The Intrarenal Renin-Angiotensin System in Hypertension Robert M. Carey The renin-angiotensin system (RAS) is a well-studied hormonal cascade controlling fluid and electrolyte balance and blood pressure through systemic actions. The classical RAS includes renin, an enzyme catalyzing the conversion of angiotensinogen to angiotensin (Ang) I, followed by angiotensin-converting enzyme (ACE) cleavage of Ang I to II, and activation of AT1 receptors, which are responsible for all RAS biologic actions. Recent discoveries have transformed the RAS into a far more complex system with several new pathways: the (des-aspartyl1)-Ang II (Ang III)/AT2 receptor pathway, the ACE-2/Ang (1-7)/Mas receptor pathway, and the prorenin-renin/prorenin receptor/mitogen-activated protein kinase pathway, among others. Although the classical RAS pathway induces Na1 reabsorption and increases blood pressure, several new pathways constitute a natriuretic/vasodilator arm of the system, opposing detrimental actions of Ang II through Ang II type 1 receptors. Instead of a simple circulating RAS, several independently functioning tissue RASs exist, the most important of which is the intrarenal RAS. Several physiological characteristics of the intrarenal RAS differ from those of the circulating RAS, autoamplifying the activity of the intrarenal RAS and leading to hypertension. This review will update current knowledge on the RAS with particular attention to the intrarenal RAS and its role in the pathophysiology of hypertension. Q 2015 by the National Kidney Foundation, Inc. All rights reserved. Key Word: Kidney, Renin, Angiotensin, Hypertension, Sodium

INTRODUCTION Hypertension (HT) is the world’s most prevalent cardiovascular disorder (approximately one third of adults have HT in most communities worldwide) and one of the leading risk factors for disability and death.1-3 The relationship between blood pressure (BP) and cardiovascular risk is continuous, consistent, and independent of other risk factors and the higher the BP, the greater the chance of cardiovascular events, stroke, and kidney disease.2 Approximately 90% of patients with HT have primary (essential) HT, the causes of which remain unknown.1-3 However, genetic and environmental factors that affect BP are currently being studied. Genetic factors include inappropriately high activity of the renin-angiotensin system (RAS) and the sympathetic nervous system and susceptibility to the effects of salt on BP (salt sensitivity of BP). Environmental factors include excess dietary salt intake, obesity, and possibly sedentary lifestyle. Of the secondary causes of HT, the most prevalent condition by far is primary aldosteronism (5%-10%), followed by CKD, renal vascular disease, sleep apnea, and a variety of uncommon endocrine conditions, including pheochromocytoma.4 The RAS is a well-studied hormonal cascade important in the control of BP.5 Angiotensin (Ang) II, the principal Ang effector peptide, binds to 1 of 2 distinct receptors, the Ang II type 1 receptor (AT1R) and Ang II type 2 recep-

From Division of Endocrinology and Metabolism, Department of Medicine, University of Virginia Health System, Charlottesville, VA. Address correspondence to Robert M. Carey, MD, Division of Endocrinology and Metabolism, Department of Medicine, University of Virginia Health System, PO Box 801414, Charlottesville, VA 22908-1414. E-mail: rmc4c@ virginia.edu Ó 2015 by the National Kidney Foundation, Inc. All rights reserved. 1548-5595/$36.00 http://dx.doi.org/10.1053/j.ackd.2014.11.004

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tor (AT2R). The vast majority of the actions of Ang II are transduced by AT1Rs, including cellular dedifferentiation and proliferation; vasoconstriction; reduced vascular compliance; cardiac contractility; increased renal tubular sodium (Na1) reabsorption; aldosterone, vasopressin, and endothelin secretion; salt appetite; thirst; and activation of the sympathetic nervous system, all of which can raise BP and contribute to the pathogenesis of HT.5 Ang II also inhibits renin secretion through AT1Rs on renal juxtaglomerular cells, serving as a short-loop negative feedback mechanism that limits the activity of the system.5 In contrast, AT2Rs generally oppose the actions of Ang II through AT1Rs under most circumstances, inducing vasodilation and natriuresis but acting in concert with AT1Rs to suppress renin secretion.6-8 Despite many years of study, the RAS continues to reveal new pathways, including additional enzymes, peptides, receptors, and actions that can contribute to HT (Fig 1).5,9 In addition to the classical renin/ACE/ Ang II-AT1R pathway, at least 3 new axes have been described, including the [des-aspartyl1]-Ang II (Ang III)/ AT2R pathway, the ACE2/Ang(1-7)/Mas receptor pathway, and the (pro)renin receptor (PRR)/mitogenactivated protein (MAP) kinase pathway. The concept that Ang II is the only active peptide of the RAS is now outmoded because angiotensinogen (Agt) can be hydrolyzed by various enzymes to generate Ang (1-7), Ang III, and alamandine, each of which has newly described biologic actions.5,9 One of the most important characteristics of the RAS is its function not only as a circulating (endocrine) system but also as an independent local tissue (paracrine) and/or cellular (autacrine or intracrine) system.5 The intrarenal RAS is such a local tissue system that has the capacity to regulate its activity independently of the circulating system. The goal of this brief review is to discuss the role of the intrarenal RAS (independently of aldosterone) in the pathophysiology of HT.

