Article

Antinatriuretic Effect of Vasopressin in Humans Is

Amiloride Sensitive, Thus ENaC Dependent
Anne Blanchard,*†‡ Michael Frank,*†§ Grégoire Wuerzner,*†‡ Severine Peyrard,† Lise Bankir, Xavier Jeunemaitre,*†§
and Michel Azizi*†‡

Summary
Background and objectives Acute infusion of the potent V2 receptor agonist 1-desamino-8-d-arginine vaso-
*Université Paris-
pressin (dDAVP) reduces sodium excretion in humans, through an effect attributed to the stimulation of Descartes, Faculté de
the amiloride sensitive epithelial sodium channel, ENaC, in ex vivo/in vivo experiments. We investigated in Médecine, Paris,
humans whether the antinatriuretic effect of dDAVP is sensitive to amiloride, a specific blocker of ENaC. France; †Assistance
Publique Hôpitaux de
Paris, Hôpital Européen
Design, setting, participants, & measurements Forty-eight healthy normotensive adult men were assigned to Georges Pompidou,
a high Na/low K (250/40 mmol/d) diet, to suppress aldosterone secretion. dDAVP (4-␮g intravenous bolus Centre d’Investigations
followed by 4 ␮g over 2 hours) was administrated before and after a 7-day administration of 20 mg/d Cliniques, Paris, France;
‡
amiloride. Urine and blood samples were collected before and at the end of the dDAVP infusion, to mea- INSERM, CIC 9201,
Paris, France; §INSERM
sure Na, K, creatinine, and osmolality concentrations. U970, Paris
Cardiovascular
Results dDAVP alone decreased the urinary flow rate by 75% and the sodium excretion rate by 19% despite Research Center, Paris,
an increase in creatinine clearance by 38 ml/min. Potassium excretion rate was unchanged and the urinary France; and 储INSERM
872, Centre de
Na/K ratio decreased by 18%. Seven-day amiloride administration had no effect on the dDAVP-induced Recherche des
decrease in the urinary flow rate (⫺71%) nor on the dDAVP-induced increase in creatinine clearance (⫹35 Cordeliers, Paris,
ml/min), but it fully prevented the dDAVP-induced decrease in both urinary sodium excretion (⫹1%) and France
urinary Na/K ratio (⫹21%).
Correspondence: Dr.
Anne Blanchard, Centre
Conclusions The antinatriuretic effect of dDAVP in humans is amiloride sensitive, and thus is related to the
d’Investigations
stimulatory effect on ENaC-mediated sodium reabsorption. This test provides a new tool to investigate Cliniques, Hôpital
ENaC function in a clinical setting. Européen Georges
Clin J Am Soc Nephrol 6: 753–759, 2011. doi: 10.2215/CJN.06540810 Pompidou, 20-40 rue
Leblanc, 75908 Paris
Cedex 15, France.
Phone: 33 1 56 09 29
Introduction ing results on the vasopressin effects on sodium re- 13; Fax: 33 1 56 09 29
Vasopressin (AVP), secreted in response to increases absorption have been reported depending on the 29; E-mail: anne.
blanchard@egp.aphp.fr
in plasma osmolality, plays a key role in water me- experimental setting (isolated collecting duct cell
tabolism by stimulating renal water conservation. Va- lines, isolated kidney, or in vivo), the concentration/
sopressin leads to a rapid increase in solute-free water dose administered, and the hydration status (3–5).
reabsorption, by increasing water permeability of These differences have been resolved by Perucca et al.,
principal cells of the collecting ducts (CD) via a V2 who showed in rats that vasopressin at physiologic
receptor–mediated (V2R-mediated) translocation of doses promotes distal sodium reabsorption in vivo via
aquaporin 2 water channels to the apical cell mem- a V2R-mediated effect, whereas at supraphysiologic
brane (1). Water reabsorption then occurs through concentrations, it induces a natriuretic effect via a
osmotic equilibration between the hypo-osmotic tu- V1aR-mediated effect (6). Moreover, acute infusion of
bular fluid delivered at the end of the diluting seg- 1-desamino-8-d-arginine-vasopressin (dDAVP), a po-
ment and, successively, the iso-osmotic cortical and tent V2R agonist, was shown in healthy humans to
hyperosmotic medullary interstitial fluids (1). There- reduce sodium excretion along with its antiaquaretic
after, water reabsorption in the outer medulla is en- effect, without affecting potassium secretion (7). The
hanced by the acute increase in osmolality of the V2R-mediated antinatriuretic effect of vasopressin
medullary interstitium, which occurs secondary to has been attributed, in ex vivo/in vitro experiments on
V2R-mediated translocation of UT-A1 and/or UT-A3 isolated CDs and various cell lines, to the stimulation
urea channels to the apical membrane of inner med- of the activity and expression of the amiloride sensi-
ullary CDs (2). tive epithelial sodium channel (ENaC) of the principal
In addition to its effect on water permeability, va- CD cells (8 –14). However, that AVP may influence
sopressin influences renal sodium handling. Conflict- ENaC activity has not been evaluated in humans.

