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Sodium Excretion in Response to and Selective Antagonists

Julie Perucca,*† Daniel G. Bichet,‡ Pascale Bardoux,* Nadine Bouby,*† and Lise Bankir*†

*INSERM, Unite´ 872, and †UMRS 872, Universite´ Paris Descartes, Centre de Recherche des Cordeliers, Paris, France; and ‡Service de Ne´phrologie, Hoˆpital du Sacre´-Coeur, De´partement de Me´decine, Universite´ de Montre´al, Montre´al, Que´bec, Canada

ABSTRACT The mechanisms by which arginine vasopressin (AVP) exerts its antidiuretic and pressor effects, via activation of V2 and V1a receptors, respectively, are relatively well understood, but the possible associated effects on sodium handling are a matter of controversy. In this study, normal conscious Wistar rats were acutely administered various doses of AVP, dDAVP (V2 agonist), furosemide, or the following selective non-peptide receptor antagonists SR121463A (V2 antagonist) or SR49059 (V1a antagonist). Urine flow and sodium excretion rates in the next 6 h were compared with basal values obtained on the previous day, after vehicle treatment, using each rat as its own control. The rate of sodium excretion decreased with V2 agonism and increased with V2 antagonism in a dose-dependent manner. However, for comparable increases in urine flow rate, the V2 antagonist induced a natriuresis 7-fold smaller than did furosemide. Vasopressin reduced sodium excretion at 1 ␮g/kg but increased it at doses Ͼ5 ␮g/kg, an effect that was abolished by the V1a antagonist. Combined V2 and V1a effects of endogenous vasopressin can be predicted to vary largely according to the respective levels of vasopressin in plasma, renal medulla (acting on interstitial cells), and urine (acting on V1a luminal receptors). In the usual range of regulation, antidiuretic effects of vasopressin may be associated with variable sodium retention. Although V2 antagonists are predominantly aquaretic, their possible effects on sodium excretion should not be neglected. In view of their proposed use in several human disorders, the respective influence of selective (V2) or mixed (V1a/V2) receptor antagonists on sodium handling in humans needs reevaluation.

J Am Soc Nephrol 19: 1721–1731, 2008. doi: 10.1681/ASN.2008010021

The mechanisms by which arginine vasopressin In contrast, the experiments intended to study (AVP) exerts its antidiuretic and its pressor effects are the effects of AVP on sodium handling in vitro or in relatively well understood. On the one hand, AVP im- vivo provide results that are difficult to reconcile. In proves water conservation by increasing the perme- the isolated microperfused CD, V2R activation in- ability to water of the renal collecting duct (CD), an creases sodium transport,1 an effect that should re- effect mediated by the V2 receptors (V2R) and per- duce sodium excretion in vivo; however, in a num- mitted by the insertion in the luminal membrane of ber of studies, AVP infusion in animals and humans principal cells of preformed aquaporin 2 (AQP2) mol- ecules. This allows more water to be reabsorbed when Received January 8, 2008. Accepted April 2, 2008. these ducts traverse the hyperosmotic medulla. On the Published online ahead of print. Publication date available at other hand, AVP increases blood pressure (BP) by in- www.jasn.org. ducing a vasoconstriction through its binding to V1a Correspondence: Dr. Lise Bankir, INSERM U 872, Centre de receptors (V1aR) expressed in vascular smooth mus- Recherche des Cordeliers, 15 rue de l’Ecole de Me´decine, 75006 cle cells. For these two different effects, in vivo studies Paris, France. Phone: 33-1-47-35-34-15; Fax: 33-1-44-07-90-40; are in good agreement with the expectations based on E-mail: [email protected] results obtained in vitro. Copyright ᮊ 2008 by the American Society of Nephrology

J Am Soc Nephrol 19: 1721–1731, 2008 ISSN : 1046-6673/1909-1721 1721 BASIC RESEARCH www.jasn.org has been shown to induce an increase in sodium excretion.2–9 It In response to V2R blockade, sodium excretion rate rose dose- is usually assumed that AVP might contribute to some forms of dependently from 0.1 mg/kg body wt (BW) onward. Note that hypertension by its vasoconstrictive effects, but an increase in water excretion already rose three-fold with the previous dose sodium excretion, if it occurred in normal life, should more (0.03 mg/kg BW), although not yet significant. With the largest likely contribute to lower BP. dose of antagonist, sodium excretion rate was increased six- In an attempt to resolve these conflicting results, we under- fold when water excretion (i.e., V) was increased 25-fold (Fig- took a series of experiments in conscious undisturbed rats to ure 1). Potassium excretion rate rose with the V2R antagonist obtain precise dose-response curves to AVP and to selective (with, however, large interindividual variation preventing agonists and antagonists of V1aR or V2R. Each rat served as its changes to reach statistical significance in most of the dose own control, receiving on separate days either the vehicle or groups); however, the transtubular potassium gradient the drug(s), and the urine produced in the next 6 h was ana- (TTKG), an index of the active secretion of potassium in the lyzed. This allowed us to evaluate separately the respective in- distal nephron, rose only with dDAVP (Figure 1). fluence of V1aR and V2R activation on urine concentration and sodium excretion and their interactions at different phys- Dose-Response Curve of AVP and Effects of the V1aR iologic and supraphysiologic levels of AVP. These new results Antagonist on the Response to AVP (Experiments C should be of special interest because nonpeptide vasopressin and D) receptor antagonists are now proposed in hyponatremia with When given at a dose of 1 ␮g/kg, the natural hormone AVP heart failure and cirrhosis, two conditions with severe water increased Uosm and reduced V by approximately 35% (Figure 10,11 12 and sodium retention, in polycystic kidney disease, and 2). The difference for Uosm did not reach significance because possibly in some forms of salt-sensitive hypertension.13 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 RESULTS line), but this effect disappears with higher doses (Figure 2). Sodium excretion rate was modestly reduced along with the For most experiments, the changes induced by the various antidiuretic effect at 1 ␮g/kg but rose markedly with higher treatments are presented as experimental day/basal day ratios doses of AVP, whereas urea excretion fell. The highest dose (Exp/Basal). One rat group always received the vehicle(s) alone increased potassium excretion rate and TTKG by 40% (Fig- on both day 1 and day 2 as an additional control for assessing ure 2). possible day-to-day variations. Absolute values obtained for To evaluate whether the loss of the antidiuretic effect of the basal day in the various experiments are shown in Table 1. AVP and the appearance of a natriuretic effect at higher doses are due to V1aR stimulation, we conducted additional experi- Dose-Response Curves of the V2R Agonist and ments with co-administration of AVP 15 ␮g/kg BW and a se- Antagonist (Experiments A and B) lective V1aR antagonist. In preliminary experiments, the ef- The V2R agonist 1-desamino 8-D-arginine vasopressin fects of the V1aR antagonist given alone at various doses (0.1, (dDAVP) showed the expected antidiuretic action, inducing a 1, and 10 mg/kg BW) were evaluated. Dose-dependent signif- marked decline in urine flow rate (V) and a rise in urine osmo- icant increases in Uosm were seen with all three doses, whereas lality (Uosm). The V2R antagonist showed the expected significant decreases in V and sodium excretion occurred in aquaretic action (Figure 1). In addition, both drugs also influ- some but not all rats with 10 mg/kg BW (data not shown). enced solute excretion rates. Urea excretion rate fell with in- These changes suggest that the V1aR antagonism suppressed a creasing V2 agonism, as already widely known. Sodium excre- modest diuretic and natriuretic influence of endogenous AVP. tion rate declined, and potassium excretion rate increased by In experiment D, two doses of AVP were tested. AVP at 3 ␮g/kg approximately 30 to 50% with the last four doses of dDAVP. Of was antidiuretic, lowering V by 27% (P Ͻ 0.01) and increasing Ͻ note, the effects of dDAVP on V and Uosm reached a maximum Uosm by 33% (P 0.001), and induced no change in sodium for the lowest dose used here, whereas the effects on solute excretion rate. With a five-fold higher dose (15 ␮g/kg), the excretion rate showed a progressive dose-dependent increase. antidiuretic effect was completely lost and a marked natriuretic

