Cardiovasc Drugs Ther (2011) 25 (Suppl 1):S67–S76
DOI 10.1007/s10557-011-6350-4
Effects of Tolvaptan on Systemic and Renal Hemodynamic
Function in Dogs with Congestive Heart Failure
Toshiyuki Onogawa & Yuki Sakamoto &
Shigeki Nakamura & Sunao Nakayama &
Hiroyuki Fujiki & Yoshitaka Yamamura
Published online: 26 November 2011
# Springer Science+Business Media, LLC 2011
Abstract
Purpose We investigated the effects of tolvaptan, a vaso-
pressin V2-receptor antagonist, on diuretic response and
systemic and renal hemodynamic characteristics in con-
scious dogs with congestive heart failure (CHF). We also
compared these effects with those of furosemide, a loop
diuretic.
Methods CHF was induced by rapid right-ventricular
pacing at 260 beats/min for at least 3 weeks, and
maintained with a pacing rate of 220–240 beats/min. CHF
dogs were orally given tolvaptan (10 mg/kg), furosemide
(10 mg/kg) and vehicle in random order during the stable
CHF state. Urine excretion, systemic and renal hemody-
namic parameters, and plasma hormone levels were
measured over 6-hour periods after drug administration.
Results Tolvaptan induced aquaresis with an increase in
free water clearance, resulting in a significant increase in
serum sodium concentrations and a decrease in cumulative
water balance. Tolvaptan also decreased pulmonary capil-
lary wedge pressure without affecting systemic vascular
resistance, glomerular filtration rate or renal blood flow.
Tolvaptan tended to increase plasma arginine vasopressin
concentrations but did not affect plasma renin activity. In
contrast, furosemide induced clear saluresis with increased
electrolyte excretion, resulting in decreased pulmonary
capillary wedge pressure. However, furosemide also de-
creased serum potassium concentration and increased
plasma arginine vasopressin concentrations and plasma
renin activity.
T. Onogawa : Y. Sakamoto : S. Nakamura : S. Nakayama :
H. Fujiki (*) : Y. Yamamura
First institute of New Drug Discovery,
Otsuka Pharmaceutical Co., Ltd,
Tokushima, Japan
e-mail: h_fujiki@research.otsuka.co.jp
Conclusion Tolvaptan elicited a potent aquaretic response
and reduced the cardiac preload without unfavorable effects
on systemic or renal hemodynamics, the renin–angiotensin–
aldosterone system, or the sympathetic nervous system in
CHF dogs. Thus, tolvaptan may offer a novel approach to
remove excess water congestion from patients with CHF.
Key words Heart failure . Hemodynamics .
Neurohormones . Vasopressin V2-receptor antagonist
Introduction
Diuretics are widely used to reduce fluid overload and
congestion in congestive heart failure (CHF). Non-
potassium (K+)-sparing diuretics, such as loop diuretics,
can cause various electrolyte disorders, including hypoka-
lemia, hypomagnesemia, and hyponatremia. Hypokalemia
and hypomagnesemia are particularly dangerous adverse
effects because they can trigger severe arrhythmias [1]. A
retrospective analysis of patients with systolic left ventric-
ular dysfunction in the SOLVD study demonstrated that
non-K+-sparing diuretics were associated with an increased
risk of arrhythmic death [2, 3]. It has also been reported that
mortality is higher in CHF patients with hyponatremia than
in those with normal blood sodium (Na+) [4]. Furthermore,
loop diuretics can unfavorably stimulate the sympathetic
nerves and the renin-angiotensin-aldosterone system
(RAAS) [5, 6], which can worsen heart failure (HF) [7].
Tolvaptan is an orally active benzazepine derivative
arginine vasopressin (AVP) V2-receptor antagonist [8].
Pharmacologically, tolvaptan inhibits AVP binding to V2-
receptors in the distal portion of the nephron, thus
inhibiting water reabsorption without an increase in urine
electrolyte excretion, i.e. inducing free water diuresis
S68
(aquaresis). It has been demonstrated that oral tolvaptan
elicits aquaresis without stimulating the RAAS or the
sympathetic nervous system in rats [9, 10]. In patients with
CHF, tolvaptan was reported to decrease body weight and
edema without causing electrolyte abnormalities or wors-
ening of renal function [11–13].
To date, no study has attempted to evaluate the effects of
tolvaptan on hemodynamics status, particularly cardiac
preload and renal hemodynamic changes. Therefore, the
present study was conducted to determine the effects of
tolvaptan versus furosemide on systemic and renal hemo-
dynamic parameters, as well as serum electrolyte and
neurohormone levels, in CHF dogs.
Methods
Animals
Eleven male beagle dogs, weighing 10–12 kg, were
purchased from Kitayama Labes Co., Ltd. (Nagano, Japan).
