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Background: Extracellular renal interstitial guanosine cyclic 3',5'-monophosphate (cGMP) inhibits renal proximal tubule (RPT) sodium (Na + ) reabsorption via Src (Src family kinase) activation. Through which target extracellular cGMP acts to induce natriuresis is unknown. We hypothesized that cGMP binds to the extracellular α1-subunit of NKA (sodium-potassium ATPase) on RPT basolateral membranes to inhibit Na + transport similar to ouabain-a cardiotonic steroid., Methods: Urine Na + excretion was measured in uninephrectomized 12-week-old female Sprague-Dawley rats that received renal interstitial infusions of vehicle (5% dextrose in water), cGMP (18, 36, and 72 μg/kg per minute; 30 minutes each), or cGMP+rostafuroxin (12 ng/kg per minute) or were subjected to pressure-natriuresis±rostafuroxin infusion. Rostafuroxin is a digitoxigenin derivative that displaces ouabain from NKA., Results: Renal interstitial cGMP and raised renal perfusion pressure induced natriuresis and increased phosphorylated Src Tyr416 and Erk 1/2 (extracellular signal-regulated protein kinase 1/2) Thr202/Tyr204 ; these responses were abolished with rostafuroxin coinfusion. To assess cGMP binding to NKA, we performed competitive binding studies with isolated rat RPTs using bodipy-ouabain (2 μM)+cGMP (10 µM) or rostafuroxin (10 µM) and 8-biotin-11-cGMP (2 μM)+ouabain (10 μM) or rostafuroxin (10 µM). cGMP or rostafuroxin reduced bodipy-ouabain fluorescence intensity, and ouabain or rostafuroxin reduced 8-biotin-11-cGMP staining. We cross-linked isolated rat RPTs with 4-N 3 -PET-8-biotin-11-cGMP (2 μM); 8-N 3 -6-biotin-10-cAMP served as negative control. Precipitation with streptavidin beads followed by immunoblot analysis showed that RPTs after cross-linking with 4-N 3 -PET-8-biotin-11-cGMP exhibited a significantly stronger signal for NKA than non-cross-linked samples and cross-linked or non-cross-linked 8-N 3 -6-biotin-10-cAMP RPTs. Ouabain (10 μM) reduced NKA in cross-linked 4-N 3 -PET-8-biotin-11-cGMP RPTs confirming fluorescence staining. 4-N 3 -PET-8-biotin-11-cGMP cross-linked samples were separated by SDS gel electrophoresis and slices corresponding to NKA molecular weight excised and processed for mass spectrometry. NKA was the second most abundant protein with 50 unique NKA peptides covering 47% of amino acids in NKA. Molecular modeling demonstrated a potential cGMP docking site in the ouabain-binding pocket of NKA., Conclusions: cGMP can bind to NKA and thereby mediate natriuresis.

Background: How signals from activated angiotensin type-2 receptors (AT 2 R) mediate inhibition of sodium ion (Na + ) reabsorption in renal proximal tubule cells is currently unknown. Protein phosphatases including PP2A (protein phosphatase 2A) have been implicated in AT 2 R signaling in tissues other than kidney. We investigated whether inhibition of protein phosphatase PP2A reduced AT 2 R-mediated natriuresis and evaluated changes in PP2A activity and localization after renal AT 2 R activation in normal 4- and 10-week-old control Wistar-Kyoto rats and 4-week-old prehypertensive and 10-week-old hypertensive spontaneously hypertensive rats., Methods and Results: In Wistar-Kyoto rats, direct renal interstitial administration of selective AT 2 R nonpeptide agonist Compound-21 (C-21) increased renal interstitial cyclic GMP (cGMP) levels, urine Na + excretion, and simultaneously increased PP2A activity ≈2-fold in homogenates of renal cortical tubules. The cyclic GMP and natriuretic responses were abolished by concurrent renal interstitial administration of protein phosphatase inhibitor calyculin A. In renal proximal tubule cells in response to C-21, PP2A subunits A, B55α and C, but not B56γ, were recruited to apical plasma membranes together with AT 2 Rs. Calyculin A treatment abolished C-21-induced translocation of both AT 2 R and PP2A regulatory subunit B55α to apical plasma membranes. Immunoprecipitation of AT 2 R solubilized from renal cortical homogenates demonstrated physical association of AT 2 R with PP2A A, B55α, and C but not B56γ subunits. In contrast, in spontaneously hypertensive rats, administration of C-21 did not alter urine Na + excretion or PP2A activity and failed to translocate AT 2 Rs and PP2A subunits to apical plasma membranes., Conclusions: In renal proximal tubule cells of Wistar-Kyoto rats, PP2A is activated and PP2A subunits AB55αC are recruited to C-21-activated AT 2 Rs during induction of natriuresis. This response is defective in prehypertensive and hypertensive spontaneously hypertensive rats, presenting a potential novel therapeutic target for treating renal Na + retention and hypertension.