Table 1. Basal values observed on day 1 in groups of rats used in the various experiments Na K Urea BW V U Experiment n osm Excretion Excretion Excretion TTKG (g) (ml/h) (mOsm/kg H O) 2 (␮mol/h) (␮mol/h) (␮mol/h) A 64 356 0.58 Ϯ 0.02 1436 Ϯ 51 57.5 Ϯ 3.0 111 Ϯ 4 431 Ϯ 14 8.36 Ϯ 0.13 B 35 281 0.48 Ϯ 0.03 1723 Ϯ 84 51.0 Ϯ 3.7 124 Ϯ 7 394 Ϯ 20 9.34 Ϯ 0.16 C 48 300 0.53 Ϯ 0.03 1643 Ϯ 74 71.5 Ϯ 3.1 126 Ϯ 5 385 Ϯ 17 9.36 Ϯ 0.14 D 70 285 0.60 Ϯ 0.03 1609 Ϯ 49 81.1 Ϯ 4.3 133 Ϯ 5 462 Ϯ 20 9.02 Ϯ 0.14 F 72 274 0.51 Ϯ 0.02 1363 Ϯ 52 51.8 Ϯ 2.5 105 Ϯ 4 387 Ϯ 16 9.48 Ϯ 0.13

1722 Journal of the American Society of Nephrology J Am Soc Nephrol 19: 1721–1731, 2008 www.jasn.org BASIC RESEARCH

V2R agonist V2R antagonist tion increased UAVP approximately 20-fold. Ad- ministration of AVP 3 ␮g/kg BW increased U to 2.02 2.02 AVP *** *** *** a similar extent, whereas dDAVP did not change it Urine *** 1.51,5 *** * 1,51.5 (Table 2). A five-fold higher dose of AVP (15 ␮g/kg osmolality 1.01 1.01 BW) increased U close to five times more. This (Exp/Basal) AVP 0.50,5 0.50,5 * high luminal AVP concentration was associated ** 0 0 ** with an increase in diuresis and natriuresis (as in 2.02 30 experiment D) and a decline in Uosm such that V was ** Urine 1,51.5 higher and Uosm was lower than those observed in 20 rats receiving a smaller dose of AVP or in water- flow rate 1.0 1 ** (Exp/Basal) 10 deprived rats. Several previous studies already ob- 0.50,5 ** * * ** *** *** ** served this simultaneous increase in sodium excre- 0 0 tion and urine flow rate after AVP infusions.4,6,8 In 2.02 2.02 Urea these experiments, it would have been difficult to evaluate the mean plasma AVP (P ) level reached excr. rate 1,51.5 1,51.5 AVP during 6 h (duration of the urine collection). Mea- (Exp/Basal) 1.01 1.01 surements of the AVP excretion rate provide more integrated information during this period. Previous 0.50,5 ** 0.50,5 *** *** *** ** studies showed that urinary AVP excretion rate is 2.02 6 * 5 roughly proportional to the plasma level when os- Sodium 1.51,5 4 ** molar excretion remains stable.16 Thus, given the excr. rate 1.01 3 * results presented in Table 2, it may be assumed that (Exp/Basal) 2 0.50,5 * administration of 1 to 3 ␮g/kg BW AVP increased * * * 1 0 0 PAVP to levels similar to those seen after 24 h of water ** 2.02 2.02 deprivation, whereas the dose of 15 ␮g/kg BW in- Potassium creased PAVP to higher values, reached only during 1,51.5 ** * 1,51.5 excr. rate * * exogenous vasopressin infusion. (Exp/Basal) 1.01 1.01 Effects of the V2R Antagonist on Sodium 0.50,5 0.50,5 and Water Excretion during the Whole 2.02 *** *** 2.02 *** *** 24 h (Experiment B)

