Renal Interstitial cGMP Research Articles

Evidence That Binding of Cyclic GMP to the Extracellular Domain of NKA (Sodium-Potassium ATPase) Mediates Natriuresis.

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. 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. Renal interstitial cGMP and raised renal perfusion pressure induced natriuresis and increased phosphorylated SrcTyr416 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-N3-PET-8-biotin-11-cGMP (2 μM); 8-N3-6-biotin-10-cAMP served as negative control. Precipitation with streptavidin beads followed by immunoblot analysis showed that RPTs after cross-linking with 4-N3-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-N3-6-biotin-10-cAMP RPTs. Ouabain (10 μM) reduced NKA in cross-linked 4-N3-PET-8-biotin-11-cGMP RPTs confirming fluorescence staining. 4-N3-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. cGMP can bind to NKA and thereby mediate natriuresis.

Identification of a Primary Renal AT2 Receptor Defect in Spontaneously Hypertensive Rats.

Previous studies identified a defect in Ang III (angiotensin III [des-aspartyl1-angiotensin II])-elicited AT2R (Ang type-2 receptor)-mediated natriuresis in renal proximal tubule cells of spontaneously hypertensive rats (SHR). This study aimed to delineate in prehypertensive SHR kidneys the receptor or postreceptor defect causing impaired AT2R signaling and renal sodium (Na+) retention by utilizing the selective AT2R agonist compound-21 (C-21). Female 4-week-old Wistar Kyoto and SHR rats were studied after 24-hour systemic AT1R (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 AT2R 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 AT2R 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 AT2R-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 AT2R recruitment. In contrast, 8-bromo-cAMP administration had no effect on natriuresis or AT2R recruitment in SHR. The results demonstrate a primary renal proximal tubule cell AT2R 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.

Abstract P616: Evidence That The Extracellular Domain Of Na + /K + Atpase Is The Receptor For Cyclic Gmp-Induced Natriuresis

Previous studies from our laboratory have shown that extracellular renal interstitial (RI) cyclic guanosine 3’5’-monophosphate (cGMP) increases urine sodium (Na + ) excretion (U Na V) at the renal proximal tubule (RPT) in rats via activation of Src family kinase. Extracellular cGMP engenders this response through an unknown receptor. We hypothesized that cGMP binds to the extracellular domain of Na + /K + -ATPase (NKA) on basolateral membranes of RPT cells inhibiting Na + transport. In the present study, we evaluated the effect of RI infusion of rostafuroxin (RF), a digitoxigenin derivative that specifically displaces oubain (OUA) binding from NKA, on U Na V in the presence of RI cGMP infusion. Volume expanded, uninephrectomized, 12-week-old female Sprague-Dawley rats received RI infusions of vehicle (D 5 W) (N=8), RI cGMP (18, 36, and 72 μg/kg/min; each dose for 30 min; N=10), or RI cGMP + RF (0.012 μg/kg/min; N=5) for 90 min following a 30 min control period with RI infusion of vehicle D 5 W. RI cGMP infusion induced a significant natriuresis from 0.39 ± 0.06 μmol/min to 1.03 ± 0.21 (P&lt;0.05), 1.17 ± 0.19 (P&lt;0.01), and 1.94 ± 0.16 (P&lt;0.001) μmol/min at 18, 36, and 72 μg/kg/min cGMP, respectively. RI co-infusion of cGMP + RF abolished the cGMP-induced natriuresis at all doses (F=16.05, P&lt;0.001). There was no change in mean arterial pressure during any infusion. To further demonstrate that cGMP binds to NKA, we performed a series of competitive binding studies in isolated RPTs from normal rat kidneys (N=4 for each) with bodipy-OUA (2 μM) + cGMP (10 μM) and 8-[Biotin]-AET-cGMP (2 μM) + OUA (10 μM). In the presence of cGMP, bodipy-OUA fluorescence intensity was reduced from 1422.1 ± 63 to 1072.5 ± 64 relative fluorescent units (RFU, P&lt;0.01). In the presence of OUA, 8-[Biotin]-AET-cGMP staining was reduced from 1916.3 ± 144 to 1492.2 ± 84 RFU (P&lt;0.05). Serving as control, biotinylated cAMP (N=2) did not demonstrate any fluorescence above background. Together, these data suggest that cGMP may compete with RF for binding on NKA and that the extracellular domain of NKA may serve as the receptor for cGMP-induced natriuresis.

