Cyclic guanosine monophosphate (cGMP) is an important second messenger in intracellular signal transduction (1). The synthesis of cGMP from guanosine triphosphate is catalyzed by guanylate cyclase (GC) which has two isoforms: particulate GC and soluble GC (2). Atrial natriuretic peptide (ANP) activates the particulate GC that is conjugated with the ANP receptor. ANP is well known to cause renal vasodilation, enhance glomerular filtration, and induce diuresis and natriuresis (3,4). Nitrovasodilators such as sodium nitroprusside (SNP) are considered to activate soluble GC through release of nitric oxide (NO) from their own molecules (5,6) It has been reported that SNP increases glomerular capillary hydraulic conductivity in isolated rat glomeruli (7) and that SNP suppresses sodium reabsorption in rabbit proximal tubules (8). These studies in vitro suggest that SNP can induce diuresis and natriuresis as well as ANP.
However, the effect of SNP on renal function in vivo has been controversial. For example, intravenous infusion of SNP reduces urine flow rate (UV) and glomerular filtration rate (GFR) but not urinary Na+ excretion (UNaV; 9), reduces UV and UNaV without affecting GFR (10), increases UV, UNaV, and GFR (11) in conscious rats, and intrarenal arterial infusion of SNP reduces UV and UNaV but not GFR in anesthetized dogs (12).
In this study, we examined the effects of intrarenal arterial infusion of SNP on renal hemodynamics and urine formation with indices of NO and cGMP production and compared the results with those obtained by using a specific NO donor, 1-hydroxy-2-oxo-3-(N-methyl-3-aminopropyl)-3-methyl 1-triazene (NOC 7) in anesthetized dogs.
Mongrel dogs of either sex weighing 8-18 kg were anesthetized with sodium pentobarbital (30 mg/kg, i.v.) and then intubated and artificially ventilated with room air. The cephalic veins were cannulated for drug administration. Decamethonium bromide (0.25 mg/kg, i.v.) was given to prevent spontaneous active respiratory movement. Anesthesia was maintained by a continuous intravenous infusion of sodium pentobarbital at a rate of 5 mg/kg/h throughout the experiments. Inulin, dissolved in 0.45% NaCl and 2.5% dextrose, was given intravenously at a prime dose of 50 mg/kg and at a maintenance dose of 1 mg/kg/min (0.1 ml/kg/min). The right brachial artery was cannulated for collection of arterial blood samples and measurement of mean arterial pressure with a pressure transducer (MPU-0.5; Nihon Kohden, Tokyo, Japan) and heart rate with a cardiotachometer (RT-5; Nihon Kohden). The right and left kidneys were exposed by retroperitoneal flank incisions. Catheters for urine collection were inserted into both the right and left ureters. Curved 18-gauge needles connected to silicone tubes were inserted into both the right and left renal veins to collect renal venous blood samples. All visible renal nerves were dissected away from the renal vessels and cut after ligation. Electromagnetic flow probes (2.5-3.5 mm in diameter; Nihon Kohden) were attached at the right and left renal arteries to measure renal blood flow with square-wave flowmeters (MF-27; Nihon Kohden). Curved 25-gauge needles connected to polyethylene tubes were inserted into both the right and left renal arteries for drug or vehicle infusion. Mean arterial pressure, heart rate, and renal blood flow were recorded with a polygraph system (RM-6000; Nihon Kohden). After completion of surgery, >90 min was allowed for stabilization.
The dogs were divided into three experimental groups. Approval for these studies was obtained from the Animal Experimentation Committee of Tohoku University Pharmaceutical Institute.