www.cjasn.org Vol 6 April, 2011 Copyright © 2011 by the American Society of Nephrology 753
754 Clinical Journal of the American Society of Nephrology
The aim of this clinical investigation was thus to inves-
tigate whether the ENaC blocker, amiloride, can fully or
partially block the antinatriuretic effects of the V2R agonist
dDAVP. We studied the effects on urine flow rate, osmo-
lality, and Na and K excretion of acute V2R stimulation by
dDAVP before and after 7-day oral administration of 20
mg oral daily (o.d.) amiloride. The patients were subjected
to a high sodium (Na)/low potassium (K) diet to suppress
endogenous aldosterone secretion, as aldosterone also in-
fluences ENaC-mediated sodium reabsorption.
Materials and Methods
Participants
Forty-eight healthy normotensive white male, nonsmok-
ing patients (age, 23.2 ⫾ 3.9 years; body mass index, 23.0 ⫾
2.3 kg/m2) were enrolled in the study after providing
written and informed consent. Data from one patient was
excluded from analysis because of inadequate urinary col-
lection.
The protocol (ClinicalTrials.gov: NCT2006-005056-32)
was approved by the Comité de Protection des Personnes
Paris, Ile de France III (France), and all procedures were in
accordance with the Declaration of Helsinki.
Study Protocol
Participants were assigned to a high Na/low K (250/40
mmol/d) diet for 14 days with protein, caloric, and water
intake kept constant. All meals were provided by the met-
abolic kitchen of the hospital and were taken in the unit.
On day 3 of the diet, patients completed a 24-hour urine
collection. On day 4 at 9:00 a.m., after a 12-hour overnight
fast, patients ingested 10 ml/kg body wt of water to
achieve a hyperhydration state. After a 1-hour period of
rest in a semirecumbent position, patients received a single
intravenous dose of dDAVP (4 ␮g injected over 30 sec-
onds) followed by a 2-hour continuous infusion of 4 ␮g of
dDAVP in 50 ml of isotonic glucose solution (33 ng/min,
0.42 ml/min). Blood samples were taken before and at the
end of the dDAVP infusion, for the measurement of
plasma Na, K, creatinine, active renin, and aldosterone
concentrations and plasma osmolality. BP was measured
before and during the dDAVP infusion with an automated
validated BP recorder (Press Mate BP 8800; Colin, Komaki-
City, Japan). Urine samples were collected before dDAVP
infusion (from 10:00 to 11:00 a.m.) and during the 2-hour
dDAVP infusion (11:00 a.m. to 1:00 p.m.), for the measure-
ment of urinary Na, K, and creatinine concentrations and
osmolality. After each urine collection, water intake was
matched to diuresis. Patients were discharged at the end of
the dDAVP infusion and continued the same high Na/low
K diet from day 4 to day 14. Amiloride (20 mg o.d.) was
then orally administered for 1 week from day 7 to day 14
and was taken everyday in the unit.
On day 13, patients again completed a 24-hour urine
collection. On day 14, they were given the last 20-mg
amiloride dose at 7:00 a.m. after an overnight fast. At 9:00
