Ghrelin-Induced Sodium Reabsorption Is Mediated by PKA and Microtubule-Dependent αENaC Translocation in Female Rats.

Intrarenal ghrelin infusion activates ghrelin receptors in the kidney collecting duct (CD) to increase α epithelial sodium (Na+) channel (αENaC)-dependent Na+ reabsorption in vivo, but the underlying mechanisms are unknown. Seventy-two hours following uninephrectomy, 12-week-old female Sprague-Dawley rats received the following renal interstitial (RI) infusions for 1 hour after a 1-hour control: vehicle (n = 10), ghrelin (3 µg/minute; n = 8), ghrelin + phosphatidylinositol 3-kinase (PI3K) inhibitor LY-294002 (0.1 µg/kg/minute; n = 7), ghrelin + protein kinase A (PKA) inhibitor adenosine 3'5'-cyclic monophosphorothioate, Rp-isomer (10 µg/kg/minute; n = 8), ghrelin + microtubule polymerization inhibitor nocodazole (0.3 µg/kg/minute; n = 7), or ghrelin + actin polymerization inhibitor cytochalasin D (0.3 µg/kg/minute; n = 6). Compared with vehicle infusion, RI ghrelin induced a significant anti-natriuresis (urine Na+ excretion was reduced by 53.7% ± 6.8%; P < 0.001). This effect was abolished during concomitant PKA or microtubule inhibition (106.4% ± 9.4% and 109.7% ± 10.6% of vehicle infusion, respectively; P < 0.01 from ghrelin) but not during concomitant PI3K or actin inhibition (reduced by 48.6% ± 3.9% and 52.8% ± 12.7%, respectively; P < 0.001 and P < 0.01 from vehicle, respectively; P = not significant from ghrelin). Infusions had no effect on mean arterial pressure. Western blot analysis demonstrated that CD membrane but not total αENaC expression increased in response to ghrelin infusion compared with vehicle, (0.39 ± 0.05 vs 0.12 ± 0.02 arbitrary units; P < 0.01). This effect was abolished during PKA or microtubule inhibition but persisted during PI3K or actin inhibition. Neural precursor cell expressed, developmentally down-regulated 4 isoform 2 (Nedd4-2) dependent internalization of αENaC was not affected by ghrelin, indicating that microtubule-dependent forward trafficking of αENaC is necessary for anti-natriuretic responses to ghrelin. Taken together, these studies highlight the importance of PKA and microtubule polymerization in ghrelin-induced αENaC-mediated Na+ reabsorption.

Intrarenal ghrelin receptor inhibition ameliorates angiotensin II-dependent hypertension in rats.

The intrarenal ghrelin receptor (GR) is localized to collecting duct (CD) cells, where it increases epithelial Na+ channel (αENaC)-dependent sodium reabsorption in rodents. We hypothesized that chronic GR inhibition with intrarenal GR siRNA lowers blood pressure (BP) in angiotensin II-dependent hypertension via reductions in αENaC-dependent sodium reabsorption. Uninephrectomized Sprague-Dawley rats ( n = 121) received subcutaneous osmotic pumps for chronic systemic delivery of angiotensin II or vehicle (5% dextrose in water). Rats also received intrarenal infusion of vehicle, GR siRNA, or scrambled (SCR) siRNA. In rats receiving intrarenal vehicle or intrarenal SCR siRNA, systemic angiotensin II infusion increased sodium retention and BP on day 1, and BP remained elevated throughout the 5-day study. These rats also demonstrated increased CD GR expression after 5 days of infusion. However, intrarenal GR siRNA infusion prevented angiotensin II-mediated sodium retention on day 1, induced a continuously negative cumulative sodium balance compared with angiotensin II alone, and reduced BP chronically. Glomerular filtration rate and renal blood flow remained unchanged in GR siRNA-infused rats. Systemic angiotensin II infusion also increased serum aldosterone levels, CD αENaC, and phosphorylated serum and glucocorticoid-inducible kinase 1 expression in rats with intrarenal SCR siRNA; however, these effects were not observed in the presence of intrarenal GR siRNA, despite exposure to the same systemic angiotensin II. These data demonstrate that chronic inhibition of intrarenal GR activity significantly reduces αENaC-dependent sodium retention, resulting in a negative cumulative sodium balance, thereby ameliorating angiotensin II-induced hypertension in rats. Renal GRs represent a novel therapeutic target for the treatment of hypertension and other sodium-retaining states.