1,51.5 *** 1,51.5 Selective V2R antagonists are usually reported to TTKG * increase markedly urine output without affecting (Exp/Basal) 1.01 1.01 solute excretion. Because a significant increase in ** sodium excretion rate was observed in these ex- 0.50,5 0,50.5 0 0.02 0.06 0.2 0.6 2 6 0 0.01 0.03 0.1 1 10 periments, it was interesting to determine g/kg BW mg/kg BW whether this change was short-lived or sustained. Collection of urine for the 18 h after the initial 6-h Figure 1. Dose-dependent effects of the V2R agonist (dDAVP) and the V2R an- tagonist (SR121463A) on fluid and solute excretion rates (Experiments A and B, n ϭ collection and calculation of the antagonist’s ef- 4 to 6 rats per dose). Results are expressed as Exp/Basal. Thin lines illustrate the fects during these two aggregated periods showed dose-dependent effects. Paired t test, experimental versus basal: *P Ͻ 0.05; **P Ͻ that the aquaretic but not the natriuretic effect 0.01; ***P Ͻ 0.001. remained detectable over 24 h (Figure 4). Most probably, thirst and thus fluid intake increased effect was observed, as depicted in Figure 3. The co-adminis- during the whole duration of V2R blockade and resulting tration of the V1aR antagonist at 10 mg/kg BW with AVP 15 aquaresis. After the initial loss of sodium, compensatory so- ␮g/kg BW largely abolished the natriuretic effect and restored dium retention occurred in the subsequent hours, bringing an antidiuretic effect close to that observed with dDAVP (Fig- back sodium balance to zero. A similar compensation also oc- ure 3) and with AVP 3 ␮g/kg. curred for potassium and urea. Note that with the highest dose of the antagonist, sodium retention occurred during the 6- to Urinary AVP Concentration in Different Experimental 24-h period despite a persisting increase in aquaresis (Figure Conditions (Experiment E) 4). It is conceivable that the natriuretic effect induced by the V2 Luminal effects of AVP in the renal CD have been described.14,15 antagonist could be sustained over the whole 24 h, along with

This prompted us to evaluate urinary AVP concentration (UAVP) the aquaresis, if the rats could compensate the initial salt loss that should, at least approximately, vary in proportion to its con- (that probably stimulates their salt appetite) by having access centration in the lumen of the CD. Twenty-four-hour dehydra- to a source of salt independent of the food, as they do for water.

J Am Soc Nephrol 19: 1721–1731, 2008 AVP Receptors and Na Excretion 1723 BASIC RESEARCH www.jasn.org

AVP excretion (both changes expressed as Exp/Basal). For both 2.02 drugs, changes in sodium and in water excretion were always ϫ Urine associated (both regression lines cross the 1 1 point), but the 1,51.5 slopes of the regression lines for the sodium-to-water relation- osmolality ship differed markedly: 1.36 for furosemide versus 0.19 for the (Exp/Basal) 1.01 Ͻ * V2R antagonist (P 0.001). Potassium excretion rate rose * 0.50,5 with both drugs but less intensely with the V2R antagonist 2.02 (slope ϭ 0.058) than with furosemide (slope ϭ 0.099), al- ϭ Urine though the difference did not reach significance (P 0.097). 1,51.5 Note that the potassium excretion rate decreased (Exp/Basal flow rate * Ͻ1) when the aquaretic effect was less than three-fold above (Exp/Basal) 1.01 basal. 0.50,5 * 2.02 Urea DISCUSSION 1,51.5 excr. rate (Exp/Basal) 1.01 AVP is mostly known for its effect on water conservation. Its possible effects on sodium excretion in usual life are rarely 0,50.5 *** addressed. This study evaluated the dose-dependent influence 2.02 of AVP on sodium excretion in vivo in conditions as close as Sodium ** possible to normal life, and dissociated the respective roles of 1,51.5 excr. rate V1aR and V2R. The main findings are that V2R effects are (Exp/Basal) 1.01 strictly antinatriuretic, whereas V1aR effects are natriuretic above a certain threshold of hormone level. For levels of the * 0.50,5 endogenous hormone prevailing in normal life, the widely 2.02 known antidiuretic effects of AVP may be associated with an- Potassium tinatriuretic effects of variable intensity. 1,51.5 * excr. rate One of the advantages of this study is that all experiments (Exp/Basal) 1.01 were conducted in conscious, unrestrained rats without any pretreatment. There was no previous oral water load or intra- 0.50,5 venous infusion of iso- or hypotonic fluid, maneuvers that 2.02 induce acute perturbations of the fluid balance. Rats under- 1,51.5 went no anesthesia and surgery that are known to stimulate TTKG *** potently endogenous AVP secretion. The use of highly selective (Exp/Basal) 1.01 nonpeptide V1aR or V2R antagonists with relatively long half- life and the building of dose-response curves allowed a better 0.50,5 00.11 1550 evaluation of AVP effects within the range occurring in usual g/kg BW physiologic and pathophysiologic situations. Finally, each rat was its own control, a favorable situation given the large inter- Figure 2. Dose-dependent effects of AVP on fluid and solute individual variability of urine flow rate and osmolality and of excretion rates (Experiment C, n ϭ 4 to 6 rats per dose). Results 17 are expressed as Exp/Basal. Thin lines illustrate the dose-depen- the response to AVP. That food and fluid intakes were not dent effects. Paired t test, experimental versus basal: *P Ͻ 0.05; measured represents a limitation in interpreting some of the **P Ͻ 0.01; ***P Ͻ 0.001. results. dDAVP is known to have some affinity for V1b recep- tors, but this affinity is much lower in rats than in humans.18,19 Comparison of V2R Antagonist and Furosemide This experimental design was not intended to address the pos- (Experiment F) sible V1b receptor-mediated effects. Because the V2R antagonist induced an increase not only of water but also of sodium excretion, we performed an addi- V2R Effects tional experiment designed to compare these effects with those Experiments performed with the V2R agonist and antagonist of a classical diuretic in the same experimental setting. Figure 5 confirm that V2R-mediated effects are not only antidiuretic shows the results obtained in 56 rats studied in parallel and but also antinatriuretic. This is consistent with observations receiving various doses of either furosemide or V2R antagonist made in isolated CD or in various mammalian or amphibian (results of 14 time-control rats receiving only the vehicles are cell culture models. In these tissues, AVP, applied to the baso- not included). The changes in sodium or potassium excretion lateral side, increases amiloride-sensitive sodium transport, an are plotted as a function of the simultaneous changes in water effect mediated by the epithelial sodium channel (ENaC).1,20