When renal blood flow and urine flow rate reached constant levels for more than three consecutive monitoring periods (10 min each), urine and blood samples for basal values were obtained in both the kidneys. Urine was collected over a 10-min period, and arterial and renal venous blood were withdrawn simultaneously at the midpoint of urine collection. Then SNP (experiment 1, n = 6: 10, 30, and 100 ng/kg/min for 40 min each; experiment 2, n = 7: 1,000 ng/kg/min for 40 min), or NOC 7 (experiment 3, n = 7:300 ng/kg/min for 40 min) was infused into the right or left renal artery. Vehicle for SNP (experiment 1 and 2, 0.9% saline) or NOC 7 (experiment 3, 0.01 M NaOH) was simultaneously infused into the renal artery (0.1 ml/min) of the contralateral control kidney. Beginning at 20 min after the start of infusion at each dose, two consecutive 10-min urine collections and blood samplings were performed in both the kidneys. Then 60 min after stopping the drug infusion, urine and blood samples for recovery values were obtained in experiments 2 and 3. The values obtained before drug infusion, during the drug infusion, and after stopping the drug infusion are expressed as "Basal," "SNP" or "NOC 7," and "Recovery," respectively, in the tables and figures. The values during drug infusion are averages of the values obtained in two consecutive sampling periods during the infusion at each dose.
Blood samples were transferred to chilled tubes containing diammonium EDTA (5-10 mg/ml blood) and then centrifuged to obtain plasma samples. Glomerular filtration rate was determined as inulin clearance (13). Inulin concentration in plasma and urine was measured by the anthrone method. Na+ and K+ were measured by flame photometry (775A; Hitachi, Tokyo, Japan). Plasma osmolarity and urine osmolarity were measured by the freezing point-depression method (OM801; Vogel, GmbH & Co KG, Postfach, Germany). Urinary nitrite + nitrate (NOx) level was determined by Griess reaction, as reported by Süto et al. (9) with slight modifications. In brief, urine samples were incubated with phosphate buffer (pH 7.5), nitrate reductase (Sigma Co., St. Louis, MO, U.S.A.) and β-NADPH (Sigma) for 1 h at room temperature. After the Griess reagent was added, samples were transferred to a 96-well plate, and absorbance at 546 nm was measured by an plate reader (ER-8100; Sanko Junyaku Co, Ltd., Tokyo, Japan). Plasma and urinary cGMP concentration were measured by radio immunoassay kit (Diagnostics Division, Yamasa Corp., Tokyo, Japan).
SNP (Sigma) was dissolved in 0.9% saline. NOC 7 (Dojindo Laboratories, Kumamoto, Japan) was dissolved in 0.01 M NaOH.
All values are expressed as means ± SEM. Data for urine formation were transformed to logarithms to obtain normal distribution before application of statistical procedures. Statistical differences were evaluated by analysis of variance (ANOVA) for single-factor repeated measures and Dunnett's test. Differences at p < 0.05 were considered to be statistically significant.
In experiment 1, SNP infused into the renal artery at 10, 30, and 100 ng/kg/min did not cause statistically significant changes in urine formation (Table 1) or urinary excretions of NOx and cGMP (UNOxV and UcGMPV, respectively, Table 2) either in the ipsilateral SNP-infused kidney or in the contralateral vehicle-infused kidney (control kidney). One animal of six had extremely high UNOxV values (>10 nmol/min) during SNP infusion (30 and 100 ng/kg/min) in the control kidney, which resulted in large mean and standard error values shown in Table 2.
Mean arterial pressure and heart rate were slightly reduced during the SNP infusion (Table 1). Because renal blood flow remained unchanged, renal vascular resistance was reduced both in the SNP-infused kidney and the control kidney during the infusion of SNP (100 ng/kg/min; Table 1).
In experiment 2, intrarenal arterial infusion of SNP at 1,000 ng/kg/min reduced mean arterial pressure with a slight increase in heart rate. Renal vascular resistance was reduced both in the SNP-infused kidney and the control kidney during the infusion of SNP (Table 3).
