Nephrol Dial Transplant (2001) 16: 725-734
© 2001 European Renal Association-European Dialysis and Transplant Association
Selective blockade of vasopressin V2 receptors reveals significant V2-mediated water reabsorption in Brattleboro rats with diabetes insipidus
1 INSERM Unité 367, 17 Rue du Fer à Moulin, Paris and 2 SANOFI, Département de Recherche Exploratoire, Toulouse, France
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Abstract |
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Background. In a previous study we observed that acute administration of the selective antagonist of vasopressin (AVP) V2 receptors, SR 121463A (SR), aggravated the symptoms of diabetes insipidus (DI) in homozygous Brattleboro rats (an AVP-deficient strain). The present study investigates in more details the acute and chronic effects of SR in DI rats.
Methods and results. In experiment A, different groups of rats received acute i.p. injections of SR (0.001–10 mg/kg) or vehicle alone, and urine was collected for the next 24 h. SR dose-dependently increased urine flow rate and decreased urine osmolality with no significant change in solute excretion, thus confirming a pure ‘aquaretic’ effect. In experiments B and C, the chronic effects of orally administered SR were evaluated over 8 days in Brattleboro DI rats (experiment B, 1 mg/kg/day) and in adult Sprague–Dawley rats with normal AVP secretion (experiment C, 3 mg/kg/day). In DI rats, the aquaretic effects of SR persisted with the same intensity over the 8 days. In Sprague–Dawley rats, SR induced a sustained, stable aquaretic effect and also increased non-renal water losses, suggesting an effect of AVP on water conservation in extrarenal sites. Because oxytocin (OT) synthesis is elevated in DI rats and OT is known to bind to V2 receptors, we evaluated the antidiuretic effects of OT in DI rats in experiment D. Chronic infusion of OT (3 µg/kg/h, i.p.) induced a marked antidiuresis, and acute SR (1 mg/kg) in OT-treated DI rats completely abolished this antidiuretic effect, thus indicating that it was due to binding of OT to V2 receptors.
Conclusion. (i) SR is a potent orally active aquaretic and induces stable effects during 1 week in rats with or without endogenous AVP secretion. (ii) Significant V2 receptor-mediated water reabsorption occurs in collecting ducts of Brattleboro DI rats because their usual urine osmolality is about twofold higher than the minimum observed during SR-induced maximum diuresis. (iii) This V2 agonism could be mediated in part by OT binding to V2 receptors. Small amounts of endogenous AVP, known to be produced by adrenal and testis in DI rats, could also contribute to this V2 agonism, as well as a possible constitutive activation of the V2 receptors. (iv) In normal rats, AVP probably reduces water losses through extrarenal sites, probably the lungs.
Keywords: antidiuretic hormone; diabetes insipidus; lung; oxytocin; urinary dilution; vasopressin receptor antagonism
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Introduction |
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The main physiological function of the hormone vasopressin (also called arginine-vasopressin (AVP), or antidiuretic hormone (ADH)) in mammals is to promote water reabsorption in renal collecting ducts (an effect mediated by V2 receptors), thus enabling the kidney to conserve water and excrete urinary solutes in a relatively low volume of hyperosmotic urine. The understanding of vasopressin actions in collecting duct cells and more largely in the urinary concentrating mechanism, has benefited from a unique model of rats presenting a spontaneous ‘knock out’ due to a single base deletion in the vasopressin gene [1]. Homozygous ‘Brattleboro’ rats are unable to synthesize functional vasopressin and thus suffer a recessive form of central diabetes insipidus (DI) [2]. Urine is typically hypo-osmotic to plasma (150–250 mOsm/kg H2O) and daily urine flow (and consequently water intake) can be as high as the rat's own body weight.