Four of the 11 CHF dogs were excluded from the study
because of death or rupture of their pacing wires. The
animals were cared for and handled in accordance with the
Guidelines for Animal Care and Use in Otsuka Pharma-
ceutical Co., Ltd. (October 1, 1994).
Animal model of CHF
The dogs were anesthetized with an intravenous injection of
pentobarbital sodium (30 mg/kg), and given supplementary
doses as needed to maintain anesthesia. They were
mechanically ventilated with a pressure-controlled respi-
rator. Under aseptic conditions, the right thorax was
opened via the fifth intercostal space. A pacing lead
placed on ventricular surface was brought out of the
chest, tunneled subcutaneously to the back of the neck,
and secured with a bandage. The dogs were given
postoperative antibiotics for about 7 days and allowed
to recover from the operation.
Two weeks after recovering from the surgery, the dogs
were subjected to ventricular pacing at 260 beats/min using
an external pacemaker (Model 240-02, Intermedics Inc.
Freeport, TX, USA) for at least 3 weeks. The development
of CHF was confirmed by measuring hemodynamic
parameters and plasma hormone levels after ventricular
pacing at 260 beats/min (CHF-1; day 1 of the CHF
experiments). Then, the pacing rate was decreased to
220–240 beats/min to maintain a stable CHF state. After
the end of CHF experiments (CHF-2; final day of the CHF
experiments), the hemodynamic parameters and plasma
hormone levels were measured to confirm the stability of
the CHF state during the CHF experiments. Twice a week,
Cardiovasc Drugs Ther (2011) 25 (Suppl 1):S67–S76
electrocardiographic monitoring was performed to ensure
continued pacing throughout the experiments.
Measurements of systemic and renal hemodynamic
parameters
The CHF dogs were sedated with intramuscular buprenor-
phine hydrochloride (0.3 mg) and placed on a table in a
lateral position. The left external jugular vein and right
femoral artery were punctured with a 7-Fr and 6-Fr cannula,
respectively, under local anesthesia (1% lidocaine). A
Swan-Ganz catheter (TC-504, Nihon Kohden, Tokyo,
Japan) was inserted into the pulmonary artery through the
jugular vein. Pulmonary capillary wedge pressure (PCWP),
right atrial pressure (RAP) and mean arterial blood pressure
(MBP) were measured using pressure transducers
connected to amplifiers (NEC San-ei, Tokyo, Japan). These
parameters were simultaneously recorded on a thermal pen
recorder (NEC San-ei). Cardiac output (CO) was measured
by the thermodilution technique.
To assess renal function, an 8-Fr urinary bladder catheter
was inserted into the bladder via the urethra, and urine was
collected. A bolus injection of 20% p-aminohippuric acid
(PAH, 200 mg) and 12.5% inulin (1,200 mg) was adminis-
tered into the left external jugular vein through a 7-Fr cannula,
followed by a continuous infusion of PAH (3 mg/min) and
inulin (12 mg/min) at a rate of 1.0 mL/min. After a 90-minute
stabilization period, pre-values were measured for 60 min.
After oral administration of tolvaptan, furosemide, or
vehicle, the hemodynamic parameters and renal function
were measured for 6 h. Urine was collected every 60 min.
Hemodynamic measurements and blood sampling were
performed in the middle of each urine sampling period.
Serum electrolyte, PAH and inulin concentrations, serum
osmolality, and plasma hormone levels were measured.
Drugs
Tolvaptan was prepared at Otsuka Pharmaceutical Co., Ltd.
(Tokushima, Japan). Furosemide was purchased from
Sigma-Aldrich Japan Co., Ltd. (Tokyo, Japan). The drugs
and vehicle were administered in a random order with at
least 5 days between each treatment. Each dog was orally
administered with 10 mg/kg tolvaptan and 10 mg/kg
furosemide contained in gelatin capsules. During the 6-
hour measurement period, the dogs received no food or
water. The experiment was performed with forced pacing
(220–240 beats/min).
Measurements of PAH, inulin, osmolality and electrolytes
PAH was measured using a commercially available assay kit,
which was modified as previously described [14]. To measure
Cardiovasc Drugs Ther (2011) 25 (Suppl 1):S67–S76
inulin, we used a modified method, as previously described
[15]. Urine and serum osmolality were determined by the
freezing point depression method using a Fiske osmometer
(Fiske Associates, Needham Heights, MA, USA). Urinary
and serum electrolyte concentrations were measured using
ion-selective glass electrodes (SYNCHRON CX3, Beckman
Instruments, Fullerton, CA, USA).
Measurement of plasma hormone levels
Plasma AVP and atrial natriuretic peptide (ANP) concen-
