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Compared with the adult, the diuretic and natriuretic response of the neonate to infusion of ANP is limited(1). Moreover, after acute volume expansion, the neonatal renal excretory response is also reduced compared with that of the adult(2). As a result of interaction with its biologically active (GC-A) receptor, ANP induces the generation of cGMP, its second messenger. cGMP, in turn, can inhibit tubular sodium reabsorption and mediate the natriuretic action of ANP(3–7). We have shown that, after either the infusion of ANP or acute volume expansion, the urinary excretion of cGMP is significantly less in the neonate than in the adult(8, 9). In addition, the extracellular generation of cGMP by glomeruli isolated from neonatal rats is significantly less than that of adults(10). This effect appears to be related to an immaturity of an organic anion transporter responsible for egression of cyclic nucleotides from the intracellular to the extracellular space(10).

The regulation of plasma ANP concentration is dependent both on the rate of release from cardiac myocytes, and on the rate of metabolic clearance from the circulation. The latter is mediated by neutral endopeptidase 24.11, and by C-receptors that greatly outnumber the GC-A receptors, and are responsible for regulation of plasma ANP in most physiologic states(11). The C-receptors are internalized and recycled in constitutive fashion, and after internalization, ANP is subjected to lysosomal hydrolysis(12). We have demonstrated previously that at least part of the attenuated neonatal renal response to ANP is due to more rapid clearance of ANP from the circulation, which is mediated by greater activity of the C-receptors(13).

After an initial postnatal natriuresis, the neonate is in a state of positive sodium balance that is necessary for normal somatic growth(1). Using a technique of artificial rearing, we have reported recently that dietary sodium supplementation in preweaned rats increases cardiac storage of ANP, and enhances the renal diuretic and natriuretic response to acute volume expansion(9, 14). Although chronic sodium loading increases basal plasma ANP concentration in neonatal rats, postexpansion plasma ANP concentration is not affected by dietary sodium intake(9). The present study was designed to test two hypotheses: first, that increased dietary sodium in the preweaned rat reduces the activity of clearance receptors, and thereby contributes to enhanced natriuresis. Because infusion of the inhibitor increases plasma ANP concentration by blocking access of endogenous ANP to the C-receptors, the experiments were designed to test also a second hypothesis: that the renal physiologic response to ANP is augmented by increased dietary sodium in the neonate.

METHODS

Experiments were performed in 51 Sprague-Dawley rats 13-16 d of age(Hilltop Laboratories, Scottsdale, PA). All studies were approved by the Animal Research Committee of the University of Virginia. Nineteen rats were used for physiologic studies, 14 for determination of plasma ANP without infusion of C-receptor inhibitor, and 18 for glomerular cGMP determinations. Gastrostomy feeding tubes were inserted into 7-d-old preweaned male and female rat pups anesthetized with methoxyflurane as described previously(14). After recovery from anesthesia, a specially formulated diet, similar to rat milk, was fed by intermittent infusion for 12 20-min periods each 24 h for 7 d as described elsewhere(14). The diet contained either normal (25 mEq/L) or high (145 mEq/L) sodium. Animals were housed in individual containers in a temperature (38-40°C) and light-controlled (12-h light/dark) environment. Rats were weighed daily, and the volume of formula was adjusted to provide 33% of the average body weight every day.

Physiologic protocols. After the period of artificial rearing, 19 animals were anesthetized with intraperitoneal pentobarbital (50 mg/kg of body weight), and were placed on a thermostatically controlled heating table. After tracheotomy, a carotid artery was cannulated for blood withdrawal and monitoring MAP as described previously(9). A jugular venous catheter was inserted for infusion of 0.85% NaCl at 3 mL/kg/h, and a suprapublic catheter was inserted into the bladder for urine collection. After a 45-min equilibration period, rat ANP (Peninsula Laboratories, Inc., Belmont, CA), 35 ng/kg/min, was infused i.v. throughout the experiment(Fig. 1). This was done to control for any variability in release of endogenous ANP into the circulation. We have reported previously that the plasma ANP concentration in rats receiving ANP at 35 ng/kg/min did not differ from that of animals receiving saline vehicle alone(13).