AT₂ receptor activation induces natriuresis and lowers blood pressure.

Compound 21 (C-21) is a highly selective nonpeptide AT2 receptor (AT2R) agonist. To test the hypothesis that renal proximal tubule AT2Rs induce natriuresis and lower blood pressure in Sprague-Dawley rats and mice. 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. 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.

Abstract 473: Compound-21 Acts at Renal Proximal Tubule AT2 Receptors to Inhibit Na+ Reabsorption via Bradykinin, Nitric Oxide, and Cyclic GMP

Angiotensin (Ang) AT2 receptors (AT2Rs) are expressed in the kidney at vascular and tubular sites. Previous studies from our lab have shown that renal AT2R activation induces natriuresis and that Ang III is the endogenous agonist for this response. Compound 21 (C21) is a highly selective non-peptide AT2R agonist with approx. 25,000-fold selectivity for AT2Rs over AT1 receptors (AT1Rs). However, C21, particularly at high concentrations, has recently been shown to have AT2R-independent effects in the vasculature. We hypothesized that C21 induces natriuresis via AT2R translocation to the apical membrane and by activating a bradykinin (BK)-nitric oxide (NO)-cyclic GMP (cGMP) signaling pathway in the renal proximal tubule (RPT). In anesthetized, uninephrectomized, volume-expanded rats, intravenous (iv) C21 infusion (100, 200 and 300 ng/kg/min; each dose for 30 min) increased urinary Na+ excretion (UNaV) from 0.16±0.03 to 1.12±0.19, 1.51±0.08 and 2.03±0.21 μmol/min, respectively; all P &lt;0.001). This natriuretic response was not altered by 24 h subcutaneous infusion of AT1R antagonist candesartan (CAND; 0.01 mg/kg/min), but was abolished by renal interstitial (RI) infusion of specific AT2R antagonist PD-123319 (PD). Blood pressure was unaltered by C21 or PD, but was lower in CAND-infused rats (P &lt;0.0001). C21 infusion increased fractional excretion of Na+ (FENa) from 0.36±0.07 to 0.90±0.18% (P&lt;0.05) and lithium (FELi) from 30.2±3.3 to 57.2±5.2% (P &lt;0.01) without altering glomerular filtration rate or renal blood flow. Compared to control, C21 infusion increased RPT cell apical membrane AT2R protein (0.11±0.006 vs. 0.08±0.004 DU respectively) without changing total cell AT2R. C21-induced natriuresis was also accompanied by an increase in RI cGMP from 3.78±0.82 to 17.1±3.39 pmol/ml (P&lt;0.01). The C21-induced increases in both UNaV and RI cGMP were abolished by RI infusion of NO synthase inhibitor L-NAME or BK B2 receptor antagonist icatibant. The results demonstrate that C21 initiates AT2R translocation to the RPT cell apical membrane inducing natriuresis in a BK-NO-cGMP-dependent manner in the absence of concurrent AT1R blockade. C21 holds promise as a natriuretic/diuretic agent for the treatment of fluid retaining states and hypertension.