trations were measured by radioimmunoassay and immunor-
adiometric assay, respectively. Plasma renin activities (PRA)
were determined by a radioimmunoassay using the formation
of angiotensin I (expressed in ng) as an index. Plasma
norepinephrine (NE) concentrations were determined by the
high-performance liquid chromatography–diphenylethylene-
diamine method.
Calculations and statistical analysis
Systemic vascular resistance (SVR) was calculated by dividing
the MBP by CO. Free water clearance was calculated as the
urine flow rate—osmolal clearance (osmolal clearance = urine
flow rate × urine osmolality/serum osmolality). Glomerular
filtration rate (GFR) and renal plasma flow (RPF) were
calculated as inulin and PAH clearance, respectively. Renal
blood flow (RBF) was calculated by compensating RPF by
hematocrit. Water balance was calculated as water intake per
hour (60 mL+9 mL: continuously infused saline containing
inulin and PAH+ injected bolus of saline to measure CO)
minus urine volume per hour.
Values are expressed as means ± standard error of the mean.
Differences in systemic and renal hemodynamic parameters,
and plasma hormone levels between the normal and baseline at
CHF-1 and CHF-2 were analyzed by analysis of variance
(ANOVA) with a randomized block design, followed by
Tukey’s test. Differences in changes from baseline in urine
parameters, systemic and renal hemodynamic parameters, and
serum and plasma parameters among the tolvaptan, furosemide
and vehicle groups were analyzed by two-way ANOVA,
followed by Tukey’s multiple-comparison test. At each time-
point, differences among groups were analyzed by one-way
ANOVA followed by Tukey’s multiple-comparison test.
Differences were considered significant at p<0.05.
Results
Development of HF and stability of induced CHF
As shown in Table 1, all seven dogs developed HF
characterized by changes in systemic and renal hemody-
S69
namic and neurohormonal parameters. After developing
CHF (i.e., at CHF-1), PCWP and RAP, which are indices of
cardiac preload, were significantly increased, while CO and
MBP were significantly decreased, and body weight and
SVR showed no changes. On the final day of the CHF
experiments (i.e., at CHF-2), the hemodynamic changes
were similar to those at CHF-1; only RAP showed a further
increase from CHF-1. The renal hemodynamic parameters
(GFR, RPF and RBF) tended to decrease at CHF-1, and
statistically significant differences were observed at CHF-2
compared with normal dogs. Serum osmolality and Na+
concentrations were not changed after developing CHF.
Plasma ANP and NE concentrations were significantly
increased in CHF dogs, whereas plasma AVP concentra-
tions and PRA were not changed. These changes were
similar to those in previous studies of a canine model of
CHF [16, 17]. Furthermore, similar hemodynamic and
neurohormonal abnormalities were obtained at both CHF-
1 and CHF-2, indicating that the entire experiment was
performed in animals with stable CHF.
Effects of tolvaptan and furosemide on urine excretion
Figure 1 shows the changes in urine parameters after oral
administration of 10 mg/kg tolvaptan, 10 mg/kg furose-
mide, or vehicle. Tolvaptan significantly increased the urine
flow rate (UFR) and decreased urine osmolality. The
increase in urine flow rate reached a peak at 2–3 h after
administration. Urinary Na+ and chloride (Cl–) excretion
were not increased by tolvaptan, resulting in a significant
increase in free water clearance, i.e. aquaresis. Although
urinary K+ excretion increased slightly during the last 2 h
of the measurement periods, the time-courses of changes in
urinary K+ excretion and urine flow rate were somewhat
different. Furosemide also increased the urinary flow rate
and decreased urine osmolality, as observed for tolvaptan.
However, the total amount of urine excreted with furose-
mide treatment (879.1±90.4 mL/6 h) was higher than that
with tolvaptan (645.2±81.1 mL/6 h). Unlike with tolvaptan,
furosemide did not increase free water clearance, and
significantly increased urine electrolyte excretion (Na+, K+
and Cl–) with a similar time-course to changes in urine flow
rate.
Effects of tolvaptan and furosemide on serum electrolytes
and neurohormones
Figure 2 shows the changes in serum osmolarity and
electrolytes after oral administration of tolvaptan, furose-
mide or vehicle. Tolvaptan significantly increased serum
osmolality and serum Na+ and Cl– concentrations compared
to the vehicle, but did not increase serum K+ concentration.
On the other hand, furosemide significantly decreased
S70 Cardiovasc Drugs Ther (2011) 25 (Suppl 1):S67–S76

