Sodium Excretion in Response to Vasopressin and Selective Vasopressin Receptor Antagonists

Dose-Response Curves of the V2R Agonist and Antagonist (Experiments A and B)

The V2R agonist 1-desamino 8-d-arginine vasopressin (dDAVP) showed the expected antidiuretic action, inducing a marked decline in urine flow rate (V) and a rise in urine osmolality (U_osm). The V2R antagonist showed the expected aquaretic action (Figure 1). In addition, both drugs also influenced solute excretion rates. Urea excretion rate fell with increasing V2 agonism, as already widely known. Sodium excretion rate declined, and potassium excretion rate increased by approximately 30 to 50% with the last four doses of dDAVP. Of note, the effects of dDAVP on V and U_osm reached a maximum for the lowest dose used here, whereas the effects on solute excretion rate showed a progressive dose-dependent increase. In response to V2R blockade, sodium excretion rate rose dose-dependently from 0.1 mg/kg body wt (BW) onward. Note that water excretion already rose three-fold with the previous dose (0.03 mg/kg BW), although not yet significant. With the largest dose of antagonist, sodium excretion rate was increased six-fold when water excretion (i.e., V) was increased 25-fold (Figure 1). Potassium excretion rate rose with the V2R antagonist (with, however, large interindividual variation preventing changes to reach statistical significance in most of the dose groups); however, the transtubular potassium gradient (TTKG), an index of the active secretion of potassium in the distal nephron, rose only with dDAVP (Figure 1).

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Figure 1.

Dose-dependent effects of the V2R agonist (dDAVP) and the V2R antagonist (SR121463A) on fluid and solute excretion rates (Experiments A and B, n = 4 to 6 rats per dose). Results are expressed as Exp/Basal. Thin lines illustrate the dose-dependent effects. Paired t test, experimental versus basal: *P < 0.05; **P < 0.01; ***P < 0.001.

Dose-Response Curve of AVP and Effects of the V1aR Antagonist on the Response to AVP (Experiments C and D)

When given at a dose of 1 μg/kg, the natural hormone AVP increased U_osm and reduced V by approximately 35% (Figure 2). The difference for U_osm did not reach significance because of large interindividual variation in the response. A tendency for a dose-dependence of the antidiuretic response is visible when considering the control and the two lowest doses (thin line), but this effect disappears with higher doses (Figure 2). Sodium excretion rate was modestly reduced along with the antidiuretic effect at 1 μg/kg but rose markedly with higher doses of AVP, whereas urea excretion fell. The highest dose increased potassium excretion rate and TTKG by 40% (Figure 2).

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Figure 2.

Dose-dependent effects of AVP on fluid and solute excretion rates (Experiment C, n = 4 to 6 rats per dose). Results are expressed as Exp/Basal. Thin lines illustrate the dose-dependent effects. Paired t test, experimental versus basal: *P < 0.05; **P < 0.01; ***P < 0.001.

To evaluate whether the loss of the antidiuretic effect of AVP and the appearance of a natriuretic effect at higher doses are due to V1aR stimulation, we conducted additional experiments with co-administration of AVP 15 μg/kg BW and a selective V1aR antagonist. In preliminary experiments, the effects of the V1aR antagonist given alone at various doses (0.1, 1, and 10 mg/kg BW) were evaluated. Dose-dependent significant increases in U_osm were seen with all three doses, whereas significant decreases in V and sodium excretion occurred in some but not all rats with 10 mg/kg BW (data not shown). These changes suggest that the V1aR antagonism suppressed a modest diuretic and natriuretic influence of endogenous AVP. In experiment D, two doses of AVP were tested. AVP at 3 μg/kg was antidiuretic, lowering V by 27% (P < 0.01) and increasing U_osm by 33% (P < 0.001), and induced no change in sodium excretion rate. With a five-fold higher dose (15 μg/kg), the antidiuretic effect was completely lost and a marked natriuretic effect was observed, as depicted in Figure 3. The co-administration of the V1aR antagonist at 10 mg/kg BW with AVP 15 μg/kg BW largely abolished the natriuretic effect and restored an antidiuretic effect close to that observed with dDAVP (Figure 3) and with AVP 3 μg/kg.