Advances in Chronic Kidney Disease, Vol 22, No 3 (May), 2015: pp 204-210

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Elegant cross-transplantation studies in mice later demonEVIDENCE FOR AN INDEPENDENT FUNCTIONAL strated the importance of, and indeed requirement for, renal INTRARENAL RAS AT1Rs in the pathophysiology of Ang II-induced HT.17-19 Although renin was identified in the brain and adrenal Kidneys from wild-type (WT) mice were transplanted into cortex in the late 1960s and early 1970s, the intrarenal mice with whole-body AT1R knockout, creating a model RAS was the first independent functional tissue RAS of exclusive renal AT1R expression, and kidneys from anito be described and characterized.10-12 The initial mals with AT1R knockout were transplanted into WT anievidence came from in vivo studies demonstrating mals, creating a model of exclusive renal AT1R that inhibition of the RAS with intrarenal infusion of knockout.17,18 When the animals were infused with Ang II angiotensin-converting enzyme (ACE) inhibitors or systemically, only the animals with intact renal AT1Rs Ang II receptor blockers, at infusion rates that did not developed sustained HT and cardiac hypertrophy.18 Thus, alter systemic BP during the experimental period, markkidney AT1Rs are necessary and sufficient for the developedly increased renal plasma flow, glomerular filtration ment of Ang II-dependent HT.18 Additional studies emrate, and sodium (Na1) and water excretion.10 These reploying Cre/Lox technology have demonstrated that sults were later confirmed using more rigorous apselective deletion of proximal tubule AT1Rs alone is suffiproaches showing that small intrarenal doses of Ang II cient to reduce basal BP despite intact systemic and renal receptor blocker, while not altering pressor responses AT1Rs outside the proximal tubule.19 Conversely, studies to systemically administered Ang II, induced marked have demonstrated that transfer of Ang II/cyan fluorescent increases in renal hemodynamic and tubular funcprotein and/or AT1 receptors selectively into the proximal tion.11,12 These results were interpreted as indicating tubule is sufficient to increase BP.20,21 Thus, the activity of that Ang II acting at AT1Rs exerts baseline tonic 1 the intrarenal RAS is critically important in the control of inhibition on Na excretion. Later, the mRNAs and BP in experimental animals, proteins for all the known and this concept is also RAS components (Agt, likely to apply to humans. renin, ACE, and AT1Rs) CLINICAL SUMMARY However, demonstrating a were found expressed in a role for the intrarenal RAS site-specific manner within  The intrarenal RAS, the most important independently in humans is replete with the kidney and it was functioning tissue RAS, is activated in hypertension. difficulty and will require shown that intrarenal  Unlike the systemic RAS, the intrarenal RAS can autothe availability of specific formation of Ang II occurs amplify Ang II production, providing a continuing urinary markers such as independently of the renal source of the peptide to maintain vasoconstriction, Ang II, Agt, or other RAS uptake of the peptide from antinatriuresis and hypertension. components. the circulation.5 Additional evidence for an independent  The intrarenal actions of Ang II via AT1Rs are opposed ROLE OF THE by several counter-regulatory RAS components, including tissue RAS included obserthe Ang III/AT2R pathway, the ACE-2/Ang (1-7)/Mas INTRARENAL RAS IN HT vations that Ang II concenreceptor pathway and possibly the newly described Currently, the intrarenal trations are elevated 1000alamandine/MrgD pathway. These pathways represent RAS is considered to be the fold higher in renal interpotential new therapeutic targets for hypertension. most important of the tissue stitial fluid compared RASs (vasculature, heart, with circulating plasma, brain, adrenal, etc.) in the intrarenal Ang II content is control of BP and HT in experimental animals.5 Indeed, markedly increased compared with circulating levels there is growing recognition that inappropriate activation in response to Na1 restriction, and this response could of the intrarenal RAS prevents the kidney from maintainbe abolished by selective intrarenal renin inhibition.13,14 ing normal Na1 balance at normal renal perfusion ROLE OF THE INTRARENAL RAS IN BP CONTROL pressures and is an important cause of HT.22-24 Several Definitive molecular studies now indicate the importance of experimental models support the concept of an the intrarenal RAS in the control of BP and the pathogenesis overactive intrarenal RAS in the development and of HT. Transgenic overexpression of Agt selectively within maintenance of HT; these include 2-kidney, 1-clip Goldblatt the mouse kidney induced chronic HT independently of HT; Ang II-infused HT; transgenic rat mRen2-27 HT with the systemic RAS.15 Transgenic mice expressing the Agt an extra renin gene; remnant kidney HT; several mouse gene selectively in the proximal tubule (through the kidney models overexpressing the renin or Agt gene; and, perhaps androgen-related promoter), when bred to mice expressing most importantly, spontaneously hypertensive rats (SHR). human renin systemically, had a major increase in BP in spite SHR are inbred rats that develop HT with increasing age of having normal circulating Ang II. The HT was Ang II and are widely employed as a model of human primary dependent as it was abolished by AT1R blockade. This HT.25 Young prehypertensive SHR exhibit increased renal constituted the first available evidence that systemic HT proximal tubule Na1 reabsorption, where normal Na1 15 excretion is achieved only at the expense of elevated renal could be induced by isolated renal tissue RAS activation. In addition, selective proximal tubule overexpression of huperfusion pressure.26,27 Over time, the kidneys reset man renin and Agt increased BP, in strong support of the to require elevated BP to excrete a normal Na1 load.28 Evidence for this pathophysiologic principle includes concept that intratubular RAS activation can induce HT.16

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Agt Unknown enzyme

Inflammatory Fibrotic

PRR

Prorenin

Renin

Cathepsin B

Ang (1-12)

ACE

Chymase (heart )

Ang I ACE

Ang II

ACE-2 ACE-2

APA

Ang (1-9) Unknown enzyme

Ang (1-7)

Alamandine

Ang III AT1R Actions

Vasoconstrictor Antinatriuretic Fibrotic Inflammatory

AT2R Vasodilator Natriuretic Antifibrotic Anti-inflammatory

MasR Vasodilator Antifibrotic Anti-inflammatory

MrgD Vasodilator Antifibrotic

Figure 1. Schematic illustration of the renin-angiotensin system. Receptors are shown in boxes, and enzymes are shown next to their respective arrows. Abbreviations: ACE, angiotensin-converting enzyme; Agt, angiotensinogen; Ang, angiotensin; APA, aminopeptidase A; AT1R, angiotensin type 1 receptor; AT2R, angiotensin type 2 receptor; MasR, Mas receptor; MrgD, Mas-related G protein-coupled receptor; PRR, (pro)renin receptor.