a.m., they ingested 10 ml/kg body wt of water and under-
went the second dDAVP test, as on day 4.
Laboratory Methods
Plasma and urine osmolality was measured by cryo-
scopy (Fiske Mark3 osmometer; Norwood, MA). The
methods used for collecting blood samples and for quan-
tifying plasma AVP (RIA), renin (immunoradiometric
assay), and aldosterone (RIA) were those as described
previously (15).
Calculations
Creatinine clearance (Ccreat), osmotic clearance (Cosm),
and free water clearance (CH2O) were calculated as
Ccreat ⫽ 共Ucreat ⫻ V兲/Pcreat
Cosm ⫽ 共Uosm ⫻ V兲/Posm
and
CH2O ⫽ V ⫺ Cosm
where Pcreat and Ucreat are plasma and urinary concentrations of
creatinine, respectively, Posm and Uosm are plasma and urinary
osmolality, respectively, and V is urinary flow.
The transtubular potassium gradient (TTKG) was calcu-
lated as follows: TTKG ⫽ (UK ⫻ Posm)/(PK ⫻ Uosm),
where PK and UK are the plasma and urinary concentra-
tions of potassium, respectively (16).
Statistical Analyses
Data were analyzed using ANOVA for repeated mea-
surements over time, taking into account amiloride admin-
istration, dDAVP infusion, and their interaction (amiloride ⫻
dDAVP) as the fixed effects and a modeling covariance
structure within patients. Unadjusted pairwise compari-
sons between days (amiloride versus control period), and
time points (before and after dDAVP), were done when the
ANOVA was significant. A nonparametric Wilcoxon test
was used for clearance parameters. Correlations between
variables were estimated using analysis of covariance after
withdrawal of the subject effect. Data are expressed as
mean ⫾ SD for normally distributed data, median [inter-
quartile range, IQR], or geometric mean (95% confidence
interval, CI) unless otherwise specified. Relative changes
from baseline are expressed as the ratio of geometric
means (95% CI). P ⬍ 0.05 was considered to be signifi-
cant except for the interaction between dDAVP and
amiloride effects that was considered significant for P ⬍
0.10. SAS statistical software version 8.2 (Cary, NC) was
used for statistical analyses.
Results
Effect of dDAVP Infusion Alone, on the Fourth Day of a
High Na/Low K Diet
After 4 days on the high Na/low K diet, sodium and
potassium balances were achieved, as reflected by the
24-hour urinary Na and K excretion rates (303 ⫾ 50 and
56 ⫾ 12 mmol/24 h, respectively). Plasma renin and
aldosterone concentrations were low and plasma osmo-
lality, plasma Na, plasma K, and creatinine values were
within the physiologic range (Table 1).
As expected, dDAVP led to a significant decrease in the
urinary flow rate by 75% (95% CI: 68 to 81%, P ⬍ 0.0001 versus
baseline; Table 2). Urine osmolality was low before dDAVP
infusion because of the water load, and increased by 246%
(199 to 301%, P ⬍ 0.0001 versus baseline; Table 2) at the end
of the infusion. Free water clearance decreased by 4.4 ml/
min (95% CI: 3.2 to 5.2 ml/min, P ⬍ 0.0001 versus baseline;
Clin J Am Soc Nephrol 6: 753–759, April, 2011 dDAVP Stimulates ENaC in Humans, Blanchard et al. 755

