Original Paper

Received: April 14, 2003

Pharmacology 2004;70:107–112
Accepted: August 19, 2003
DOI: 10.1159/000074675

Gender-Related Differences in the

Pharmacodynamics of Furosemide
in Rats
Anabel Brandoni Silvina R. Villar Adriana M. Torres
Area Farmacologı́a, Facultad de Ciencias Bioquı́micas y Farmacéuticas, Universidad Nacional de Rosario, CONICET,
Rosario, Argentina

Key Words Introduction

Sex, furosemide pharmacodynamics W Furosemide W
Pharmacodynamics, sex differences W Na-K-2Cl
cotransporter
Abstract
The possibility of gender differences in the relationship
between dose and efficacy of drugs has historically been
limited. The aim of the present study was to evaluate the
influence of sex on the pharmacodynamics of furose-
mide (FUR) in adult Wistar rats. Conventional renal clear-
ance studies were performed in the absence and in the
presence of a FUR dose necessary to produce identical
urine excretion rates of FUR in both sexes. Fractional
excretions of sodium, potassium, and water were similar
in male and female rats in the absence of FUR. Natriuret-
ic, kaliuretic, and diuretic responses to FUR, when ex-
pressed in fractional terms, were significantly increased
in female rats. Diuretic, natriuretic, and kaliuretic efficien-
cies of FUR were also higher in female rats as compared
with males. This might be explained by the lower abun-
dance of the Na-K-2Cl cotransporter observed in homog-
enates from the medullae of female kidneys as com-
pared with male ones.
Copyright © 2004 S. Karger AG, Basel
The availability of information about pharmacokinet-
ics in women and about the possibility of gender differ-
ences in the relationships between dose and efficacy or
between dose and adverse drug reactions has historically
been limited. From the pharmacodynamic point of view,
only few studies could clearly identify clinically important
gender effects [1–3]. Because approximately 50% of the
population of any species are females, it is appropriate to
consider the influence of sex on the pharmacokinetics and
pharmacodynamics of drugs.
Studies recently done in our laboratory demonstrated
that sex modifies the pharmacokinetics of organic anions.
Female rats display a lower p-aminohippurate and furose-
mide (FUR) clearance as a consequence of their lower
renal clearance [4, 5]. This is mediated, at least in part, by
the lower abundance of the renal organic anion transport-
er (OAT1) in female rats as compared with male rats [6].
FUR is a well-known organic anion which exerts its
diuretic action by binding to the Na-K-2Cl cotransporter
(NKCC2) in the thick ascending limb (TAL) and blocking
ion transport. FUR is commonly used to treat edematous
conditions, congestive heart failure, cirrhosis of the liver,
and nephrotic syndrome. Of the adverse reactions to FUR
hypotension, circulatory collapse, thromboembolic epi-
sodes, ototoxicity, hearing impairment, deafness, vertigo,
and tinnitus have been reported [7, 8]. In order to contin-
ue our previous studies about the gender differences in
FUR pharmacokinetics, it was of interest to examine the
influence of sex on the pharmacodynamics of FUR.