1724 Journal of the American Society of Nephrology J Am Soc Nephrol 19: 1721–1731, 2008 www.jasn.org BASIC RESEARCH

Urine flow rate Urine osmolality Sodium excretion rate 190 190 190

HÑ *** 160 160 *** 160 Figure 3. Effects of AVP (AVP-15,15 ␮g/kg J ** BW) on urine flow rate and osmolality and on sodium excretion rate without or with the 130 130 130 D *** co-administration of the V1aR antagonist (Ex- J H periment D, n ϭ 4 to 6 rats per dose). The HDÑJ HDÑJ HDÑJ 100 AVP-15 100 100 J D effects of the antagonist alone and of dDAVP D * V1a antag are also shown. Results of the experimental Per cent of basal day Ñ *** 70 H 70 70 day are expressed as percentage of values AVP-15 + V1a antag * Ñ observed during the basal day in the same *** dDAVP rats. Paired t test, experimental versus basal: 40 40 40 *P Ͻ 0.05; **P Ͻ 0.01; ***P Ͻ 0.001. Basal Experimental Basal Experimental Basal Experimental

Table 2. Urinary AVP and other urinary characteristics in Experiment E

Uosm Osmolar V UNa Na Excretion UAVP AVP Excretion Treatment n (mOsm/kg Excretion ␮ (ml/h) ␮ (mmol/L) ( mol/h) (pg/ml) (pg/h) H2O) ( Osm/h) Vehicle 5 0.54 Ϯ 0.06 1699 Ϯ 150 921 Ϯ 129 142 Ϯ 37 75 Ϯ 19 272 Ϯ 102 149 Ϯ 57 AVP-3 5 0.52 Ϯ 0.06C 2105 Ϯ 141c 1069 Ϯ 103 195 Ϯ 17c 100 Ϯ 12 7269 Ϯ 1646 3489 Ϯ 435 AVP-15 5 0.87 Ϯ 0.09A,B,C 1192 Ϯ 61b,C 1035 Ϯ 113 204 Ϯ 5 176 Ϯ 15A,B,C 29,028 Ϯ 7314A,B,C 25,275 Ϯ 7891A,B,C 24-h dehydration 8 0.24 Ϯ 0.02A 2914 Ϯ 213A 702 Ϯ 68 295 Ϯ 25A 71 Ϯ 8 5329 Ϯ 1072 1322 Ϯ 220 dDAVP 5 0.31 Ϯ 0.03a 2461 Ϯ 139a 769 Ϯ 114 172 Ϯ 34c 54 Ϯ 12 269 Ϯ 53 84 Ϯ 19 One-way ANOVA followed by Fisher post hoc test was used to compare the various groups. Letters indicate the significance of the different comparisons as follows: a or A: comparison with the time-control group (vehicle); b or B, comparison with the AVP-3 group; c or C, comparison with the 24-h dehydrated Ͻ Ͻ group. Lower case letters: P 0.01; capital letters: P 0.001. UNa, urinary Na concentration.

V2R are also expressed in the thick ascending limb of Henle’s Figure 4, the natriuretic effects observed here were not appar- loop, but previous studies suggested that the influence of AVP ent in 24-h urine because compensatory sodium retention oc- on sodium transport in this segment is negligible in normal curred after the initial loss. In two previous studies in con- conditions.21 Micropuncture experiments in rats receiving scious rats receiving daily injections of a V2R antagonist, no SR121463A confirmed that the aquaretic effect was located effects on 24-h sodium excretion were reported, but distinct downstream of the early distal tubule and thus did not involve and significant three-fold increases in sodium excretion rate the thick ascending limb.22 In humans, acute dDAVP admin- are visible (although not commented on) in the first 4 or 6 h istration also induced a two-fold reduction in sodium excre- after daily dosing in figures of both articles (Figure 6 in refer- tion rate that was observed in healthy individuals and in indi- ence25 and Figure 2 in reference26). A significant three- and viduals with nephrogenic diabetes insipidus as a result of five-fold increase in sodium excretion with 10 and 30 but not mutations of AQP2 but not in those with mutations of the with 1 and 3 mg/kg BW was also reported in the previous V2R.23 In the isolated, erythrocyte-perfused kidney (a model in studies of the first nonpeptide V2R antagonist, OPC-31260.27 which a good oxygenation allows an efficient urine concentra- VPA 985, another V2R antagonist, increased sodium excretion tion), dDAVP induced a five-fold decrease in the fractional significantly in patients with hyponatremia and cirrhosis as- excretion of sodium.24 cites28,29 but not in patients with the syndrome of inappropri- Noteworthy, in the present experiments, the antidiuretic ate secretion of antidiuretic hormone.28 SR121463A was also effects of dDAVP reached their maximum for low doses, shown to increase sodium excretion chronically in rats with whereas the effects on solute excretion rate rose progressively cirrhosis.30 In agreement with the natriuretic effect of V2R an- with increasing doses (Figure 1). This suggests a very sensitive tagonists, it is interesting to note that the fractional excretion influence of V2R activation on AQP2 and resulting water per- of sodium was 50% higher in healthy individuals when they meability in the CD and a less sensitive action on ENaC and on were studied during high oral hydration (likely reducing their the urea transporter UT-A1, in agreement with in vitro find- endogenous AVP level) than when they received a low fluid ings.23 intake31 and that the excretion of a sodium load was two-fold Most studies describing the effects of selective nonpeptide faster.32 V2R antagonists in experimental animals or humans con- Even if aquaretics increase sodium excretion to some ex- cluded that these drugs behave as pure “aquaretics” with no tent, it should be kept in mind that their natriuretic effect re- effect on electrolyte excretion.10 Why was the natriuretic effect mains far smaller than their aquaretic effect, at variance with of aquaretics not disclosed in previous studies? As shown in the action of classical diuretics, as shown here in Figure 5. For