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Figure 3.

Effects of AVP (AVP-15,15 μg/kg BW) on urine flow rate and osmolality and on sodium excretion rate without or with the co-administration of the V1aR antagonist (Experiment D, n = 4 to 6 rats per dose). The effects of the antagonist alone and of dDAVP are also shown. Results of the experimental day are expressed as percentage of values observed during the basal day in the same rats. Paired t test, experimental versus basal: *P < 0.05; **P < 0.01; ***P < 0.001.

Urinary AVP Concentration in Different Experimental Conditions (Experiment E)

Luminal effects of AVP in the renal CD have been described.^14,15 This prompted us to evaluate urinary AVP concentration (U_AVP) that should, at least approximately, vary in proportion to its concentration in the lumen of the CD. Twenty-four-hour dehydration increased U_AVP approximately 20-fold. Administration of AVP 3 μg/kg BW increased U_AVP to a similar extent, whereas dDAVP did not change it (Table 2). A five-fold higher dose of AVP (15 μg/kg BW) increased U_AVP close to five times more. This high luminal AVP concentration was associated with an increase in diuresis and natriuresis (as in experiment D) and a decline in U_osm such that V was higher and U_osm was lower than those observed in rats receiving a smaller dose of AVP or in water-deprived rats. Several previous studies already observed this simultaneous increase in sodium excretion and urine flow rate after AVP infusions.^4,6,8 In these experiments, it would have been difficult to evaluate the mean plasma AVP (P_AVP) level reached during 6 h (duration of the urine collection). Measurements of the AVP excretion rate provide more integrated information during this period. Previous studies showed that urinary AVP excretion rate is roughly proportional to the plasma level when osmolar excretion remains stable.¹⁶ Thus, given the results presented in Table 2, it may be assumed that administration of 1 to 3 μg/kg BW AVP increased P_AVP to levels similar to those seen after 24 h of water deprivation, whereas the dose of 15 μg/kg BW increased P_AVP to higher values, reached only during exogenous vasopressin infusion.

Effects of the V2R Antagonist on Sodium and Water Excretion during the Whole 24 h (Experiment B)

Selective V2R antagonists are usually reported to increase markedly urine output without affecting solute excretion. Because a significant increase in sodium excretion rate was observed in these experiments, it was interesting to determine whether this change was short-lived or sustained. Collection of urine for the 18 h after the initial 6-h collection and calculation of the antagonist's effects during these two aggregated periods showed that the aquaretic but not the natriuretic effect remained detectable over 24 h (Figure 4). Most probably, thirst and thus fluid intake increased during the whole duration of V2R blockade and resulting aquaresis. After the initial loss of sodium, compensatory sodium retention occurred in the subsequent hours, bringing back sodium balance to zero. A similar compensation also occurred for potassium and urea. Note that with the highest dose of the antagonist, sodium retention occurred during the 6- to 24-h period despite a persisting increase in aquaresis (Figure 4). It is conceivable that the natriuretic effect induced by the V2 antagonist could be sustained over the whole 24 h, along with the aquaresis, if the rats could compensate the initial salt loss (that probably stimulates their salt appetite) by having access to a source of salt independent of the food, as they do for water.

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Figure 4.

Dose-dependent effects of the V2R antagonist on urine flow rate and osmolality and on sodium excretion rate in the first 6 h after drug administration, in the subsequent 18 h (6 to 24 h), and in the whole 24 h during the experimental day (basal day not shown; Experiment B, n = 4 to 6 rats per dose). Paired t test, experimental versus basal: *P < 0.05; **P < 0.01; ***P < 0.001.

