the reader up-to-date on the important new findings and the currently controversial topics in the field. Then, novel translatable strategies to attenuate the upregulation of brain RAS activity in human resistant hypertension will be also discussed.

Although more than 50 years of research supports the important role of the brain RAS in modulating several physiological functions, it is not completely clear how angiotensin peptides are generated within the central nervous system (CNS). There is extensive evidence indicating that angiotensinogen is highly expressed in astrocytes and in some specific populations of neurons, which suggests that the extracellular space of the CNS has abundant renin substrate [19, [24] [25] [26] [27] [28] . The distribution of the two main ANG-II receptors, AT 1 R and AT 2 R, was mapped initially by autoradiography and subsequently confirmed by either in situ hybridization or utilizing transgenic mice expressing reporter genes under the control of either AT 1 R or AT 2 R promoters [29, 30, 31•] . AT 1 R is highly expressed in most of the circumventricular organs such as the subfornical organ, the organum vasculosum laminae terminalis (OVLT), and the area postrema. However, the elevated expression of AT 1 R in some regions behind the bloodbrain barrier such as the paraventricular nucleus of the hypothalamus (PVN), the nucleus tractus solitarius (NTS), the rostral and caudal ventrolateral medulla (RVLM and CVLM, respectively), the medial preoptic nucleus (MnPO), and some neurons within the arcuate nucleus (ARC) suggests a role for ANG-II as a neuromodulator. Expression of AT 2 R in the brain predominates over AT 1 R expression during fetal development [32] . However, recent studies from De Kloet et al. utilizing bacterial artificial chromosome transgenic AT 2 R-enhanced green fluorescent protein (eGFP) reporter mouse confirmed the presence of AT 2 R in adult brains particularly in neurons and/or fiber terminals in circumventricular organs, hypothalamic nuclei, and the hindbrain [30] .

Although most of the components of the RAS have been identified in the brain, the lack of a reliable method to detect renin in small neuroanatomical structures had led some to question whether the central generation of ANG-II requires renin. Indeed, renin-independent ANG-II generating biochemical pathways involving tonin and cathepsin D have been proposed [33] [34] [35] [36] . Others have performed experiments which they interpret as a refutation of the brain RAS [37] . The difficulties in measuring renin stem from several factors. First, considering that renin is the rate-limiting enzyme for the generation of ANG-I, and given the elevated bioavailability of angiotensinogen in the extracellular space of the brain, it is expected renin must be tightly controlled and secreted from specific cells within selected neuroanatomical nuclei. Supporting this, minimal elevation of ANG-II levels in discrete neuroanatomical regions is expected to elicit extremely profound effects. Indeed, evidence indicates that ablation or stimulation of angiotensinergic signaling within a few cells in the brain leads to extremely prominent cardiovascular and metabolic phenotypes [38] [39] [40] . Second, we and others have described the existence of an alternative renin isoform termed renin-b, which is the predominant transcribed renin isoform in the brain, but is absent in other tissues [41, 42] . Renin-b lacks the signal peptide and the first third of the pro-segment which implies that (1) renin-b protein is catalytically active and (2) is predicted to remain in an intracellular compartment [43] . This observation was particularly compelling as an intracrine RAS had been previously proposed [44, 45] . Notably, evidence that ANG-II can be generated intracellularly in presynaptic neurons and subsequently released to the synaptic terminal upon presynaptic depolarization would provide a strong argument to define ANG-II as a neurotransmitter. To test the hypothesis that renin-b is involved in the generation of intracellular ANG-II in the brain, we generated transgenic mice lacking the alternative renin-b, while renin-a was preserved [46] . Paradoxically, mice lacking renin-b exhibited a mild increase in blood pressure during the light cycle, which was attenuated by intracerebroventricular infusions of AT 1 R blockers or ACE inhibitors [47•] . These data indicate that intracellular renin-b might play a regulatory role on the brain RAS rather than generating intracellular ANG-II as initially proposed [46] . Even though our data do not support the existence of an intracellular ANG-II generating mechanism, they neither disprove it.