Selective antagonists of V2 receptors could represent powerful tools for the treatment of several pathological states associated with disorders of water balance and/or vasopressin secretion. Promising peptidic analogues of vasopressin displaying potent V2 antagonist activity in vitro and in vivo in several animal models have been found to present significant agonistic effects when tested in humans [3]. Moreover, the therapeutic utility of peptidic drugs has been severely hampered by their lack of oral activity. Recently, powerful selective non-peptidic antagonists of vasopressin have been designed [4–9]. Administration of these drugs (so-called aquaretic agents) results in a marked increase in diuresis and a fall in urinary osmolality, without alteration in osmolar or sodium and potassium excretion, in experimental animals and humans [5–7]. During the development of these new drugs, Brattleboro rats with DI were widely used to reveal possible agonistic effects [7]. This model is particularly convenient for the disclosure of partial agonistic properties of putative aquaretic agents, because the lack of endogenous vasopressin favours the disclosure of even a modest agonist effect, which could be obscured by endogenous vasopressin in normal rats. Actually the agonist activity of early peptidic vasopressin antagonists observed in healthy humans, but not in healthy rats, is very apparent in Brattleboro DI rats [4,7].
The study of Serradeil-Le Gal et al. [7] revealed that the non-peptide V2 receptor antagonist, SR 121463A (SR) markedly worsened the DI symptoms of Brattleboro rats, doubling their urine flow rate and decreasing their urine osmolality by half. These effects lasted for several hours after drug administration. This observation suggests that in spite of their inability to secrete vasopressin, a significant amount of water is reabsorbed by the collecting duct in homozygous Brattleboro rats, in response to the occupancy of V2 receptors. This prompted us to study in more details the mechanism of this residual antidiuretic activity of Brattleboro DI rats and to see if it could be antagonized chronically without desensitization.
In the present paper we confirm that this antidiuretic activity is V2 receptor-mediated and can be blocked in a chronic fashion. Sustained stable maximal urinary dilution was achieved pharmacologically in conscious Brattleboro rats and sustained positive free-water clearance was induced in vasopressin-replete rats. In addition, we bring indirect arguments in favour of a role for oxytocin in V2-mediated water reabsorption in Brattleboro rats. Finally, the present studies reveal that vasopressin contributes to water conservation not only by reducing water excretion through the kidneys, but also by reducing water losses through non-renal sites, i.e. probably through the lungs and airways.
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Subjects and methods |
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Animals and general procedures
The whole study included three different experiments performed in Brattleboro rats (experiments A, B, and D) and one (experiment C) in normal Sprague–Dawley rats. Male adult homozygous Brattleboro rats (initial body weight 220–300 g) with diabetes insipidus (DI) used in experiments A1, A2, and D were bred at INSERM Unit 90 (Necker Hospital, Paris) and those used in experiment B were bought from Harlan Sprague–Dawley (Indianapolis, USA). Experiment C was performed in normal male Sprague–Dawley rats bought from Iffa Credo (France) (initial body weight 280–300 g). All rats were housed in individual metabolic cages (Tecniplast, Varese, Italy) at 20–22°C with a 12/12 h light/dark cycle. They had free access to normal powdered laboratory rat food (M25, Pietrement, Paris) and tap water at all times, during all experiments. They were accustomed to the cages for 8 days before the beginning of any measurement. In each experiment, rats were divided into groups of equivalent body weight, diuresis and osmolality (based on 2x24 h urine collections), in order to test different doses of drug, or vehicle alone, in groups of rats with comparable basal conditions.
Drugs
SR, a potent and selective non-peptide vasopressin V2-receptor antagonist has been shown to bind very selectively to rat and human kidney V2 receptors. Its affinity for other related receptors (vasopressin V1a, V1b, or oxytocin) is 3000–10 000-fold lower than that for the V2 receptor [7]. Thus any effect of this drug can be interpreted as a consequence of its selective binding to V2 receptors.
SR and OPC 31260 (OPC), another published non-peptide vasopressin V2/V1a compound [7], were synthesized in SANOFI (Toulouse, France). The structures of SR and OPC were determined by 1H and 13C NMR and infrared spectroscopy. The molecular weights, determined by mass spectrometry, are 736.6 and 427.5 for SR and OPC respectively. Melting points of 172 and 207.8°C respectively were obtained. The purity, measured by high-pressure liquid chromatography, thin-layer chromatography, and elemental analysis, was >98%. The analytical parameters reported above for OPC are identical to those initially described for this molecule [7]. SR was dissolved in dimethylformamide (DMF) (experiment A) or saline (experiment D) for intraperitoneal injections and in 0.6% cellulose in water for oral administration (experiment B and C). OPC was solubilized in DMF for i.p. treatment. Oxytocin (OT) was purchased from Sigma (France).