Comparison of V2R Antagonist and Furosemide (Experiment F)

Because the V2R antagonist induced an increase not only of water but also of sodium excretion, we performed an additional experiment designed to compare these effects with those of a classical diuretic in the same experimental setting. Figure 5 shows the results obtained in 56 rats studied in parallel and receiving various doses of either furosemide or V2R antagonist (results of 14 time-control rats receiving only the vehicles are not included). The changes in sodium or potassium excretion are plotted as a function of the simultaneous changes in water excretion (both changes expressed as Exp/Basal). For both drugs, changes in sodium and in water excretion were always associated (both regression lines cross the 1 × 1 point), but the slopes of the regression lines for the sodium-to-water relationship differed markedly: 1.36 for furosemide versus 0.19 for the V2R antagonist (P < 0.001). Potassium excretion rate rose with both drugs but less intensely with the V2R antagonist (slope = 0.058) than with furosemide (slope = 0.099), although the difference did not reach significance (P = 0.097). Note that the potassium excretion rate decreased (Exp/Basal <1) when the aquaretic effect was less than three-fold above basal.

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Figure 5.

Influence of the V2R antagonist (○ and dotted line, n = 27) or furosemide (• and solid line, n = 29) at various doses (0.1, 0.3, 1.0, 10.0, and 30.0 mg/kg BW for furosemide and 0.1, 0.3, 1.0, and 10.0 mg/kg BW for the V2R antagonist) on sodium (top) and potassium (bottom) excretion rates as a function of the simultaneous changes in fluid excretion rate (urine flow rate). Results are expressed as Exp/Basal for each rat, and regression lines are shown. For both the abscissa and the ordinate, a value of 1 means no change. The equations of the regression lines and corresponding correlation coefficients are as follows. For sodium with furosemide y = 1.360x − 0.54; r = 0.88, P < 0.001. For sodium with the V2R antagonist y = 0.189x + 0.82; r = 0.82, P < 0.001. For potassium with furosemide y = 0.099x + 0.88; r = 0.45, P < 0.05. For potassium with the V2R antagonist y = 0.058x + 0.86; r = 0.77, P < 0.001.

V2R Effects

Experiments performed with the V2R agonist and antagonist confirm that V2R-mediated effects are not only antidiuretic but also antinatriuretic. This is consistent with observations made in isolated CD or in various mammalian or amphibian cell culture models. In these tissues, AVP, applied to the basolateral side, increases amiloride-sensitive sodium transport, an effect mediated by the epithelial sodium channel (ENaC).^1,20 V2R are also expressed in the thick ascending limb of Henle's loop, but previous studies suggested that the influence of AVP on sodium transport in this segment is negligible in normal conditions.²¹ Micropuncture experiments in rats receiving SR121463A confirmed that the aquaretic effect was located downstream of the early distal tubule and thus did not involve the thick ascending limb.²² In humans, acute dDAVP administration also induced a two-fold reduction in sodium excretion rate that was observed in healthy individuals and in individuals with nephrogenic diabetes insipidus as a result of mutations of AQP2 but not in those with mutations of the V2R.²³ In the isolated, erythrocyte-perfused kidney (a model in which a good oxygenation allows an efficient urine concentration), dDAVP induced a five-fold decrease in the fractional excretion of sodium.²⁴

Noteworthy, in the present experiments, the antidiuretic effects of dDAVP reached their maximum for low doses, whereas the effects on solute excretion rate rose progressively with increasing doses (Figure 1). This suggests a very sensitive influence of V2R activation on AQP2 and resulting water permeability in the CD and a less sensitive action on ENaC and on the urea transporter UT-A1, in agreement with in vitro findings.²³