observations that transplantation of prehypertensive kidneys from SHR to their normotensive Wistar-Kyoto (WKY) controls induces HT in WKY and that both humans with genetic HT and SHR excrete less Na1 and water than normotensive controls when renal perfusion pressure is lowered to normotensive levels.29,30 In addition, chronic relationships between arterial pressure and urinary Na1 and water output are shifted toward higher BP in SHR compared with WKY, reflecting the ability of the kidney to adapt to a higher perfusion pressure.30 Most importantly, numerous studies have demonstrated that the intrarenal RAS is activated in SHR, and elevated intrarenal Ang II content likely causes increased Na1 retention through activation of renal AT1Rs.31-36 In many forms of HT, inappropriate activation of the intrarenal RAS limits the ability of the kidney to maintain Na1 balance, when perfused at normal arterial pressure. In addition to Na1 and fluid retention and progressive HT, other long-term consequences of an inappropriately activated intrarenal RAS include renal vascular, glomerular, tubular, and interstitial inflammation and fibrosis. INTRARENAL MECHANISMS OF CLASSICAL RAS ACTION Several interesting mechanisms have been shown to mediate increased intrarenal RAS content and action in HT. Agt is the only known precursor of Ang peptides and its regulation may be of key importance to the activity of the intrarenal RAS.5 The vast majority of renal Agt mRNA and protein is localized to the proximal tubule cells, suggesting that intratubular Ang II is derived from locally synthesized Agt. Both Agt and its metabolite Ang II are secreted from the proximal tubule cell into the tubule lumen, indicating that intracellular formation of Ang II may occur. Importantly, Ang II upregulates Agt in proximal tubule cells.37-39 Therefore, a positive feedforward loop exists whereby increased intrarenal RAS activity may reinforce itself by Ang II stimulation of its requisite

precursor, leading to continuous tissue RAS stimulation and persistence of HT. Indeed, prorenin, renin, Agt, and ACE have now been shown to be upregulated within the kidneys of rodents undergoing long-term Ang II infusion. This concept may fit with the aforementioned observations that the intrarenal RAS is continuously activated in SHR. Renin is synthesized not only in juxtaglomerular cells but also in connecting tubules, so that renin is likely to be secreted into the distal nephron.39-41 In addition, collecting duct renin expression is upregulated by Ang II through AT1Rs, suggesting that distal nephron Ang II formation may occur as well.39-41 Taken altogether, several intrarenal mechanisms contribute feedforward control to enhance Ang II concentrations at both proximal and distal nephron sites, where Ang II has potent Na1 reabsorptive actions. Another important feedforward concept is the interrelationship of AT1Rs to the intrarenal content of Ang II. When Ang II binds to AT1Rs, the intracellular accumulation of Ang II increases, particularly in the proximal tubule.42-45 This is the opposite of usual thinking in receptor pharmacology in which exposure of a G protein-coupled receptor to its ligand on the cell surface internalizes and desensitizes the biologic response while preparing the complex for lysosomal degradation. In the case of Ang II action in the kidney through AT1Rs, desensitization is absent.42-45 Therefore, AT1R-mediated cellular uptake of Ang II, at least in part, contributes to the high levels of Ang II in the kidney during Ang II infusion. The reason for elevated renal Ang II levels in models of HT has been debated for years. Agt protein principally exists in the proximal tubule and concurrent increases in Agt mRNA, and protein in renal tissue homogenates has been interpreted as indicating that transcriptional activation of the renal Agt gene is responsible for increases in renal Ang II content. Unexpectedly, current studies demonstrate definitively that hepatic biosynthesis of Agt accounts for the majority of renal Ang II content under basal conditions.46,47 Kidney-specific Agt knockout mice were shown to have markedly reduced renal Agt mRNA, but renal Agt protein and Ang II content were unchanged. In contrast, liver-specific Agt knockout abolishes renal Agt protein levels and markedly reduces Ang II content.46 Although this observation was interpreted as applying only under basal conditions, recent studies indicate that enhanced renal Ang II content in a model of severe podocyte damage is because of the loss of glomerular barrier function allowing a massive influx of circulating Agt, specifically not because of transcriptional activation of Agt within the kidney.47 Thus, it appears that both under basal and stimulated (disease) conditions, liver Agt provides the renal substrate source for Ang II. Whether these considerations apply also to animal models of HT requires further research. In addition to the tissue intrarenal RAS, there is emerging evidence that subcellular independent RASs exist within cells and may contribute to BP regulation and HT.48-50 Such considerations apply to both the nuclear and mitochondrial compartments. Within the nucleus, Ang II is coupled to the 3 main Ang receptors (AT1R, AT2R, and