AT2 Receptor Activation Prevents Sodium Retention and Reduces Blood Pressure in Angiotensin II-Dependent Hypertension.

RATIONALE: Compound 21 (C-21) is a highly selective nonpeptide angiotensin AT2 receptor (AT2R) agonist. OBJECTIVE: To test the hypothesis that chronic AT2R activation with C-21 induces natriuresis via an action at the renal proximal tubule (RPT) and lowers blood pressure (BP) in experimental angiotensin II (Ang II)-dependent hypertension. METHODS AND RESULTS: In rats, Ang II infusion increased both sodium (Na(+)) retention and BP on day 1, and BP remained elevated throughout the 7-day infusion period. Either intrarenal or systemic administration of C-21 prevented Ang II-mediated Na(+) retention on day 1, induced continuously negative cumulative Na(+) balance compared with Ang II alone, and reduced BP chronically. The effects of C-21 are likely to be mediated by action on the RPT as acute systemic C-21-induced natriuresis was additive to that induced by chlorothiazide and amiloride. At 24 hours of Ang II infusion, AT2R activation with C-21, both intrarenally and systemically, translocated AT2Rs from intracellular sites to the apical plasma membranes of RPT cells without altering the total cellular pool of AT2Rs and internalized/inactivated major RPT Na(+) transporters Na(+)-H(+)-exchanger-3 and Na(+)/K(+)ATPase. C-21 lowered BP to a similar degree whether administered before or subsequent to the establishment of Ang II-dependent hypertension. CONCLUSIONS: Chronic AT2R activation initiates and sustains receptor translocation to RPT apical plasma membranes, internalizes/inactivates Na(+)-H(+)-exchanger-3 and Na(+)/K(+)ATPase, prevents Na(+) retention resulting in negative cumulative Na(+) balance, and lowers BP in experimental Ang II-induced hypertension. Acting uniquely at the RPT, C-21 is a promising candidate for the treatment of hypertension and Na(+)-retaining states in humans.

Adrenalectomy for adrenal-mediated hypertension: National Surgical Quality Improvement Program analysis of an institutional experience.

Adrenal-mediated hypertension (AMH) has been increasingly treated by laparoscopic adrenalectomy (LA). Metabolic derangements in patients with AMH could result in perioperative complications and mortality. Long-term operative and clinical outcomes after laparoscopic treatment of AMH have not been evaluated using large clinical databases. The institutional National Surgical Quality Improvement Program (NSQIP) data for patients undergoing adrenalectomy for AMH between 2002 and 2012 were reviewed. Patient demographics, perioperative variables, and outcomes were analyzed and compared with national NSQIP adrenalectomy data. Improvement in AMH was recorded when discontinuation or reduction of antihypertensive medication occurred or with a decrease of blood pressure on the preoperative antihypertensive regimen. Ninety-four patients underwent adrenalectomy. There were 48 patients with pheochromocytoma (PHE) and 46 patients with aldosterone-producing adenoma (APA). Eighty-five patients (90%) were taking antihypertensive medications preoperatively compared with 36 patients (38%) postoperatively (P < 0.0001). Patients with PHE were more likely to discontinue all medications compared with the patients with APA (80 vs 20%, respectively, P < 0.0001). Patients with PHE and APA, respectively, took an average of 2.0 and 3.2 antihypertensive medications preoperatively compared with 0.3 and 1.2 postoperatively. There were no conversions to open procedures or 30-day mortality. Our results were 0 per cent for cerebral vascular accident, 0 per cent for myocardial infarction, and 0.5 per cent for transfusions compared with the national NSQIP data of 0.2, 0, and 6.7 per cent, respectively. Patients presenting with significant AMH including PHE and APA can be effectively and safely treated with LA with minimal complications and with a significant number of patients eliminating or decreasing their need for antihypertensive medications.

