Nancy L. Howell

Renal Interstitial Fluid Angiotensin : Modulation by Anesthesia, Epinephrine, Sodium Depletion, and Renin Inhibition

Using a microdialysis technique, we monitored changes in right and left renal interstitial fluid angiotensins in anesthetized and conscious dogs (both n = 5) in response to right renal interstitial epinephrine (0.2 mg/kg per minute) administration. Renal interstitial and plasma angiotensin levels also were monitored in conscious dogs (n = 4) in response to dietary sodium deprivation (10 mmol/d) for 5 consecutive days. Changes in renal interstitial and plasma angiotensins in response to interstitial administration of a specific renin inhibitor, ACRIP (0.5 micrograms/kg per minute for 20 minutes), were monitored on day 5 of sodium depletion. At basal levels, there were no significant differences between the right and left renal interstitial immunoreactive angiotensin levels in anesthetized dogs. Renal interstitial epinephrine administration caused a significant increase in renal interstitial immunoreactive angiotensin concentrations in both anesthetized and conscious dogs (P < .01). However, anesthetized dogs had significantly higher renal interstitial immunoreactive angiotensin levels basally and in response to epinephrine than conscious dogs (P < .05). Renal interstitial immunoreactive angiotensin concentrations increased significantly and progressively during exposure to a low sodium diet from 3.9 +/- 1 nmol on day 1 to 740 +/- 332 nmol on day 5 (P < .01). Renal interstitial immunoreactive angiotensin decreased significantly to 124 +/- 37 nmol (P < .01) in response to intrarenal renin inhibition at the end of day 5 of sodium depletion. Plasma immunoreactive angiotensin increased significantly (P < .01) in response to sodium depletion, and no change occurred during intrarenal renin inhibition.(ABSTRACT TRUNCATED AT 250 WORDS)

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 &mgr;mol/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 &mgr;mol/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 UNaV from 0.08±0.01 &mgr;mol/min to 0.18±0.04 &mgr;mol/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.

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 (AT2R)-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 (AT1R) 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 (UNaV) was calculated separately from experimental and control kidneys for each period. In contrast to Ang II-infused kidneys, UNaV from Ang II+PC-18-infused kidneys increased from a baseline of 0.03±0.01 to 0.09±0.02 &mgr;mol/min (P<0.05). MAP was unchanged by either infusion. RI addition of PD-123319, an AT2R 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 AT2R, and that conversion of Ang II to Ang III is critical for this response.

Intrarenal Dopamine D1-Like Receptor Stimulation Induces Natriuresis via an Angiotensin Type-2 Receptor Mechanism

We explored the effects of direct renal interstitial stimulation of dopamine D1-like receptors with fenoldopam, a selective D1-like receptor agonist, on renal sodium excretion and angiotensin type-2 (AT2) receptor expression and cellular distribution in rats on a high-sodium intake. In contrast to vehicle-infused rats, sodium excretion increased in fenoldopam-infused rats during each of three 1-hour experimental periods (<0.001). Blood pressure was unaffected by vehicle or fenoldopam. In plasma membranes of renal cortical cells, fenoldopam increased D1 receptor expression by 38% (P<0.05) and AT2 receptor expression by 69% (P<0.01). In plasma membranes of renal proximal tubule cells, fenoldopam increased AT2 receptor expression by 108% (P<0.01). In outer apical membranes of proximal tubule cells, fenoldopam increased AT2 receptor expression by 59% (P<0.01). No significant change in total AT2 receptor protein expression was detectable in response to fenoldopam. Fenoldopam-induced natriuresis was abolished when either PD-123319, a specific AT2 receptor antagonist, or SCH-23390, a potent D1-like receptor antagonist, was coinfused with F (P<0.001). In summary, direct renal D1-like receptor activation increased urinary sodium excretion and the plasma membrane expression of AT2 receptors in renal cortical and proximal tubule cells. D1-like receptor–induced natriuresis was abolished by intrarenal AT2 receptor inhibition. These findings suggest that dopaminergic regulation of sodium excretion involves recruitment of AT2 receptors to the outer plasma membranes of renal proximal tubule cells and that dopamine-induced natriuresis requires AT2 receptor activation.

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 &mgr;mol/min in stepwise fashion to 0.11±0.01 &mgr;mol/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 &mgr;mol/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.

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-N6-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.