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Mas receptor).49 Functionally, the nuclear AT1R is coupled to phosphoinositol-3 kinase and protein kinase C and activation leads to production of reactive oxygen species. In contrast, both the nuclear AT2R and Mas receptor are linked to nitric oxide (NO) formation. Aging and steroid administration alter the balance among nuclear RAS receptors leading to increased reactive oxygen species, reduced NO, and development of fetal-programmed HT. Recently, a mitochondrial Ang system was also identified and characterized.50 In this system, Ang II/Ang III are coupled predominantly to AT2Rs, and activation of the system is also coupled to NO production and mitochondrial respiration. The role of the intracellular RAS has been at least partially elucidated using transgenic mice overexpressing a nonsecreted form of intracellular Ang II.51 This animal model is characterized by HT and microangiopathy of glomerular capillaries and small vessels. Overexpression of intracellular Ang II restricted to the proximal tubule also leads to HT accompanied by antinatriuresis. The role of intracellular RASs in the pathophysiology of HT is currently unknown and will be an area of exciting investigation in the future. NEW PATHWAYS OF THE INTRARENAL RAS IN HT Ang III/AT2 Receptor Pathway AT2Rs are expressed predominantly in the renal proximal tubule and to a less extent in glomeruli and intrarenal vasculature.52 These receptors are generally accepted as opposing many of the detrimental actions of AT1Rs within the kidney and also systemically.5,9 Although AT2Rs clearly induce vasodilation in small resistance vessels, for example, in the heart, adrenal gland, and mesenteric circulation, most studies have demonstrated no substantial role for systemic vascular AT2Rs in the control of BP.53 However, recent studies have demonstrated a significant natriuretic response to AT2R activation within the kidney and indicate that AT2Rs likely control BP largely by regulating renal Na1 excretion and volume.54-56 The endogenous intrarenal agonist for AT2R-induced natriuresis appears to be des-aspartyl1-Ang II (Ang III), the heptapeptide metabolite of Ang II through aminopeptidase A.57,58 In the kidney, Ang II does not invoke a natriuretic response, even at high doses, but Ang III induces a robust natriuresis. Ang III is 30-fold more selective for AT2Rs over AT1Rs, consistent with a role of endogenous Ang III through AT2R activation.57,58 Employing specific blockers of APA and the major Ang III-metabolizing enzyme aminopeptidase N, recent studies have shown that Ang II must be converted to Ang III within the kidney to induce a natriuretic response.57,58 Ang III activates AT2Rs largely in proximal tubules by increasing the renal production of bradykinin, NO, and cyclic GMP, and AT2R activation is coupled to translocation of the receptors from intracellular sites to apical plasma membranes. Novel AT2R agonists are now available, including single b-amino acid-substituted Ang II and Ang III that are characterized by dramatically increased selectivity for AT2Rs

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over AT1Rs.59,60 One of these, compound 21 (C-21), is the first available nonpeptide agonist with approximately 25,000-fold selectivity for AT2Rs over AT1Rs.60 C-21 induces natriuresis in a renal AT2R-dependent manner in male and female Na1-loaded and volume-expanded normal Sprague-Dawley rats.61 In Na1-loaded rats, this effect is enhanced by concurrent systemic AT1R blockade. Similar to Ang III-induced natriuresis, C-21-induced natriuresis is because of proximal tubular inhibition of Na1 reabsorption in a bradykinin-, NO-, and cyclic GMP-dependent manner. C-21 induces natriuresis chronically in WT mice, and this effect does not occur in AT2R knockout mice consuming an identical amount of Na1. AT2R-induced natriuresis is accompanied by internalization and inhibition of the 2 major proximal tubule Na1 transporters, apical membrane Na1-hydrogen exchanger-3 and basolateral membrane Na1/K1ATPase, the latter by phosphorylation of signaling molecules, Src-family kinase (Src) and extracellular signal-related kinase (ERK).61 Renal AT2R expression is markedly increased by low Na1 intake, and AT2Rs control BP especially in situations when the RAS is activated and/or AT1Rs are blocked. Intrarenal AT2R activation by C-21 markedly inhibits the longterm BP rise in response to systemic Ang II infusion in the rat.61 Although Ang II infusion induced Na1 reabsorption on the first infusion day, this response was blocked with concurrent intrarenal C-21 administration, which also markedly inhibited the pressor response to Ang II not only on the first day but for 7 days of infusion. These findings, taken together, suggest that AT2R activation is a new therapeutic target for HT and states of fluid retention in humans. ACE-2/Ang (1-7)/Mas Receptor Pathway One of the major counter-regulatory arms of the RAS, which generally opposes the actions of Ang II through AT1Rs, is the ACE-2/Ang (1-7)/Mas receptor pathway.5,62,63 The most important step is the cleavage of phenylalanine from Ang II by the homolog of ACE, ACE-2.64 Ang (1-7) activates the Mas receptor, inducing phosphatidylinositol 3-kinases/Akt pathway, leading to activation of endothelial NO synthase.65-67 The consequent release of NO induces vasodilation. Ang (1-7) may also directly inhibit pathways, such as cSrc and ERK, that are stimulated by Ang II.62,63 The kidney is one of the most important organs in which Ang (1-7) is generated from the metabolism of Ang II by ACE-2, and the proximal tubule exhibits a high level of ACE-2 activity.62,68 Ang (1-7) is present in the proximal tubule but can be rapidly hydrolyzed to smaller inactive peptides by ACE and neprilysin.62,68 The exact biologic role of Ang (1-7) in the kidney is still a matter of debate. However, both renal hemodynamic and tubular effects have been demonstrated. Acute intravenous infusion of Ang (1-7) induces renal vasodilation, natriuresis, and diuresis.69 The diuretic and natriuretic effects of Ang (1-7) are at least partially because of renal vasodilation but may also be because of inhibition of Na1 and water reabsorption along the nephron. Studies in which Ang (1-7) was infused interstitially within the