AT2 receptor activation induces natriuresis and lowers blood pressure.

RATIONALE: Compound 21 (C-21) is a highly selective nonpeptide AT2 receptor (AT2R) agonist. OBJECTIVE: To test the hypothesis that renal proximal tubule AT2Rs induce natriuresis and lower blood pressure in Sprague-Dawley rats and mice. METHODS AND RESULTS: In rats, AT2R activation with intravenous C-21 increased urinary sodium excretion by 10-fold (P<0.0001); this natriuresis was abolished by direct renal interstitial infusion of specific AT2R antagonist PD-123319. C-21 increased fractional excretion of Na(+) (P<0.05) and lithium (P<0.01) without altering renal hemodynamic function. AT2R activation increased renal proximal tubule cell apical membrane AT2R protein (P<0.001) without changing total AT2R expression and internalized/inactivated Na(+)-H(+) exchanger-3 and Na(+)/K(+)ATPase. C-21-induced natriuresis was accompanied by an increase in renal interstitial cGMP (P<0.01); C-21-induced increases in urinary sodium excretion and renal interstitial cGMP were abolished by renal interstitial nitric oxide synthase inhibitor l-N(6)-nitroarginine methyl ester or bradykinin B2 receptor antagonist icatibant. Renal AT2R activation with C-21 prevented Na(+) retention and lowered blood pressure in the angiotensin II infusion model of experimental hypertension. CONCLUSIONS: AT2R activation initiates its translocation to the renal proximal tubule cell apical membrane and the internalization of Na(+)-H(+) exchanger-3 and Na(+)/K(+)ATPase, inducing natriuresis in a bradykinin-nitric oxide-cGMP-dependent manner. Intrarenal AT2R activation prevents Na(+) retention and lowers blood pressure in angiotensin II-dependent hypertension. AT2R activation holds promise as a renal proximal tubule natriuretic/diuretic target for the treatment of fluid-retaining states and hypertension.

Intrarenal ghrelin receptor antagonism prevents high-fat diet-induced hypertension in male rats.

Excess weight gain contributes up to 65% of the risk of primary hypertension, and the increase in blood pressure in response to high-fat diet (HFD) is preceded by significant increases in renal tubular sodium (Na(+)) reabsorption. In normal rats, intrarenal ghrelin infusion increases distal nephron-dependent Na(+) reabsorption via activation of the intrarenal ghrelin receptor (GHSR). This study focusses on the role of intrarenal GHSR-mediated Na(+) reabsorption in HFD-induced hypertension. Dahl salt-sensitive rats received standard diet or HFD for 6 weeks. Rats underwent uninephrectomy and osmotic minipump implantation for chronic intrarenal delivery of vehicle (0.25 µL/h × 28 d), selective GHSR antagonist [D-Lys-3]-growth hormone releasing peptide-6 (0.2µM/d), or GHSR inverse agonist [D-Arg(1), D-Phe(5), D-Trp(7,9), Leu(11)]-substance P (SUB-P) (3.6µM/d). HFD rats with vehicle pumps had significantly increased renal GHSR expression compared with standard diet (0.092 ± 0.005 vs 0.065 ± 0.004 arbitrary units; P < .05), whereas acyl ghrelin levels were similar (16.3±6.2 vs 15.7±8.7 pg/g tissue). HFD rats with vehicle pumps became hypertensive after 2 weeks (P < .05) and showed a significant reduction in 24-hour urine Na(+) before hypertension. At this time, these rats showed an increase in collecting duct α-epithelial Na(+) channel, thereby providing a potential mechanism for the excess Na(+) reabsorption. In contrast, HFD rats with [D-Lys-3]-growth hormone releasing peptide-6 or SUB-P pumps never became hypertensive and did not show the reduction in urine Na(+). Because SUB-P blocks the constitutive, but not ghrelin-dependent, activity of the GHSR, and HFD-induced α-epithelial Na(+) channel up-regulation was abolished during GHSR antagonism, these data suggest that HFD increases the constitutive activity of renal GHSR to increase Na(+) reabsorption and induce hypertension in rats.