Table 1. Biochemical and hormonal changes in response to dDAVP administered alone (control period, day 4) or after 7-day
pretreatment with amiloride (amiloride period, day 14) in 47 healthy patients on a high Na/low K diet

Control Period (Day 4) Amiloride (Day 14)

Plasma osmolality (mosmol/kg H2O) Before dDAVP 284 ⫾ 4 283 ⫾ 3

After dDAVP 285 ⫾ 4 282 ⫾ 4
Plasma sodium (mmol/L) Before dDAVP 139 ⫾ 2 137 ⫾ 2
After dDAVP 139 ⫾ 3 137 ⫾ 2
Plasma potassium (mmol/L) Before dDAVP 3.6 ⫾ 0.2 4.1 ⫾ 0.3
After dDAVP 3.6 ⫾ 0.2 4.1 ⫾ 0.3
Plasma creatinine (␮mol/L) Before dDAVP 81 ⫾ 10 81 ⫾ 10
After dDAVP 81 ⫾ 11 80 ⫾ 10
Plasma active renin (pg/ml) Before dDAVP 13 (12, 15) 43 (39, 48)
After dDAVP 15 (14, 17)a 51 (46, 57)a
Plasma aldosterone (pg/ml) Before dDAVP 20 (16, 25) 180 (159, 203)
After dDAVP 16 (13, 19)a 183 (165, 204)

Data are mean ⫾ SD or geometric mean (95% CI).

a
P ⬍ 0.001 after versus before dDAVP.

Table 2. Changes in urine in response to dDAVP in response to dDAVP administered alone (control period, day 4) or after 7-day
pretreatment with amiloride (amiloride period, day 14) in 47 healthy subjects on a high Na/low K diet

Control Period (Day 4) Amiloride (Day 14)

Flow rate (ml/min) Before dDAVP 5.9 [4.1, 8.0] 5.6 [3.9, 7.0]
After dDAVP 1.2 [1.1, 1.6]b 1.3 [1.0, 1.7]b
Uosm (mosmol/kg H2O) Before dDAVP 137 [117, 211] 165 [126, 222]
After dDAVP 588 [487, 636]b 630 [545, 723]b
UcreatV (␮mol/min) Before dDAVP 9.5 [8.0, 10.7] 9.2 [7.7, 10.6]
After dDAVP 12.1 [10.9, 14.0]b 11.6 [10.7, 13.1]b
UNaV (␮mol/min) Before dDAVP 249 [186, 308] 254 [194, 343]
After dDAVP 193 [139, 236]a 234 [190, 300]
UKV (␮mol/min) Before dDAVP 75 [62, 90] 61 [46, 76]
After dDAVP 70 [60, 87] 53 [39, 75]
UNa/UK (mmol/mmol) Before dDAVP 3.2 [2.7, 3.9] 4.1 [3.3, 5.2]
After dDAVP 2.5 [2.2, 3.2]b 4.4 [3.3, 6.2]
Ccreat (ml/min) Before dDAVP 118 [98, 138] 113 [94, 134]
After dDAVP 153 [138, 178]b 144 [136, 165]b
Cosm (ml/min) Before dDAVP 3.1 [2.5, 3.8] 3.5 [2.6, 3.8]
After dDAVP 2.6 [2.2, 3.0]b 2.9 [2.6, 3.5]a
CH2O (ml/min) Before dDAVP 3.0 [0.9, 4.4] 2.4 [0.4, 3.3]
After dDAVP ⫺1.3 [⫺1.5, ⫺1.0]b ⫺1.6 [⫺1.9, ⫺1.2]b
TTKG Before dDAVP 6.6 [5.3, 7.6] 4.5 [3.7, 5.7]
After dDAVP 7.8 [6.3, 8.9]b 4.2 [3.4, 5.7]

Uosm, urinary osmolality; UNaV, urinary sodium excretions; UKV, urinary potassium excretions; UcreatV, urinary creatinine
excretions; Ccreat, creatinine clearances, Cosm, osmolar clearances; CH2O, free water clearances; TTKG, transtubular potassium
gradient (see Materials and Methods section). Data are median [IQR].
a
P ⬍ 0.05 after versus before dDAVP.
b
P ⬍ 0.001 after versus before dDAVP.