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kidney have not been able to demonstrate a natriuretic action, even when intrarenal Ang (1-7) concentrations were augmented by ACE inhibition.58 However, other studies demonstrate that intrarenal arterial administration of Ang (1-7) increases Na1 and water excretion, because of inhibition of Na1 reabsorption in the proximal tubule.62,66,70 This effect appears to be dependent on the Na1 balance state as Ang (1-7) administration increased GFR, Na1, and water excretion in animals with low Na1 intake compared with those on a high Na1 diet.69 Ang (1-7) demonstrates biphasic effects on Na1-hydrogen exchanger-3 and may inhibit Na1/ K1ATPase, but this effect may also be mediated through AT2Rs.71-73 Molecular overexpression of Ang (1-7) or ACE-2 did not alter BP, GFR, or urine Na1 excretion.74,75 In addition, Mas receptor knockout did not produce any significant change in BP, although there was a reduction in renal blood flow.76,77 However, salt sensitivity of BP was significantly increased in knockout mice.77,78 Taken together, the evidence for substantial intrarenal effects of the ACE-2/Ang (1-7)/Mas receptor pathway is controversial, and more research clearly needs to be performed in this important area. (Pro)renin Receptor/MAP Kinase Pathway Another emerging pathway of the RAS is the prorenin/ renin/MAP kinase pathway.5 Before the cloning of the PRR, renin was considered simply as an enzyme catalyzing the conversion of Agt into Ang I as the ratelimiting initial step in the RAS peptide cascade and prorenin was considered an inactive precursor. Now we know that prorenin and/or active renin can bind directly to PRR, a 350 amino acid single transmembrane receptor, increasing the catalytic conversion of Agt by at least 4-fold.79,80 PRR is expressed in glomerular mesangial cells and the subendothelium of renal arteries and the apical membrane of intercalated cells in the collecting ducts.79,80 Activation of PRR directly by prorenin or renin activates the ERK 1/2 axis to increase vacuolar H1-ATPase (VATPase) activity.81 V-ATPases are ATP-dependent proton pumps that acidify intracellular compartments, including lysosomes, endosomes, and synaptic vesicles. Activation of PRR also increases plasminogen activator inhibitor-1 and collagen formation and potentially contributes to tissue fibrosis through generation of TGF-b.82,83 The major potential significance of PRR may lie in its increased activation by high levels of prorenin or renin, for example, during renin or ACE inhibition or Ang receptor blocker administration, because of inhibition of the negative feedback inhibition of renin release at juxtaglomerular cells. However, the specific roles of this pathway in the physiological regulation of cardiovascular and renal function and BP remain to be confirmed. In particular, availability of cell- and tissue-specific inducible prorenin/ renin receptor knockout animals and/or antagonists of prorenin/renin binding to the prorenin receptor without interfering with V-ATPases are required to separate the direct effects of prorenin/renin from effects mediated by V-ATPase.81

Other Pathways An additional new pathway of the RAS is the alamandine/ Mas receptor G protein-coupled receptor (MrgD) pathway.9 Alamandine is an Ang peptide that differs from Ang (1-7) by the presence of an N-terminal alanine (Ala1-Ang (1-7)).84 Alamandine can be synthesized from perfused Ang (1-7), but the enzyme responsible for endogenous alamandine formation remains unknown.84 Alamandine induces NO-dependent vasorelaxation and has been demonstrated to have antifibrotic activity.84 Current evidence indicates that alamandine exerts its biologic actions through its own receptor, the MrgD.85 The role of alamandine and the MrgD in the intrarenal RAS is currently unknown. In addition, Ang (1-9), which is generated from Ang I by the action of ACE-2, may counter-regulate the activity of the classic RAS.9 SUMMARY AND CONCLUSIONS Our understanding of the RAS has been transformed from that of a simple circulating/endocrine system with the classical Ang II/AT1R pathway to a complex paracrine, autacrine, and intracrine system with multiple new enzymes, peptides, receptors, and intracellular targets, including the (pro)renin/PRR/MAP kinase pathway and the AT1Rcounterregulatory Ang III/AT2R, ACE-2/Ang (1-7)/Mas receptor, alamandine/MrgD pathways, and newly discovered nuclear and mitochondrial systems. The intrarenal RAS, functioning as an independent tissue system, is clearly important in the regulation of BP and renal function in experimental animals, and this concept is highly likely to apply also to humans. Overactivation of the RAS leads to increased Ang II levels, which unlike the systemic RAS can lead to autoamplification of Ang II production. AT1R-dependent Ang II cellular internalization and upregulated renin synthesized in the distal nephron may provide a sufficient source of continuing intrarenal Ang II formation to maintain vasoconstriction. The precise role of the new counter-regulatory pathways in dampening the intrarenal effects of Ang II through the AT1R are currently being investigated and may provide new therapeutic targets for the treatment of HT in the future. Independent nuclear and/or mitochondrial systems may also regulate renal function and contribute to the pathogenesis of HT. REFERENCES 1. James PA, Oparil S, Carter BL, et al. 2014 Evidence-based guideline for the management of high blood pressure in adults: report from the panel members appointed to the Eighth Joint National Committee (JNC8). JAMA. 2014;311(5):507-520. 2. Chobanian AV, Bakris GL, Black HR, et al. Seventh report of the Joint National Committee on prevention, detection, evaluation and treatment of high blood pressure. Hypertension. 2003;42(6): 1206-1252. 3. Weber MA, Schiffrin EL, White WB, et al. Clinical practice guidelines for the management of hypertension in the community. A statement by the American Society of Hypertension and the International Society of Hypertension. J Clin Hypertens. 2014;16(1):14-26. 4. Funder JW, Carey RM, Fardella C, et al. Case detection, diagnosis and treatment of patients with primary aldosteronism: an Endocrine

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5.

6.

7.

8.

9. 10.

11.

12.

13.

14.

15.

16.

17.

18.

19.

20.

21.

22.

23. 24.

25.