Urine flow rate was reduced both in the SNP-infused kidney and the control kidney during the SNP infusion (Fig. 1), but the reduction in the SNP-infused kidney (18 ± 9% reduction from the basal values) was significantly smaller than that observed in the control kidney (33 ± 6%; p < 0.01). The SNP infusion did not affect urinary Na+ excretion or fractional Na+ excretion and tended to increase urinary NOx excretion in the SNP-infused kidney (Figs. 1 and 2), but the values of these urinary parameters decreased in the control kidney (Figs. 1 and 2).
The SNP infusion increased urinary cGMP excretion (UcGMPV) and renal venous plasma cGMP concentration (PcGMPv) in the SNP-infused kidney (Fig. 2). UcGMPV and PcGMPv in the control kidney and arterial plasma cGMP concentration tended to be increased during the SNP infusion, but the changes were not statistically significant (Fig. 2).
There was no statistically significant change in glomerular filtration rate, filtration fraction, or free water reabsorption in experiments 1 and 2 (Tables 1-3). Osmolar clearance was decreased during the SNP infusion in the control kidney but not in the SNP-infused kidney. Urine osmolarity was increased during the SNP infusion at 1,000 ng/kg/min (Table 3).
In experiment 3, NOC 7 infused into the renal artery at 300 ng/kg/min reduced mean arterial pressure (Table 4). Renal blood flow was increased during the infusion of NOC 7 in the ipsilateral NOC 7-infused kidney but not in the control kidney. Renal vascular resistance was decreased during the NOC 7 infusion in both the NOC 7-infused kidney and the control kidney (Table 4).
Urine flow rate, urinary Na+ excretion, and fractional Na+ excretion in the control kidney were reduced during the NOC 7 infusion, whereas in the NOC 7-infused kidney, these values remained unchanged (Fig. 3).
Urinary NOx and cGMP excretion were increased during the infusion of NOC 7 in the NOC 7-infused kidney but not in the control kidney. Renal venous plasma cGMP concentration was increased during the NOC 7 infusion in both the NOC 7-infused kidney and the control kidney (Table 4), but the increase in the NOC 7-infused kidney (120 ± 27% increase from the basal values) was significantly higher than the increase observed in the control kidney (18 ± 6%; p < 0.05).
Osmolar clearance and free water reabsorption were decreased in the control kidney but not in the NOC 7-infused kidney (Table 4). Arterial plasma cGMP concentration, glomerular filtration rate, filtration fraction, and urine osmolarity were not significantly changed during the NOC 7 infusion (Table 4).
The aim of this study was to examine whether an authentic nitrovasodilator, SNP, releases NO to enhance cGMP production and thereby induces natriuresis in the dog denervated kidney. SNP was infused into the renal artery, and renal responses in the ipsilateral SNP-infused kidney were compared with the responses in the contralateral vehicle-infused kidney (control kidney).
SNP infusion at increasing doses of 10, 30, and 100 ng/kg/min (experiment 1) did not cause a statistically significant change in urinary parameters or NO-cGMP productions, although SNP infusion at 100 ng/kg/min slightly reduced mean arterial pressure and renal vascular resistance in both the SNP-infused and the control kidneys. The reduction in vascular resistance may be due to the autoregulation of renal circulation, which is not related to renal effects of SNP. SNP at these doses does not seem to affect renal NO or cGMP level.
We therefore applied the higher dose of SNP (1,000 ng/kg/min) to clarify the renal effects of SNP (experiment 2). This dose of SNP significantly reduced mean arterial pressure with reflex tachycardia. These findings are in agreement with earlier works (14,15). SNP escaped from the renal circulation may cause systemic vasodilation via the NO-cGMP pathway. Despite the systemic hypotension, statistically significant increase of arterial plasma cGMP concentration was not observed during the SNP infusion. It is possible that cGMP production occurs at the tissue level, which could not be detected by the measurement of cGMP in arterial plasma samples (16).