J Am Soc Nephrol 19: 1721–1731, 2008 AVP Receptors and Na Excretion 1725 BASIC RESEARCH www.jasn.org

150 15 ** Furosemide

Urine volume 100 10 (mL/period) ** ** V2R antagonist 50 * * 5 * *

0 Sodium excr. rate (Exp/Basal) 3000 0 0510152025 Urine flow rate (Exp/Basal) Urine 2000 * 2.52,5 osmolality * ** Furosemide (mosm/kg H2O) 2.02 1000 ** ** 1.51,5 * *** *** ** V2R antagonist 0 1.01

0.50,5 2000 Sodium Potassium excr. rate (Exp/Basal) 0 excretion 0 5 10 15 20 25 * ( mol/period) 1000 * Urine flow rate (Exp/Basal) * ** Figure 5. Influence of the V2R antagonist (E and dotted line, n ϭ 27) or furosemide (F and solid line, n ϭ 29) at various doses (0.1, 0 1 1 0 0 0 1 10 10 10 0.1 0.3, 1.0, 10.0, and 30.0 mg/kg BW for furosemide and 0.1, 0.3, 0.1 Dose (mg/kg BW) 0.1 0.01 0.01 0.01 0.03 0.03 0.03 1.0, and 10.0 mg/kg BW for the V2R antagonist) on sodium (top) 0 - 6 h 6 h - 24 h Whole 24 h and potassium (bottom) excretion rates as a function of the simul- taneous changes in fluid excretion rate (urine flow rate). Results Figure 4. Dose-dependent effects of the V2R antagonist on are expressed as Exp/Basal for each rat, and regression lines are urine flow rate and osmolality and on sodium excretion rate in the shown. For both the abscissa and the ordinate, a value of 1 means first 6 h after drug administration, in the subsequent 18 h (6 to no change. The equations of the regression lines and correspond- 24 h), and in the whole 24 h during the experimental day (basal ing correlation coefficients are as follows. For sodium with furo- day not shown; Experiment B, n ϭ 4 to 6 rats per dose). Paired t test, semide y ϭ 1.360x Ϫ 0.54; r ϭ 0.88, P Ͻ 0.001. For sodium with experimental versus basal: *P Ͻ 0.05; **P Ͻ 0.01; ***P Ͻ 0.001. the V2R antagonist y ϭ 0.189x ϩ 0.82; r ϭ 0.82, P Ͻ 0.001. For ϭ ϩ ϭ Ͻ comparable increases in V, the V2R antagonist induced a na- potassium with furosemide y 0.099x 0.88; r 0.45, P 0.05. ϭ ϩ ϭ triuresis approximately seven-fold smaller than did furo- For potassium with the V2R antagonist y 0.058x 0.86; r 0.77, P Ͻ 0.001. semide. Thus, even if their effects on electrolyte excretion should not be neglected, V2R antagonists remain predomi- nantly aquaretic. by the CD.35,36 This is confirmed here by the dose-dependent Several mechanisms may contribute to the natriuretic effect increase in TTKG seen in rats receiving dDAVP (Figure 1, bot- of the V2R antagonist. First, the antagonist probably inhibits tom left). Second, large increases in V are known to increase endogenous V2R-mediated stimulation of ENaC in the CD. potassium excretion by a flow-dependent mechanism.37 As Second, an increase in endogenous AVP level has been well seen in Figure 1 (bottom right), this physical effect is not due to documented in response to the rise in plasma osmolality in- an increase in potassium secretion, because there is no signifi- duced by the water loss after administration of aquaretics.33,34 cant increase in TTKG. Of note, the influence of dDAVP on Thus, the increased natriuresis could also be due to enhanced potassium secretion was also seen in healthy humans in some38 V1aR effects because these effects are natriuretic, as shown in but not all studies.23 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 V1aR Effects antagonist resulted in changes in V, osmolality, and sodium In agreement with a number of previous studies of rats, excretion very similar to those observed with the V2 antagonist dogs, sheep, and humans, we observed that AVP increased alone (data not shown). sodium excretion.2–9 The dose-response curve performed in The effects of the V2R agonist and antagonist on potassium this study shows that this effect occurred only for high doses ␮ handling deserve some comments. Although both drugs have (15 and 50 g/kg BW) that probably increased PAVP levels opposite effects on all other variables, they both increased po- distinctly above those involved in water conservation, even tassium excretion rate. These puzzling results are easily ex- after 24 h of water deprivation, as judged here by the urinary plained by two widely known previous observations. First, AVP data (Table 2). With lower doses (0.1 and 1 ␮g/kg BW), stimulation of V2R is known to increase potassium secretion likely corresponding to the range of usual osmotic stimuli,