Figure 1
figure 1

Scheme of experimental protocol. After an equilibration period, four 20-min urine collections were obtained. Rat ANP was infused throughout all four periods at 35 ng/kg/min to control for variability in endogenous ANP release. After the first two baseline urine collections, the C-receptor inhibitor, ANF(4-23), was infused at 50 μg/kg/min throughout the third and fourth urine collections. In separate groups of rats, blood was obtained for ANP concentration either at the end of the second or fourth period.

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Two 20-min urine collections were obtained, after which an i.v. infusion of C-receptor inhibitor, C-ANF(4-23) (Peninsula Laboratories, Inc., Belmont, CA), was begun at 50 μg/kg/min, and was continued for the remaining two urine collection periods (Fig. 1). This dose was selected because our previous studies indicated that it effectively blocks C-receptor activity in the neonatal rat(13). Variables measured during each collection period included MAP, heart rate, and urine volume. Blood (100 μL) was withdrawn in heparinized capillary tubes over 10-15 s from the arterial catheter at the midpoint of each 20-min urine collection, and hematocrit was determined by centrifugation. Plasma was stored at-70°C until assayed. An equal volume of 4% BSA was infused to replace all blood withdrawn. At the end of the fourth period, 1 mL of blood was collected in EDTA, centrifuged at 4°C, and stored at -70°C for plasma ANP determination. Blood (1 mL) was also collected in 14 additional rats(n = 7, normal and high sodium respectively), following the identical protocol described in Fig. 1, with the exception that animals were killed at the end of the second period, and C-receptor inhibitor was not infused. Although the volume of blood withdrawn(1 mL) represents a significant fraction of the animal's blood volume, we have shown previously that, after infusion of C-ANF or acute volume expansion, the expected changes in plasma ANP concentration can be detected in plasma samples obtained from neonatal rats in this manner(13, 15). Animals were killed by infusion of an overdose of sodium pentobarbital, and kidney weights were measured after renal decapsulation.

Studies in isolated glomeruli. Eighteen additional rats underwent the same artificial rearing procedure used for physiologic studies, but were killed by sodium pentobarbital injection for removal of the kidneys. All glomerular cGMP assays were performed on the day of harvesting the kidneys. Kidneys were excised, placed in 4°C modified Dulbecco's PBS, decapsulated, and demedullated. Renal cortex was minced, incubated with collagenase and DNase I, and subjected to sieving to isolate glomeruli as described previously(10). Kidneys from three rats in each group were pooled for each determination. Briefly, isolated glomeruli were maintained at 4°C throughout the sieving procedure, and were resuspended in fresh, enzyme-free Dulbecco's buffer. For each determination, aliquots of 2000 glomeruli each were preincubated for 15 min at 37°C in the presence of 0.5 mM 3-isobutyl-1-methylxanthine. Glomeruli were then incubated for 60 min with either 0.1 μM ANP or buffer. The dose of ANP was chosen based on dose-response studies previously reported, which revealed that maximal stimulation of cGMP release is achieved at 0.1 μM(16).

Assays. Urine and plasma sodium concentrations were measured by flame photometer as described previously(9). Plasma ANP and urinary cGMP concentrations were measured by RIA as described elsewhere(8). The intraassay coefficient of variation for the ANP assay was 6.7 ± 0.3%, and the interassay coefficient of variation was 12.7%. In preliminary studies, the anti-ANP antibody used (Peninsula Laboratories, Belmont, CA) was found to have less than 1% cross-reactivity with C-ANF. Extracellular and intracellular concentrations of cGMP in isolated glomeruli were measured by RIA as described previously(10). The intraassay coefficient of variation for the cGMP assay was 11.3 ± 1.4%.