Most studies describing the effects of selective nonpeptide V2R antagonists in experimental animals or humans concluded that these drugs behave as pure “aquaretics” with no effect on electrolyte excretion.¹⁰ Why was the natriuretic effect of aquaretics not disclosed in previous studies? As shown in Figure 4, the natriuretic effects observed here were not apparent in 24-h urine because compensatory sodium retention occurred after the initial loss. In two previous studies in conscious rats receiving daily injections of a V2R antagonist, no effects on 24-h sodium excretion were reported, but distinct and significant three-fold increases in sodium excretion rate are visible (although not commented on) in the first 4 or 6 h after daily dosing in figures of both articles (Figure 6 in reference²⁵ and Figure 2 in reference²⁶). A significant three- and five-fold increase in sodium excretion with 10 and 30 but not with 1 and 3 mg/kg BW was also reported in the previous studies of the first nonpeptide V2R antagonist, OPC-31260.²⁷ VPA 985, another V2R antagonist, increased sodium excretion significantly in patients with hyponatremia and cirrhosis ascites^28,29 but not in patients with the syndrome of inappropriate secretion of antidiuretic hormone.²⁸ SR121463A was also shown to increase sodium excretion chronically in rats with cirrhosis.³⁰ In agreement with the natriuretic effect of V2R antagonists, it is interesting to note that the fractional excretion of sodium was 50% higher in healthy individuals when they were studied during high oral hydration (likely reducing their endogenous AVP level) than when they received a low fluid intake³¹ and that the excretion of a sodium load was two-fold faster.³²

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Figure 6.

Schematic representation of the dose-dependent effects of AVP on sodium excretion rate and dissociation of these effects into V2R- and V1aR-mediated responses. The abscissa represents increasing levels of plasma AVP from left (undetectable) to right. A corresponds to the lowest values of AVP which reduce urine flow rate but not sodium excretion rate. V2R antinatriuretic and V1aR natriuretic effects are depicted as sigmoid curves with different thresholds (B for V2R and C for V1aR effects). B′ and C′ correspond to the maximum effects depending on each receptor type, respectively. M corresponds to the value of plasma AVP for which the antinatriuretic and the natriuretic effects compensate each other. This value probably fluctuates according to a number of factors influencing the intensity of the responses mediated by each of the two receptor types.

Even if aquaretics increase sodium excretion to some extent, it should be kept in mind that their natriuretic effect remains far smaller than their aquaretic effect, at variance with the action of classical diuretics, as shown here in Figure 5. For comparable increases in V, the V2R antagonist induced a natriuresis approximately seven-fold smaller than did furosemide. Thus, even if their effects on electrolyte excretion should not be neglected, V2R antagonists remain predominantly aquaretic.

Several mechanisms may contribute to the natriuretic effect of the V2R antagonist. First, the antagonist probably inhibits endogenous V2R-mediated stimulation of ENaC in the CD. Second, an increase in endogenous AVP level has been well documented in response to the rise in plasma osmolality induced by the water loss after administration of aquaretics.^33,34 Thus, the increased natriuresis could also be due to enhanced V1aR effects because these effects are natriuretic, as shown in this study; however this does not seem to be the case because the co-injection of 10 mg/kg V1a antagonist with 10 mg/kg V2 antagonist resulted in changes in V, osmolality, and sodium excretion very similar to those observed with the V2 antagonist alone (data not shown).

The effects of the V2R agonist and antagonist on potassium handling deserve some comments. Although both drugs have opposite effects on all other variables, they both increased potassium excretion rate. These puzzling results are easily explained by two widely known previous observations. First, stimulation of V2R is known to increase potassium secretion by the CD.^35,36 This is confirmed here by the dose-dependent increase in TTKG seen in rats receiving dDAVP (Figure 1, bottom left). Second, large increases in V are known to increase potassium excretion by a flow-dependent mechanism.³⁷ As seen in Figure 1 (bottom right), this physical effect is not due to an increase in potassium secretion, because there is no significant increase in TTKG. Of note, the influence of dDAVP on potassium secretion was also seen in healthy humans in some³⁸ but not all studies.²³

V1aR Effects

In agreement with a number of previous studies of rats, dogs, sheep, and humans, we observed that AVP increased sodium excretion.^2–9 The dose-response curve performed in this study shows that this effect occurred only for high doses (15 and 50 μg/kg BW) that probably increased P_AVP levels distinctly above those involved in water conservation, even after 24 h of water deprivation, as judged here by the urinary AVP data (Table 2). With lower doses (0.1 and 1 μg/kg BW), likely corresponding to the range of usual osmotic stimuli, the antidiuretic effects of AVP were accompanied by a small but significant reduction in sodium excretion, as with dDAVP. Because the natriuretic effect of AVP 15 μg/kg BW was largely abolished by the co-administration of the selective V1aR antagonist, these experiments clearly establish that the natriuresis induced by AVP is mostly due to V1aR-mediated actions.