Table 1 Baseline systemic and renal hemodynamic parameters, and plasma hormone levels in CHF dogs

Normal dogs pre pacing CHF-1 CHF-2

BW (kg) 10.65±0.37 10.51±0.41 10.68±0.38

CO (L/min) 1.58±0.12 1.29±0.09* 1.29±0.07*
MBP (mmHg) 103.4±2.7 90.7±3.2* 91.1±1.4*
SVR (mmHg·min/L) 68.0±5.3 71.9±4.6 71.8±3.3
PCWP (mmHg) 9.4±0.7 19.1±0.7** 20.6±1.0**
RAP (mmHg) 5.4±0.6 8.5±0.8** 10.0±0.8** †
GFR (mL/min) 44.1±5.1 39.7±2.1 34.7±2.3*
RPF (mL/min) 127.1±15.0 110.4±12.4 92.7±9.2*
Serum Osm (mOsm/kg) 296±1 298±2 298±1
Serum Na+ (mEq/L) 146.8±0.6 145.7±0.7 146.5±0.5
RBF (mL/min) 208.4±25.5 172.0±20.4 135.8±14.1**
Plasma AVP (pg/mL) 8.3±3.5 14.9±4.5 9.9±2.5
PRA (ng/mL/h) 0.50±0.23 0.48±0.17 0.67±0.19
Plasma ANP (pg/mL) 25.8±2.2 148.6±13.5** 167.0±21.6**
Plasma NE (ng/mL) 0.153±0.034 0.416±0.040** 0.546±0.087** †

Data are means ± SEM of seven animals. Differences were analyzed by ANOVA with a randomized block design, followed by Tukey’s test.*p<
0.05 and **p<0.01 vs normal dogs; † p<0.05 vs CHF-1. CHF-1day 1 of the CHF experiments; CHF-2 final day of the CHF experiments; ANP
atrial naturetic peptide; AVP arginine vasopressin; BW body weight; CHF congestive heart failure; CO cardiac output; GFR glomerular filtration
rate; HR heart rate; MBP mean arterial blood pressure; NE norephinephrine; Osm osmolality; PCWP pulmonary capillary wedge pressure; PRA
plasma renin activities; RAP right atrial pressure; RBF renal blood flow; RPF renal plasma flow; SVR systemic vascular resistance

serum K+ and Cl– levels compared with vehicle, but did not
affect serum osmolality or Na+ levels.
Figure 3 shows the changes in plasma hormones after
oral administration of tolvaptan, furosemide or vehicle.
Tolvaptan did not affect PRA or plasma ANP and NE
concentrations. However, tolvaptan slightly increased plas-
ma AVP concentrations, although this increase was not
statistically significant. In contrast, furosemide significantly
increased PRA and plasma AVP concentrations, and
slightly but not significantly increased plasma NE.
Effects of tolvaptan and furosemide on systemic and renal
hemodynamic parameters
Figure 4 shows the changes in systemic and renal
hemodynamic parameters after oral administration of
tolvaptan, furosemide or vehicle. Tolvaptan did not signif-
icantly affect the renal hemodynamic parameters GFR, RPF
or RBF. Tolvaptan slightly increased the MBP compared
with vehicle, but no significant differences were observed
at any time-point. Furthermore, there were no significant
differences in other systemic hemodynamic parameters,
including CO and SVR, indicating that tolvaptan does not
affect cardiac afterload. Similarly, furosemide did not affect
any of the renal or systemic hemodynamic parameters.
Figure 5 shows the changes in cardiac preload parame-
ters, including PCWP and RAP, and the cumulative water
balance and body weight after oral administration of
tolvaptan, furosemide or vehicle. In the vehicle group,
PCWP and RAP gradually increased after continuous
infusion of saline containing inulin and PAH, followed by
an increase in cumulative water balance. Consistent with its
potent aquaretic effects, tolvaptan decreased cumulative
water balance and body weight at 6 h after administration,
and significantly reduced the increases in PCWP and RAP.
Similarly, furosemide decreased cumulative water balance
and body weight because of its diuretic effects. The effects
of furosemide on these parameters were greater than those
with tolvaptan because diuresis was more pronounced with
furosemide. Because of the larger decreases in cumulative
water balance, furosemide elicited a greater decrease in
PCWP and RAP compared with tolvaptan.
Discussion
Effects of tolvaptan on urinary electrolyte excretion
and serum electrolyte concentrations
Tolvaptan did not increase urinary Na+ excretion in CHF
dogs, despite its potent diuretic action. Consequently, the
free water clearance was markedly increased, suggesting
that tolvaptan enhanced the excretion of almost solute-free
water from the body. Because of the water diuresis
associated with tolvaptan, the serum Na+ concentrations
were markedly and continuously elevated during the
Cardiovasc Drugs Ther (2011) 25 (Suppl 1):S67–S76 S71