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Experimental protocols |
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Acute effects of SR in DI rats (Experiments A1 and A2)
The aim of this series of experiments was to evaluate the time-course of the diuretic effects of a high dose of SR (10 mg/kg, i.p.) (experiment A1) and to establish a dose–response curve of these effects (experiment A2) in DI rats. Drug dissolved at the appropriate concentration in DMF, or DMF alone was injected i.p. (100 µl per rat) with a 500 µl Hamilton glass syringe at
9:00 a.m.±15 min, and urine was collected as indicated below. In experiment A1, urine was collected for the preceding 24 h to verify that urine flow rate and osmolality were normal in all rats. Five DI rats received SR, 10 mg/kg, and four rats vehicle only. Urine was then collected in periods of 2 h from the time of injection to 5.00 p.m. (4 samples) and from 5.00 p.m. to 9.00 a.m. on the next day (to complete a 24-h period after injection). Urine volume and osmolality, as well as concentrations of urea, sodium, and potassium were measured as described below. Means of results obtained in rats injected with either SR or vehicle were compared by Student's t-test.
In experiment A2, 24 DI rats divided in four equivalent groups were used simultaneously for several acute i.p. injections of SR (0.001–10 mg/kg) or vehicle, in a random fashion. The same rats received different doses of drug or vehicle on different days, with successive injections separated by at least a 3-day wash-out period (or 1 week after the highest dose of 10 mg/kg). For comparison, five rats also received, once, 10 mg/kg of OPC 31260. Each series of injections included the following steps. Collection of 24-h urine of the preceding day (to ensure that urine flow rate and osmolality were normal in all rats); injection of drug or vehicle, at 9.00 a.m.±15 min; urine collection in periods of 2 h from the time of injection to 5.00 p.m. and from 5.00 p.m. to 9.00 a.m. of the next day (to complete a 24-h period after injection). Urine volume and osmolality were measured as described below.
Results of two or three different injections of the same doses or vehicle in four–six rats each were pooled making 10–12 rats per dose (except for the lower dose which was only given once in six rats). Cumulated urine volumes over the first 6 and 24 h after drug or vehicle, and corresponding urine osmolality were calculated. Vehicle was given in 16 rats in all. Differences observed between the different doses of SR and vehicle were analysed by one-way ANOVA followed by Fisher post hoc test.
Chronic effects of orally administered SR in DI rats (experiment B)
The aim of this experiment was to evaluate if chronic V2 antagonism induces sustained changes in diuresis in homozygous Brattleboro rats. Eighteen DI rats were divided into two equivalent groups of nine rats each. Rats of the two groups underwent gastric gavage every morning at 9.00 a.m. for 8 days with either SR, 1 mg/kg in 0.6% methylcellulose, or 0.6% methylcellulose alone (0.6 ml/rat). Food and fluid intakes, urine flow rate, and osmolality were measured daily. Results were analysed by two-way ANOVA to evaluate the effects of the drug and the influence of time (days of treatment).
Chronic effects of orally administered SR in Sprague–Dawley rats (experiment C)
The influence of chronic blockade of V2 receptors on renal and extrarenal water losses was evaluated in rats with normal vasopressin secretion. Two modes of oral administration were compared, i.e. gastric gavage once daily, and addition of the drug to the food. Eighteen adult male Sprague–Dawley rats were divided in three equivalent groups of six rats each. Rats were gavaged once daily for 7 days (0.6 ml/rat), at 9.00 a.m. with either SR at a dose of 3 mg/kg in 0.6% methylcellulose, for one of the three groups, or with 0.6% methylcellulose alone for the two other groups. In one of the latter two groups, SR was added to the food to achieve a daily intake of 3 mg/kg (equivalent to the dose given by gavage to the other group). In order to ensure homogenous concentration of the drug in the food, a pasty food was prepared by mixing normal powdered rat food (same as used in experiments A and B) with an agar gel (270 g dry food and 1 g agar in 100 ml water, heated at 70°C, and then cooled at room temperature) [10]. A similarly prepared pasty food, but without drug, was given to the other two groups. All rats were offered only 20.5 g pasty food per day (somewhat less than their spontaneous intake) so as to achieve the same drug intake in all treated rats, and the same total food intake in all rats (20.5 g pasty food provided 15 g dry food and 5.5 ml water). It was verified that all rats emptied their food dispenser every day. Rats had free access to tap water during the entire experiment. Urine was collected every day and urine volume and osmolality measured. Results obtained for each rat during days 4–7 were averaged and data from the three groups were compared by one way ANOVA.