Several possible renal and nonrenal mechanisms may account for this natriuretic effect. V1aR and V2R are expressed in multiple sites throughout the body and throughout the kidney itself. Although these experiments cannot disclose the mechanisms involved in the observed responses or the organs/tissues/cell types contributing to these changes, we can briefly mention some possible pathways. (1) In several previous studies, the AVP-induced natriuresis was assumed to result from the volume expansion induced by fluid loads given orally and/or intravenously before and/or during AVP administration. The authors proposed that this expansion induced the release of another mediator inhibiting renal tubular sodium transport^4,39; however, our experiments show that AVP increased sodium excretion even in the absence of any fluid load. (2) Prostaglandins are known to reduce sodium transport in the CD⁴⁰ and to increase medullary blood flow,^41,42 two effects that each will contribute in different ways to increased sodium excretion. Actually, prostaglandins have been shown to contribute to the AVP-induced natriuresis.⁹ AVP stimulates prostaglandin production in interstitial medullary cells,⁴⁰ which express V1aR,⁴³ and in the cortical CD.⁴⁰ V1aR in the CD are mostly located in the luminal membrane^14,15; therefore, urinary, rather than peripheral AVP could be involved here. Of note, AVP concentration is known to be higher in the CD lumen and in the medullary interstitium than in peripheral blood.¹⁷ (3) Finally, the natriuretic effects of AVP could be due to pressure-natriuresis resulting from the vasopressor effects of the hormone, either within the whole circulation or selectively within the kidney vasculature.

Integration of V2R and V1aR Effects

This functional in vivo dissection of the respective influence of V1aR and V2R on sodium handling by the kidney provides for the first time several clues for a better understanding of the integrated actions of AVP in vivo. Combined effects mediated by V2R and V1aR can be predicted to vary greatly according to the levels of plasma and urinary AVP. They will provide different sets of water and sodium responses. The changes in sodium excretion as a result of the algebraic sum of V2 and V1a effects over progressively increasing plasma levels of AVP are represented schematically in Figure 6. V2R antinatriuretic and V1aR natriuretic effects are depicted as sigmoid curves with different thresholds (B for V2R and C for V1aR effects) and different AVP levels inducing the maximum effects for each receptor type (B′ and C′, respectively).

In the range of very low plasma levels of AVP, only the V2 effects on water permeability of the CD are apparent, and AVP will reduce V without any effect on sodium excretion rate (Figure 6, range A to B). Such purely antidiuretic action is visible in the study of Andersen et al.,⁴⁴ who gave subpicomolar doses of AVP to individuals undergoing water diuresis. Although these doses did not increase P_AVP above the detection limit of the assay, V fell and U_osm rose significantly without any change in sodium excretion rate. Another example is found in individuals with central diabetes insipidus, in whom dDAVP induced a marked decline in V independent of any change in sodium excretion rate (see Figure 4 in reference²³). This action of AVP in the low range of plasma concentrations can induce the reabsorption of very large amounts of solute-free water.¹⁷

When P_AVP rises a little more (beyond B in Figure 6), V2R effects stimulate sodium reabsorption and should thus reduce sodium excretion rate in addition to reducing V, as seen in healthy humans who received an infusion of AVP at a dose of 25 μg/min per kg BW⁴⁴ or with dDAVP²³ and in the isolated rat kidney perfused with a medium containing erythrocytes and dDAVP.²⁴ This sodium-retaining effect probably explains why, in normal individuals (with normal fluid and food intake) who provided multiple urine samples, sodium excretion rate declined in parallel with V in the samples in which U_osm exceeded 600 mOsm/kg H₂O but was not altered in those under this limit despite wide differences in V (from 60 to 300 ml/h).⁴⁵ This is in agreement with in vitro data showing that the effect of AVP of sodium transport in isolated perfused rat CD requires higher levels of peritubular AVP than the effect on water permeability.¹⁷