Table 2), and osmotic clearance decreased by 0.8 ml/min (0.5
to 1.1 ml/min, P ⫽ 0.0004 versus baseline; Table 2).
In addition to its effects on urinary flow rate and
osmolality, dDAVP infusion was associated with a sig-
nificant decrease in urine sodium excretion by 18.9% (4.5
to 31.1%, P ⫽ 0.0124; Table 2 and Figure 1) and a significant
increase in creatinine clearance by 38 ml/min (31 to 48
ml/min, P ⬍ 0.0001; Table 2). At the end of the dDAVP
infusion, there was a slight increase in the plasma renin
concentration (Table 1), and a slight decrease in the plasma
aldosterone concentration (Table 1) and in the systolic BP
(⫺2.1 mmHg, 95% CI: ⫺3.9 to ⫺0.3, P ⫽ 0.0228, not
shown). Changes in urine sodium excretion were corre-
lated with changes in the urine flow rate (r ⫽ 0.62, n ⫽ 47,
P ⬍ 0.0001), but not with changes in BP (not shown).
Despite the decrease in sodium and water excretion,
potassium excretion remained unaffected (Table 2). The
urinary Na/K ratio decreased by 18% (7 to 27%, P ⬍
0.0001; Table 2 and Figure 2) and TTKG increased by
⫹21% (9 to 34%, P ⫽ 0.0004; Table 2), suggesting that
potassium secretion at the distal nephron was stimu-
lated.
756 Clinical Journal of the American Society of Nephrology

Figure 1. | Interaction between amiloride and the dDAVP effects on urinary sodium excretion. Forty-seven patients received twice a single
intravenous dose of dDAVP (4 ␮g injected over 30 seconds) followed by a 2-hour continuous infusion of 4 ␮g of dDAVP in 50 ml isotonic
saline solution alone (day 4) or after a 7-day administration of 20 mg o.d. amiloride (day 14). The left panel shows the individual UNaV.
The box plot in right panel shows changes from baseline in UNaV (box, median and IQR; whiskers, 95% CI). UNaV, urine sodium excretion
rate; *P ⬍ 0.05; $, significant interaction between amiloride and dDAVP effects (P ⬍ 0.10).

Figure 2. | Effect of dDAVP on urine potassium excretion rate (1) and (2) urinary Na/K ratio in the presence or absence of amiloride
pretreatment (individual data of the 47 patients). ***P ⬍ 0.0001 dDAVP versus baseline.