The brain is one of the most vascularized systems because it receives 8.6-20.4% of total cardiac output; thus, distinguishing renin levels generated within brain tissues (autocrine/paracrine) from the circulating renin (endocrine) is extremely challenging [48] . Despite evidence that renin activity and ANG-II have been identified in the cerebrospinal fluid (CSF) and brain tissues from nephrectomized animals, those who resist the existence of the brain RAS postulated that samples must be contaminated with traces of trapped blood [49] [50] [51] [52] [53] [54] [55] [56] [57] . Following this rationale, Van Thiel and colleagues attempted to provide evidence that the brain lacks the capacity to generate ANG-II by measuring renin activity (angiotensin-generating activity) in brain structures before and after organ buffer perfusion [37] . It is not surprising that there was a significant decrease in renin activity within several brain structures after buffer perfusion because (1) the brain has blood, and (2) neurons unlikely store renin within granules as do specialized renal juxtaglomerular cells. Indeed, the most direct and accurate conclusion from this study was that the brain indeed contains blood. It is surprising the authors decided to ignore their own data that renin activity remained at detectable levels in some buffer-perfused tissues including the brainstem. This is particularly interesting because in a double-transgenic mouse in which enhanced green fluorescence protein (GFP) is expressed under the control of the renin promoter and β-galactosidase is controlled by the angiotensinogen promoter, we identified unique reninexpressing cells in close proximity to angiotensinogenexpressing cells specifically within the RVLM, a brainstem nucleus [58, 59] .

It is of utmost importance that one contrasts a classical endocrine system and views the brain RAS as a neuroendocrine, autacoid, or neurotransmitter system where single neurons or small collections of neurons mediate output from the CNS. Thus, cellular, neuroanatomical, and molecular specificities are the key aspects to be considered when neuroendocrine systems, such as the brain RAS, are studied. It remains unfortunate that equivocal interpretations from experiments lacking sensitivity and neuroanatomical selectivity, those which are not required when studying the classical RAS, re-emerge from time to time to question the existence of the brain RAS [60] . In the final section of this article, we will discuss how the development of state-of-the-art technology to study precise molecular signaling and neuronal circuits within the CNS are appropriate to elucidate the key mechanisms regulating generation and action of angiotensin peptides within different neuroanatomical regions.

The proteolytic activation of prorenin that normally occurs in secretory granules in renal JG cells is unlikely to occur in extrarenal tissues, including the brain [61] . Thus, it has been proposed that activation of renin in extrarenal tissues requires its binding to ATPase H(+)-transporting lysosomal accessory protein 2, also known as the prorenin receptor (PRR), encoded by the ATP6ap2 gene. PRR has the unique capability to bind prorenin and induce its activation without prosegment removal [62] . PRR is highly expressed in neurons and some microglia cells and has been detected in several brain regions implicated in cardiovascular and autonomic control including the subfornical organ (SFO), paraventricular nucleus (PVN), nucleus of the solitary tract (NTS), and the rostral ventrolateral medulla (RVLM) [63•, 64] . Despite initial reports indicating that PRR might be involved in hypertension through local generation of ANG-II, other roles that are independent of RAS activation have been described (reviewed in [65] ). For instance, the transmembrane domain of the PRR interacts with the vacuolar H(+)-ATPase and plays an important role in lysosomal function and neuronal development [66] . In humans, a unique mutation (c.321C>T, p.D107D) in the PRR locus is associated with X-linked mental retardation and epilepsy [67] . Although many RAS-independent functions of PRR are well defined in kidney, the RAS-independent functions of PRR in the brain are not completely understood [49, 66, [68] [69] [70] .