With further increases in P_AVP, V1aR-dependent natriuretic effects appear and increase progressively, but the net effect of AVP is still antinatriuretic (P_AVP between C and M in Figure 6). Finally, with much higher levels of AVP, such as those induced by exogenous AVP infusion at a rate higher than 5 μg/kg, the V1a natriuretic effects overcome the V2 effects, leading to increased natriuresis (P_AVP beyond point M in Figure 6). We want to underline that the dose of AVP inducing either negative or positive effects on sodium excretion was quite variable among rats and experiments between 1 and 5 μg/kg BW (data not shown). This variability may be due to different sensitivities of the two receptors and/or of subsequent signal transduction, and to differences in AVP concentrations in luminal fluid of the CD and/or in medullary tissue (determining V1a effects) with respect to peripheral AVP concentration (determining V2 effects).

The secretion of AVP is not known to depend on sodium intake. As discussed previously,^20,23 the V2R-dependent stimulation of sodium reabsorption likely serves the purpose of fluid conservation rather than that of regulating sodium excretion. A stronger sodium reabsorption in the AVP-sensitive distal nephron, which expresses both ENaC and AQP2, will drive more water out of the lumen and will thus increase the concentration of all other solutes but sodium. Thus, AVP will help conserve water at the expense of a less efficient sodium excretion. V1aR-dependent effects that increase sodium excretion may be viewed as a safeguard mechanism limiting the risk for too intense sodium retention. An imbalance between V1aR and V2R sensitivity or signal transduction may thus influence the ability of the kidney to excrete sodium and may, indirectly, be responsible for inappropriate sodium retention and resulting increase in BP. Actually, it has long been known that highly selective peptide and nonpeptide inhibitors of V1aR block exogenous AVP's pressor action but that they have little effect on the basal levels of arterial BP.⁴⁶ Thus, under normal conditions of cardiovascular homeostasis and appropriate extracellular fluid volume, AVP and its V1aR seem to play only a minor role in maintaining normal cardiovascular function. In contrast, several observations link BP and the antidiuretic V2R-dependent actions of AVP. Long-term dDAVP infusion in normal rats has been shown to increase BP by approximately 10 mmHg.^47,48 Salt-sensitive hypertension-prone Sabra rats exhibit higher AVP mRNA, higher P_AVP, and twofold higher U_osm and lower V than their salt-resistant counterparts.⁴⁹ In humans, significant negative correlations were observed between 24-h V and BP.^13,50

In the usual range of P_AVP, the antidiuretic effect of AVP is possibly associated with an antinatriuretic effect of variable intensity depending on the level of urine concentration. This will delay the excretion of sodium and could thus result in some degree of sodium and volume retention, which could favor an increase in BP. The natriuretic effects induced by the V1aR stimulation may counteract this tendency. Thus, instead of the usually assumed pressor effect, V1a activation within the physiologic range may actually reduce the risk for V2R-induced sodium retention. Moreover, the balance between V1aR- and V2R-mediated actions in cells possessing the two types of receptors (e.g., cortical CD) may be further amplified by a downregulation of the V2R induced by a V1a-mediated pathway.⁵¹

That V1aR effects counteract V2R tubular effects could explain several puzzling observations. (1) In pathologic models in which AVP levels are known to be elevated (nitric oxide–deprived hypertensive rats and diabetic rats), the long-term administration of a selective V1aR nonpeptide antagonist worsened BP and albuminuria⁵² and did not prevent or even aggravated diabetes-related vascular damage.⁵³ (2) Several other studies suggested that V1a effects attenuate or counteract several direct and indirect V2R-dependent effects in normal rats and in rats with renal failure.^54–57 (3) In patients with chronic renal failure, the increased urinary excretion of AVP per remaining nephron correlated positively and more significantly than for any other hormone with the fractional excretion of sodium, suggesting that luminal AVP contributed to ensure an appropriate natriuresis through V1aR-mediated luminal effects.⁵⁸ (4) Interestingly, mice with deletion of the V1aR exhibit a lower BP as a result not of a reduced vasoconstriction but of a lower extracellular fluid volume.⁵⁹