Abstract 216: Mechanism of Pressure-Natriuresis: Evidence That Na+/K+-ATPase is a Renal Extracellular Cyclic Guanosine 3’,5’-Monophosphate Receptor

Pressure-natriuresis (P-N) is the main compensatory mechanism by which the kidney excretes sodium (Na+) in response to an increase in blood pressure. Extracellular renal interstitial (RI) cyclic guanosine 3’,5’-monophosphate (cGMP) modulates P-N by binding to an unidentified receptor on the basolateral membrane of renal proximal tubule cells and activating the Src tyrosine kinase signaling cascade. In this study, we hypothesized that the cGMP receptor is the extracellular domain of Na+/K+-ATPase (NKA). In uninephrectomized Sprague Dawley rats, the renal perfusion pressure (RPP) was increased from 110 to 140 mmHg by ligating the infrarenal aorta, celiac, and mesenteric arteries, in a standard P-N model. In response to increased RPP, urinary Na+ excretion (UNaV) increased from 0.35±0.065 to 2.19±0.31 umol/min (P&lt;0.0001; N=14). Rostafuroxin, a digitoxigenin derivative that specifically displaces ouabain binding from NKA without inhibiting NKA pump activity, significantly decreased UNaV in a dose-dependent manner, from 2.19±0.31 to 1.38±0.18 (P&lt;0.05; N=8) and to 0.34±0.080 umol/min (P&lt;0.0001; N=6), at RI infusion rates of 2.4 and 12 ng/kg/min, respectively. In contrast, at higher infusion rates, of 60 and 120 ng/kg/min, rostafuroxin increased UNaV to 2.49±0.45 (P=0.60; N=8) and to 3.68±0.49 umol/min (P&lt;0.05; N=8), respectively. Thus, rostafuroxin is a pharmacologic agent with NKA antagonist/agonist activity, depending on dose. To show that cGMP-induced natriuresis depends upon NKA binding, we microinfused cGMP directly into the RI compartment of uninephrectomized Sprague Dawley rats without changing the RPP. We compared UNaV in the second hour period of a 2-hour RI microinfusion of vehicle (0.66±0.086 umol/min; N=5), to rostafuroxin 12 ng/kg/min (0.69±0.11 umol/min; P=0.81; N=6), cGMP 72 ng/kg/min (3.41±0.57 umol/min; P&lt;0.005; N=7), or cGMP together with rostafuroxin (0.64±0.21 umol/min; P=0.93; N=6). The rostafuroxin dose that abolished P-N also nullified the natriuretic response to cGMP. In summary, RI microinfusion of rostafuroxin blocked both pressure- and cGMP-induced natriuresis. These data provide strong evidence that the extracellular domain of NKA serves as a receptor for RI cGMP, which mediates P-N.

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.

Role of Src Family Kinase in Extracellular Renal Cyclic Guanosine 3′,5′-Monophosphate- and Pressure-Induced Natriuresis

cGMP functions as an extracellular (paracrine) messenger acting at the renal proximal tubule and is an important modulator of pressure-natriuresis (P-N). The signaling pathway activated by cGMP in the tubule cell basolateral membrane remains unknown. We hypothesized that renal interstitial microinfusion of cGMP (50 nmol/kg per minute) or P-N would be accompanied by increased renal protein levels of phospho-Src (Tyr 416) and that the natriuresis would be decreased by Src inhibition. Renal interstitial cGMP-induced natriuresis was blocked by Src inhibitor PP2 (2.0±0.4 versus 0.5±0.01 μEq/g per minute; P<0.001). The inactive analog of PP2, PP3, had no effect on cGMP-induced natriuresis. SU6656, another Src inhibitor, also inhibited cGMP-induced natriuresis (2.0±0.4 versus 1.02±0.01 μEq/g per minute; P<0.001). Renal interstitial cGMP infusion increased phospho-Src protein levels 5.6-fold at 15 minutes and 6.8-fold at 30 minutes compared with vehicle infusion but returned toward basal levels after 60 minutes. PP2 also blunted P-N (3.1±0.1 versus 1.1±0.3 μEq/g per minute; P<0.01) despite a similar increase in blood pressure. PP3 had no effect on P-N. Phospho-Src protein levels increased during P-N in vehicle- (1.8-fold) and PP3-treated (2.1-fold) groups compared with the sham-operated group. PP2 blocked the pressure-induced increase in renal phospho-Src protein levels. PP2 had no effect on renal hemodynamics but decreased both fractional excretion of Na(+) and lithium. Both extracellular cGMP and increased renal perfusion pressure increased renal phospho-Src protein levels and induced natriuresis in an Src-dependent manner, demonstrating that Src is an important downstream signaling molecule for extracellular cGMP-induced natriuresis.