Intrarenal ghrelin receptors regulate ENaC-dependent sodium reabsorption by a cAMP-dependent pathway.

The main distal nephron segment sodium transporters are the distal tubule chlorothiazide-sensitive sodium chloride cotransporter (NCC) and the collecting duct amiloride-sensitive epithelial sodium channel (ENaC). The infusion of ghrelin into the renal interstitium stimulates distal nephron-dependent sodium reabsorption in normal rats, but the mechanism is unknown. Here we localize renal ghrelin receptors (GR) to the cortical collecting duct (CCD). Ghrelin significantly increased phosphorylated serum/glucocorticoid-regulated kinase-1 (pSGK1), a major upstream signaling intermediate regulating ENaC. To test whether increased apical membrane αENaC induced the antinatriuresis, ghrelin was infused in the presence of acute and chronic amiloride, a selective inhibitor of ENaC. In the presence of amiloride, renal interstitial ghrelin failed to reduce urine sodium excretion, suggesting that ghrelin-induced sodium reabsorption is dependent on intact ENaC activity. While the main sodium transporter of the CCD is ENaC, NCC is also present. In response to renal interstitial ghrelin infusion, neither total nor phosphorylated NCC levels are altered. Ghrelin-induced sodium reabsorption persisted in the presence of chlorothiazide (selective inhibitor of NCC), indicating that intact NCC activity is not necessary for ghrelin-induced antinatriuresis. Finally, renal interstitial ghrelin infusion significantly increased interstitial cAMP levels and adenylyl cyclase blockade abolished ghrelin-induced antinatriuresis. Thus, GRs expressed in the CCD regulate sodium reabsorption by cAMP-induced trafficking of ENaC to the apical membrane.

Role of angiotensin AT(2) receptors in natriuresis: Intrarenal mechanisms and therapeutic potential.

The renin-angiotensin system is a coordinated hormonal cascade critical for the regulation of blood pressure (BP) and kidney function. Angiotensin (Ang) II, the major angiotensin effector peptide, binds to two major receptors, namely AT1 and AT2 receptors. The AT1 receptors engender antinatriuresis and raise BP, whereas AT2 receptors oppose these effects, inducing natriuresis and reducing BP. There is high AT2 receptor expression in the adult kidney, especially in the proximal tubule. In AT2 receptor-null mice, long-term AngII infusion results in pressor and antinatriuretic hypersensivivity compared with responses in wild-type mice. The major endogenous receptor ligand for AT2 receptor-mediated natriuretic responses appears to be des-aspartyl(1) -AngII (AngIII) instead of AngII. Recent studies have demonstrated that AngII requires metabolism to AngIII by aminopeptidase A to induce natriuresis and that inhibition of aminopeptidase N increases intrarenal AngIII and augments AngIII-induced natriuresis. The renal dopaminergic system is another important natriuretic pathway. Renal proximal tubule the D1 and D5 receptor subtypes (D1 -like receptors (D1LIKE R)) control approximately 50% of basal sodium excretion. Recently, we have found that natriuresis induced by proximal tubule D1LIKE R requires AT2 receptor activation and that D1LIKE R stimulation induces recruitment of AT2 receptors to the apical plasma membrane via a cAMP-dependent mechanism. Initial studies using the potent AT2 receptor non-peptide agonist Compound 21 demonstrate natriuresis in both the presence and absence of AT1 receptor blockade, indicating the therapeutic potential of this compound in fluid-retaining states and hypertension.

AT2 receptors: beneficial counter-regulatory role in cardiovascular and renal function.