In summary, these results provide a better knowledge of the respective V2R- and V1aR-dependent effects of AVP on sodium excretion in rats. Whether a similar balance between V1aR- and V2R-dependent effects occurs in humans remains to be determined. These results are potentially important in view of the proposed use of AVP antagonists in several human disorders.^10–12 In patients with hypervolemic hyponatremia as a result of heart failure or cirrhosis, the additional effect of V2 antagonism on sodium excretion may be beneficial and may decrease diuretic use. V2R antagonists may also be beneficial in some forms of salt-sensitive hypertension^13,60; however, more precise information is required regarding V1a and V2 effects in humans to know whether selective rather than mixed antagonists are more appropriate in each of these disorders.

Animals and General Procedures

Adult male Wistar rats (Charles River, L'Arbresle, France) of initial body weight 230 to 240 g were housed individually in metabolic cages with free access to tap water and powdered food (A03; Safe, Villemoisson/Orge, France) at all times before and during the experiments (except for one rat group in experiment E, discussed later). They were accustomed to the cages for 5 d before the experiments. All experiments were carried out in conscious, undisturbed rats and involved no previous water load, no instrumentation, and no pretreatment. Each experiment included 24 to 36 rats studied in parallel on two successive days. On day 1 (basal), rats were administered an injection at time 0 (approximately 10 a.m.) of the vehicle(s) only, and on day 2 (experimental) with AVP and/or various drugs (see details that follow). Urine was collected for 6 h starting just after the injection(s). Urine volume was determined gravimetrically, assuming the density of urine was equal to unity. U_osm was measured with a freezing point osmometer (Roebling, Berlin, Germany) and urinary concentration of sodium and potassium by flame photometry (IL 943, Instrument Laboratory, Lexington, MA). Urine urea concentration was measured with a standard kit (Urea Kit S 1000; BioMérieux, Lyon, France).

In each experiment, on the basis of values obtained during the basal day, the rats were divided into several groups of equivalent urine volume and osmolality (n = 4 to 6 per group) to test, during the experimental day, the effects of the various doses of hormones or drugs in groups of rats with equivalent urine concentrating activity. Usually, three or four independent experiments were performed in the same series of rats at 1-wk intervals (to allow washout from the preceding experiment). All animal procedures were conducted in accordance with the European guidelines for the care and use of laboratory animals.

V2R Agonist dDAVP: Dose-Response Curve (Experiment A)

dDAVP (Minirin; Ferring Pharmaceuticals AB, Malmö, Sweden) is a selective peptidic V2R agonist.⁶¹ The dDAVP solution was emulsified in oil (1:9 vol/vol) to prolong its bioavailability, and injected subcutaneously (under the skin of the back of the neck) in seven rat groups (600 μl emulsion/kg BW) at concentrations in the aqueous phase designed to deliver 0.00 (vehicle alone), 0.02, 0.06, 0.20, 0.60, 2.00, or 6.00 μg/kg BW (corresponding approximately to 0.02, 0.06, 0.20, 0.60, 2.00, or 6.00 nmol/kg BW).

V2R Antagonist SR121463A: Dose-Response Curve (Experiment B)

SR121463A (Satavaptan; Sanofi-Aventis, Toulouse, France) is a selective nonpeptide V2R antagonist with a relatively long half-life.^62,63 On the experimental day, the V2R antagonist dissolved in DMSO (10% final volume) and saline was injected intraperitoneally (1 ml/kg BW) in six equivalent rat groups at various concentrations delivering 0 (vehicle alone), 0.01, 0.03, 0.10, 1.00, or 10.00 mg/kg BW. In this experiment, urine was collected not only for the first 6 h after the drug administration but also in separate tubes for the subsequent 18 h (i.e., from 6 to 24 h after the injection) so as to evaluate the delayed effects of the drug.