Antidiuretic effects of oxytocin and influence of V2 antagonism (experiment D)
The aim of this experiment was to see if OT can exert an antidiuretic effect in Brattleboro DI rats and if this effect is abolished by selective V2-receptor antagonism. Nineteen DI rats were divided into three equivalent groups of six–seven rats each. Rats of two of the groups were implanted with osmotic minipumps (Alzet, Charles River, France, model 2002) placed in the peritoneal cavity under brief ether anaesthesia, and delivering OT at a rate of 3 µg/kg/h. This dose was chosen because of its demonstrated antidiuretic effect [11]. Rats of the third group served as controls and were sham-operated (anaesthesia and laparotomy, but no minipump implanted). On the 4th day of the chronic OT treatment, one of the two treated groups received an acute i.p. injection of SR at 9.00 a.m., at a dose of 1 mg/kg dissolved in 100 µl saline. Rats of the two other groups (OT and control) received an acute i.p. injection of saline alone. Urine was collected for the next 3 days in periods of 24 h and urine volume and osmolality were measured.
Measurements and calculations
Urine volume was determined gravimetrically, assuming density of urine was equal to unity. Urine osmolality (Uosm) was measured by the freezing point method (Roebling, Berlin, Germany). Sodium and potassium concentrations were measured by flame photometry (IL-243-05, Instrumentation Laboratory, USA) and urea with a standard Kit (Kit S 180, BioMérieux, Lyon, France). Urine flow rate (V), osmolar excretion, excretion of the different solutes, and solute-free water clearance (CH2O) were calculated according to standard formulae. Plasma osmolality was arbitrarily considered to be 300 mOsm/kg H2O in all rats, for the calculation of CH2O. Extrarenal water losses were calculated as the difference between daily water intake and urine flow rate.
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Results |
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Acute effects of 10 mg/kg i.p. SR in Brattleboro DI rats (experiments A1 and A2)
Because of their genetic defect in the vasopressin gene, DI rats produce large volumes (10–15 ml/2 h or
180 ml/day) of hypo-osmolar urine (150–250 mOsm/ kg H2O). Acute i.p. administration of SR, 10 mg/kg BW, induced an aggravation of DI symptoms with further decline in Uosm down to 100 mOsm/kg H2O and doubling of urine flow rate, leading to a marked increase in solute-free water clearance. In the first 2 h, the fall in urine osmolality preceded the increase in urine flow rate. Thereafter, a steady state was maintained for at least 6 h, during which free-water clearance was on average three-fold higher in SR-treated than in control rats (Table 1
). The diuretic effect declined during the following 16-h period (not shown). The marked influence of SR on water excretion was not accompanied by any change in total solute excretion, nor in any of the main individual urinary solutes, Na, K, and urea, either during the whole 24 h following drug administration (not shown), or during the first 6 h (Table 1). This short period of observation is more appropriate to disclose a possible early influence of the drug on solute excretion, which could have been compensated for in subsequent hours, when the diuretic effects of the drug declined.
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The influence of acute SR was dose-dependent as shown in Figure 1
. In the first 6 h after drug administration (Figure 1, left), a significant effect on Uosm and CH2O was detected with as low as 0.01 mg/kg. Urine flow rate was also slightly increased after this dose, but the difference reached statistical significance only with a 10-fold higher dose, 0.1 mg/kg. One order of magnitude higher doses were required to induce significantly different changes over a 24-h period (Figure 1, right). Note that Uosm was more sensitive to the drug than urine flow rate. The minimum Uosm attainable during the first 6 h seems to be reached with 1 mg/kg.