500
5 ΔUFR UOsm 4 FWC

4 0
3
**

(mOsm/kg)
** -500 ‡

(mL/min)
3
(mL/min)

**
* 2 ‡
** ‡
2 ** ** -1000 **
**
** **
* * 1
* -1500 † †
1
* * *
-2000 0
0 C vs. T: p<0.01 C vs. T: p<0.01
C vs. F: p<0.01 C vs. F: p<0.01 C vs. T: p<0.01
-1
1 T vs. F: p<0.01 -2500 -1 C vs
vs. F: p<0 01
p 0.01
T vs. F: NS
T vs. F: NS

60 UNaEx 10 UKEx 60 UClEx

‡ ‡ ‡
50 50 ‡
** ** 8 **
**
0)

0)
mEq/min × 100

mEq/min × 100
‡
mEq/min × 100)

40 ‡ 40
† 6 ** †
**
30 ** ‡ * 30 **
† ** * †
* **
4 **
20 * **
20 * *
(m

(m
* 2
(m

10 10

0 0 0
C vs. T: NS C vs. T: p<0.01 C vs. T: NS
C vs. F: p<0.01 C vs. F: p<0.01 C vs. F: p<0.01
-10 T vs. F: p<0.01 -2 T vs. F: p<0.01
-10 T vs. F: p<0.01

-60 0 60 120 180 240 300 360 -60 0 60 120 180 240 300 360 -60 0 60 120 180 240 300 360
Time after administration (min) Time after administration (min) Time after administration (min)

Fig. 1 Effects of tolvaptan and furosemide on urine flow rate (UFR),
urine osmolality (UOsm), free-water clearance (FWC), and urinary
Na+ (UNaEx), K+ (UKEx) and Cl– (UClEx) excretion in congestive
heart failure (CHF) dogs. q: differences from baseline are expressed
as means ± SEM (n=7). p values show the differences among three
groups analyzed by two-way ANOVA, followed by Tukey’s multiple-
comparison test. *p<0.05 and **p<0.01 vs. control; †p<0.05 and ‡p<
0.01 vs. tolvaptan or furosemide at each time-point. C: control (○); T:
tolvaptan (●); F: furosemide (r)

measurement period. This effect is clinically important for cortical collecting duct [19]. Amorim et al. showed that
the treatment of water-retaining diseases, such as CHF and luminal application of AVP enhanced K+ secretion in the rat
cirrhosis, because dilutional hyponatremia frequently devel- initial collecting duct, and that this action was abolished by
ops secondarily to these diseases. In particular, hypona- luminal V1-receptor blockers but not by V2-receptor blockers
tremia in CHF is strongly correlated with poor outcomes [20]. Therefore, we speculate that the enhanced urinary K+
[18]. In the Acute and Chronic Therapeutic Impact of excretion induced by tolvaptan is not a direct effect, but may
Vasopressin Antagonist in Congestive Heart Failure be a secondary effect caused by compensatory secretion of
(ACTIV in CHF) clinical trial, 67 patients (21.3%) AVP through luminal V1 receptors.
hospitalized with acute decompensated HF had hyponatre- As a consequence of its saluretic effect, furosemide
mia (Na+ levels <136 mEq/L). After taking tolvaptan, these significantly and continuously decreased serum K+ concen-
patients showed a rapid increase, and in many patients trations during the measurement period. This finding
normalization, of the serum Na+ levels was sustained suggests that furosemide may deplete K+ levels, resulting
throughout the study (60 days) [12]. in hypokalemia. It is well known that hypokalemia can
Tolvaptan significantly increased urinary K+ excretion in predispose patients to serious cardiac arrhythmias, particu-
CHF dogs. Interestingly, these increases were only observed larly in the presence of digitalis therapy [2, 21]. These
during the last 2 h of the measurement period, and closely results indicate that tolvaptan will be useful for the
resembled the changes in plasma AVP concentrations rather treatment of volume-overload states associated with CHF
than changes in urine flow rate. It has been reported that because it does not cause plasma electrolytes abnormalities,
AVP stimulates K+ secretion in the cortical distal tubule and such as hyponatremia and hypokalemia.
S72 Cardiovasc Drugs Ther (2011) 25 (Suppl 1):S67–S76