1726 Journal of the American Society of Nephrology J Am Soc Nephrol 19: 1721–1731, 2008 www.jasn.org BASIC RESEARCH the antidiuretic effects of AVP were accompanied by a small ure 6, range A to B). Such purely antidiuretic action is visible in but significant reduction in sodium excretion, as with the study of Andersen et al.,44 who gave subpicomolar doses of dDAVP. Because the natriuretic effect of AVP 15 ␮g/kg BW AVP to individuals undergoing water diuresis. Although these was largely abolished by the co-administration of the selec- doses did not increase PAVP above the detection limit of the tive V1aR antagonist, these experiments clearly establish assay, V fell and Uosm rose significantly without any change in that the natriuresis induced by AVP is mostly due to V1aR- sodium excretion rate. Another example is found in individu- mediated actions. als with central diabetes insipidus, in whom dDAVP induced a Several possible renal and nonrenal mechanisms may ac- marked decline in V independent of any change in sodium count for this natriuretic effect. V1aR and V2R are expressed in excretion rate (see Figure 4 in reference23). This action of AVP multiple sites throughout the body and throughout the kidney in the low range of plasma concentrations can induce the re- itself. Although these experiments cannot disclose the mecha- absorption of very large amounts of solute-free water.17 nisms involved in the observed responses or the organs/tissues/ When PAVP rises a little more (beyond B in Figure 6), V2R cell types contributing to these changes, we can briefly mention effects stimulate sodium reabsorption and should thus reduce some possible pathways. (1) In several previous studies, the sodium excretion rate in addition to reducing V, as seen in AVP-induced natriuresis was assumed to result from the vol- healthy humans who received an infusion of AVP at a dose of ume expansion induced by fluid loads given orally and/or in- 25 ␮g/min per kg BW44 or with dDAVP23 and in the isolated rat travenously before and/or during AVP administration. The kidney perfused with a medium containing erythrocytes and authors proposed that this expansion induced the release of dDAVP.24 This sodium-retaining effect probably explains another mediator inhibiting renal tubular sodium trans- why, in normal individuals (with normal fluid and food in- port4,39; however, our experiments show that AVP increased take) who provided multiple urine samples, sodium excretion sodium excretion even in the absence of any fluid load. (2) rate declined in parallel with V in the samples in which Uosm Prostaglandins are known to reduce sodium transport in the exceeded 600 mOsm/kg H2O but was not altered in those un- CD40 and to increase medullary blood flow,41,42 two effects that der this limit despite wide differences in V (from 60 to 300 each will contribute in different ways to increased sodium ex- ml/h).45 This is in agreement with in vitro data showing that the cretion. Actually, prostaglandins have been shown to contrib- effect of AVP of sodium transport in isolated perfused rat CD ute to the AVP-induced natriuresis.9 AVP stimulates prosta- requires higher levels of peritubular AVP than the effect on glandin production in interstitial medullary cells,40 which water permeability.17 43 40 express V1aR, and in the cortical CD. V1aR in the CD are With further increases in PAVP, V1aR-dependent natri- mostly located in the luminal membrane14,15; therefore, uri- uretic effects appear and increase progressively, but the net nary, rather than peripheral AVP could be involved here. Of effect of AVP is still antinatriuretic (PAVP between C and M in note, AVP concentration is known to be higher in the CD Figure 6). Finally, with much higher levels of AVP, such as lumen and in the medullary interstitium than in peripheral those induced by exogenous AVP infusion at a rate higher than blood.17 (3) Finally, the natriuretic effects of AVP could be due 5 ␮g/kg, the V1a natriuretic effects overcome the V2 effects, to pressure-natriuresis resulting from the vasopressor effects of leading to increased natriuresis (PAVP beyond point M in Fig- the hormone, either within the whole circulation or selectively ure 6). We want to underline that the dose of AVP inducing within the kidney vasculature. either negative or positive effects on sodium excretion was quite variable among rats and experiments between 1 and 5 Integration of V2R and V1aR Effects ␮g/kg BW (data not shown). This variability may be due to This functional in vivo dissection of the respective influence of different sensitivities of the two receptors and/or of subsequent V1aR and V2R on sodium handling by the kidney provides for signal transduction, and to differences in AVP concentrations the first time several clues for a better understanding of the in luminal fluid of the CD and/or in medullary tissue (deter- integrated actions of AVP in vivo. Combined effects mediated mining V1a effects) with respect to peripheral AVP concentra- by V2R and V1aR can be predicted to vary greatly according to tion (determining V2 effects). the levels of plasma and urinary AVP. They will provide differ- The secretion of AVP is not known to depend on sodium ent sets of water and sodium responses. The changes in sodium intake. As discussed previously,20,23 the V2R-dependent stim- excretion as a result of the algebraic sum of V2 and V1a effects ulation of sodium reabsorption likely serves the purpose of over progressively increasing plasma levels of AVP are repre- fluid conservation rather than that of regulating sodium excre- sented schematically in Figure 6. V2R antinatriuretic and V1aR tion. A stronger sodium reabsorption in the AVP-sensitive natriuretic effects are depicted as sigmoid curves with different distal nephron, which expresses both ENaC and AQP2, will thresholds (B for V2R and C for V1aR effects) and different drive more water out of the lumen and will thus increase the AVP levels inducing the maximum effects for each receptor concentration of all other solutes but sodium. Thus, AVP will type (BЈ and CЈ, respectively). help conserve water at the expense of a less efficient sodium In the range of very low plasma levels of AVP, only the V2 excretion. V1aR-dependent effects that increase sodium excre- effects on water permeability of the CD are apparent, and AVP tion may be viewed as a safeguard mechanism limiting the risk will reduce V without any effect on sodium excretion rate (Fig- for too intense sodium retention. An imbalance between V1aR

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Figure 6. Schematic representation of the dose-dependent effects of AVP on sodium excretion rate and dissociation of these ef- fects into V2R- and V1aR-mediated re- sponses. 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). BЈ and CЈ correspond to the maximum effects depend- ing 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 re- sponses mediated by each of the two recep- tor types. and V2R sensitivity or signal transduction may thus influence administration of a selective V1aR nonpeptide antagonist the ability of the kidney to excrete sodium and may, indirectly, worsened BP and albuminuria52 and did not prevent or even be responsible for inappropriate sodium retention and result- aggravated diabetes-related vascular damage.53 (2) Several ing increase in BP. Actually, it has long been known that highly other studies suggested that V1a effects attenuate or counteract selective peptide and nonpeptide inhibitors of V1aR block exog- several direct and indirect V2R-dependent effects in normal enous AVP’s pressor action but that they have little effect on the rats and in rats with renal failure.54–57 (3) In patients with basal levels of arterial BP.46 Thus, under normal conditions of chronic renal failure, the increased urinary excretion of AVP cardiovascular homeostasis and appropriate extracellular fluid per remaining nephron correlated positively and more signif- volume, AVP and its V1aR seem to play only a minor role in icantly than for any other hormone with the fractional excre- maintaining normal cardiovascular function. In contrast, several tion of sodium, suggesting that luminal AVP contributed to observations link BP and the antidiuretic V2R-dependent actions ensure an appropriate natriuresis through V1aR-mediated lu- of AVP. Long-term dDAVP infusion in normal rats has been minal effects.58 (4) Interestingly, mice with deletion of the shown to increase BP by approximately 10 mmHg.47,48 Salt- V1aR exhibit a lower BP as a result not of a reduced vasocon- sensitive hypertension-prone Sabra rats exhibit higher AVP striction but of a lower extracellular fluid volume.59 mRNA, higher PAVP, and twofold higher Uosm and lower V In summary, these results provide a better knowledge of the than their salt-resistant counterparts.49 In humans, significant respective V2R- and V1aR-dependent effects of AVP on so- negative correlations were observed between 24-h V and BP.13,50 dium excretion in rats. Whether a similar balance between