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As already reported [7], the V2-receptor antagonist OPC was much less potent than SR. Ten milligrams per kilogram OPC increased V and CH2O, and decreased Uosm, but the differences did not reach statistical significance, even over the first 6 h (not shown). The difference in mean CH2O between drug- and vehicle-injected rats amounted to 39.7 ml/ 6 h for SR and to only 15.4 ml/6 h for OPC. Values for the entire 24 h were 161 and 32 ml/24 h respectively.
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Chronic effects of orally administered SR in Brattleboro DI rats (experiment B)
Because SR has been shown to be a potent orally active compound, the convenient oral route was chosen for the chronic study. Previous experiments showed that the drug is about five times less potent orally than when administered i.v. or i.p. [7]. Repeated administration of SR, 1 mg/kg, by daily gavage, over 8 days induced a sustained increase in urine flow rate to about 240 ml/day and a fall in Uosm to about 110 mOsm/kg H2O, resulting in a twofold increase in CH2O compared to vehicle-treated DI rats (Figure 2
). All parameters remained fairly stable during the 8 days of treatment, thus showing no attenuation of the aquaretic effect of SR with time. Two-way ANOVA disclosed a highly significant effect of the drug, no effect of time, and no interaction.
Chronic SR administration in DI rats increased daily water intake (WI) by the same amount as the increase in urine flow rate, thus disclosing no influence of SR on non-renal water losses (amounting to about 18 ml/day per rat or 6.7 ml/day per 100 g BW in both groups). Rats treated with vehicle only gained 18 g BW in 8 days whereas rats receiving the drug gained almost no weight. Food intake was slightly lower in SR- than in vehicle-treated rats although this difference was not statistically significant. However, it probably accounts for the slightly but significantly lower osmolar excretion observed in these rats (-9%, P<0.01).
Chronic effects of oral SR in Sprague–Dawley rats (experiment C)
Experiment C investigated the effects on water excretion of chronic administration of SR in Sprague–Dawley rats exhibiting normal vasopressin secretion. As shown in our previous study [7], 3 mg/kg SR per os in rats with normal vasopressin secretion induces an aquaretic effect which is lower than the maximum attainable with this drug. This intermediate dose was chosen in the present experiment in order not to induce an excessive diuresis and thus excessive need to drink which could perturb the rats' behaviour and feeding pattern. As shown in Table 2, the resulting Uosm was close to iso-osmolality, and urine flow rate was increased 3.5–4 fold above that in control rats, without any change in osmolar excretion (and thus probably in food intake). Notably, aquaretic effects of SR were 30% and 20% more intense during days 1 and 2 of drug administration (not shown) than in subsequent days, and plateaued thereafter to values shown in Table 2. This can be explained by a rise in endogenous secretion of vasopressin, subsequent to the initial dehydration that followed the first exposure to V2 antagonism.
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The intensity of the aquaretic effects was similar when the drug was administered by once-daily gavage in the morning, or when it was mixed with the food and thus ingested progressively, with maximal consumption at night (because rats are nocturnal). Interestingly, extrarenal water losses were more than doubled by chronic SR administration (P<0.01), suggesting that V2 receptor blockade affects water turnover in organs other than the kidney (Table 2
). Also note that extrarenal water losses were about twofold lower per unit body weight in SD rats than in Brattleboro DI rats (3.5 ml/day per 100 g BW vs 6.7 in DI rats).