Fig. 2 Effects of tolvaptan and 30 SOsm 15 SNa

furosemide on serum osmolality ‡
(SOsm) and serum Na+ (SNa), ‡ ‡
20 ‡ ‡ ‡ 10 ‡ ** **
**
K+ (SK) and Cl– (SCl) concen- ** ** **
‡ **

kg)
trations in congestive heart fail-
**

L)
(mOsm/k
ure (CHF) dogs. q: differences

(mEq/L
10 ‡ 5 †
from baseline are expressed as ** **
means ± SEM (n=7). p values *
0 0
show the differences among
three groups analyzed by two-
-10 C vs. T: p
p<0.01 -5 C vs. T: p<0.01
way ANOVA, followed by
C vs. F: NS C vs. F: NS
Tukey’s multiple-comparison T vs. F: p<0.01
T vs. F: p<0.01
test. *p<0.05 and **p<0.01 vs. -20 -10
control; †p<0.05 and ‡p<0.01
vs. tolvaptan or furosemide at
each time-point. C: control (○); 2 SK 15 SCl
T: tolvaptan (●); F: furosemide ‡ ‡
‡ ** **
(r) 10
1 ‡ **
**

Eq/L)
Eq/L)
5 †

(mE
(mE

0
0
* ** *
C vs. T: p<0.01 ** ** **
-1 **
‡ **
‡
C vs. F: p<0.01 -5 C vs. T: p<0.01
T vs. F: p<0.01 C vs. F: p<0.01
T vs.
vs F: p<0
p<0.01
01
-2 -10

-60 0 60 120 180 240 300 360 -60 0 60 120 180 240 300 360
Time after administration (min) Time after administration (min)

Fig. 3 Effects of tolvaptan and 4

furosemide on neurohormones 60 ‡
AVP PRA
levels in congestive heart failure 3 **
(CHF) dogs. q: differences from 40
baseline are expressed as means ‡
hr)

20 2
(ng/mL/h

**
(pg/mL)

± SEM (n=7). p values show the

differences among three groups 1
0
analyzed by two-way ANOVA,
followed by Tukey’s multiple- 0
-20
comparison test. **p<0.01 vs.
control; ‡p<0.01 vs. tolvaptan at -40
C vs. T: NS -1 C vs. T: NS
each time-point. C: control (○); C vs. F: p<0.01 C vs. F: p<0.01
T: tolvaptan (●); F: furosemide T vs. F: NS -2 T vs. F: p<0.01
-60
(r). ANP = atrial naturetic
peptide; AVP = arginine vaso- 300 0.4
pressin; NE = norephinephrine; ANP NE
PRA = plasma renin activities
200 0.2
g/mL)
g/mL)

0 0
0.0
(ng

100
(pg

0 -0.2
C vs. T: NS C vs. T: NS
C vs. F: NS C vs. F: NS
T vs. F: NS T vs. F: NS
-100 -0.4

-60 0 60 120 180 240 300 360 -60 0 60 120 180 240 300 360
Time after administration (min) Time after administration (min)
Cardiovasc Drugs Ther (2011) 25 (Suppl 1):S67–S76 S73

1.0 CO 30 MBP 60
SVR
0 8
0.8
20
0.6 40

mHg min/L)
0.4
10

mmHg)
L/min)

0.2 20
0.0
0 0 0

(m
(L

(mm
-0.2 0
-10
-0.4
C vs. T: NS C vs. T: p<0.05 -20 C vs. T: NS
-0.6
C vs. F: NS -20 C vs. F: NS C vs. F: NS
-0.8 T vs. F: NS T vs. F: NS T vs. F: NS
-1.0 -30 -40

40 150 200
GFR RPF RBF
150
100
20 100
(mL/min)

(mL/min)
((mL/min)

50 50

0 0
0 -50

-20 C vs. T: NS C vs. T: NS -100 C vs. T: NS

-50 C vs. F: NS
C vs. F: NS C vs. F: NS
T vs. F: NS T vs. F: NS
-150 T vs. F: NS

-40 -100
100 -200
200

-60 0 60 120 180 240 300 360 -60 0 60 120 180 240 300 360 -60 0 60 120 180 240 300 360
Time after administration (min) Time after administration (min) Time after administration (min)