In the usual range of PAVP, the antidiuretic effect of AVP is V1aR- and V2R-dependent effects occurs in humans remains possibly associated with an antinatriuretic effect of variable to be determined. These results are potentially important in intensity depending on the level of urine concentration. This view of the proposed use of AVP antagonists in several human will delay the excretion of sodium and could thus result in disorders.10–12 In patients with hypervolemic hyponatremia as some degree of sodium and volume retention, which could a result of heart failure or cirrhosis, the additional effect of V2 favor an increase in BP. The natriuretic effects induced by the antagonism on sodium excretion may be beneficial and may V1aR stimulation may counteract this tendency. Thus, instead decrease diuretic use. V2R antagonists may also be beneficial in of the usually assumed pressor effect, V1a activation within the some forms of salt-sensitive hypertension13,60; however, more physiologic range may actually reduce the risk for V2R-induced precise information is required regarding V1a and V2 effects in sodium retention. Moreover, the balance between V1aR- and humans to know whether selective rather than mixed antago- V2R-mediated actions in cells possessing the two types of re- nists are more appropriate in each of these disorders. ceptors (e.g., cortical CD) may be further amplified by a down- regulation of the V2R induced by a V1a-mediated pathway.51 That V1aR effects counteract V2R tubular effects could ex- CONCISE METHODS plain several puzzling observations. (1) In pathologic models Animals and General Procedures in which AVP levels are known to be elevated (nitric oxide– Adult male Wistar rats (Charles River, L’Arbresle, France) of initial deprived hypertensive rats and diabetic rats), the long-term body weight 230 to 240 g were housed individually in metabolic cages

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with free access to tap water and powdered food (A03; Safe, Villem- dDAVP (600 ␮l emulsion/kg BW), in five groups of rats. The concen- oisson/Orge, France) at all times before and during the experiments tration of AVP in the aqueous phase was designed to deliver 0.0 (ve- (except for one rat group in experiment E, discussed later). They were hicle alone), 0.1, 1.0, 15.0, or 50.0 ␮g/kg BW (corresponding approx- accustomed to the cages for 5 d before the experiments. All experi- imately to 0.1, 1.0, 15.0, or 50.0 nmol/kg BW). As in previous ments were carried out in conscious, undisturbed rats and involved studies,54,57 the dose range for AVP was approximately five times no previous water load, no instrumentation, and no pretreatment. higher than that for dDAVP because of the faster degradation of AVP Each experiment included 24 to 36 rats studied in parallel on two in vivo. 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 Natural AVP Plus V1aR Antagonist SR49059 (experimental) with AVP and/or various drugs (see details that fol- (Experiment D) low). Urine was collected for 6 h starting just after the injection(s). For evaluation of the role of V1aR in the response to the natural ␮ Urine volume was determined gravimetrically, assuming the density hormone AVP, rats of this series were administered AVP (15 g/kg BW, same procedure as in Experiment C) without or with the con- of urine was equal to unity. Uosm was measured with a freezing point osmometer (Roebling, Berlin, Germany) and urinary concentration comitant administration of the selective nonpeptide V1aR antagonist of sodium and potassium by flame photometry (IL 943, Instrument SR49059 (Relcovaptan; Sanofi-Aventis) at a dose of 10 mg/kg BW, 64 Laboratory, Lexington, MA). Urine urea concentration was measured shown in previous studies to block the pressor response to AVP. with a standard kit (Urea Kit S 1000; BioMe´rieux, Lyon, France). This drug was dissolved in DMSO and cremophor (each 10% of final In each experiment, on the basis of values obtained during the volume) and then in saline, and was injected intraperitoneally (1 basal day, the rats were divided into several groups of equivalent urine ml/kg BW). For comparison, additional rats received vehicle alone, ␮ volume and osmolality (n ϭ 4 to 6 per group) to test, during the the V1aR antagonist alone, AVP at a lower dose (3 g/kg BW), or ␮ experimental day, the effects of the various doses of hormones or dDAVP (0.6 g/kg BW). drugs in groups of rats with equivalent urine concentrating activity. Urinary AVP Concentration in Various Situations Usually, three or four independent experiments were performed in (Experiment E) the same series of rats at 1-wk intervals (to allow washout from the To evaluate the concentration of AVP prevailing in the lumen of the preceding experiment). All animal procedures were conducted in ac- CD in different situations, we performed additional experiments in cordance with the European guidelines for the care and use of labo- which urine was collected for 6 h, as in the other experiments, under ratory animals. various experimental conditions, including injection of isotonic sa- line, injection of dDAVP 0.6 ␮g/kg BW, injection of AVP 3 or 15 V2R Agonist dDAVP: Dose-Response Curve ␮ g/kg BW, and 24-h dehydration. Urine volume and Uosm were mea- (Experiment A) Ϫ dDAVP (Minirin; Ferring Pharmaceuticals AB, Malmo¨, Sweden) is a sured, and aliquots of urine were stored at 20°C for further AVP 65 selective peptidic V2R agonist.61 The dDAVP solution was emulsified assay. UAVP was measured as described previously. The antibody in oil (1:9 vol/vol) to prolong its bioavailability, and injected subcu- used does not recognize dDAVP. taneously (under the skin of the back of the neck) in seven rat groups (600 ␮l emulsion/kg BW) at concentrations in the aqueous phase Comparison of V2R Antagonist and Furosemide designed to deliver 0.00 (vehicle alone), 0.02, 0.06, 0.20, 0.60, 2.00, or Effects (Experiment F) Classical diuretics and aquaretics (V2R antagonist) increase V by dif- 6.00 ␮g/kg BW (corresponding approximately to 0.02, 0.06, 0.20, ferent mechanisms. To compare the respective effects of the two types 0.60, 2.00, or 6.00 nmol/kg BW). of drugs on water and sodium excretion, we performed an additional V2R Antagonist SR121463A: Dose-Response Curve experiment in which different groups of rats received either furo- (Experiment B) semide (Lasix; Sanofi-Aventis) or the V2R antagonist (prepared and SR121463A (; Sanofi-Aventis, Toulouse, France) is a selec- injected as in Experiment B) at various doses (0.0, 0.1, 0.3, 1.0, 10.0, tive nonpeptide V2R antagonist with a relatively long half-life.62,63 On and 30.0 mg/kg BW for furosemide and 0.0, 0.1, 0.3, 1.0, and 10.0 the experimental day, the V2R antagonist dissolved in DMSO (10% mg/kg BW for the V2R antagonist). Urine was collected for 6 h after final volume) and saline was injected intraperitoneally (1 ml/kg BW) the injections. 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 Statistical Analysis experiment, urine was collected not only for the first 6 h after the drug V and the excretion rate of the different solutes were calculated administration but also in separate tubes for the subsequent 18 h (i.e., according to standard formulas. The TTKG, reflecting the inten- from 6 to 24 h after the injection) so as to evaluate the delayed effects sity of potassium secretion, was calculated by dividing the urine/ of the drug. plasma potassium concentration ratio by the urine/plasma osmo- lality ratio.66 Plasma osmolality was arbitrarily considered to be