Antidiuretic effects of oxytocin and influence of V2 antagonism in Brattleboro DI rats (experiment D)
Experiment D was designed to investigate if OT could induce a significant antidiuresis by binding to V2 receptors. If this were indeed the case, this antidiuresis should be reversed by a selective V2 antagonist. Figure 3 shows that OT given in supraphysiological amounts was indeed a powerful antidiuretic hormone, bringing Uosm to more than 1500 mOsm/kg H2O and reducing V to 20 ml/day vs 200 mOsm/kg H2O and 150 ml/day respectively, in sham-treated rats. This effect did not decline over time for 6 days. In one of the two OT-treated groups, SR administered acutely i.p. (1 mg/kg) on day 4 completely abolished the antidiuretic action of OT, bringing Uosm down to values observed in DI rats without OT treatment. Urine flow rate and free-water clearance increased to even higher values than those seen in control DI rats (sham-treated rats for OT). Vehicle of SR given to the other OT-treated group was without effect (Figure 3). These observations indicate that the antidiuretic effect of OT involves specific binding of OT to vasopressin V2 receptors.
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Discussion |
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In a previous study, acute i.p. administration of a selective V2-receptor antagonist SR 121463A in Brattleboro rats lacking vasopressin revealed that these rats actually exhibit some significant V2-mediated water reabsorption, because this drug induced a marked further increase in urine flow rate and fall in urine osmolality [7]. The present study shows that this effect is dose-dependent and is sustained without tachyphylaxis during chronic administration of the drug for at least 8 days. In addition, this study confirms that OT may act as a very potent antidiuretic hormone when administered in supraphysiological amounts and shows that this antidiuretic action is counteracted by selective antagonism to V2 receptors. Finally, experiments in Sprague–Dawley rats (with normal endogenous vasopressin secretion) revealed that chronic administration of a selective V2-receptor antagonist not only increased diuresis but also enhanced extrarenal water losses, thus disclosing a V2-mediated influence of vasopressin on water reabsorption in extra-renal sites.
SR appears to be a potent ‘aquaretic’ drug even in homozygous Brattleboro rats. A significant effect over 24 h was obtained with 0.1 mg/kg, i.p., i.e. only threefold more than the minimum dose required to induce a significant effect over 24 h after i.v. administration in Sprague–Dawley rats with normal vasopressin secretion [7]. Although Brattleboro rats already exhibit a large positive free-water clearance (75 ml/day for a 250 g rat), SR at 1 and 10 mg/kg respectively doubled and tripled this parameter.
Over a 6-h period of time, the minimum effective dose in Brattleboro rats was 0.01 mg/kg and the maximum effect was reached for 1.0 mg/kg, i.e. a range spanning about 2 orders of magnitude. Increasing the dose further did not induce a more intense effect but increased its duration. For example, 1 and 10 mg/kg elicited the same change in CH2O over 6 h, but 10 mg/kg induced a twofold higher increase in CH2O over 24 h than did 1 mg/kg. Note that CH2O is the most sensitive index of the aquaretic effect of SR. A significant increase in CH2O was already detectable over 24 h for 0.01 mg/kg, when no significant changes were noted in either urine flow rate or osmolality. Urine flow rate is a less sensitive index than urine osmolality, both over the first 6 h and over 24 h.
In Brattleboro rats, SR induced a selective increase in water excretion but did not influence solute excretion, as already observed in rats with normal vasopressin secretion [7]. Thus, this drug is a very specific ‘aquaretic’ at variance with so called ‘diuretics’ which primarily influence solute transport along the nephron and only secondarily water transport. The V2-receptor antagonist, OPC, although aquaretic in normal rats, did not reveal any aquaretic effect in Brattleboro rats in the study of Yamamura et al. [4] and only a weak aquaretic effect in our own hands, in agreement with a much lower affinity (about 20-fold) than SR for rat renal V2 receptors [7].
In Sprague–Dawley rats, SR was given orally at a dose that did not produce the maximal possible effect but brought Uosm close to iso-osmolality. In these rats with normal vasopressin secretion, the aquaretic effect of SR was sustained for more than a week without any sign of desensitization and with no influence on solute excretion. The drug was well tolerated and the once-daily dose induced the same effect over 24 h as the more diffuse administration achieved by incorporation of the drug in the food. This suggests that this drug could be used in chronic treatments without a risk of tachyphylaxis and with a convenient pharmacokinetic profile. Using a different V2 antagonist, OPC 41061, Yamamura et al. [9] indicated that the level of diuresis remained stable during 4 weeks of daily administration. Unfortunately, 24-h urine osmolality and urine flow rate are not reported in their study and the paper only describes the changes observed during the first 4 h post-dosing [9].