Effect of dDAVP Infusion After a 7-Day Administration of
20 mg o.d. Amiloride (Day 14 of the High Na/Low K
Diet)
The 7-day administration of amiloride led to a 1.6-kg
(95% CI: 1.0 to 2.2 kg) decrease in body weight (P ⬍
0.0001, day 14 versus day 4) and to a significant increase
in plasma renin and aldosterone concentrations (Table
1), reflecting the net negative sodium balance. Twenty-
four-hour Na and K excretion rates (246 ⫾ 72 and 45 ⫾
16 mmol/24 h, respectively) matched Na and K intakes,
suggesting that a new steady state was reached. Systol-
ic/diastolic BP did not significantly change with
amiloride (122 ⫾ 10/66 ⫾ 9 versus 120 ⫾ 9/62 ⫾ 7
mmHg, P ⫽ NS day 14 versus day 4). Amiloride in-
creased the plasma potassium concentration by 0.50
mmol/L (0.40 to 0.59 mmol/L, P ⬍ 0.0001), reflecting its
potassium sparing effect. The plasma sodium concentra-
tion was 2.2 mmol/L (1.5 to 2.9 mmol/L, P ⬍ 0.0001)
lower, without any change either in plasma creatinine
concentration or in osmolality (Table 1).
Amiloride pretreatment did not influence the dDAVP
effects on urine flow rate, free water clearance, urinary
osmolarity, and creatinine clearance (Table 2): dDAVP de-
creased urinary flow rate by 71% (63 to 77%, P ⬍ 0.0001
dDAVP versus baseline and P ⫽ 0.41 for amiloride ⫻
dDAVP interaction) and increased creatinine clearance by
35 ml/min (23 to 43 ml/min, P ⬍ 0.0001 versus baseline
and P ⫽ 0.37 for amiloride ⫻ dDAVP interaction). These
figures were similar to those observed on day 4.
In contrast, the antinatriuretic effect of dDAVP was al-
most abolished by amiloride pretreatment (Table 2 and
Figure 1), as shown by the nonsignificant dDAVP-induced
change in sodium excretion (⫹1%, 95% CI: ⫺14.3 to
⫹18.9%, P ⫽ 0.91 dDAVP versus baseline and P ⫽ 0.0626
for amiloride ⫻ dDAVP interaction). The dDAVP-induced
decrease in osmolar clearance was significantly smaller
than that on day 4 (⫺0.43 ml/min, 95% CI: ⫺0.74 to ⫺0.10
ml/min, P ⫽ 0.0268 dDAVP versus baseline and P ⫽ 0.0487
for amiloride ⫻ dDAVP interaction).
Clin J Am Soc Nephrol 6: 753–759, April, 2011
As on day 4, potassium excretion did not significantly
change after dDAVP infusion (⫺4.2%, 95% CI: ⫺20.5 to
⫹15.5%, P ⫽ 0.65 dDAVP versus baseline and P ⫽ 0.57 for
amiloride ⫻ dDAVP interaction; Table 2 and Figure 2).
Amiloride prevented dDAVP effects on TTGK (⫺2.7%,
95% CI: ⫺12.4 to ⫹8.0%, P ⫽ 0.60 dDAVP versus baseline
and P ⫽ 0.004 for amiloride ⫻ dDAVP interaction) and on
urinary Na/K ratio (21%, 95% CI: ⫺11 to ⫹52%, P ⫽ 0.81
dDAVP versus baseline and P ⫽ 0.004 for amiloride ⫻
dDAVP interaction; Table 2 and Figure 2).
Finally, the plasma renin concentration increased
slightly, as on day 4 (Table 1), but the plasma aldosterone
concentration (Table 1) and systolic BP (⫺0.7 mmHg, 95%
CI: ⫺2.5 to 1.1 mmHg, P ⫽ 0.44, not shown) showed no
change.
Discussion
We showed that the acute antinatriuretic effect of the
V2R agonist dDAVP was fully prevented by a 7-day ad-
ministration of 20 mg o.d. amiloride, a selective ENaC
inhibitor. However, dDAVP-induced antidiuretic effects
were maintained. We also showed that the stimulatory
effect of dDAVP on distal potassium secretion in humans
was amiloride sensitive. These data confirm the major
physiologic role of ENaC in the V2R-mediated antinatri-
uretic effect of vasopressin in humans, a phenomenon
currently described only in vitro.
We compared renal responses to dDAVP before and
after a 7-day administration of a high dose of amiloride (20
mg o.d.). We selected this dose of amiloride because it was
previously shown to be more potent than 100 mg of spi-
ronolactone, the mineralocorticoid receptor antagonist, for
reversing hydrochlorothiazide-induced hypokalemia in
healthy patients (17). We studied the effects of dDAVP on
a high Na and low K diet, to suppress endogenous aldo-
sterone secretion, the main stimulus of ENaC activity and
expression, and induced water diuresis, before dDAVP
infusion, to decrease endogenous vasopressin secretion. To
minimize the hemodynamic effects of supraphysiologic
doses of the V2R agonist, we selected lower doses of
dDAVP than those previously used (7). Indeed, we ob-
served only a small nonsignificant decrease in BP, a mar-