© 2004 S. Karger AG, Basel Adriana Mónica Torres, PhD

ABC 0031–7012/04/0702–0107$21.00/0 Suipacha 531
Fax + 41 61 306 12 34 Rosario 2000 (Argentina)
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 Materials and Methods
Experimental Animals
Adult male and female Wistar rats aged 110–130 days were used
throughout the study. The animals were allowed free access to a stan-
dard laboratory chow and tap water and were housed in a constant
temperature and humidity environment with regular light cycles
(12 h). The animals were cared for in accordance with institutional
guidelines for the care and use of laboratory animals.
Renal Function Studies
Renal clearance studies: Four experimental groups were em-
ployed for these studies: (1) females; (2) males; (3) females plus FUR,
and (4) males plus FUR. FUR was administered besides the dose of
inulin at a priming dose (10 mg/kg) and in the infusion solution
(20 g%). Considering the sex differences related to the pharmacoki-
netics of FUR [5], we have previously demonstrated that this dosage
causes an identical excreted load of FUR in both sexes.
These studies were performed as previously described [4, 9].
Briefly, the animals were anesthetized with sodium thiopental
(70 mg/kg i.p.). Femoral vein and femoral artery were cannulated
(PE50 tubing; Intramedic), and a bladder catheter (3 mm inner diam-
eter) was inserted through a suprapubic incision. The animals were
maintained in restraining cages throughout the experiments to facili-
tate collection of urine. A priming dose of inulin (0.6 mg/kg) in 1 ml
of saline solution was administered through the venous catheter.
Then, a solution containing inulin (1.8 g%) and saline (0.3 g%) was
infused through the venous catheter employing a constant-infusion
pump (Pump 22; Harvard Apparatus) at a rate of 1 ml/h/100 g. After
equilibration for 60 min, urine was collected during two 30-min peri-
ods. Blood from the femoral artery was obtained at the midpoint of
each clearance period. The arterial blood pressure was estimated
throughout the experiments using a manometer inserted in the femo-
ral artery. The glomerular filtration rate (GFR) was calculated from
the clearance of inulin. Clearance of FUR, the urinary excretion rates
of potassium (UKV), sodium (UNaV), water, and FUR (UFURV), and
the fractional excretions of water (FE% H2O), sodium (FE% Na+),
and potassium (FE% K+) were calculated by conventional formulae
for each animal. The FUR concentrations in serum and urine were
determined by the method of Bratton and Marshall [10]. The inulin
concentrations in the same samples were determined by the proce-
dure of Roe [11]. Sodium and potassium were measured by flame
photometry and the volume of urine by gravimetry.
The natriuretic, kaliuretic, and diuretic efficiencies of FUR (FS)
were calculated as:
FUReffFENa = [FE Na% (group with FS) – FE Na%
(group without FS)]/UFURV (1)
FUReffFEK = [FE K% (group with FS) – FE K%
(group without FS)]/UFURV (2)
FUReffFEH2O = [FE H2O% (group with FS) – FE H2O%
(group without FS)]/UFURV (3)
The relative effect of FUR on the GFR was also calculated as:
FUReffGFR = [GFR (group with FS) – GFR
(group without FS)]/UFURV (4)
The mean values of FE Na%, FE K%, FE H2O%, and GFR of the
group without FS was subtracted from the individual values of FE
Na%, FE K%, FE H2O%, and GFR for each rat from the group with
FS, respectively, divided by the excreted load of FUR of each rat.
108 Pharmacology 2004;70:107–112
NKCC2 Abundance Assay
In other groups of male and female rats, the kidneys were rapidly
removed and dissected to obtain medulla and cortex. The tissues
were homogenized in ice-cold solution containing 250 mmol/l su-
crose, 10 mmol/l triethanolamine, 1 Ìg/ml leupeptin, and 0.1 mg/ml
phenylmethylsulfonyl fluoride.
Homogenate samples were boiled for 3 min in the presence of 1%
2-mercaptoethanol/2% SDS. Samples containing 50 Ìg of proteins
were applied to a 5% gel, separated by SDS-PAGE, and then electro-
blotted to nitrocellulose membranes. The membranes were stained
with ponceau red to confirm equal protein loading and transfer
between lanes as previously described [6, 12–14]. The nitrocellulose
membranes were incubated for 24 h with a commercial polyclonal
antibody to NKCC2 (1.25 Ìg/ml). The membranes were incubated
for 1 h with a peroxidase-coupled goat antirabbit IgG (Bio-Rad) after
being rinsed with PBST. Then, the blots were processed for detection
using a commercial kit (Opti-4CN; Bio-Rad).
Materials
Chemicals were purchased from Sigma Chemical and were of
pure analytical grade.
Statistics
Statistical analysis was performed using an unpaired t test. When
variances were not homogeneous a Welch correction was employed.
p ! 0.05 was considered significant. Whenever more than two mean
values were to be compared, analysis of variance was used. When the
F value was significant (p ! 0.05), the group mean values were ana-
lyzed using the Newman-Keuls test for significant differences. Values
are expressed as mean values B SEM. For these analyses a GraphPad
software was used.
Results
FUR is secreted in the proximal tubules and is deliv-
ered to its site of action in the TAL by the tubule fluid.
Because FUR is not reabsorbed in nephron segments fur-
ther downstream, the urinary FUR excretion rate
(UFURV) reflects the amount of FUR delivered to the
NKCC2 at the luminal site of the TAL. As gender differ-
ences in pharmacokinetics of FUR have been recently
demonstrated [5], we administered to the rats the FUR
dose necessary to achieve a similar UFURV in both sexes.
Urine and plasma levels of FUR and UFURV are shown in
table 1.
Body weight and mean arterial blood pressure (MAP)
are summarized in table 2. The MAP decreased as a con-
sequence of FUR administration in both male and female
rats, reaching only statistical significance in females.
The effects of FUR in both male and female rats on
sodium and potassium excretion rates, urine flow, and
sodium, potassium, and water fractional excretions are
shown in table 3. No gender differences were observed in
FE% Na, FE% K, and FE% H2O in the absence of FUR,
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Table 1. Urine and plasma levels of FUR ([FUR]u; [FUR]p) and Table 2. Body weight and MAP in male and female rats in the pres-
urinary FUR excretion rates (UFURV) in male (n = 6) and female (n = ence and absence of intravenous infusions of FUR (mean B SEM)
6) rats (mean B SEM) (each group n = 6)