The usual Uosm of homozygous Brattleboro rats (150–250 mOsm/kg H2O) is not the lowest possible Uosm a rat can achieve. Intense water loading in normal rats is reported to bring Uosm to as low as 70 mOsm/kg H2O, at least in acute studies [12,13]. In the present study, Uosm was maintained at a daily average of 120 mOsm/kg H2O during several days (range 90–140 mOsm/kg H2O). Even lower values (down to 77 mOsm/kg H2O) may be reached in Brattleboro rats, after discontinuation of a chronic treatment with dDAVP, a potent peptidic V2 receptor agonist of AVP (1-deamino, 8-D-arginine vasopressin), as shown in Table 3 [14]. The production of highly diluted urine was attributed, in this case, to increased diluting capacity of the kidney resulting from hypertrophy of the diluting segment (the thick ascending limb of the loop of Henle) induced by prior chronic dDAVP treatment [15].
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That an antagonist of vasopressin V2 receptors is able to increase markedly CH2O in Brattleboro rats reveals that these rats still possess some significant V2-dependent water reabsorption in their distal nephron. This ‘concentrating’ activity, even if not bringing urine to become hyperosmotic to plasma, usually masks the maximum ‘obligatory’ dilution achieved in the rat kidney. This maximum dilution results from sodium reabsorption in the distal tubule (thiazide sensitive) and collecting ducts (amiloride sensitive), which further dilutes the hypo-osmotic fluid exiting the ascending limb of the loop of Henle.
SR exhibits high affinity and selectivity for V2 receptors and has a very low affinity for other related receptors including V1a, V1b, and OT receptors [7]. Thus, the aquaretic activity of this drug most probably results from the displacement of endogenous hormone(s) from V2 receptors. Two hormones might possibly be responsible for the V2-mediated water reabsorption in Brattleboro rats, namely AVP and OT. Although AVP cannot be synthesized and processed normally in the neurohypophysis of these rats, small amounts of immuno-reactive AVP or of AVP mRNA have been detected in the hypothalamus, adrenal, ovary, and testis (see review in [16]). Very low (undetectable) levels of circulating AVP can exert some significant influence on water reabsorption in collecting ducts [17], and probably even more so in DI rats because their V2 receptors may become hypersensitive in response to chronic exposure to very low levels of endogenous vasopressin. In this regard, patients with central DI exhibit an enhanced antidiuretic response to low levels of vasopressin [18], and Brattleboro rats have been recognized to be more sensitive than other rats in the bioassay of antidiuretic hormone [19].
It has recently been proposed that many G protein-coupled receptors may exist in an active form in the absence (or in the presence of very low amounts) of agonist [20,21]. These receptors may be constitutively activated, resulting in significant V2 agonism in the vasopressin-deficient rat, and thus to some water reabsorption. In this context, the aquaretic effect of SR in vasopressin-deficient rats may reflect its recently described ‘V2-inverse agonist’ properties [22].
Early studies in Brattleboro rats showed that their pituitary content of OT was about one-third of normal and returned to normal values after chronic vasopressin administration, suggesting a stronger release of OT and depletion of OT stores in untreated Brattleboro rats [23]. In addition, OT secretion has been shown to respond to osmotic stimuli and to contribute to the increase in Uosm observed in water-deprived Brattleboro rats [24]. Although the affinity of OT for V2 receptors is at least a hundredfold less than that of vasopressin [25], it may be assumed that significant OT binding to V2 receptors occurs in the absence (or in the presence of very low levels) of vasopressin in Brattleboro rats. OT can promote water reabsorption in the collecting duct in vitro [26], and produce significant antidiuretic activity in vivo [11]. Our study confirms that OT can exert potent antidiuretic effects when pharmacological doses are given in vivo. Average Uosm in rats with chronic OT infusion reached 1500 mOsm/kg H2O. This antidiuretic effect was due to the occupancy of V2 receptors because it was abolished by the administration of a highly selective non peptide V2-receptor antagonist, thus confirming in vivo the results obtained in isolated tubules in vitro with a peptidic antagonist [27]. The fact that exogenous OT administration was able to increase urine osmolality to such a high level suggests that only a small fraction of V2 receptors are occupied by endogenous OT in Brattleboro DI rats because their urine remains hypo-osmotic, reaching an osmolality only about twice the minimum achieved when all V2 receptors are blocked by the antagonist.