Society Clinical Practice Guideline. J Clin Endocrinol Metab. 2008;93(9):3266-3281. Siragy HM, Carey RM. Newly recognized components of the reninangiotensin system: potential roles in cardiovascular and renal regulation. Endocr Rev. 2003;24(3):261-271. Carey RM. Cardiovascular and renal regulation by the angiotensin type-2 receptor: the AT2 receptor comes of age. Hypertension. 2005;45(5):840-844. Jones ES, Vinh A, McCarthy CA, Gaspari TA, Widdop RE. AT2 receptors: functional relevance in cardiovascular disease. Pharmacol Ther. 2008;120(3):292-316. Siragy HM, Xue C, Abadir P, Carey RM. Angiotensin subtype-2 receptors inhibit renin biosynthesis and angiotensin II formation. Hypertension. 2005;45(1):133-137. Carey RM. Newly discovered components and actions of the reninangiotensin system. Hypertension. 2013;62(5):818-822. Kimbrough HM, Vaughan ED Jr, Carey RM, Ayers CR. Effect of intrarenal angiotensin II blockade on renal function in conscious dogs. Circ Res. 1977;40(2):174-178. Levens NR, Freedlender AE, Peach MJ, Carey RM. Control of renal function by intrarenal angiotensin II. Endocrinology. 1983;112(1):43-49. Levens NR, Peach MJ, Carey RM. Role of the intrarenal reninangiotensin system in the control of renal function. Circ Res. 1981;48(2):157-167. Navar LG, Lewis L, Hymel A, Braam B, Mitchell KD. Tubular fluid concentrations and kidney contents of angiotensins I and II in anesthetized rats. J Am Soc Nephrol. 1994;5(4):1153-1158. Siragy HM, Howell NL, Peach MJ, Carey RM. Renal interstitial fluid angiotensin. Modulation by anesthesia, epinephrine, sodium depletion and renin inhibition. Hypertension. 1995;25(5):1021-1024. Davisson RL, Ding Y, Stee DE, Caterall JP, Sigmund CD. Novel mechanism of hypertension revealed by cell-specific targeting of human angiotensinogen in transgenic mice. Physiol Genomics. 1999;1(1):3-9. Lavoie JL, Lake-Bruse KD, Sigmund CD. Increased blood pressure in transgenic mice expressing both human renin and angiotensinogen in the proximal tubule. Am J Physiol Renal Physiol. 2004;286(5):F965-F971. Crowley SD, Gurley SB, Oliverio MI, et al. Distinct roles for the kidney and systemic tissues in blood pressure regulation by the renin-angiotensin system. J Clin Invest. 2005;115(4):1092-1099. Crowley SD, Gurley SB, Herrera MJ, et al. Angiotensin II causes hypertension and cardiac hypertrophy through its receptors in the kidney. Proc Natl Acad Sci U S A. 2006;103(47): 17985-17990. Gurley SB, Riquier-Brison AD, Schnerman J, et al. AT1A angiotensin receptors in the renal proximal tubule regulate blood pressure. Cell Metab. 2011;13(4):469-475. Li XC, Cook JL, Rubera I, Tauc M, Zhang F, Zhuo JL. Intrarenal transfer of an intracellular cyan fluorescent fusion of angiotensin II selectively in proximal tubules increases blood pressure in rats and mice. Am J Physiol Renal Physiol. 2011;300(5):F1076-F1088. Li XC, Zhuo JL. Proximal tubule-dominant transfer of AT1a receptors induces blood pressure responses to intracellular angiotensin II in AT1a receptor-deficient mice. Am J Physiol Regul Integr Comp Physiol. 2013;304(8):R588-R598. Navar LG, Harrison-Bernard LM, Nishiyama A, Kobori H. Regulation of intrarenal angiotensin II in hypertension. Hypertension. 2002;39(8):316-322. Navar LG, Kobori H, Prieto-Carrasquero M. Intrarenal angiotensin II and hypertension. Curr Hypertens Rep. 2003;5(2):135-143. Navar LG. Translational studies on augmentation of intratubular renin-angiotensin system in hypertension. Kidney Int Suppl. 2013;3(4):321-325. Trippodo NC, Frohlich ED. Similarities of genetic (spontaneous) hypertension: man and rat. Circ Res. 1981;48(3):309-319.