Natural AVP: Dose-Response Curve (Experiment C) 300 mOsm/kg H2O and plasma potassium concentration 5 Synthetic [Arg8]-vasopressin acetate salt (Sigma-Aldrich, Chemie, mmol/L in all rats. Steinheim, Germany; i.e. natural AVP), dissolved in saline, was emul- Results are expressed as means Ϯ SEM. In Experiments A through sified in oil (1:9 vol/vol) and injected subcutaneously, as described for D, each rat was its own control. Thus, for each variable, the Exp/Basal

J Am Soc Nephrol 19: 1721–1731, 2008 AVP Receptors and Na Excretion 1729 BASIC RESEARCH www.jasn.org provides a quantitative estimate of the changes induced by the hor- 9. Kompanowska-Jezierska E, Emmeluth C, Grove L, Christensen P, Sa- mone and/or drug. The statistical significance of these changes was dowski J, Bie P: Mechanism of vasopressin natriuresis in the dog: Role evaluated by paired t test comparing results of the experimental day of vasopressin receptors and prostaglandins. Am J Physiol 274: R1619–R1625, 1998 with those of the basal day in each rat. In Experiment E, comparison 10. Verbalis JG: Vasopressin V2 receptor antagonists. J Mol Endocrinol between the various groups was performed by one-way ANOVA fol- 29: 1–9, 2002 lowed by Fisher post hoc test. In Experiment F, linear regressions were 11. Lemmens-Gruber R, Kamyar M: Vasopressin antagonists. Cell Mol Life calculated between the changes in sodium or potassium excretion and Sci 63: 1766–1779, 2006 those in water excretion. 12. Torres VE: Vasopressin antagonists in polycystic kidney disease. Kid- ney Int 68: 2405–2418, 2005 13. Bankir L, Perucca J, Weinberger MH: Ethnic differences in urine con- centration: Possible relationship to blood pressure. Clin J Am Soc ACKNOWLEDGMENTS Nephrol 2: 304–312, 2007 14. Ando Y, Asano Y: Functional evidence for an apical V1 receptor in rabbit cortical collecting duct. Am J Physiol 264: F467–F471, 1993 Financial support for these studies was provided by the INSERM an- 15. Ikeda M, Yoshitomi K, Imai M, Kurokawa K: Cell Ca2ϩ response to nual budget of Unit 872 (ex Unit 652 and 367; to L.B.) and by Canada luminal vasopressin in cortical collecting tubule principal cells. Kidney Research Chair Program (to D.G.B.). J.P. received a fellowship from Int 45: 811–816, 1994 the French Society of Nephrology in 2006 to 2007. 16. Robertson GL: The regulation of vasopressin function in health and disease. Recent Progr Horm Res 33: 333–385, 1977 Part of this work was presented at the annual meeting of the Amer- 17. Bankir L: Antidiuretic action of vasopressin: Quantitative aspects and ican Society of Nephrology; October 29 through November 1, 2004; interaction between V1a and V2 receptor-mediated effects. Cardio- St. Louis, MO; and published in abstract form (J Am Soc Nephrol 16: vasc Res 51: 372–390, 2001 87A, 2004). 18. Serradeil-Le Gal C, Derick S, Brossard G, Manning M, Simiand J, We thank Sanofi-Aventis for supplying the antagonists used in the Gaillard R, Griebel G, Guillon G: Functional and pharmacological characterization of the first specific agonist and antagonist for the V1b experiments described in this article. receptor in mammals. Stress 6: 199–206, 2003 We thank Claudine Serradeil-Le Gal (Sanofi-Aventis, Toulouse, 19. Saito M, Tahara A, Sugimoto T: 1-Desamino-8-D-arginine vasopressin France) for fruitful scientific discussions, Marie-Franc¸oise Gonzales (DDAVP) as an agonist on V1b vasopressin receptor. Biochem Phar- for pilot experiments, and Marie-Franc¸oise Arthus and Miche`le Lon- macol 53: 1711–1717, 1997 ergan (Hoˆpital du Sacre´-Coeur, Montre´al, Que´bec, Canada) for AVP 20. Nicco C, Wittner M, DiStefano A, Jounier S, Bankir L, Bouby N: Chronic exposure to vasopressin upregulates ENaC and sodium trans- measurements. port in the rat renal collecting duct and lung. Hypertension 38: 1143– 1149, 2001 21. Kim JK, Summer SN, Erickson AE, Schrier RW: Role of arginine vaso- DISCLOSURES pressin in medullary thick ascending limb on maximal urinary concen- tration. Am J Physiol 251: F266–F270, 1986 None. 22. Huang DY, Pfaff I, Serradeil-Le Gal C, Vallon V: Acute renal response to the non-peptide vasopressin V2-receptor antagonist SR 121463B in anesthetized rats. 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