Abstract-Whereas angiotensin (Ang) II is the major effector peptide of the renin-angiotensin system, its metabolite, des-aspartyl 1 -Ang II (Ang III), may also have biologic activity. We investigated the effects of renal interstitial (RI) administration of candesartan (CAND), a specific Ang II type 1 receptor (AT 1 ) blocker, with and without coinfusion of PD-123319 (PD), a specific Ang II type 2 receptor (AT 2 ) blocker, on Na ϩ excretion (U Na V) in uninephrectomized rats. We also studied the effects of unilateral RI infusion of Ang II or Ang III on U Na V with and without systemic infusion of CAND with the noninfused kidney as control. In rats receiving normal Na ϩ intake, RI CAND increased U Na V from 0.07Ϯ0.08 to 0.82Ϯ0.17 mol/min (PϽ0.01); this response was abolished by PD. During Na ϩ restriction, CAND increased U Na V from 0.06Ϯ0.02 to 0.1Ϯ0.02 mol/min (PϽ0.05); this response also was blocked by PD. In rats with both kidneys intact, in the absence of CAND, unilateral RI infusion of Ang III did not significantly alter U Na V. However, with systemic CAND infusion, RI Ang III increased U Na V from 0.08Ϯ0.01 mol/min to 0.18Ϯ0.04 mol/min (PϽ0.01) at 3.5 nmol/kg per minute, and U Na V remained elevated throughout the infusion; this response was abolished by PD. However, RI infusion of Ang II did not significantly alter U Na V at any infusion rate (3.5 to 80 nmol/kg per minute) with or without systemic CAND infusion. These results suggest that intrarenal AT 1 receptor blockade engenders natriuresis by activation of AT 2 receptors. AT 2 receptor activation via Ang III, but not via Ang II, mediates the natriuretic response in the presence of systemic AT 1 receptor blockade. Key Words: angiotensin Ⅲ sodium Ⅲ natriuresis Ⅲ angiotensin Ⅲ receptors, angiotensin II A ngiotensin (Ang) II, the primary transducer peptide of the renin-Ang system (RAS), acts at 2 major Ang II receptors, type 1 (AT 1 ) and type 2 (AT 2 ). 1 The majority of Ang II actions are believed to occur via the AT 1 receptor, including antinatriuresis. 2 The role of the AT 2 receptor is less clearly understood. 3 Furthermore, whereas Ang II has been considered the major effector peptide of the RAS, its direct metabolite, des-aspartyl 1 -Ang II (Ang III) also has biologic activity. 4 Indeed, some actions originally attributable to Ang II, such as vasopressin release, are mediated at least in part by Ang III. 5 Recent reports suggest that Ang III could be involved in renal physiological processes as well. 6 -8 Studies involving AT 2 receptors and the regulation of sodium (Na ϩ ) excretion have been limited. In vitro studies have demonstrated that the AT 2 receptor may decrease renal proximal tubule bicarbonate reabsorption via phospholipase A 2 and arachidonic acid release. 9 In vivo studies in mice lacking the AT 2 receptor (AT 2 -null), have demonstrated a shift to the right (less sensitive) in the pressure-natriuresis curve. 10 In addition, antinatriuretic and pressor hypersensitivity to Ang II has been documented in AT 2 -null mice. 11 However, th...