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 (AT(1)R) and the angiotensin type-2 (AT(2)R) receptor. The vast majority of the physiological actions of Ang II, almost all of them detrimental, are mediated by AT(1)Rs. In contrast, AT(2)Rs negatively modulate the actions of AT(1)Rs under the majority of circumstances and generally possess beneficial effects. AT(2)Rs induce vasodilation in both resistance and capacitance vessels, mediating natriuresis directly and via interactions with dopamine D1 receptors in the renal proximal tubule. AT(2)Rs inhibit renin biosynthesis and secretion and protect the kidneys from inflammation and ischemic injury. Our understanding of the exact role of AT(2)Rs 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 AT(2)Rs at the organ, tissue, cellular, and subcellular levels with emphasis on the vascular and renal actions that bear on blood pressure regulation and hypertension.

Intrarenal angiotensin III is the predominant agonist for proximal tubule angiotensin type 2 receptors.

In angiotensin type 1 receptor-blocked rats, renal interstitial (RI) administration of des-aspartyl(1)-angiotensin II (Ang III) but not angiotensin II induces natriuresis via activation of angiotensin type 2 receptors. In the present study, renal function was documented during systemic angiotensin type 1 receptor blockade with candesartan in Sprague-Dawley rats receiving unilateral RI infusion of Ang III. Ang III increased urine sodium excretion, fractional sodium, and lithium excretion. RI coinfusion of specific angiotensin type 2 receptor antagonist PD-123319 abolished Ang III-induced natriuresis. The natriuretic response observed with RI Ang III was not reproducible with RI angiotensin (1-7) alone or together with angiotensin-converting enzyme inhibition. Similarly, neither RI angiotensin II alone or in the presence of aminopeptidase A inhibitor increased urine sodium excretion. In the absence of systemic angiotensin type 1 receptor blockade, Ang III alone did not increase urine sodium excretion, but natriuresis was enabled by the coinfusion of aminopeptidase N inhibitor and subsequently blocked by PD-123319. In angiotensin type 1 receptor-blocked rats, RI administration of aminopeptidase N inhibitor alone also induced natriuresis that was abolished by PD-123319. Ang III-induced natriuresis was accompanied by increased RI cGMP 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 aminopeptidase N inhibition. These data demonstrate that endogenous intrarenal Ang III but not angiotensin II or angiotensin (1-7) induces natriuresis via activation of angiotensin type 2 receptors in the proximal tubule via a cGMP-dependent mechanism and suggest aminopeptidase N inhibition as a potential therapeutic target in hypertension.

Mechanisms of dopamine D(1) and angiotensin type 2 receptor interaction in natriuresis.

Renal dopamine D(1)-like receptors (D(1)Rs) and angiotensin type 2 receptors (AT(2)Rs) are important natriuretic receptors counterbalancing angiotensin type 1 receptor-mediated tubular sodium reabsorption. Here we explore the mechanisms of D(1)R and AT(2)R interactions in natriuresis. In uninephrectomized, sodium-loaded Sprague-Dawley rats, direct renal interstitial infusion of the highly selective D(1)R agonist fenoldopam induced a natriuretic response that was abolished by the AT(2)R-specific antagonist PD-123319 or by microtubule polymerization inhibitor nocodazole but not by actin polymerization inhibitor cytochalasin D. By confocal microscopy and immunoelectron microscopy, fenoldopam translocated AT(2)Rs from intracellular sites to the apical plasma membranes of renal proximal tubule cells, and this translocation was abolished by nocodazole. Because D(1)R activation induces natriuresis via an adenylyl cyclase/cAMP signaling pathway, we explored whether this pathway is responsible for AT(2)R recruitment and AT(2)R-mediated natriuresis. Renal interstitial coinfusion of the adenylyl cyclase activator forskolin and 3-isobutly-1-methylxanthine induced natriuresis that was abolished either by PD-123319 or nocodazole but was unaffected by specific the D(1)R antagonist SCH-23390. Coadministration of forskolin and 3-isobutly-1-methylxanthine also translocated AT(2)Rs to the apical plasma membranes of renal proximal tubule cells; this translocation was abolished by nocodazole but was unaffected by SCH-23390. The results demonstrate that D(1)R-induced natriuresis requires AT(2)R recruitment to the apical plasma membranes of renal proximal tubule cells in a microtubule-dependent manner involving an adenylyl cyclase/cAMP signaling pathway. These studies provide novel insights regarding the mechanisms whereby renal D(1)Rs and AT(2)Rs act in concert to promote sodium excretion in vivo.