Statistical analysis. Data are expressed as mean ± SEM. Comparisons between periods within each group were made by Friedman repeated measures analysis of variance followed by Student-Newman-Keuls pairwise multiple comparison. For variables measured in physiologic studies, comparisons between groups for each period were made by unpaired t test or by the Mann-Whitney rank sum test for data not normally distributed. Comparisons between groups for measurements of cGMP in isolated glomeruli were made by Wilcoxon signed rank test, as assays were performed in pairs of normal and high salt groups.

RESULTS

Body weight at the time of study was 30.9 ± 1.0 g for control normal sodium diet rats, and 31.3 ± 0.6 g for the high sodium group(p = not significant). There was also no difference in kidney weight between groups: the left kidney weight was 169 ± 5 mg and 179 ± 4 mg for control and high sodium groups, whereas right kidney weight was 174± 4 and 185 ± 4 mg for control and high sodium groups, respectively. As shown in Table 1, heart rate did not differ between groups and did not change throughout the experiment in either group. Hematocrit did not differ between groups and decreased in both groups during periods 3 and 4. Although, as shown in Table 1, the differences were not significant for each period, throughout the experiment (mean of periods 1-4), the plasma sodium concentration was greater in high sodium (148 ± 2 mEq/L) than normal sodium groups (136 ± 2 mEq/L) (p < 0.001). There was no significant change in plasma sodium concentration during the experiment in either group.

Table 1 Characteristics of rats
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As shown in Figure 2, MAP tended to be higher in normal than in high sodium rats throughout the experiment, but differences were not significant. The MAP decreased during infusion of C-receptor inhibitor regardless of sodium intake.

Figure 2
figure 2

MAP during each urine collection period. Solid line, normal sodium diet; dashed line, high sodium diet.*p < 0.05 vs periods 1 or 2, same group.

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As shown in Figure 3, infusion of C-receptor inhibitor resulted in a 10-fold increase in plasma ANP concentration (p < 0.001). Although levels tended to be higher in control than in high sodium groups, differences between dietary treatment groups were not statistically significant. The antibody used in the ANP assay demonstrated less than 1% cross-reactivity with C-ANF (data not shown).

Figure 3
figure 3

Plasma ANP concentration immediately after urine collection 2 (Baseline); and after urine collection 4(C-receptor inhibitor). Solid bar, normal sodium diet;hatched bar, high sodium diet. n = 7 each group.

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Compared with baseline periods 1 and 2, there was a significant increase in urine flow immediately after the beginning of the C-ANF infusion (period 3) in the high sodium, but not the normal sodium, group (Fig. 4). Median urine sodium excretion throughout the experiment (periods 1-4) was higher in the high than the normal sodium groups (p < 0.001). Urine sodium excretion increased with C-ANF infusion (period 3versus 2) in each of the 10 rats receiving a high sodium diet(p < 0.05), but increased in only 4 of 9 in the normal sodium group (p = not significant) (Fig. 5). Urinary cGMP excretion also was significantly greater after C-receptor inhibitor infusion only in rats receiving high sodium intake (Fig. 6).

Figure 4
figure 4

Urine flow rate during each urine collection period.Solid line, normal sodium diet; dashed line, high sodium diet. *p < 0.05 vs periods 1 or 2, same group.

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Figure 5
figure 5

Urinary sodium excretion during each urine collection period. Solid line, normal sodium diet; dashed line, high sodium diet. *p < 0.05 vs periods 1 or 2, same group.

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Figure 6
figure 6

Urinary cGMP excretion during each urine collection period. Solid line, normal sodium diet; dashed line, high sodium diet. *p < 0.05 vs periods 1 or 2, same group;#p < 0.05 vs normal sodium group, same period.