Rationale Compound 21 (C-21) is a highly selective non-peptide AT2 receptor (AT2R) agonist. Objective To test the hypothesis that renal proximal tubule AT2Rs induce natriuresis and lower blood pressure (BP) in Sprague-Dawley rats and mice. Methods and Results In rats, AT2R activation with intravenous C-21 increased urinary sodium (Na+) excretion (UNaV) by 10-fold (P<0.0001); this natriuresis was abolished by direct renal interstitial (RI) infusion of specific AT2R antagonist PD-123319 (PD). C-21 increased fractional excretion of Na+ (FENa; P<0.05) and lithium (FELi; P<0.01) without altering renal hemodynamic function. AT2R activation increased renal proximal tubule cell (RPTC) apical membrane AT2R protein (P<0.001) without changing total AT2R expression and internalized/inactivated Na+- H+ exchanger-3 (NHE-3) and Na+/K+ATPase (NKA). C-21-induced natriuresis was accompanied by an increase in RI cyclic GMP (cGMP; P<0.01); C-21-induced increases in UNaV and RI cGMP were abolished by RI nitric oxide (NO) synthase inhibitor L-NAME or bradykinin (BK) B2 receptor antagonist icatibant. Renal AT2R activation with C-21 prevented Na+ retention and lowered BP in the angiotensin II (Ang II) infusion model of experimental hypertension. Conclusions AT2R activation initiates its translocation to the RPTC apical membrane and the internalization of NHE-3 and NKA inducing natriuresis in a BK-NO-cGMP-dependent manner. Intrarenal AT2R activation prevents Na+ retention and lowers BP in Ang II-dependent hypertension. AT2R activation holds promise as a RPT natriuretic/diuretic target for the treatment of fluid retaining states and hypertension.

The renin–angiotensin system (RAS) is a coordinated hormonal cascade intimately involved in cardiovascular and renal control and blood pressure regulation. Angiotensin II (Ang II), the major RAS effector peptide, binds two distinct receptors, the angiotensin type-1 receptor (AT1R) and the angiotensin type-2 (AT2R) receptor. The vast majority of the physiological actions of Ang II, almost all of them detrimental, are mediated by AT1Rs. In contrast, AT2Rs negatively modulate the actions of AT1Rs under the majority of circumstances and generally posess beneficial effects. AT2Rs induce vasodilation in both resistance and capacitance vessels, mediate natriuresis directly and via interactions with dopamine D1 receptors in the renal proximal tubule. AT2Rs inhibit renin biosynthesis and secretion and protect the kidneys from inflammation and ischemic injury. Our understanding of the exact role of AT2Rs in physiology and pathophysiology continues to expand; the purpose of this review is to provide an up-to- date summary of the functional role of AT2Rs at the organ, tissue, cellular and subcellular levels with emphasis on the vascular and renal actions that bear on blood pressure regulation and hypertension.

In AT1 receptor (AT1R)-blocked rats, renal interstitial (RI) administration of des-aspartyl1-angiotensin II (Ang III), but not angiotensin II (Ang II), induces natriuresis via activation of angiotensin type-2 receptors (AT2R). In the present study, renal function was documented during systemic AT1R blockade with candesartan in Sprague-Dawley rats receiving unilateral RI infusion of Ang III. Ang III increased urine sodium excretion (UNaV), fractional excretion of sodium (FENa), and fractional excretion of lithium (FELi). RI co-infusion of specific AT2R antagonist PD-123319 (PD) abolished Ang III-induced natriuresis. The natriuretic response observed with RI Ang III was not reproducible with RI Ang (1–7) alone or together with angiotensin converting enzyme (ACE) inhibition. Similarly, neither RI Ang II alone nor in the presence of aminopeptidase A (APA) inhibitor to prevent degradation increased UNaV. In the absence of systemic AT1R blockade, Ang III alone did not increase UNaV, but natriuresis was enabled by the co-infusion of aminopeptidase N (APN) inhibitor and subsequently blocked by PD. In AT1R-blocked rats, RI administration of APN inhibitor alone also induced natriuresis that was abolished by PD. Ang III-induced natriuresis was accompanied by increased RI cyclic GMP levels and was abolished by inhibition of soluble guanylyl cyclase. RI and renal tissue Ang III levels increased in response to Ang III infusion and were augmented by APN inhibition. These data demonstrate that endogenous intrarenal Ang III, but not Ang II or Ang (1–7), induces natriuresis via activation of AT2Rs in the proximal tubule via a cyclic GMP-dependent mechanism and suggest APN inhibition as a potential therapeutic target in hypertension.