Reinforcing Feedback Loop of Renal Cyclic Guanosine 3′ 5′ -Monophosphate and Interstitial Hydrostatic Pressure in Pressure-Natriuresis

This study addresses the hypothesis that renal interstitial (RI) cGMP, a modulator of pressure-natriuresis, exerts its effect through a relationship with renal interstitial hydrostatic pressure (RIHP). Increasing renal perfusion pressure in Sprague-Dawley rats led to increases in RIHP (5.2+/-0.6 to 10.9+/-1.6 mm Hg; P<0.01), urine sodium excretion (0.062+/-0.009 to 0.420+/-0.068 micromol/min per gram; P<0.01), and RI cGMP (3.5+/-0.8 to 9.5+/-1.7 fmol/min; P<0.01), and these effects were blocked by partial renal decapsulation. Infusion of cGMP into the RI compartment of decapsulated animals restored natriuresis (0.067+/-0.010 to 0.310+/-0.061 micromol/min per gram; P<0.01). These changes were independent of changes in glomerular filtration rate . Artificially increasing RIHP in normotensive animals increased RI cGMP (4.1+/-0.6 to 6.9+/-0.7 fmol/min; P<0.01) and urine sodium excretion (0.071+/-0.013 to 0.179+/-0.039 micromol/min per gram; P<0.05). Coinfusion of organic anion transport-inhibitor probenecid, or soluble guanylyl cyclase inhibitor 1-H(1,2,4) oxadiazolo-(4,2)quinoxalin-1-one, abolished these effects. Infusion of cGMP into the RI compartment of normotensive animals increased RIHP (6.7+/-0.4 to 10.3+/-0.9 mm Hg; P<0.001). Exogenous RI cGMP delivery did not affect total, cortical, or medullary renal blood flow. These studies suggest that extracellular RI cGMP is required for the natriuresis observed after increases in renal perfusion pressure and RIHP and that cGMP acts via a tubule mechanism. The results support an intrarenal positive-feedback loop wherein RI cGMP increases RIHP, which, in turn, increases RI cGMP, contributing to the reinforcement of pressure-natriuresis.

Intact Microtubules Are Required for Natriuretic Responses to Nitric Oxide and Increased Renal Perfusion Pressure

Extracellular renal interstitial (RI) cGMP modulates NO- and pressure-induced natriuresis in vivo in the rat. The present study objective was to test the hypothesis that an intact microtubulin network is required for transport of cGMP from intracellular sites into the extracellular compartment in vivo and that this transport is required for natriuresis induced by NO and increased renal perfusion pressure. After a 1-hour control period, uninephrectomized rats received an RI infusion of NO donor S-nitroso-N-acetylpenicillamine (SNAP), SNAP+microtubule inhibitor nocodazole (NOC), SNAP+NOC+cGMP, or NOC alone for 2 consecutive 1-hour collection periods. SNAP alone increased RI cGMP (P<0.05 during both experimental periods) and urinary sodium excretion (P<0.05 at 1 hour and P<0.005 at 2 hours). In contrast, when SNAP+NOC were coinfused, there was no increase in either RI cGMP or urinary sodium excretion. However, when cGMP was coinfused with SNAP+NOC, the natriuretic response to SNAP was fully restored. Similarly, NOC abolished SNAP-induced increases in the fractional excretion of Na(+) and Li(+). NOC also prevented the increase in both RI cGMP and natriuresis engendered by raising renal perfusion pressure in uninephrectomized rats, and pressure-natriuresis was re-established by coadministration of RI cGMP. As demonstrated by confocal microscopy after in vivo renal perfusion fixation, beta-tubulin was disrupted in renal cortical nephrons of kidneys infused intrarenally with NOC. These observations indicate that a functioning microtubulin network is required for the transport of cGMP into the extracellular space to modulate NO- and pressure-induced natriuresis.