Intrarenal ghrelin infusion stimulates distal nephron-dependent sodium reabsorption in normal rats.

Ghrelin is a 28-amino acid peptide hormone that exerts powerful orexigenic effects. Ghrelin receptor expression has been reported in the kidney, but the role of ghrelin in the kidney is unknown. The present studies confirmed ghrelin receptor mRNA expression in the kidneys of normal Sprague Dawley rats (n=6) using reverse transcription polymerase chain reaction (PCR) and sequencing of the 588-bp PCR product. To test intrarenal ghrelin action, uninephrectomized rats received 3 cumulative 1-hour renal interstitial (RI) infusions of 5% dextrose in water (vehicle, n=21), ghrelin (n=10), ghrelin plus specific ghrelin receptor antagonist [D-Lys-3]-GHRP-6 (n=24), or [D-Lys-3]-GHRP-6 alone (n=32). Mean arterial pressure (MAP), urine sodium excretion rate (U(Na)V), glomerular filtration rate (GFR), fractional excretion of sodium (FE(Na)), and fractional excretion of lithium (FE(Li)) were calculated for each period. RI ghrelin infusion significantly decreased U(Na)V to 86 ± 4.9% (P<0.05), 74 ± 6.5% (P<0.01), and 62 ± 10% (P<0.01) of baseline during periods 1 to 3, respectively. Ghrelin also significantly decreased FE(Na) to 68 ± 11% (P<0.05), 57 ± 8.6% (P<0.001), and 59 ± 12% (P<0.01) of baseline, without changing GFR or FE(Li). Identical ghrelin infusions in the presence of [D-Lys-3]-GHRP-6 failed to permit reductions in U(Na)V or FE(Na). Following [D-Lys-3]-GHRP-6 infusion alone, U(Na)V increased from 0.06 ± 0.01 to 0.18 ± 0.03 µmol/min (P<0.0001). Concomitant increases in FE(Na) were also observed (0.23 ± 0.03% to 0.51 ± 0.06% [P<0.001]), without changes in MAP, GFR, or FE(Li). Together, these data introduce a novel intrarenal ghrelin-ghrelin receptor system, which, on activation, significantly increases Na(+) reabsorption at the level of the distal nephron.

Intrarenal aminopeptidase N inhibition restores defective angiontesin II type 2-mediated natriuresis in spontaneously hypertensive rats.

The preferred ligand of angiotensin (Ang) II type 2 (AT(2)R)-mediated natriuresis is Ang III. The major enzyme responsible for the metabolism of Ang III is aminopeptidase N, which is selectively inhibited by compound PC-18. In this study, urine sodium excretion rates (U(Na)V), fractional excretion of sodium, fractional excretion of lithium, glomerular filtration rate, and mean arterial pressures were studied in prehypertensive and hypertensive spontaneously hypertensive rats (SHRs) and compared with age-matched Wistar-Kyoto rats (WKYs). Although renal interstitial infusion of Ang II type 1 receptor blocker candesartan increased U(Na)V in WKYs from a baseline of 0.05+/-0.01 to 0.17+/-0.04 micromol/min (P<0.01), identical infusions failed to increase U(Na)V in hypertensive SHRs. Coinfusion of AT(2)R antagonist PD-123319 abolished the natriuretic responses to candesartan in WKYs, indicating an AT(2)R-mediated effect. AT(2)R-mediated natriuresis was enabled in hypertensive SHRs by inhibiting the metabolism of Ang III with PC-18 (0.05+/-0.01 to 0.11+/-0.03 micromol/min; P<0.05). The defects in sodium excretion were present before the onset of hypertension in SHRs, because young WKYs demonstrated double the U(Na)V of SHRs (0.04+/-0.006 versus 0.02+/-0.003 micromol/min; P<0.01) at baseline. The increased U(Na)V of young WKYs was attributed to reduced renal proximal tubule sodium reabsorption, because increases in fractional excretion of sodium were paralleled by increases in fractional excretion of lithium. Renal interstitial PC-18 infusion ameliorated defective AT(2)R-mediated natriuresis in young SHRs by increasing fractional excretion of sodium and fractional excretion of lithium without changing the glomerular filtration rate. Thus, increased renal proximal tubule sodium retention is observed before the onset of hypertension in SHRs, and inhibition of the metabolism of Ang III ameliorates this pathophysiologic defect in sodium excretion.