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26. Biollaz J, Waeber B, Diezi J, Burnier M, Brunner HR. Lithium infusion to study sodium handling in unanesthetized hypertensive rats. Hypertension. 1986;8(2):117-121. 27. Beierwaltes WH, Arendshorst WJ, Klemmer PJ. Electrolyte and water balance in young spontaneously hypertensive rats. Hypertension. 1982;4(6):908-915. 28. Guyton AC, Coleman TG, Young DB, Lohmeier TE, DeClue JW. Salt balance and long-term blood pressure control. Annu Rev Med. 1980;31(2):15-27. 29. Arendshorst WJ, Beierwaltes WH. Renal tubular reabsorption in spontaneously hypertensive rats. Am J Physiol Renal Physiol. 1979;237(1):F38-F47. 30. Omvik P, Tarazi RC, Brave EL. Regulation of sodium balance in hypertension. Hypertension. 1908;2(4):515-523. 31. Meng QC, Durand J, Chen Y-F, Oparil S. Effects of dietary salt on angiotensin peptides in the kidney. J Am Soc Nephrol. 1995;6(4):1209-1215. 32. Cheng H-F, Wang J-L, Vinson GP, Harris RC. Young SHR express increased type 1 angiotensin II receptors in renal proximal tubule. Am J Physiol Renal Physiol. 1998;274(1 pt 2):F10-F17. 33. Yoneda M, Sanada H, Yatabe J, et al. Differential effects of angiotensin II type 1 receptor antisense oligonucleotides on renal function in spontaneously hypertensive rats. Hypertension. 2005; 46(1):58-65. 34. Kobori H, Ozawa Y, Suzuke Y, Nishiyama A. Enhanced intrarenal angiotensinogen contributes to early renal injury in spontaneously hypertensive rats. J Am Soc Nephrol. 2005;16(7):2073-2080. 35. Landgraf SS, Wengert M, Silva JS, et al. Changes in angiotensin receptor expression play a pivotal role in the renal damage observed in spontaneously hypertensive rats. Am J Physiol Renal Physiol. 2011;300(2):F499-F510. 36. Lee H-A, Cho H-M, Lee D-Y, Kim K-C, Han HS, Kim IK. Tissue-specific upregulation of angiotensin converting enzyme 1 in spontaneously hypertensive rats through histone code modifications. Hypertension. 2012;59(3):621-626. 37. Kobori H, Harrison-Bernard LM, Navar LG. Enhancement of angiotensinogen expression in angiotensin II-dependent hypertension. Hypertension. 2001;37(5):1329-1335. 38. Kobori H, Nangaku M, Navar LG, Nishiyama A. The intrarenal renin-angiotensin system: from physiology to pathobiology of hypertension and kidney disease. Pharmacol Rev. 2007;59(3):251-287. 39. Navar LG, Kobori H, Prieto MC, Gonzalez-Villalobos RA. Intratubular renin-angiotensin system in hypertension. Hypertension. 2011;57(3):355-362. 40. Prieto-Carrasquero MC, Kobori H, Ozawa Y, Guiterez A, Seth D, Navar LG. AT1 receptor-mediated enhancement of collecting duct renin in angiotensin II-dependenthypertensive rats. Am J Physiol Renal Physiol. 2005;289(3):F632-F637. 41. Prieto-Carrasquero MC, Williams DE, Liu L, Kavanaugh KI, Mullins JJ, Mitchell KD. Enhancement of renin and prorenin receptor in collecting duct of Cyp1a1-Ren 2 rats may contribute to development and progression of malignant hypertension. Am J Physiol Renl Physiol. 2011;300(2):F581-F588. 42. Zhuo JL, Imig JD, Hammond TG, Orengo S, Benes E, Navar LG. Ang II accumulation in rat renal endosomes during Ang IIinduced hypertension: role of AT1 receptor. Hypertension. 2002; 39(1):116-121. 43. Zou LX, Hymel A, Imig JD, Navar LG. Renal accumulation of circulating angiotensin II in angiotensin II-infused rats. Hypertension. 1996;27(3 pt 2):658-662. 44. Zou LX, Imig JD, Hymel A, Navar LG. Renal uptake of circulating angiotensin II in Val5-angiotensin II-infused rats is mediated by AT1 receptor. Am J Hypertens. 1998;11(5):570-578. 45. Li XC, Navar LG, Shao Y, Zhuo JL. Genetic deletion of AT1a receptors attenuates intracellular accumulation of angiotensin II in the kidney of AT1a receptor-deficient mice. Am J Physiol Renal Physiol. 2007;293(2):F586-F593.

210

Carey

46. Matsusaka T, Niimura F, Shimizu A, et al. Liver angiotensinogen is the primary source of renal angiotensin II. J Am Soc Nephrol. 2012;23(7):1181-1189. 47. Matsusaka R, Niimura F, Pastan I, Shintani A, Nishiyama A, Ichikawa I. Podocyte injury enhances filtration of liver-derived angiotensinogen and renal angiotensin II generation. Kidney Int. 2014;85(5):1068-1077. 48. Abadir PM, Walston JD, Carey RM. Subcellular characteristics of functional intracellular renin-angiotenisn systems. Peptides. 2012;38(2):437-445. 49. Gwathmey TM, Alzayadneh EM, Pendergrass KD, Chappell MC. Novel roles of nuclear angiotensin receptors and signaling mechanisms. Am J Physiol Regul Integr Comp Physiol. 2012;302(5):R518-R530. 50. Abadir PM, Foster DB, Crow M, et al. Identification and characterization of a functional mitochondrial angiotensin system. Proc Natl Acad Sci U S A. 2011;108(36):14849-14854. 51. Li XC, Zhuo JL. Proximal tubule-dominant transfer of AT(1a) receptors induces blood pressure responses to intracellular angiotensin II in AT(1a) receptor-deficient mice. Am J Physiol Regul Integr Comp Physiol. 2013;304(8):R588-98. 52. Miyata N, Park F, Li XF, Cowley AW Jr. Distribution of angiotensin AT1 and AT2 receptor subtypes in the rat kidney. Am J Physiol. 1999;277(3 pt 2):F437-F446. 53. Danyel LA, Schmerier P, Paulis L, Unger T, Steckelings UM. Impact of AT2 receptor stimulation on vascular biology, kidney function and blood pressure. Integr Blood Press Control. 2013;6(6):153-161. 54. Padia SH, Carey RM. AT2 receptors: beneficial counter-regulatory role in natriuresis: intrarenal mechanisms and therapeutic potential. Pflugers Arch. 2013;120(1):476-479. 55. Carey RM, Padia SH. Role of AT2 receptors in natriuresis: intrarenal mechanisms and therapeutic potential. Clin Exp Pharmacol Physiol. 2013;40(8):527-534. 56. Carey RM. The intrarenal renin-angiotensin and dopaminergic systems: control of renal sodium excretion and blood pressure. Hypertension. 2013;61(3):673-680. 57. Padia SH, Kemp BA, Howell NL, Fournie-Zaluski M-C, Roques BP, Carey RM. Conversion of renal angiotensin II to angiotensin III is critical for AT2 receptor-nediated natriuresis in rats. Hypertension. 2008;51(2):460-465. 58. Kemp BA, Bell JF, Rottkamp DM, et al. Intrarenal angiotensin III is the predominant agonist for proximal tubule angiotensin type-2 receptors. Hypertension. 2012;60(2):387-395. 59. Jones ES, Del Borgo MP, Kirsch JF, et al. A single b-amino acid substitution to angiotensin II confers AT2 receptor selectivity and vascular function. Hypertension. 2011;57(3):570-576. 60. Bosnyak S, Jones ES, Christopolous A, Aguilar M-I, Thomas WG, Widdop RE. Relative affinity of angiotensin peptides and novel ligands at AT1 and AT2 receptors. Clin Sci. 2011;121(7):297-303. 61. Kemp BA, Howell NL, Gildea JJ, Keller SR, Padia SH, Carey RM. AT2 receptor activation induces natriuresis and lowers blood pressure. Circ Res. 2014;115(3):388-399. 62. Chappell MC. Emerging evidence for a functional angiotensinconverting enzyme 2-angiotensin (1-7)-MAS receptor axis: more than regulation of blood pressure. Hypertension. 2007;50(4):596-599. 63. Ferrario CM. Angiotensin-converting enzyme 2 and angiotensin (17): an evolving story in cardiovascular regulation. Hypertension. 2006;47(3):515-521. 64. Vickers C, Hales P, Kaushik V, et al. Hydrolysis of biological peptides by human angiotensin-converting enzyme-related carboxypeptidase. J Biol Chem. 2002;277(17):14838-14843. 65. Santos RA, Simoes E, Silva AC, et al. Angiotensin (1-7) is an endogenous ligand for the G protein-coupled receptor MAS. Proc Natl Acad Sci U S A. 2003;100(14):8258-8263. 66. Ferrario CM, Varagic J. The Ang-(1-7)/ACE-2/Mas axis in the regulation of nephron function. Am J Physiol Renal Physiol. 2010;298(6): F1297-F1305.