Males Females Males Males Females Females

plus FUR plus FUR plus FUR plus FUR

[FUR]u, Ìg/ml 738B79 782B89 Body weight, g 373B13 346B13 232B2a, b 264B13a, b
[FUR]p, Ìg/ml 105B13 173B7* MAP, mm Hg 131B4 108B11 130B5 92B10a, c
UFURV, Ìg/min/100 g body weight 30B2 28B2
UFURV, Ìg/min/g kidney weight 41B4 40B3 Anova plus Newman-Keuls test was performed (a vs. males, b vs.
males plus FUR, c vs. females: p ! 0.05).
* p ! 0.05.

Table 3. Sodium and potassium urine

excretion rates (UNaV, UKV), urine flow Males Males Females Females
(V), and sodium, potassium, and water plus FUR plus FUR
fractional excretion (FE% Na, FE% K, FE%
H2O) in male and female rats in the UNaV, mEq/min/100 g 0.72B0.09 7.42B1.00a, c 0.48B0.04b, d 7.16B0.46a, c
presence and absence of intravenous FE% Na 0.76B0.08 12.85B1.24 0.89B0.08b, d 18.33B1.55a, b, c
infusions of FUR (mean B SEM) (each UKV, mEq/min/100 g 0.52B0.06 1.11B0.08 0.32B0.02 1.28B0.08a, c
group n = 6) FE% K 21B3 99B9 23B2 189B8a, b, c
V, Ìl/min/100 g 2.60B0.26 39.5B3.7a, c 1.98B0.08 37.0B2.5a, c
FE% H2O 0.44B0.05 11.55B0.82a, c 0.69B0.09 15.96B0.46a, b, c

Anova plus Newman-Keuls test was performed (a vs. males, b vs. males plus FUR, c vs.
females, d vs. females plus FUR: p ! 0.05).