Extracellular Renal Guanosine Cyclic 3′5′-Monophosphate Modulates Nitric Oxide– and Pressure-Induced Natriuresis

This study addresses the hypothesis that NO- and pressure-induced natriuresis are inhibited when guanosine cyclic 3',5'-monophosphate (cGMP) is prevented from being transported outside its renal synthesizing cells in vivo. Rats received a renal interstitial (RI) infusion of NO donor S-nitroso-N-acetylpenicillamine (SNAP) or SNAP+organic anion transporter inhibitor probenecid (PB) or SNAP+PB+cGMP. SNAP alone increased U(Na)V (P<0.05 at 1 hour and P<0.005 at 2 hours). In contrast, SNAP failed to increase U(Na)V when coinfused with PB, but cGMP coinfused with SNAP+probenecid restored the natriuretic response. SNAP alone increased RI cGMP (P<0.05) during the second experimental period. PB abolished the increase in RI cGMP in response to SNAP (P<0.01), but cGMP levels were restored by coinfusion with cGMP. PB also abolished SNAP-induced increases in fractional excretion of Na(+) (FE(Na)) and lithium (FE(Li)) (both P<0.01). PB also abolished the rise in RI cGMP and natriuresis induced by raising renal perfusion pressure (RPP) from 100 to 160 mm Hg in rats subjected to a standard pressure-natriuresis protocol and the natriuretic response was rescued by coinfusion with cGMP. RI administration of phosphodiesterase type V (PDE V) reduced both RIcGMP and U(Na)V in parallel (both P<0.01) without altering RIcAMP. The data demonstrate that export of cGMP from its renal synthesizing cells into the extracellular RI compartment is critical for the natriuretic action of NO donor SNAP or increased RPP and that RI cGMP controls basal Na(+) excretion. Extracellular cGMP modulates NO- and pressure-induced natriuresis.

Role of cGMP in natriuresis and pressure-natriuresis

Cyclic GMP (cGMP) is synthesized by renal proximal tubule (RPT) cells and is exported from these cells into the renal interstitial (RI) compartment by an organic anion transporter system. We have demonstrated that RI gGMP inhibits renal Na+ reabsorption at the renal tubule. We have further demonstrated that cGMP-induced natriuresis can be blocked by intrarenal pharmacological inhibition of protein kinase G (PKG). In order to demonstrate that RI cGMP is a potent physiological regulator of Na+ excretion, we selectively decreased RI cGMP using an RI infusion of cGMP-specific phosphodiesterase (PDE), which does not cross the cell membrane and is confined to the extracellular space because of its molecular size. cGMP PDE selectively decreased extracellular RI cGMP levels, without influencing cAMP levels, and caused significant antinatriuresis. On the other hand, blockade of cGMP degradation with selective cGMP PDE inhibitor, zaprinast, increased both RI cGMP and sodium excretion, independently of systemic or renal hemodynamic changes. Therefore, extracellular RI cGMP plays an important role in the regulation of renal Na+ excretion.

Renal Interstitial Guanosine Cyclic 3′, 5′-Monophosphate Mediates Pressure-Natriuresis Via Protein Kinase G