Abstract-In the kidney, angiotensin II (Ang II) is metabolized to angiotensin III (Ang III) by aminopeptidase A (APA).In turn, Ang III is metabolized to angiotensin IV by aminopeptidase N (APN T he intrarenal renin-angiotensin system (RAS) is separate from the peripheral RAS and contributes independently to the regulation of blood pressure and sodium excretion. [1][2][3][4] All of the precursors of angiotensin peptide synthesis are located within the renal proximal tubule (RPT), including angiotensinogen, renin, and angiotensin converting-enzyme mRNA. [5][6][7][8] Interstitial fluid concentrations of angiotensin II (Ang II) and angiotensin III (Ang III) are far greater (nanomolar) than can be explained solely on the basis of equilibration with circulating concentrations (femtomolar). 9,10 These findings suggest that important influences are exerted by locally generated angiotensins. Additionally, the 2 major receptor subtypes that mediate actions of RAS, the angiotensin type-1 receptor (AT 1 R) and the angiotensin type-2 receptor (AT 2 R), are both present in RPT cells, consistent with a primary role for tubular angiotensins as paracrine substances in the control of renal function. 11,12 The enzymes responsible for the metabolism of Ang II are also highly expressed in the kidney. Aminopeptidase A (APA), a membrane-bound zinc-dependent aminopeptidase, is distributed at the surface of glomerular endothelial and mesangial cells and podocytes and all along the nephron, with highest expression in the RPT. 13,14 As shown in Figure 1, APA preferentially cleaves the N-terminal acidic amino acid (aspartate) from Ang II to generate Ang III, which serves as the rate-limiting step in Ang II metabolism. 15 Aminopeptidase N (APN) is also a membrane-bound, zinc-dependent aminopeptidase and is a major constituent of RPT cell brush border membranes. 16,17 The metabolism of Ang III to Ang IV is mediated by APN, which preferentially releases neutral amino acids from the N-terminal end of oligopeptides. 18 Specific pharmacological inhibitors of APA and APN have been used to elucidate the role of Ang II and Ang III within local tissue RAS. Selective APA inhibitor, EC-33 (3-amino-4-thio-butyl-sulfonic acid, inhibition constant K i ϭ0

Abstract-The renal angiotensin angiotensin type 2 receptor has been shown to mediate natriuresis, and angiotensin III, not angiotensin II, may be the preferential angiotensin type 2 receptor activator of this response. Angiotensin III is metabolized to angiotensin IV by aminopeptidase N. The present study hypothesizes that inhibition of aminopeptidase N will augment natriuretic responses to intrarenal angiotensin III in angiotension type 1 receptor-blocked rats. Rats received systemic candesartan for 24 hours before the experiment. After a 1-hour control, cumulative renal interstitial infusion of angiotensin III at 3.5, 7, 14, and 28 nmol/kg per minute (each dose for 30 minutes) or angiotensin III combined with aminopeptidase N inhibitor PC-18 was administered into 1 kidney.

Renal dopamine D1-like receptors (D1R) and angiotensin type-2 receptors (AT2R) are important natriuretic receptors counterbalancing angiotensin type-1 receptor-mediated tubular sodium (Na+) reabsorption. Here we explore the mechanisms of D1R and AT2R interaction in natriuresis. In uninephrectomized, Na+-loaded Sprague-Dawley rats, direct renal interstitial (RI) infusion of highly selective D1R agonist fenoldopam (FEN) induced a natriuretic response that was abolished by AT2R specific antagonist PD-123319 (PD) or by microtubule polymerization inhibitor nocodazole (NOC) but not by actin polymerization inhibitor cytochalasin D. By confocal microscopy and immuno-electron microscopy, FEN translocated AT2Rs from intracellular sites to the apical plasma membranes (AM) of renal proximal tubule cells (RPTCs) and this translocation was abolished by NOC. Since D1R activation induces natriuresis via an adenylyl cyclase/cyclic adenosine monophosphate (cAMP) signaling pathway, we explored whether this pathway is responsible for AT2R recruitment and AT2R-mediated natriuresis. RI co-infusion of adenylyl cyclase activator forskolin (FSK) and 3-isobutly-1-methylxanthine (IBMX) induced natriuresis that was abolished either by PD or NOC but was unaffected by specific D1R antagonist SCH-23390 (SCH). Co-administration of FSK and IBMX also translocated AT2Rs to the AMs of RPTCs; this translocation was abolished by NOC but was unaffected by SCH. The results demonstrate that D1R-induced natriuresis requires AT2R recruitment to the AMs of RPTCs in a microtubule-dependent manner involving an adenylyl cyclase/cAMP signaling pathway. These studies provide novel insights regarding the mechanisms whereby renal D1Rs and AT2Rs act in concert to promote Na+ excretion in vivo.