In mice where neuronal PRR was ablated at embryonic daỹ 18.5 (neuron filament promoter-CRE crossed with PRRfloxed mice; Nefh-PRRKO), central generation of ANG-II in response to intracerebroventricular administration of recombinant prorenin was attenuated. To study the role of neuronal PRR in the pathogenesis of hypertension, Feng's lab utilized the model of low-renin hypertension induced by deoxycorticosterone acetate (DOCA) infusion and high dietary sodium. Intracerebroventricular infusion of ACE inhibitor prevents and reverses high blood pressure, demonstrating that production of ANG-II in the brain is required for DOCA-salt hypertension even though circulating RAS activity is suppressed [71] . Moreover, two separate studies have shown that central infusion of an AT 1 R blocker mimics the effects of central delivery of ACE inhibitors [72, 73] . Thus, it is well accepted that the blood pressure elevation in DOCA-salt hypertension is strongly driven by a neurogenic mechanism involving activation of the brain RAS [74] .

Li et al. reported that the selective ablation of PRR in neurons attenuated the elevation of blood pressure in DOCA-salt hypertension when 0.9 M NaCl was the only fluid offered [75] . In contrast, experiments where DOCA-treated Nefh-PRRKO mice were exposed to two-bottle choice paradigm for the assessment of sodium preference revealed that the ablation of PRR in neurons is insufficient to decrease blood pressure but suppressed DOCA-induced saline intake [76] . These data suggest expression of PRR in specific brain regions controlling drinking behavior, such as the SFO is of physiological relevance. Recently, Souza and colleagues examined the role of PRR in the PVN, a key integratory nucleus involved in blood pressure control [77] . In this study, PVNtargeted ablation of PRR was induced in PRR-floxed mice by bilateral stereotactic microinjection of adeno-associated virus (AAV)-CRE-GFP. The reduction of PRR expression in the PVN by less than 50% was sufficient to attenuate DOCA-salt induced blood pressure elevations, cardiac and vasomotor sympathetic over-activity, and improved cardiac parasympathetic function [63• ]. In addition, live-cell calcium recordings utilizing a novel calcium biosensor (GCaMP6) revealed PVNtargeted ablation of PRR attenuates calcium influx in response to ANG-II in DOCA-salt hypertension. Despite the growing evidence indicating the importance of PRR in the CNS to control cardiovascular function, there is no generalized consensus whether the underlying mechanisms require local ANG-II generation. It is likely that both ANG-II-dependent and ANG-II-independent mechanisms might occur simultaneously at different degrees depending on the neuroanatomical localization and cell types, as well as different physiological and pathological circumstances [78] .

There is a growing interest in the soluble PRR fragment (sPRR) which arises from the proteolytic cleavage of PRR by furin or site-1 protease to generate a 10-kD transmembrane/ cytoplasmic fragment and a 28-kD soluble PRR form [79, 80] . Growing evidence supports that elevation of circulating sPRR levels are associated with high blood pressure, chronic kidney disease, preeclampsia, and obstructive sleep apnea [81] [82] [83] [84] . However, the biological function and the physiological relevance of sPRR were completely unknown until recently. Many functions of sPRR controlling renal function have been reported. For instance, sPRR exerts antidiuretic actions in part by inducing frizzled 8-dependent stimulation of aquaporin 2 expression in the collecting duct [49] . Studies specifically aiming to elucidate the role of sPRR in the CNS have not been reported. Recently, Gatineau et al. demonstrated that the selective deletion of adipose tissue PRR elevates systolic blood pressure concomitant with increased circulating sPRR levels in high fat diet-fed mice [85•] . In males, systemic infusion of recombinant mouse epitope tagged sPRR resulted in blood pressure elevation and this increase was attenuated by ganglionic blockade, but not administration of AT 1 R blockers, indicating that autonomic dysfunction, but not circulating RAS overactivation, is a key mechanism underlying sPRR-mediated blood pressure elevation in obese male mice. In contrast, infusion of sPRR in females failed to induce autonomic dysfunction but it induced elevated vasopressin levels and plasma renin indicating the existence of sex differences in sPRR-mediated responses [86] . Given that the source of vasopressin is exclusively from the PVN and supraoptic nucleus (SON), these observations support that sPRR is biologically active in the CNS and elevations of sPRR in the brain might be implicated in certain forms of neurogenic hypertension linked to obesity.