The SNP-induced hypotension makes it difficult to evaluate direct renal effects of SNP. This may have caused conflicting observations reported in previous studies (9-12). In our study, however, comparison of the data between the SNP-infused kidney and the contralateral control kidney (although the drug is delivered to some extent), which has not been done in the previous studies, could detect possible renal actions of SNP.
Urine flow rate was reduced both in the SNP-infused kidney and the control kidney during the SNP infusion. In this study, these kidneys were surgically denervated to rule out the possibility that changes in renal sympathetic nerve activity, which may be increased by systemic hypotension, influence renal functions. Previous studies from our laboratory suggest that the surgical denervation abolishes the changes in the renal nerve activity, because systemic hypotension during prazosin or ANP infusion did not alter basal renal norepinephrine release (17,18). Therefore the antidiuretic response during systemic hypotension may result from a reduction in renal perfusion pressure rather than reflex activation of the renal sympathetic nervous system. The reduction in renal perfusion pressure reduces both the glomerular filtration pressure and the peritubular capillary pressure. Because the glomerular filtration rate remained unchanged, the antidiuretic response observed in this study may be due to a reduction in peritubular capillary pressure, which could facilitate tubular reabsorption of water and sodium. However, the antinatriuretic response in the SNP-infused kidney was significantly smaller than that observed in the control kidney. Neither urinary Na+ excretion nor fractional Na+ excretion was reduced in the infused kidney, whereas in the control kidney, significant reductions in urinary Na+ excretion and fractional Na+ excretion were observed during the SNP infusion. These results suggest that SNP maintains urine formation against the antinatriuresis resulting from the SNP-induced systemic hypotension.
It should be noted, however, that the intrarenal arterial infusion of SNP at 1,000 ng/kg/min may increase SNP concentration over the range that would be achieved by intravenous infusion at usual clinical doses. Thus the clinical significance of these results is unclear.
SNP is considered to produce NO and thereby activate soluble Gc, thus increasing cGMP (2,5,6). In our study, urinary NOx excretion tended to be increased in the infused kidney but was decreased in the control kidney during the SNP infusion at 1,000 ng/kg/min. It is possible that SNP can release NO in the kidney, but the systemic hypotension enhances tubular NOx reabsorption, as suggested by Süto et al. (9).
The SNP infusion increased urinary cGMP excretion and renal venous plasma cGMP concentration in the SNP-infused kidney, whereas these values did not change in the control kidney. Although it has been unclear whether the changes in urinary and plasma cGMP levels correctly reflect the intracellular cGMP production, this result at least indicates that the SNP infusion can activate the renal cGMP system. Maintenance of the urine formation during the SNP infusion could be ascribable to the enhanced renal NO and cGMP production. It has been reported that NO inhibits sodium flux in the cortical collecting duct by inhibition of amiloride-sensitive sodium channels (19) and that cGMP attenuates sodium reabsorption in renal tubule by inhibition of Na+,K+-ATPase (20). Thus it is possible that SNP attenuates sodium reabsorption by activating the NO-cGMP-mediated mechanisms.
It was reported that SNP is not a specific NO releaser (21,22). Therefore we also examined the effects of a specific spontaneous NO donor NOC 7 (23; experiment 3). The intrarenal arterial infusion of NOC 7 (300 ng/kg/min) reduced mean arterial pressure by the same degree as SNP (1,000 ng/kg/min) and also retained sodium and water excretion against the hypotension with activating cGMP production. NOC 7 increased renal blood flow and urinary NOx excretion in the ipsilateral NOC 7-infused kidney, although SNP did not cause these changes. It is reported that NOC 7 produces NO more potently than the other classic NO donors (23). This may explain these difference between SNP and NOC 7.
In summary, our study demonstrates that in anesthetized dogs (a) SNP can produce NO and activate cGMP production in the kidney, (b) SNP attenuates sodium reabsorption, and (c) the natriuretic property of SNP may be masked by systemic hypotension.
Acknowledgment: This work was supported in part by Grant-in-Aid for Scientific Research (Japan) No. 08457632.
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