Abstract-In Sprague-Dawley rats, renal angiotensin (Ang) type 2 receptors (AT 2 Rs) mediate natriuresis in response to renal interstitial (RI) D 1 -like receptor stimulation or RI Ang III infusion. After D 1 -like receptor activation, apical membrane (AM) but not total renal proximal tubule cell AT 2 R expression is increased, suggesting that AM AT 2 R translocation may be important for natriuresis. Key Words: sodium Ⅲ natriuresis Ⅲ angiotensin III Ⅲ AT 2 receptor Ⅲ AT 1 receptor Ⅲ translocation T he renin-angiotensin system is a coordinated hormonal cascade of crucial importance in cardiovascular and renal function. In recent years, an emphasis has been placed on delineating the role of the intrarenal renin-angiotensin system in the regulation of blood pressure (BP) 1,2 and sodium (Na ϩ ) balance. [3][4][5] The majority of the effects of the intrarenal reninangiotensin system are mediated by 2 angiotensin receptors, angiotensin (Ang) type 1 (AT 1 R) and Ang II type II (AT 2 R). Renal AT 1 Rs, as a consequence of their antinatriuretic actions, are required for the development of Ang II-dependent hypertension, because the presence of systemic extrarenal AT 1 Rs alone is not sufficient to sustain hypertensive responses to Ang II infusion. 1 Furthermore, AT 1 Rs in renal proximal tubule cells (RPTCs), as opposed to other sites along the nephron, are primarily responsible for this response. 6 In Sprague-Dawley rats, renal AT 2 Rs have been reported to mediate natriuresis in response to renal interstitial (RI) AT 1 R blockade or Ang III infusion. 3 Inhibition of the conversion of Ang II to Ang III in the kidney abolishes natriuresis mediated by renal AT 2 Rs, indicating that Ang III is the preferred agonist of this response. 4 Thus, RPTC AT 1 Rs and AT 2 Rs are major determinants of BP and Na ϩ responses in normal and hypertensive animals.The intrarenaldopaminergic system also plays an important role in the regulation of Na ϩ balance. Dopamine, synthesized by the RPTCs, mediates diuresis and natriuresis via D 1 -like receptor (D 1 R) activation. 7,8 A physiological interaction between the intrarenal renin-angiotensin system and dopaminergic systems has been reported in normal Sprague-Dawley rats. In response to a high-salt diet, RI D 1 R activation with fenoldapam results in natriuresis and diuresis that is abolished by selective pharmacological inhibition of renal AT 2 Rs with PD-123319 (PD). 9 Furthermore, fenoldapam-induced natriuresis is accompanied by an increase in apical plasma membrane (AM) but not total RPTC AT 2 R expression, as quantified by Western blot analysis. 9 Thus, D 1 R-mediated natriuresis depends on functional renal AT 2 Rs, and one of the mechanisms involves RPTC AT 2 R translocation.Spontaneously hypertensive rats (SHRs) develop hypertension as they age and are widely used as a model to study the development and maintenance of human primary (essential) hypertension. 10 Before the onset of hypertension, SHRs