It has been proposed that many of the effects of ANG-(1-7) in the CNS oppose many of the AT 1 R-mediated actions of ANG-II in the brain [87] . ANG-(1-7) induces its effects mainly through Mas receptor activation, although it has been reported to also act through AT 2 R [88] . Other studies proposed that Mas receptors form heterodimers with AT 2 R in astrocytes [89] . Teixeira et al. recently reported that ANG-(1-7) binds to AT 1 R but fails to engage its canonical G protein signaling [90] . Instead, ANG-(1-7) triggers β-arrestin recruitment and intracellular signaling, suggesting it may have the properties of a biased agonist for AT 1 R. Several synthetic AT 1 R biased agonists have been designed, but the existence of an endogenous functional biased ligand for AT 1 R has not been reported. ANG-(1-7) can be generated from the direct cleavage of ANG-II by angiotensin converting enzyme 2 (ACE2), but ACE2 can also catalyze the conversion of angiotensin I to Angiotensin-(1-9), which subsequently is converted to ANG-(1-7) by ACE or neutral endopeptidase [91] . Downregulation of ACE2 and suppression of central ANG-(1-7) levels are thought to be one of the underlying mechanisms causing low renin hypertension [20, 92] .

In recent years, significant progress has been achieved particularly on the molecular mechanisms controlling brain ACE2 activity. Lambert et al. described a process termed "ACE2 shedding" in which a disintegrin and metalloprotease 17 (ADAM17) catalyze the cleavage of membrane anchored ACE2 [93] . However, advances on the physiological and pathophysiological role of ADAM17-mediated ACE2 shedding in the brain have only been recently demonstrated. Xia and Sriramula et al. demonstrated that neuron-targeted overexpression of ACE2 is sufficient to ameliorate elevated blood pressure, autonomic dysfunction, and vasopressin release in response to DOCA-salt hypertension [94] . Moreover, braintargeted ablation of ADAM17 utilizing central infusions of siRNA suppressed DOCA-salt induced hypertension concomitant with blunted reduction of ACE2 activity in the hypothalamus and cerebrospinal fluid, indicating that ACE2 shedding by ADAM17 in the brain is a relevant mechanism contributing to neurogenic hypertension. New evidence indicates that activation of AT 1 R is required for ADAM17-mediated ACE2 shedding possibly via reactive oxygen species and phosphorylation of extracellular signal-regulated kinase in neurons [92] . It has been previously shown that a reduction of ACE2 expression in the RVLM is a contributing factor in the development of hypertension in spontaneously hypertensive rats [95] . Mukerjee et al. provided evidence of the importance of ACE2/ADAM17 pathway in pre-sympathetic neurons within the PVN [96•] . Interestingly, ACE2 is expressed in GABAergic inhibitory neurons projecting onto the hypothalamus. Thus, ACE2 is thought to maintain a normal GABAergic inhibitory tone to the presympathetic neurons in the PVN in normal physiological conditions, while disinhibition of this pathway might lead to hypertension. In contrast, ADAM17 is expressed in single-minded family basic helixloop helix transcription factor 1 (Sim1)-positive excitatory neurons within the PVN and promotes excitatory activity. Ablation of ADAM17 in the PVN blunts pressor responses to acute PVN-targeted microinjection of ANG-II. This accumulating evidence suggests the importance of the opposing roles of ACE2 and ADAM17 in modulating of sympathetic activity and central control of blood pressure. Finally, it has been recognized that ADAM17 has a role in processing the proinflammatory cytokine, tumor necrosis factor alpha (TNF-α). Indeed, upregulation of the brain ADAM17 was associated with elevated TNF-α implicating that attenuation of TNF-α-related mechanisms could be mediating part of the phenotype observed in mice lacking hypothalamic ADAM17. Numerous reports suggest elevation of TNF-α and activation of inflammatory cells (microglia) within certain brain regions triggers hypertensive responses [97] [98] [99] . Therefore, future studies are expected to clarify whether TNF-α plays a regulatory role in ACE2 activity and other RAS components.