Fig. 4 Effects of tolvaptan and furosemide on systemic (upper panel)
and renal (lower panel) hemodynamic parameters in CHF dogs. q:
differences from baseline are expressed as mean ± SEM (n=7). p values
show the differences among three groups analyzed by two-way
ANOVA, followed by Tukey’s multiple comparison test. C: control
(○); T: tolvaptan (●); F: furosemide (r). CO = cardiac output; GFR =
glomerular filtration rate; MBP = mean arterial blood pressure; RBF =
renal blood flow; RPF = renal plasma flow; SVR = systemic vascular
resistance

Effects of tolvaptan on plasma neurohormones
In this study, tolvaptan tended to increase the plasma AVP
concentrations (Fig. 3), although these were not statistically
significant. Tolvaptan did not affect PRA or plasma NE
concentrations. These observations are consistent with those
in previous studies in rats [9, 10], and in patients with CHF
[13]. Under physiological conditions, AVP secretion is
primarily controlled by osmoreceptors in the brain and by
pressure-sensitive receptors in the left atrium and large
arteries of the chest and neck [22]. Therefore, the increase
in plasma osmolality and/or the reduction in cardiac preload
caused by tolvaptan are likely to contribute to the increase in
plasma AVP observed in this study. Interestingly, the
increase in plasma AVP concentrations caused by tolvaptan
in normal dogs (data not shown) was greater than those in
the CHF dogs, while the increase in urine excretion and
serum osmolality caused by tolvaptan were similar in normal
and CHF dogs. When we compared the relationships
between serum osmolality and plasma AVP concentrations
after tolvaptan administration in normal and CHF dogs,
the CHF dogs showed blunted AVP secretion in response
to the osmotic stimuli of tolvaptan, as compared with
normal dogs (Fig. 6). It is well known that changes in
blood volume can influence the relationship between
serum osmolality and plasma AVP concentrations, and
that volume depletion exaggerates, and volume expansion
blunts AVP secretion in response to osmotic stimuli [22–
24]. Moreover, Wang et al. reported that most of the
volume influences on the relationship between plasma
osmolality and plasma AVP concentrations were mediated
via reflex effects elicited by cardiac receptors, particularly
those located in the left atrium [24]. The baseline value of
PCWP, an index of left atrial pressure, was much greater
in CHF dogs than in normal dogs (Table 1). Therefore, it
seems possible that the blunted AVP secretion in CHF
dogs may be caused by the reflex effects elicited by left
atrial distention. These observations suggest that compen-
satory secretion of AVP after tolvaptan treatment may be
reduced in volume-overload diseases such as CHF.
S74 Cardiovasc Drugs Ther (2011) 25 (Suppl 1):S67–S76

Fig. 5 Effects of tolvaptan and Change in body weight

furosemide on cumulative water 400 Cumulative water balance 0.4
balance and body weight (upper
panel) and cardiac preload 200 0.2
(lower panel) in congestive
heart failure (CHF) dogs. In

g)
* 00
0.0

(kg
0 ** ** **

(mL)
upper panel, values are
expressed as the mean ± SEM
** -0.2
(n=7). p values show the differ- -200
ences among three groups ana-
** Control **
lyzed by repeated measures -400 -0.4
C vs. T: p<0.01 **
ANOVA, followed by Tukey’s
C vs. F: p<0.01 **
multiple comparison test. Tolvaptan
-600 T vs. F: p<0.01 ** -0.6
*p<0.05 and **p<0.01 vs.
control. In lower panel, q: **
Furosemide
differences from baseline are 4
4 PCWP RAP
expressed as means ± SEM 3
3
(n=7). p values show the differ- 2
ences among three groups 2
1

mmHg)
analyzed by two-way ANOVA, 1 *
mHg)

followed by Tukey’s multiple 0 ** *

0 *
(mm

(m
comparison test. *p<0.05 and -1
1
**
**p<0.01 vs. control; †p<0.05 -1
-2
and ‡p<0.01 vs. tolvaptan at -2 ** **
each time-point. C: control (○); † ** -3 † **
-3 ‡ **
T: tolvaptan (●); F: furosemide C vs. T: p<0.01 ** -4 C vs. T: p<0.01 ‡ **
-4 † ‡
(r). PCWP = pulmonary capil- C vs. F: p<0.01
01 -5
5 C vs. F: p<0.01
lary wedge pressure; RAP = right -5 T vs. F: p<0.01 T vs. F: p<0.01
atrial pressure
-60 0 60 120 180 240 300 360 -60 0 60 120 180 240 300 360
Time after administration (min) Time after administration (min)