Rationale: Previous studies identified a defect in Ang III (angiotensin III [des-aspartyl 1 -angiotensin II])-elicited AT 2 R (Ang type-2 receptor)-mediated natriuresis in renal proximal tubule cells of spontaneously hypertensive rats (SHR)., Objective: This study aimed to delineate in prehypertensive SHR kidneys the receptor or postreceptor defect causing impaired AT 2 R signaling and renal sodium (Na + ) retention by utilizing the selective AT 2 R agonist compound-21 (C-21)., Methods and Results: Female 4-week-old Wistar Kyoto and SHR rats were studied after 24-hour systemic AT 1 R (Ang II type-1 receptor) blockade. Left kidneys received 30-minute renal interstitial infusions of vehicle followed by C-21 (20, 40, and 60 ng/[kg·min], each dose 30 minutes). Right kidneys received vehicle infusions. In Wistar Kyoto, C-21 dose-dependently increased urine Na + excretion from 0.023±0.01 to 0.064±0.02, 0.087±0.01, and 0.089±0.01 µmol/min ( P =0.008, P <0.0001, and P <0.0001, respectively) and renal interstitial fluid levels of AT 2 R downstream signaling molecule cGMP (cyclic guanosine 3',5' monophosphate) from 0.91±0.3 to 3.1±1.0, 5.9±1.2 and 5.3±0.5 fmol/mL ( P =nonsignificant, P <0.0001, and P <0.0001, respectively). In contrast, C-21 did not increase urine Na + excretion or renal interstitial cGMP in SHR. Mean arterial pressure was slightly higher in SHR but within the normotensive range and unaffected by C-21. In Wistar Kyoto, but not SHR, C-21 induced AT 2 R translocation to apical plasma membranes of renal proximal tubule cells, internalization/inactivation of NHE-3 (sodium-hydrogen exchanger-3) and Na + /K + ATPase (sodium-potassium-atpase) and phosphorylation of AT 2 R-cGMP downstream signaling molecules Src (Src family kinase), ERK (extracellular signal-related kinase), and VASP (vasodilator-stimulated phosphoprotein). To test whether cGMP could bypass the natriuretic defect in SHR, we infused 8-bromo-cGMP. This restored natriuresis, Na + transporter internalization/inactivation, and Src and VASP phosphorylation, but not apical plasma membrane AT 2 R recruitment. In contrast, 8-bromo-cAMP administration had no effect on natriuresis or AT 2 R recruitment in SHR., Conclusions: The results demonstrate a primary renal proximal tubule cell AT 2 R natriuretic defect in SHR that may contribute to the development of hypertension. Since the defect is abrogated by exogenous intrarenal cGMP, the renal cGMP pathway may represent a viable target for the treatment of hypertension. Visual Overview: An online visual overview is available for this article.

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., (© 2016 American Heart Association, Inc.)

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., (© 2014 American Heart Association, Inc.)