Pressure-natriuresis is the physiological protective mechanism whereby elevation of blood pressure induces a rapid increase in renal sodium (Na+) excretion. Pressure-natriuresis abnormalities are common to all forms of hypertension. We tested the hypothesis that pressure-natriuresis is mediated by renal interstitial (RI) cGMP and protein kinase G (PKG). We used anesthetized, uninephrectomized Sprague-Dawley rats and a standard pressure-natriuresis model in which bilateral adrenalectomy and renal denervation was done on rats. Renal perfusion pressure (RPP) was adjusted by manipulating clamps above and below the renal artery, and RI cGMP was quantified by microdialysis. RI cGMP increased from 3.1+/-0.5 to 5.5+/-0.4 fmol/min (P<0.05) when RPP was raised from 100 to 140 mm Hg. This increase in RI cGMP was eliminated by RI infusion of soluble guanylyl cyclase inhibitor 1H-[1,2,4]oxadiazolo[4,2-alpha]quinoxalin-1-one (ODQ). Raising RPP from 100 to 140 mm Hg increased urinary sodium excretion from 0.2+/-0.1 to 0.8+/-0.1 micromol/min, fractional sodium excretion from 0.2+/-0.1% to 0.8+/-0.1%, and fractional lithium excretion from 20.1+/-3.0% to 62.7+/-3.7% (all P<0.05). These responses were eliminated by RI infusion of nitric oxide synthase inhibitor N-nitro-l-arginine methyl ester, ODQ, and PKG inhibitors Rp-8-pCPT-cGMP and Rp-8-Br-cGMP. Increasing RPP from 100 to 140 mm Hg decreased fractional proximal sodium reabsorption without influencing fractional distal Na+ reabsorption or glomerular filtration rate. In conclusion, pressure-natriuresis is mediated by RI cGMP and a PKG signaling pathway in target renal proximal tubule cells.

Blood flow-dependent changes in renal interstitial guanosine 3',5'-cyclic monophosphate in rabbits.

We examined responses of renal interstitial guanosine 3',5'-cyclic monophosphate (cGMP) to changes in renal perfusion pressure (RPP) within and below the range of renal blood flow (RBF) autoregulation. A microdialysis method was used to monitor renal cortical and medullary interstitial cGMP levels in anesthetized rabbits. RPP was reduced in two steps: from ambient pressure (89 +/- 3 mmHg) to 70 +/- 2 mmHg (step 1) and then to 48 +/- 3 mmHg (step 2). RBF was maintained in step 1 but was significantly decreased in step 2 from 2.94 +/- 0.23 to 1.47 +/- 0.08 ml x min(-1) x g(-1). Basal interstitial concentrations of cGMP were significantly lower in the cortex than in the medulla (12.1 +/- 1.4 and 19.9 +/- 0.4 nmol/l, respectively). Cortical and medullary cGMP did not change in step 1 but were significantly decreased in step 2, with significantly less reduction in cGMP concentrations in the medulla than in the cortex (-25 +/- 3 and -44 +/- 3%, respectively). Over this pressure range, changes in cortical and medullary cGMP were highly correlated with changes in RBF (r = 0.94, P < 0.005 for cortex; r = 0.82, P < 0.01 for medulla). Renal interstitial nitrate/nitrite was not changed in step 1 but was significantly decreased in step 2 (-38 +/- 2% in cortex and -20 +/- 2% in medulla). Nitric oxide synthase inhibition with N(G)-nitro-L-arginine methyl ester (L-NAME, 30 mg/kg bolus, 50 mg x kg(-1) x h(-1) i.v. infusion) significantly decreased RBF (by -46 +/- 4%) and interstitial concentrations of cGMP (-27 +/- 4% in cortex and -22 +/- 4% in medulla, respectively). During L-NAME treatment, renal interstitial concentrations of cGMP in the cortex and medulla were similarly not altered in step 1. However, L-NAME significantly attenuated cGMP responses to a reduction in RPP in step 2. These results indicate that acute changes in RBF result in alterations in nitric oxide-dependent renal interstitial cGMP levels, with differential effects in the medulla compared with the cortex.

Renal interstitial cGMP mediates natriuresis by direct tubule mechanism.