Natural AVP: Dose-Response Curve (Experiment C)

Synthetic [Arg⁸]-vasopressin acetate salt (Sigma-Aldrich, Chemie, Steinheim, Germany; i.e. natural AVP), dissolved in saline, was emulsified in oil (1:9 vol/vol) and injected subcutaneously, as described for dDAVP (600 μl emulsion/kg BW), in five groups of rats. The concentration of AVP in the aqueous phase was designed to deliver 0.0 (vehicle alone), 0.1, 1.0, 15.0, or 50.0 μg/kg BW (corresponding approximately to 0.1, 1.0, 15.0, or 50.0 nmol/kg BW). As in previous studies,^54,57 the dose range for AVP was approximately five times higher than that for dDAVP because of the faster degradation of AVP in vivo.

Natural AVP Plus V1aR Antagonist SR49059 (Experiment D)

For evaluation of the role of V1aR in the response to the natural hormone AVP, rats of this series were administered AVP (15 μg/kg BW, same procedure as in Experiment C) without or with the concomitant administration of the selective nonpeptide V1aR antagonist SR49059 (Relcovaptan; Sanofi-Aventis) at a dose of 10 mg/kg BW, shown in previous studies to block the pressor response to AVP.⁶⁴ This drug was dissolved in DMSO and cremophor (each 10% of final volume) and then in saline, and was injected intraperitoneally (1 ml/kg BW). For comparison, additional rats received vehicle alone, the V1aR antagonist alone, AVP at a lower dose (3 μg/kg BW), or dDAVP (0.6 μg/kg BW).

Urinary AVP Concentration in Various Situations (Experiment E)

To evaluate the concentration of AVP prevailing in the lumen of the CD in different situations, we performed additional experiments in which urine was collected for 6 h, as in the other experiments, under various experimental conditions, including injection of isotonic saline, injection of dDAVP 0.6 μg/kg BW, injection of AVP 3 or 15 μg/kg BW, and 24-h dehydration. Urine volume and U_osm were measured, and aliquots of urine were stored at −20°C for further AVP assay. U_AVP was measured as described previously.⁶⁵ The antibody used does not recognize dDAVP.

Comparison of V2R Antagonist and Furosemide Effects (Experiment F)

Classical diuretics and aquaretics (V2R antagonist) increase V by different mechanisms. To compare the respective effects of the two types of drugs on water and sodium excretion, we performed an additional experiment in which different groups of rats received either furosemide (Lasix; Sanofi-Aventis) or the V2R antagonist (prepared and injected as in Experiment B) at various doses (0.0, 0.1, 0.3, 1.0, 10.0, and 30.0 mg/kg BW for furosemide and 0.0, 0.1, 0.3, 1.0, and 10.0 mg/kg BW for the V2R antagonist). Urine was collected for 6 h after the injections.

Statistical Analysis

V and the excretion rate of the different solutes were calculated according to standard formulas. The TTKG, reflecting the intensity of potassium secretion, was calculated by dividing the urine/plasma potassium concentration ratio by the urine/plasma osmolality ratio.⁶⁶ Plasma osmolality was arbitrarily considered to be 300 mOsm/kg H₂O and plasma potassium concentration 5 mmol/L in all rats.

Results are expressed as means ± SEM. In Experiments A through D, each rat was its own control. Thus, for each variable, the Exp/Basal provides a quantitative estimate of the changes induced by the hormone and/or drug. The statistical significance of these changes was evaluated by paired t test comparing results of the experimental day with those of the basal day in each rat. In Experiment E, comparison between the various groups was performed by one-way ANOVA followed by Fisher post hoc test. In Experiment F, linear regressions were calculated between the changes in sodium or potassium excretion and those in water excretion.