Intrarenal angiotensin III infusion induces natriuresis and angiotensin type 2 receptor translocation in Wistar-Kyoto but not in spontaneously hypertensive rats.

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. The onset of hypertension in spontaneously hypertensive rats (SHRs) is preceded by defects in renal sodium excretion. The present study examines AT(2)R-mediated natriuresis in response to RI Ang III infusion in Wistar-Kyoto rats (WKYs) and SHRs. WKYs and SHRs received RI Ang III infusion after 24 hours of systemic AT(1)R blockade with candesartan. In WKYs, urine sodium excretion rate increased from 0.043+/-0.01 to 0.191+/-0.06 micromol/min (P<0.05) in response to Ang III infusion, but identical conditions failed to increase the urine sodium excretion rate in SHRs. The increase in the urine sodium excretion rate was blocked by coinfusion of PD-123319, a selective AT(2)R antagonist. On confocal microscopy images, Ang III-infused WKYs demonstrated greater renal proximal tubule cell AM AT(2)R fluorescence intensity compared with SHRs (5385+/-725 versus 919+/-35; P<0.0001), and Western blot analysis demonstrated increased AM (0.050+/-0.003 versus 0.038+/-0.003; P<0.01) but not total cell AT(2)R expression in WKYs. In SHRs, AM AT(2)R expression remained unchanged in response to RI Ang III infusion. Thus, RI Ang III infusion elicits natriuresis and renal proximal tubule cell AT(2)R translocation in WKYs. Identical manipulations fail to induce natriuresis or AT(2)R translocation in SHRs, suggesting that defects in AT(2)R-mediated natriuresis and trafficking may be important to the development of hypertension in SHRs.

Angiotensin AT2 receptors: control of renal sodium excretion and blood pressure.

The renin-angiotensin system is a coordinated hormonal cascade of crucial importance in cardiovascular and renal function. The primary effector peptide angiotensin II functions at two major receptors, the AT1 and AT2 receptors. AT2 receptors mediate vasodilation and natriuresis. Regarding vasodilator actions, AT2 receptors oppose the AT1 receptor-mediated vasoconstrictor action of angiotensin II. Regarding the natriuretic actions of AT2 receptors, des-aspartyl 1-angiotensin II, rather than angiotensin II, is the preferred agonist. Regarding both the vasodilator and natriuretic properties of AT2 receptors, the beneficial blood pressure reduction and natriuretic responses to AT1 receptor blockade are mediated, at least in part, by AT2 receptor activation. In addition, AT2 receptor activation suppresses renin biosynthesis and release at renal juxtaglomerular cells. Therefore, AT2 receptors are potential therapeutic targets in hypertension.

Conversion of renal angiotensin II to angiotensin III is critical for AT2 receptor-mediated natriuresis in rats.