Extrarenal water losses in homozygous DI rats were about twofold higher than those in Sprague–Dawley rats. Administration of SR did not further increase non-renal water losses in DI rats but doubled them in Sprague–Dawley rats with normal vasopressin secretion. Experiments performed for another purpose in our laboratory had shown that the high extrarenal water losses of DI rats could be significantly reduced by chronic V2 agonism (dDAVP infusion) [14]. Abrupt discontinuation of the dDAVP treatment resulted in a marked overshoot of all symptoms of DI, including extrarenal water losses (Table 3) [14]. These observations suggest that endogenous as well as exogenous vasopressin contributes to promote water reabsorption in some non-renal tissues through V2 receptors. This possibility had already been suggested in humans [28] and rats [29]. Vasopressin V2 receptor mRNA is expressed in the adult rat and human lung [30], but the vasopressin-sensitive aquaporin (AQP2) has not been identified in this organ. Nonetheless, vasopressin has been shown to slow lung liquid production or even to cause liquid reabsorption in sheep and guinea-pig fetuses, an effect which is thought to contribute to drain the lungs at birth [31,32]. Because this effect is reversed by amiloride, it indicates that vasopressin activates the luminal amiloride-sensitive sodium channel (ENaC) in the lung, as it does in the renal collecting duct [33]. Moreover, in a recent study, we have shown that chronic dDAVP infusion in DI rats (similar protocol as in the present study) increases the abundance of ENaC mRNA in the kidney and the lung, but not in the colon, an organ which does not express V2 receptors [34]. Thus, vasopressin probably enhances water conservation through the adult lung by enhancing ENaC abundance and activity. The resulting increase in sodium transport creates an additional osmotic driving force leading to more intense passive water absorption in the lung [31] as in the kidney [35].
Salivary secretion may also be reduced by vasopressin because this hormone has been shown to decrease sodium and chloride concentrations and the osmolality of submaxillary saliva in the dog in a dose dependent fashion [36]. This suggests that vasopressin stimulates sodium reabsorption in salivary glands through ENaC (also known to be expressed in these glands) as it does in the lung. Rats have no sweat glands, but they frequently lick their fur and may thus lose significant amounts of water in saliva. An enhanced salivation may thus also participate to the higher extrarenal water losses observed during SR treatment in Sprague–Dawley rats.
In conclusion, this study reveals that a significant V2-mediated water reabsorption is present in homozygous Brattleboro rats and that this water reabsorption limits the intensity of their diabetes insipidus symptoms. The corresponding water reabsorption might result from both a hypersensitivity of AVP receptors to low levels of endogenous AVP and to their occupancy by increased levels of OT. The present experiments also demonstrate the powerful and selective influence of SR on the renal excretion of free water. These aquaretic effects are sustained in both Brattleboro and normal rats for several days. Finally, this study disclosed a significant V2-mediated action on water conservation by organs other than the kidney.
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TopAbstractIntroductionSubjects and methodsExperimental protocolsResultsDiscussionNote added in proofReferences |
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A recently published study described the influence of a chronic oral treatment with SR121463 (3 mg/kg/day) in Sprague Dawley rats. They observed a stable diuresis and no change in osmolar excretion, for 4 weeks.
Lacour C, Galindo G, Canals F et al. Aquaretic and hormonal effects of a vasopressin V2 receptor antagonist after acute and long-term treatment in rats. Eur J Pharmacol 2000; 394: 131–138
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The authors thank Hélène Martin (INSERM Unit 90, Hôpital Necker, Paris, France) for her contribution to some parts of this study.
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Correspondence and offprint requests to: Lise Bankir, INSERM Unité 367, 17 Rue du Fer à Moulin, F-75005 Paris, France.
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Accepted in revised form: 15. 9.00
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