67. Chappell MC. Nonclassical renin-angiotensin system and renal function. Compr Physiol. 2012;2(4):2733-2752. 68. Shaltout HA, Westwood BM, Averill DB, et al. Angiotensin metabolism in renal proximal tubules, urine and serum of sheep: evidence for ACE 2-dependent processing of angiotensin II. Am J Physiol Renal Physiol. 2007;292(1):F82-F91. 69. O’Neill J, Corbett A, Johns EJ. Dietary sodium intake modulates renal excretory responses to intrarenal angiotensin (1-7) administration in anesthetized rats. Am J Physiol Regul Integr Comp Physiol. 2013;304(3): R260-R266. 70. Ferrario CM, Chappell MC, Tallant EA, Brosnihan KB, Diz DI. Counterregulatory actions of angiotensin (1-7). Hypertension. 1997;30(3 Pt 2):535-541. 71. De Souza AM, Lopes AG, Pizzino CP, et al. Angiotensin II and angiotensin (1-7) inhibit the inner cortex Na1-ATPase activity through AT2 receptor. Regul Pept. 2004;120(1-3):167-175. 72. Lara LS, Cavaleante F, Axelband F, DeSouza AM, Lopes AG, Caruso-Deves C. Involvement of the Gi/o/cGMP/PKG pathway in the AT2-mediated inhibition of outer cortex proximal tubule Na1ATPase by Ang (1-7). Biochem J. 2006;395(1):183-190. 73. Castelo-Branco RC, Leite-Delova DC, deMello-Aires M. Dosedependent effects of angiotensin (1-7) on the NHE3 exchanger and [Ca(210](i) in in vivo proximal tubules. Am J Physiol Renal Physiol. 2013;304(10):F1258-F1265. 74. Botelho-Santos GA, Sampaio WO, Reudelhuber TL, Bader M, Campagnole-Santos MR, Santos RA. Exression of an amgoptmesom-(17)-producing fusion protein in rats induced marked changes in regional vascular resistance. Am J Physiol Heart Circ Physiol. 2007;292(5):H2485-H2490. 75. Santos RA, Ferreira AJ, Nadu AP, et al. Expression of an angiotensin-(1-7) fusion protein produces cardioprotective effects in rats. Physiol Genomics. 2004;17(3):292-299. 76. Botelho-Santos GA, Bader M, Alenina N, Santos RA. Altered regional blood flow distribution in Mas-deficient mice. Ther Adv Cardiovasc Dis. 2013;6(5):201-211. 77. Walther T, Wessel N, Kang N, et al. Altered heart rate and blood pressure variability in mice lacking the Mas protooncogene. Braz J Med Biol Res. 2000;33(1):1-9. 78. Heringer-Walther S, Gembardt R, Perschel FH, Katz N, Schulthsiss HP, Walther T. The genetic deletion of Mas abolishes salt induced hypertension in mice. Eur J Pharmacol. 2012;689(13):147-153. 79. Nguyen F, Delarue R, Burckle C, Bouzhir L, Giller T, Sraer JD. Pivotal role of the renin/prorenin receptor in angiotensin II production and cellular responses to renin. J Clin Invest. 2002;109(11): 1417-1427. 80. Nguyen G, Delarue F, Berrou J, Rondeau E, Sraer JD. Specific receptor binding of renin on human mesangial cells in culture increases plasminogen activator inhibitor-1 antigen. Kidney Int. 1996;50(6): 1897-1903. 81. Advani A, Kelly DJ, Cox AJ, et al. The (Pro) renin receptor: sitespecific and functional linkage to the vacuolar H1-ATPase in the kidney. Hypertension. 2009;54(2):261-269. 82. Huang Y, Novle NA, Zhang J, Xu C, Border WA. Renin-stimulated TGF-beta 1 expression is regulated by a mitogen-activated protein kinase in mesangial cells. Kidney Int. 2007;72(1):45-52. 83. Zhang J, Wu J, Gu C, Noble NA, Border WA, Huang Y. Receptormediated nonproteolytic activation of prorenin and induction of TGF-b1 and PAI-1 expression in renal mesangial cells. Am J Physiol Renal Physiol. 2012;303(1):F11-F20. 84. Lautner RQ, Villela DC, Fraga-Silva RA, et al. Discovery and characterization of alamandine: a novel component of the reninangiotensin system. Circ Res. 2013;112(8):1104-1111. 85. Gambardt F, Grajewske S, Vahl M, Schultheiss HP, Walther R. Angiotensin metabolites can stimulate receptors of the Mas-related genes family. Mol Cell Biochem. 2008;319(1-2):115-123.