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After incubation of isolated glomeruli in buffer, there was no difference between groups in the accumulation of either extracellular or intracellular cGMP (Fig. 7). However, after incubation with 0.1 μM ANP, stimulation of extracellular cGMP generation was significantly greater in high sodium than normal sodium rats (Fig. 7A). There were no differences in intracellular cGMP sodium generation after stimulation with ANP (Fig. 7B).

Figure 7
figure 7

(A) Extracellular generation of cGMP by isolated glomeruli from preweaned rat pups receiving normal (solid bars) or high sodium (hatched bars). Glomeruli were incubated for 60 min with either buffer alone or buffer containing ANP, 0.1 μM.*p < 0.05 vs glomeruli from normal sodium group.(B) Intracellular generation of cGMP.

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DISCUSSION

The major finding of the present study was a significant increase in diuresis and natriuresis after infusion of C-receptor inhibitor in the high sodium, but not the normal sodium, neonatal rats. This was associated with an increase in glomerular cGMP release and in urinary cGMP excretion. These effects occurred in response to marked elevations in plasma ANP concentration, which tended to be lower in high sodium than in normal sodium rat pups. These results indicate that, although sodium intake may not modulate C-receptor activity, sodium intake affects the renal excretory response to circulating ANP in the neonate.

The first hypothesis for the present study was that increased sodium intake in the neonate decreases C-receptor activity. Infusion of C-ANF resulted in an 8-10-fold increase in plasma ANP concentration in both groups of rat pups. This indicates a high level of C-receptor activity in the neonatal rat and confirms previous findings(13). Although C-receptor inhibition tended to increase the plasma ANP concentration more in normal sodium than in high sodium rat pups, the difference was not significant. Thus, although the results suggest a decreased C-receptor activity in the high sodium group, additional studies will be needed to confirm this. The measured increase in plasma ANP concentration did not spuriously result from antibody cross-reactivity of ANP and C-ANF, as C-ANF was not detected by the RIA. Although there are no data regarding pharmacokinetics of the volume of distribution of ANP in neonatal compared with adult rats subjected to inhibition of C-receptors, infusion of C-ANF at 50 μg/kg/min results in plasma ANP concentration in neonatal rats that is double that in adults(13). It is unlikely that this dose of C-ANF is significantly interfering with degradation of ANP by neutral endopeptidase 24.11, the other known pathway for clearance of ANP, because combined infusion of C-ANF at this dose and phosphoramidon (an inhibitor of neutral endopeptidase) results in a further 3-fold increase in plasma ANP concentration(13).

The second hypothesis for the present study was that the renal physiologic response to ANP in the neonate is augmented by increased dietary sodium. The greater diuretic and natriuretic responses to increased circulating ANP after C-ANF infusion in high sodium rat pups supports this. It has been well established that the natriuretic actions of ANP are mediated by cGMP(3–7). In the present study, ANP-stimulated glomerular release and urinary excretion of cGMP were significantly greater in the high sodium group. Others have shown that sodium supplementation of cultured endothelial cells decreases C-receptor number, and greatly increases the ANP-stimulated production of cGMP(17). This occurs independently of binding of the C-receptors with C-ANF, so that the effect is not due to the“clearance” function of the C-receptor(17). It is also independent of change in GC-A receptor number(17).

We have shown that, in the presence of 3-isobutyl-1-methylzanthine, intracellular cGMP increases 5 min after addition of 0.1 μM ANP to isolated glomeruli(10). However, after 1 h of incubation with 0.1 μM ANP (without 3-isobutyl-1-methylzanthine), extracellular, but not intracellular, cGMP is increased(10). We have shown previously that, compared with the adult, reduced release of cGMP by neonatal glomeruli stimulated by ANP is due in part to slower extrusion of cGMP out of glomerular cells due to immaturity of an organic anion transporter(10). The increased extracellular accumulation of cGMP by glomeruli from high sodium rat pups therefore may be due to stimulation of the transporter that is responsible for extrusion of cGMP out of the cells(10). Conversely, the lack of effect of sodium intake on ANP-stimulated glomerular intracellular cGMP in the present study is likely due to greater extrusion of cGMP out of glomeruli from high sodium rats. We have reported recently that extracellular cGMP can reduce sodium transport by LLCPK1 renal tubular epithelial cells, and that inhibition of cGMP extrusion out of the cells can abolish the effect(18). This raises the possibility that glomerular cGMP released after stimulation with ANP may inhibit sodium transport by tubular segments downstream. Therefore, in high sodium rat pups, the greater ANP-stimulated glomerular cGMP release, and the greater C-ANF-induced urinary cGMP excretion may be due at least in part to increased extracellular cGMP levels.