ginal increase in plasma renin concentrations without any
change in the plasma aldosterone concentration.
Under these experimental conditions, infusion of
dDAVP induced a marked decrease in urinary flow rate.
This was associated with a decrease in urine sodium ex-
cretion in healthy patients, consistent with results obtained
previously under different experimental conditions (7,18).
The antinatriuretic effect of dDAVP was associated with a
significant decrease in osmolar clearance. The magnitude
of the decrease in sodium excretion in our study was
smaller than those previously reported by Bankir et al.
(⫺20% versus ⫺60%, respectively) (7), probably because of
the lower dose of dDAVP used in our study and/or of the
initial washout of the medullary osmotic gradient due to
the water load (19 –21). In both studies, individual changes
in the urine flow rate were significantly correlated with
simultaneous individual changes in urine sodium excre-
tion but not with those in BP, illustrating the dependence
of the urinary flow rate on the change in “effective” solute
excretion rates.
dDAVP Stimulates ENaC in Humans, Blanchard et al. 757
The decrease in the urinary sodium excretion rate with
dDAVP alone occurred despite an increase in the filtered
load of sodium, demonstrating that dDAVP has a potent
stimulatory effect on renal tubular sodium reabsorption.
Indeed, creatinine clearance, a GFR index, increased by
approximately 30% after dDAVP infusion independently
of amiloride administration. Although the mechanism of
such V2R-induced rise in GFR has not yet been elucidated,
a similar effect was previously reported in rats (19,22) as in
water-loaded patients (5,23). Accordingly, GFR was shown
to be significantly higher during low-hydration regimes
than during high-hydration regimens in healthy patients
(5). In the presence of amiloride, the dDAVP-induced in-
crease in the filtered load of sodium without a change in
sodium excretion suggests that sodium overload has been
compensated in amiloride-insensitive segments. The mech-
anism of this adaptation is however beyond the scope of
our study.
Amiloride pretreatment did not affect the antidiuretic
effect of dDAVP (decrease in urinary flow rate) nor its
antiaquaretic effect (decrease in water clearance), but fully
prevented its antinatriuretic effect and blunted the de-
crease in osmolar clearance. This effect was independent of
aldosterone that was suppressed by the high Na/low K
diet. Altogether, these results suggest that dDAVP in-
creases sodium reabsorption in a nephron segment that
expresses V2R and is permeable to water in the presence of
vasopressin, that is, in the CD, and are in agreement with
the results of ex vivo/in vitro experiments (for review, see
reference 11). Several studies have been published on va-
sopressin and its effects on tubular sodium reabsorption
with conflicting results (3,4,7,11,24,25). Actually, vasopres-
sin displays biphasic effects on sodium excretion in rats,
resulting from a balance between the V2R-mediated anti-
natriuretic effect of lower concentrations and the V1aR-
mediated natriuretic effect achieved at higher concentra-
tions, which overrides the sustained action on V2R (6).
Acute V2R activation stimulates the activity of ENaC in CD
by inducing translocation of ENaC from intracellular stor-
age vesicles to the apical membrane, by increasing its open
probability, and by enhancing its stability, increasing in
turn the number of functional ENaCs at that membrane
(13,26). In addition, chronic treatment with dDAVP in rats
also leads to a large increase in the expression of both the
␤- and ␥-ENaC subunits in the kidney (for review, see
references 11,27). This effect is different from that of aldo-
sterone, which induces only a modest increase in ␣-ENaC
and no change in ␤- and ␥-ENaC expression (28). How-
ever, in addition to its effect on ENaC, acute administration
of dDAVP has been shown in rats to induce translocation
of the Na-K-2Cl cotransporter (NKCC2) to the apical mem-
brane in the thick ascending limb via a V2R-mediated
effect (29), whereas chronic administration of dDAVP to
Brattleboro rats (genetically devoid of vasopressin) in-