A quarter of hypertensive patients exhibit low-renin hypertension [100] . It has been suggested that low-renin hypertension is in part driven by elevated angiotensinergic signaling in the brain [71, 101] . Therefore, the development of novel drugs modulating the brain RAS might represent an effective solution to treat resistant hypertension coincident with elevated sympathetic activity and suppressed circulating renin activity. Two decades ago, Llorens-Cortes's laboratory demonstrated that the conversion of ANG-II into angiotensin III (ANG-III) in the brain is catalyzed by aminopeptidase A (APA), a zinc metalloprotease [102] . Importantly, ANG-III has been hypothesized to be the major biologically active peptide of the brain RAS. This is based on the observation that inhibition of the brain APA completely prevents elevated blood pressure in models of neurogenic hypertension with elevated brain RAS [103] .

It took years to translate the observation that pharmacological inhibition of APA can be used as a therapeutic tool to treat resistant hypertension in humans. Recently, a new multicenter, open-label, phase II study was released evidencing the efficacy of a brain penetrating inhibitor of APA, firibastat (previously named RB150), in reducing blood pressure in overweight patients of multiple ethnic origins without angioedema [104•] . Moreover, an additional pilot double-blinded randomized placebo-controlled study in hypertensive patients demonstrated that blood pressure in firibastat-treated patients trended to be decreased compared to placebo controls without affecting systemic RAS activity [105] . Firibastat is the first oral medication that may target the brain RAS with promising clinical application. Current efforts in designing new brainpenetrating APA inhibitors led to a 10-fold more potent new prodrug, NI956/QGC006, which has been shown to exert powerful antihypertensive effects in rats treated with DOCA-salt [106] .

In recent years, multiple pleiotropic roles of the brain RAS, namely neuroinflammation, autophagy, ER stress, and mitochondrial dysfunction, have emerged. These findings resulted in considerable advances in utilizing brain RAS blockade or RAS modulation as a therapeutic strategy to treat diseases beyond neurogenic hypertension. This is specifically relevant in stroke and cerebrovascular diseases, Alzheimer's disease, cognitive dysfunction, Parkinson disease, aging, and others [107] [108] [109] [110] [111] [112] [113] [114] [115] . Numerous other new molecules or administration routes to target the brain RAS are currently under investigation. For instance, new classes of AT 1 R biased agonists, which can selectively activate β-arrestin without activating the classical G protein-coupled signaling, might represent potential tools to modulate angiotensin signaling within the brain. For example, Carvalho-Galvão et al. demonstrated that intracerebroventricular infusion of TRV027, a β-arrestin-biased AT 1 R-agonist effectively attenuated autonomic dysfunction and decreased arterial pressure in spontaneously hypertensive rats (SHR) [116] . Thus, the development of brain-penetrating AT 1 R-biased agonists would be a promising strategy to treat resistant hypertension. Finally, Torika et al. demonstrated that intranasal administration of telmisartan is effective at reducing brain inflammation and ameliorating the progression of Alzheimer's disease in mice indicating that novel routes of administration can also be employed to inhibit the brain RAS without systemic off targets effects [117] .

The development of several cutting-edge technologies to study the CNS predicts that we might witness a profound advance in this field in the near future. Several laboratories are currently utilizing novel in situ hybridization techniques with significantly higher specificity and sensitivity. These are powerful tools to identify the anatomical and cellular localization of cells expressing components of the RAS within the CNS and to query the molecular and/or neural significance of these cells by multiplexing with different probes. We used RNAscope® technology to confirm the abundance of AGT and the presence of AT 1 R and PRR in discrete cells within the brain. Using this same technique, we and others have identified the distribution of RAS genes in specific NeuN+ neurons, GFAP+ astrocytes, and Iba-1+ microglia cells in the SFO, PVN, ARC, and the RVLM (unpublished). In addition, the development of an extensive array of transgenic mice carrying conditional alleles of RAS genes as well as mice expressing tamoxifen-inducible and/or cell-specific CRE recombinase expression facilitates further exploration of novel molecular and physiological functions of the RAS within specific cell types within the brain. Novel and previously generated animals expressing CRE recombinase under the control of specific promotors including AT 1 R-CRE or Ren1-CRE mice, which can be crossed with mice expressing CRE-dependent TdTomato (or other reporter genes) as well as mice expressing a fluorescent reporter gene under the control of RAS genes such as AT 2 R-eGFP or AT 1a R-EGFP (NZ44), allow fluorescent labelling of cells expressing RAS genes [30, 31•, 118, 119] .