Selective Inhibition of the Renal Angiotensin Type 2 Receptor Increases Blood Pressure in Conscious Rats

Abstract—The angiotensin II type 2 (AT2) receptor is present in rat kidney; however, its function is not well understood. The purpose of this study was to evaluate the role of the AT2 receptor in blood pressure (BP) regulation. The effects of selective inhibition of the renal AT2 receptor with phosphorothioated antisense oligodeoxynucleotide (AS-ODN) were examined in conscious uninephrectomized rats. Oligodeoxynucleotides (AS-ODN or scrambled [S-ODN]) were infused directly into the renal interstitial space by using an osmotic pump at 1 &mgr;L/h for 7 days. Texas red-labeled AS-ODN was distributed in renal tubules in the infused but not the contralateral kidney of normal rats. Continuous renal interstitial infusion of the AS-ODN, but not S-ODN, caused a significant (P <0.01) increase in BP 1 to 5 days after the initiation of the infusion. AS-ODN-treated rats experienced an increase in systolic BP from 109±4 to 130±4 mm Hg (n=8, P <0.01), whereas S-ODN-treated (n=8) and vehicle-treated (n=8) rats did not show any significant change in BP. On day 5 of the oligodeoxynucleotide infusion, AS-ODN-treated rats exhibited a greater pressor response to systemic angiotensin II infusion (30 ng/kg per hour) than did S-ODN-treated rats (P <0.01). Renal interstitial fluid cGMP decreased from 11.9±0.8 to 3.6±0.5 pmol/mL (P <0.001), and bradykinin decreased from 0.05±0.05 to 0.18±0.03 ng/mL (P <0.001) in response to AS-ODN, but they were not significantly changed in response to S-ODN. To evaluate the effects of AS-ODN and S-ODN on AT2 receptor expression, Western Blot analysis was performed on treated kidneys. Kidneys treated with AS-ODN had ≈40% less expression of AT2 receptor than did kidneys treated with S-ODN or vehicle (P <0.05). These results suggest that AS-ODN directed selectively against the renal AT2 receptor decreased receptor expression and caused an increase in BP. We conclude that the renal AT2 receptor plays an important role in the regulation of BP via a bradykinin/cGMP vasodilator signaling cascade.

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-aspartyl1-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.

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 (AT2Rs) mediate natriuresis in response to renal interstitial (RI) D1-like receptor stimulation or RI Ang III infusion. After D1-like receptor activation, apical membrane (AM) but not total renal proximal tubule cell AT2R expression is increased, suggesting that AM AT2R 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 AT2R-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 AT1R blockade with candesartan. In WKYs, urine sodium excretion rate increased from 0.043±0.01 to 0.191±0.06 &mgr;mol/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 AT2R antagonist. On confocal microscopy images, Ang III–infused WKYs demonstrated greater renal proximal tubule cell AM AT2R 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 AT2R expression in WKYs. In SHRs, AM AT2R expression remained unchanged in response to RI Ang III infusion. Thus, RI Ang III infusion elicits natriuresis and renal proximal tubule cell AT2R translocation in WKYs. Identical manipulations fail to induce natriuresis or AT2R translocation in SHRs, suggesting that defects in AT2R-mediated natriuresis and trafficking may be important to the development of hypertension in SHRs.

Molecular basis for the modulation of native T-type Ca2+ channels in vivo by Ca2+ /calmodulin-dependent protein kinase II

Ang II receptor activation increases cytosolic Ca2+ levels to enhance the synthesis and secretion of aldosterone, a recently identified early pathogenic stimulus that adversely influences cardiovascular homeostasis. Ca2+/calmodulin-dependent protein kinase II (CaMKII) is a downstream effector of the Ang II-elicited signaling cascade that serves as a key intracellular Ca2+ sensor to feedback-regulate Ca2+ entry through voltage-gated Ca2+ channels. However, the molecular mechanism(s) by which CaMKII regulates these important physiological targets to increase Ca2+ entry remain unresolved. We show here that CaMKII forms a signaling complex with alpha1H T-type Ca2+ channels, directly interacting with the intracellular loop connecting domains II and III of the channel pore (II-III loop). Activation of the kinase mediated the phosphorylation of Ser1198 in the II-III loop and the positive feedback regulation of channel gating both in intact cells in situ and in cells of the native adrenal zona glomerulosa stimulated by Ang II in vivo. These data define the molecular basis for the in vivo modulation of native T-type Ca2+ channels by CaMKII and suggest that the disruption of this signaling complex in the zona glomerulosa may provide a new therapeutic approach to limit aldosterone production and cardiovascular disease progression.