In contrast to tolvaptan, furosemide significantly increased
the PRA and plasma AVP concentrations during the measure-
ment period. In addition, furosemide tended to increase
plasma NE concentrations. It is well known that furosemide
stimulates renin release, and possible mechanisms include a
direct intrarenal effect on the macula densa, activation of the
baroreceptor mechanism, and diuretic-induced volume con-
traction [25]. Neurohormonal activation may enhance the
frequency and severity of electrolyte depletion for the short
term, and may increase the risk of disease progression for the
long term in patients with CHF [26].
Effects of tolvaptan on systemic and renal hemodynamics

100 Oral administration of tolvaptan reduced the increase in

pg/mL)

Normal dogs cardiac preload (PCWP and RAP) caused by rapid right
CHF dogs
80 ventricular pacing. Similarly, furosemide also reduced the
oncentration (p

cardiac preload in CHF dogs. Furosemide (10 mg/kg) elicited

60 greater decreases in PCWP and RAP than tolvaptan because
y=1.6207x–471.2 furosemide had a greater diuretic effect than tolvaptan (10 mg/
R2=0.4826
kg) in CHF dogs. The decrease in ventricular preload with
Plasma AVP co

40
each drug corresponded to the decrease in cumulative water
20 y=0.498x–139.42 balance and the decrease in body weight (Fig. 5, upper panel).
R2=0.2547
Therefore, it seems possible that the aquaretic effect of
tolvaptan is not inferior to the saluretic effect of furosemide
0
in terms of reducing the cardiac preload increase in CHF. In
patients with CHF, similar results have been reported;
-20
280 285 290 295 300 305 310 315 320 325 tolvaptan decreased PCWP in association with a significant
Serum osmolality (mOsm/kg) increase in urine output [27].
This study also showed that the compensatory increase in
Fig. 6 Relationship between serum osmolality and plasma arginine
vasopressin (AVP) concentrations after administration of tolvaptan in plasma AVP concentration caused by tolvaptan did not affect
normal (○) and congestive heart failure (CHF) (●) dogs CO or SVR, or aggravate renal hemodynamic parameters such
Cardiovasc Drugs Ther (2011) 25 (Suppl 1):S67–S76
as GFR, RBF and RPF (Fig. 4). These findings are consistent
with an earlier report in patients with CHF [13, 27]. Similar
results have been reported using another vasopressin V2-
antagonist, OPC-31260, in normal and CHF dogs [28].
Moreover, long-term OPC-31260 administration did not
aggravate cardiac remodeling, cardiac function, or survival,
despite significantly increasing the plasma AVP concentra-
tion in rat models of postinfarction-induced [29] and
adriamycin-induced [30] CHF. Therefore, a compensatory
increase in plasma AVP by the V2-antagonist may not
adversely affect cardiac or renal function during short- or
long-term treatment in patients with CHF.
Study limitation
In the present study, CHF dogs clearly showed haemodynamic
and neurohormonal abnormalities, but serum osmolality and
Na+ concentrations were minimally altered in CHF dogs,
suggesting that water retention in the CHF dogs was not
substantially large. These observations indicated that CHF
model was not severe but moderate heart failure. In fact, our
CHF dogs showed neither ascites nor symptom of edema.
However, tolvaptan induced aquaresis and PCWP was
decreased as much as with furosemide. To clarify whether
tolvaptan improves edematous disorder in severe CHF, the
animal models with more severe CHF associated with ascites
or symptom of edema may be needed for further study.
Conclusions
This study showed that tolvaptan elicits a potent aquaretic
response accompanied by a significant increase in free
water clearance in CHF dogs. Because of the reduction in
body fluid, tolvaptan decreased PCWP and RAP, indices of
cardiac preload, without unfavorable effects on systemic or
renal hemodynamic parameters. Furthermore, unlike furo-
semide, tolvaptan did not decrease serum electrolyte
concentrations nor affect the RAAS in CHF dogs. These
results suggest that tolvaptan offers a novel approach to
remove congestion and edema in patients with CHF.
Acknowledgments The authors thank Mr. S Kinoshita for his
excellent technical assistance and Dr. Y Liu for his assistance in
preparing this manuscript.
Disclosures None of the authors have any conflicts of interest
associated with this study.
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