After an initial postnatal diuresis, the neonate, whose sodium intake is limited to that present in maternal milk, avidly retains sodium which is necessary for somatic growth(1). Infusion of ANP in humans with chronic sodium deficiency results in blunted excretion of cGMP and attenuated diuresis and natriuresis(19). Chronic salt deprivation in adult rats can prevent the expected actions of ANP on sodium reabsorption in the medullary collecting duct(20). As this response is similar to that of rat pups receiving normal sodium intake, the present findings are consistent with the hypothesis that the normal neonate is in a state of relative sodium deprivation. Moreover, water-deprived rats develop an increase in glomerular C-receptor density, whereas activity of the biologically active receptor is unchanged(21).

With respect to the renal response to ANP, there are remarkable similarities between the normal neonate and other sodium-retaining states. Natriuresis after infusion of ANP is markedly blunted in rats with experimental nephrosis(22), and glomerular generation of cGMP is also attenuated(23). Natriuresis after ANP infusion is also suppressed in rats with experimental heart failure(24), and glomerular cGMP production is significantly reduced(25). Density and binding affinity of biologically active renal ANP receptors are unaffected either by nephrosis or congestive heart failure(23, 24). It would appear that a reduction in ANP-stimulated renal generation or release of cGMP contributes to sodium retention in response to a variety of stimuli.

The renin-angiotensin system is activated in the neonate as well as in other sodium-retaining states(26). The attenuation of natriuresis could therefore result also from the inhibitory effect of angiotensin II on cGMP accumulation produced by ANP(27–29). In addition, the renin-angiotensin system in neonatal rats is more responsive to chronic sodium loading than that of the adult(30). This may account for the dramatic effects of chronic sodium loading on the renal response to C-receptor inhibition in the present study. An attenuated glomerular response to ANP in sodium-deprived rats may in fact result from increased angiotensin II, which increases intracellular calcium concentration(31).

Preweaned rat pups receiving high sodium intake were hypernatremic. Sodium loading of weanling rats also results in hypernatremia, although extracellular volume is not increased(30). In the present study, because animals in both groups received the same fluid intake, and body weights were not different, the increased serum sodium concentration is not due to volume contraction caused by greater water losses in the high sodium group. It is likely, therefore, that hypernatremia results from persistently enhanced renal sodium reabsorption despite the increased natriuretic response to ANP.

In summary, inhibition of ANP C-receptors increased plasma ANP concentration in normal and sodium-loaded neonatal rats. The elevation in plasma ANP concentration did not affect urine flow, sodium excretion, or urinary cGMP excretion in rat pups receiving a normal intake. However, in animals receiving a high sodium diet, the increased plasma ANP levels due to C-receptor inhibition were associated with a transient diuresis, sustained natriuresis, and increased urinary cGMP excretion. In addition, glomeruli isolated from rat pups receiving high sodium intake released greater amounts of cGMP after stimulation with ANP. It is likely that the greater renal response to ANP resulting from sodium loading contributes to the enhanced diuretic and natriuretic response to acute volume expansion reported by us previously in similarly treated preweaned rat pups(9). Additional studies will be required to elucidate the regulatory mechanisms underlying this response to dietary sodium. Because previous studies failed to show any effect of sodium or cGMP on binding characteristics of the GC-A receptor(17, 32, 33), a postreceptor effect is likely.