The objective of this study was to test the hypothesis that renal interstitial (RI) cGMP is natriuretic in vivo. In conscious rats (n=8), urinary sodium excretion (U(Na)V) was significantly greater on days 3 and 4 of RI infusion of cGMP (1.17+/-0.14 and 1.61+/-0.11 mmol/24 h, respectively) than during vehicle infusion (0.56+/-0.15 and 0.70+/-0.17 mmol/24 h, respectively) (P<0.01). Similarly, U(Na)V was greater on days 3 and 4 of RI infusion of 8-bromo-cGMP (2.15+/-0.42 and 2.16+/-0.1 mmol/24 h, respectively). Protein kinase G inhibitor Rp-8-pCPT-cGMPS reduced cGMP-induced and 8-bromo-cGMP-induced U(Na)V to control levels. Acute RI infusion of L-arginine (L-Arg, 40 mg. kg(-1). min(-1)), but not D-arginine, caused an increase in U(Na)V from 1.65+/-0.11 to 4.07+/-0.1 micromol/30 min (P<0.01). This increase was blocked by RI infusion of N(G)-nitro-L-arginine methyl ester (100 ng. kg(-1). min(-1)) by the phosphodiesterase (PDE II) activator 5,6DMcBIMP (0.01 micromol/microL), by PDE II (0.03 U. kg(-1). min(-1)) itself, or by the soluble guanylyl cyclase inhibitor 1-H-[1,2,4]oxadiazolo-[4,2-alpha]quinoxalin-1-one (ODQ, 0.12 mg. kg(-1). min(-1)). The PDE II activator also blocked L-Arg-stimulated cGMP levels. The NO donor S-nitroso-N-acetylpenicillamine (SNAP, 0.12 micromol. L(-1). kg(-1). min(-1)) increased U(Na)V from 1.65+/-0.11 to 2.93+/-0.08 micromol/30 min (P<0.01), and this response was blocked completely by ODQ. Renal arterial but not RI administration of the heat-stable enterotoxin of Escherichia coli induced natriuresis. RA infusion of cGMP (3 microg/min) increased U(Na)V, renal blood flow (RBF), and glomerular filtration rate (GFR). Renal cortical interstitial cGMP infusion increased U(Na)V with no effect on total RBF, renal cortical blood flow, or GFR. Similarly, the natriuretic actions of renal interstitial L-Arg or SNAP were not accompanied by any change in RBF or GFR. Medullary cGMP infusion had no effect on U(Na)V, total RBF, or medullary blood flow. Texas red-labeled cGMP infused via the RI space was distributed exclusively to cortical renal tubular cells. The results demonstrate that RI cGMP inhibits renal tubular sodium absorption via protein kinase G independently of hemodynamic changes. These observations indicate that the cortical interstitial compartment provides a potentially important domain for cell-to-cell signaling within the kidney.

Endothelium-derived relaxing factor modulates renal interstitial cyclic GMP.

The arterial vasodilator activity of endothelium-derived relaxing factor (EDRF) is mediated by activation of the soluble form of guanylate cyclase, causing increased levels of guanosine-3',5'-cyclic monophosphate (cGMP). Because of its extreme lability, the actions of EDRF are local. The ability to monitor changes in renal interstitial fluid cGMP would be of great advantage in clarification of local mechanisms controlling renal function. Utilizing a renal interstitial microdialysis technique, we investigated changes in renal interstitial and urinary cGMP in response to right intrarenal arterial administration of the EDRF inhibitor, NG-monomethyl-L-arginine (L-NMMA), in anesthetized dogs (n = 5) in metabolic balance at a sodium intake of 40 mEq/day. Urine was collected directly from the right and left ureter. L-NMMA at 20-60 micrograms/kg/min significantly decreased right renal interstitial and right urinary cGMP levels (p < 0.01) without changing left renal interstitial and urinary cGMP levels (p < 0.01). L-NMMA at 100 micrograms/kg/min decreased both right and left renal interstitial and urinary cGMP levels (p < 0.01). These data demonstrate the ability to monitor renal interstitial cGMP in vivo. There was a dose-dependent decrease in renal interstitial and urinary cGMP in response to intrarenal EDRF inhibition. Additionally, they suggest that EDRF acts as a renal paracrine substance through the modulation of renal interstitial cGMP.