creased protein abundance of both the NKCC2 and the
thiazide-sensitive NCC cotransporter (30). Moreover, if no
stimulation of adenylate cyclase production by vasopressin
in Thick Ascending Limb (TAL) was initially observed,
suggesting no V2R expression in this segment in humans
(31,32), a recent study demonstrated the presence of sig-
nificant amounts of V2R mRNA and protein along the
758 Clinical Journal of the American Society of Nephrology
human nephron (from medullary TAL to CDs) (33).
Altogether, these data suggest that transporters other
than ENaC may also contribute to the V2R-mediated
antinatriuresis in humans, but the importance of their
contribution in vivo is not known. Nevertheless, our
results suggest that the ENaC-dependent mechanisms
may be predominant because the dDAVP-induced anti-
natriuretic effect was reversed by amiloride. However,
as in all human studies, it is not possible to affirm that
the amiloride-sensitive decrease in sodium excretion re-
sults from a direct action of vasopressin on ENaC activ-
ity or is a consequence of an indirect mechanism. Recent
patch clamp studies on the luminal membrane of collect-
ing duct principal cells in vitro bring support for a direct
action of vasopressin on ENaC (14).
As in previous studies in humans (7), potassium excretion
rate did not change with dDAVP infusion despite a marked
decrease in the urinary flow rate and sodium excretion. This
suggests that distal potassium excretion was maintained by
compensatory mechanisms. Accordingly, two clinical indices
of distal potassium secretion (TTKG and urinary Na/K ratio)
were both significantly increased by dDAVP, confirming the
known stimulatory effect of V2R agonists on distal potassium
secretion (6,8,34). These effects were prevented by amiloride,
suggesting the indirect involvement of an ENaC-dependent
mechanism.
This study has some limitations including the use of an
asymmetrical sequential design instead of a randomized
crossover design and the absence of time control experi-
ments before each dDAVP infusion due to blood volume
constraints, time constraints, and cost limitations. The fact
that each subject was studied twice and was its own con-
trol can partially compensate for this lack of control study.
Despite these limitations, our study design allowed us to
show neutralization by amiloride of the antinatriuretic ef-
fects of dDAVP, which was the primary objective of the
study. Finally, we used dDAVP, a selective V2 agonist, and
not the natural hormone AVP, which acts on several re-
ceptor subtypes. Indeed, it has been shown that V1aR-
mediated effects may partially blunt the renal V2R-medi-
ated effects of vasopressin in rats, however, only at higher
concentrations of the hormone (6,27). Therefore, there is
likely a certain range of vasopressin concentrations in
which an antinatriuretic effect will occur along with the
antidiuretic action of the hormone.
In conclusion, this study shows that, in addition to its
well-known effect on water reabsorption, vasopressin
significantly reduces urinary sodium excretion in hu-
mans by stimulating ENaC-mediated sodium reabsorp-
tion. It may thus be assumed that ENaC stimulation by
vasopressin might induce, along with many other fac-
tors, some degree of sodium retention (27). The contri-
bution of this effect to pathophysiologic variations in BP
and to the pathophysiology of edematous states, in
which vasopressin secretion can be significantly stimu-
lated, remains to be addressed (27).
Acknowledgments
This work was supported by the “Programme Hospitalier de
Recherche Clinique from the Assistance Publique des Hôpitaux de
Paris” (Grant: AOR0641). Dr. A. Blanchard is a recipient of a
“Contrat d’Interface” grant from INSERM.
We thank the nursing staff of the Clinical Investigation Center
who ran the protocol and Mrs. Christiane Dollin who performed
the renin and the aldosterone measurements.
This work was previously reported in abstract form (Blanchard
et al. J Hypertens 27: S374, 2009).
Disclosures
None.
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Received: August 2, 2010 Accepted: November 22, 2010
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