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). Renal interstitial (RI) infusion of Ang III, but not Ang II, results in angiotensin type-2 receptor (AT(2)R)-mediated natriuresis. This response is augmented by coinfusion of PC-18, a specific inhibitor of APN. The present study addresses the hypotheses that Ang II conversion to Ang III is critical for the natriuretic response. Sprague-Dawley rats received systemic angiotensin type-1 receptor (AT(1)R) blockade with candesartan (CAND; 0.01 mg/kg/min) for 24 hours before and during the experiment. After a control period, rats received either RI infusion of Ang II or Ang II+PC-18. The contralateral kidney received a RI infusion of vehicle in all rats. Mean arterial pressure (MAP) was monitored, and urinary sodium excretion rate (U(Na)V) was calculated separately from experimental and control kidneys for each period. In contrast to Ang II-infused kidneys, U(Na)V from Ang II+PC-18-infused kidneys increased from a baseline of 0.03+/-0.01 to 0.09+/-0.02 micromol/min (P<0.05). MAP was unchanged by either infusion. RI addition of PD-123319, an AT(2)R antagonist, inhibited the natriuretic response. Furthermore, RI addition of EC-33, a selective APA inhibitor, abolished the natriuretic response to Ang II+PC-18. These data demonstrate that RI addition of PC-18 to Ang II enables natriuresis mediated by the AT(2)R, and that conversion of Ang II to Ang III is critical for this response.

Intrarenal aminopeptidase N inhibition augments natriuretic responses to angiotensin III in angiotensin type 1 receptor-blocked rats.

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. The contralateral control kidney received renal interstitial infusion of vehicle. In kidneys infused with angiotensin III alone, renal sodium excretion rate increased from 0.05+/-0.01 micromol/min in stepwise fashion to 0.11+/-0.01 micromol/min at 28 nmol/kg per minute of angiotensin III (overall ANOVA F=3.68; P<0.01). In angiotensin III combined with PC-18, the renal sodium excretion rate increased from 0.05+/-0.01 to 0.32+/-0.08 mumol/min at 28 nmol/kg per minute of angiotensin III (overall ANOVA F=6.2; P<0.001). The addition of intrarenal PD-123319, an angiotensin type 2 receptor antagonist, to renal interstitial angiotensin III plus PC-18 inhibited the natriuretic response. Mean arterial blood pressure and renal sodium excretion rate from control kidneys were unchanged by angiotensin III +/- PC-18 + PD-123319. Angiotensin III plus PC-18 induced a greater natriuretic response than Ang III alone (overall ANOVA F=16.9; P=0.0001). Aminopeptidase N inhibition augmented the natriuretic response to angiotensin III, suggesting that angiotensin III is a major agonist of angiotensin type 2 receptor-induced natriuresis.

Renal angiotensin type 2 receptors mediate natriuresis via angiotensin III in the angiotensin II type 1 receptor-blocked rat.

Whereas angiotensin (Ang) II is the major effector peptide of the renin-angiotensin system, its metabolite, des-aspartyl1-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 (AT1) blocker, with and without coinfusion of PD-123319 (PD), a specific Ang II type 2 receptor (AT2) blocker, on Na+ excretion (UNaV) in uninephrectomized rats. We also studied the effects of unilateral RI infusion of Ang II or Ang III on UNaV with and without systemic infusion of CAND with the noninfused kidney as control. In rats receiving normal Na+ intake, RI CAND increased UNaV from 0.07+/-0.08 to 0.82+/-0.17 micromol/min (P<0.01); this response was abolished by PD. During Na+ restriction, CAND increased UNaV from 0.06+/-0.02 to 0.1+/-0.02 micromol/min (P<0.05); 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 UNaV. However, with systemic CAND infusion, RI Ang III increased U(Na)V from 0.08+/-0.01 micromol/min to 0.18+/-0.04 micromol/min (P<0.01) at 3.5 nmol/kg per minute, and UNaV remained elevated throughout the infusion; this response was abolished by PD. However, RI infusion of Ang II did not significantly alter UNaV at any infusion rate (3.5 to 80 nmol/kg per minute) with or without systemic CAND infusion. These results suggest that intrarenal AT1 receptor blockade engenders natriuresis by activation of AT2 receptors. AT2 receptor activation via Ang III, but not via Ang II, mediates the natriuretic response in the presence of systemic AT1 receptor blockade.