Significant progress has been made in developing techniques to identify the distinct roles of specific neurons in the brain. These techniques include optogenetics and Designer Receptors Exclusively Activated by Designer Drugs (DREADD), a chemogenetic technique, in which neuronal activity can be stimulated or suppressed in specific brain nuclei utilizing light or designer drugs, respectively. Using these techniques, several laboratories recently showed the distinct roles of specific cells within selected nuclei controlling cardiovascular, metabolic, and autonomic function. For instance, De Kloet el al. recently reported that optogenetic stimulation of AT 1 R expressing neurons in the PVN promotes blood pressure elevation and activation of the hypothalamic-pituitary-adrenal axis [31•] . Similarly, Nation et al. utilized DREADDs to study the role of SFO neurons in thirst and salt appetite [120•] . Stimulation of neuronal firing and activation of Gq signaling in mice receiving SFO-targeted microinjection of a virus (AAV2-CaMKII-HA-hM3D(Gq)-IRES-mCitrine) to induce selective neuronal expression of Gαq via a designer receptor (hM3D) that is exclusively activated by clozapine N-oxide, resulted in strong dipsogenic responses and preference to 0.3 M saline. The combination of these novel techniques with the array of transgenic mice described above are powerful tools to inquire the responses to stimulating or inactivating different neuronal circuits in the CNS.

Finally, emerging "omics" techniques are becoming more accessible and reliable to study the transcriptome profile in different neuronal populations. There is particular interest in single-cell and single-nuclear RNA sequencing technology to identify different clusters expressing RAS genes to evaluate the molecular signature of cells. Although studies utilizing these techniques to specifically evaluate the brain RAS are not yet available, Sapouckey recently reported an in silico re-analysis of hypothalamic single-cell RNA sequencing datasets revealing that AT 1 R is expressed in a specific cluster of neurons expressing both Agouti-related peptide (AgRP) and leptin receptors [121• ]. This seminal discovery may illuminate the underlying mechanisms by which the brain RAS controls resting metabolic rate and sympathetic activity in obesity-related hypertension.

Although the RAS in the brain has been studied for decades, interesting and seminal discoveries continue to be made to this day. These include assessing the functional significance of newly identified components of the RAS (such as prorenin receptor), the action of unconventional RAS peptides (such as ANG III), enzymes which modify RAS components (such as ADAM17), and new therapeutic tools to combat neurogenic hypertension. New technologies are making it easier to answer old questions-what is the localization of angiotensin receptors-and investigate new ones-what are function of specific subsets of AT 1 R-containng neurons? New genomic technologies such as single cell sequencing will provide novel platforms to understand the diversity of neuronal types which respond to RAS activation or mediate downstream RAS signaling.

Conflict of Interest The authors declare no conflicts of interest relevant to this manuscript.

Human and Animal Rights and Informed Consent This article does not contain any studies with human or animal subjects performed by any of the authors.

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Soluble form of the (pro)renin receptor generated by intracellular cleavage by furin is secreted in plasma

Site-1 protease is required for the generation of soluble (pro)renin receptor

Elevated plasma levels of soluble (pro)renin receptor in patients with obstructive sleep apnea syndrome: association with polysomnographic parameters

Serum level of soluble (pro)renin receptor is modulated in chronic kidney disease

Soluble (pro)renin receptor and blood pressure during pregnancy: a prospective cohort study

Serum soluble (pro)renin receptor levels in patients with essential hypertension

Provides key evidence that soluble prorenin receptor induces blood pressure elevation possibly through a neurogenic mechanism in diseases associated with obesity

Losartan prevents the elevation of blood pressure in adipose-PRR deficient female mice while elevated circulating sPRR activates the renin-angiotensin system

Protective axis of the renin-angiotensin system in the brain

Angiotensin-(1-7) is an endogenous ligand for the G protein-coupled receptor mas

Evidence for heterodimerization and functional interaction of the angiotensin type 2 receptor and the receptor MAS

Ang-(1-7) is an endogenous betaarrestin-biased agonist of the AT1 receptor with protective action in cardiac hypertrophy

Angiotensinconverting enzyme 2, angiotensin-(1-7) and mas: new players of the renin-angiotensin system

Angiotensin II type 1 receptor-mediated reduction of angiotensin-converting enzyme 2 activity in the brain impairs baroreflex function in hypertensive mice

Tumor necrosis factor-alpha convertase (ADAM17) mediates regulated ectodomain shedding of the severe-acute respiratory syndrome-coronavirus (SARS-CoV) receptor, angiotensinconverting enzyme-2 (ACE2)

Brain angiotensin-converting enzyme type 2 shedding contributes to the development of neurogenic hypertension

Overexpression of angiotensin-converting enzyme 2 in the rostral ventrolateral medulla causes long-term decrease in blood pressure in the spontaneously hypertensive rats

ACE2 and ADAM17 interaction regulates the activity of presympathetic neurons

Stressinduced sensitization of angiotensin II hypertension is reversed by blockade of angiotensin-converting enzyme or tumor necrosis factor-alpha

Central inhibition of tumor necrosis factor alpha reduces hypertension by attenuating oxidative stress in the rostral ventrolateral medulla in renovascular hypertensive rats

Central leptin and tumor necrosis factor-alpha (TNFalpha) in diurnal control of blood pressure and hypertension

Diagnosis and treatment of low-renin hypertension

Angiotensinergic signaling in the brain mediates metabolic effects of deoxycorticosterone (DOCA)-salt in C57 mice

Aminopeptidase a inhibitors as potential central antihypertensive agents

Brain renin-angiotensin system blockade by systemically active aminopeptidase a inhibitors: a potential treatment of salt-dependent hypertension

Efficacy and safety of firibastat, a first-in-class brain aminopeptidase A inhibitor, in hypertensive overweight patients of multiple ethnic origins

A pilot double-blind randomized placebo-controlled crossover pharmacodynamic study of the centrally active aminopeptidase A inhibitor, firibastat, in hypertension

NI956/QGC006, a potent orally active, brainpenetrating aminopeptidase a inhibitor for treating hypertension

Attenuation of stroke damage by angiotensin II type 2 receptor stimulation via peroxisome proliferator-activated receptor-gamma activation

Protective effects of the angiotensin II AT2 receptor agonist compound 21 in ischemic stroke: a nose-tobrain delivery approach

Central application of Aliskiren, a renin inhibitor, improves outcome after experimental stroke independent of its blood pressure lowering effect

Angiotensin-III is increased in Alzheimer's disease in association with amyloidbeta and tau pathology

Pleiotropic benefits of the angiotensin receptor blocker candesartan in a mouse model of Alzheimer disease

An integrative genomewide transcriptome reveals that candesartan is neuroprotective and a candidate therapeutic for Alzheimer's disease

Memory is preserved in older adults taking AT1 receptor blockers

Cognitive benefits of angiotensin IV and angiotensin-(1-7): a systematic review of experimental studies

Angiotensin II-and Alzheimer-type cardiovascular aging

Central administration of TRV027 improves baroreflex sensitivity and vascular reactivity in spontaneously hypertensive rats

Intranasal telmisartan ameliorates brain pathology in five familial Alzheimer's disease mice

Distribution of angiotensin type 1a receptorcontaining cells in the brains of bacterial artificial chromosome transgenic mice

Renin cells are precursors for multiple cell types that switch to the renin phenotype when homeostasis is threatened

Demonstrated that DREADD-mediated stimulation of neurons within the subfornical organ leads to prominent dipsogenic responses and salt appetite

In silico re-analysis of hypothalamic single-cell RNA sequencing datasets reveals that AT 1 R is expressed in a specific subcluster of neurons expressing both Agouti-related peptide (AgRP) and leptin receptors

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