as was previously described in our laboratory [4]. But the
diuretic, natriuretic, and kaliuretic responses to FUR,
when expressed in fractional terms, were significantly
increased in females rats. Diuretic, natriuretic, and ka-
liuretic efficiencies of FUR were also higher in female rats
as compared with male rats (fig. 1).
The GFR before and during FUR administration in
male and female rats can be observed in figure 2a. The
GFR was significantly lower in female rats, as previously
described [4]. FUR administration decreased the GFR in
both male and female rats, failing to reach statistical sig-
nificance in females. When the data were expressed in rel-
ative terms as the efficiency of FUR in decreasing the
GFR, FUR induced a lower effect in female rats (fig. 2b).
Homogenates from male and female kidney cortices
and medullae were subject to immunoblot analysis for
NKCC2 protein. A primary band with a size of 161 kD
was detected. Figure 3 shows the NKCC2 protein levels in
male and female kidney cortices and medullae. A similar
abundance of NKCC2 was detected in renal cortex ho-
mogenates from male and female rats. On the other hand,
a decrement in the abundance of NKCC2 in renal medul-
la homogenates from female rats was observed. The
NKCC2 protein of 161 kD disappeared when the anti-
body was preabsorbed to the synthetic antigen peptide
(data not shown).
Discussion
In the past, women were underrepresented as partici-
pants in clinical drug studies. In the majority of the cases,
when both women and men were studied, the results were
grouped together, and there was no analysis of sex differ-
ences. Although the FDA and other regulatory authorities
required more participation of women in clinical trials
after recognizing the problem of underrepresentation of
women, little is known about drug effects in women when
compared to men. For a number of drugs it is well recog-
nized that women suffer more frequently from side ef-
fects; however, it is often not clear whether this is due to
gender differences in the pharmacokinetics or pharmaco-
dynamics of the responsible drug. Very little is known
about these gender-related differences and about the pos-

Furosemide Effects in Male and Female Pharmacology 2004;70:107–112 109

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Fig. 1. Natriuretic (a), kaliuretic (b), and diuretic (c) efficiencies of
FUR (FUReffFE% Na, FUReffFE% K, and FUReffFE% H2O) in
male (n = 6) and female (n = 6) rats. The results are expressed as
mean B SEM. # p ! 0.05.
sibility that women may show a treatment response differ-
ent from that of men. As a result, drug approval authori-
ties now require more data on the pharmacokinetics of
novel drugs in women as well as a sufficient accrual of
women in efficacy and outcome trials. Pharmacodynamic
differences could potentially be clinically important for
drugs having either narrow or wide therapeutic ranges. It
is certainly possible that these sex differences may be
related to the higher incidence of adverse reactions to
drugs observed in women as compared with men [1–3].
Further investigations are urgently needed in this area.
We have recently demonstrated that female rats have a
lower systemic and renal clearance of FUR [5]. As FUR is
a loop diuretic commonly employed in clinical practice, it
110 Pharmacology 2004;70:107–112
Fig. 2. a GFR in male and female rats in the presence and in the
absence of intravenous infusions of FUR. The results are expressed
as mean B SEM. ap ! 0.05 vs. males. b Efficiency of FUR in decreas-
ing the GFR (FUReffGFR) in male and female rats. The results are
expressed as mean B SEM. # p ! 0.05 vs. males plus FUR.
was of interest to complete the previous pharmacokinetic
studies, determining whether gender-related differences
are also observed in the pharmacodynamics of the diuret-
ic. In order to achieve this objective, we administered the
FUR dosage necessary to produce identical urine excre-
tion rates of FUR in both male and female rats. The uri-
nary excretion rate of a loop diuretic has been shown to be
a reliable measure of amounts of a diuretic reaching the
site of action and can be used as a surrogate concentration
in a typical concentration-response analysis of diuretic
action [15, 16]. The urinary concentration has not proven
to be a useful measure, because the concentration of a
diuretic in the final urine does not represent it at the site of
action. Simplistically, the more diuretic reaching its site of
action, the greater the response, so that the net result is
that the diuretic concentration in the final urine is con-
stant. Therein, the diuretic excretion rate is a better reflec-
tion of the amount of diuretic that is able to interact with
the Na-K-2Cl transporter. At this place, FUR inhibits the
active NaCl transport. Stoichiometry and mechanism of
active NaCl transport in the TAL have been elucidated
above all by Greger and Schlatter [17, 18]. These authors
showed that NaCl transport across the apical plasma
membrane is mediated by a NKCC2 that is directly inhib-
ited by FUR. The K entering the cell on the cotransporter
is recycled into the lumen via an apical K channel. The Na
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Fig. 3. a Renal medulla homogenates (50 Ìg proteins) and renal cor-
tex homogenates (50 Ì proteins) from kidneys of male (M) and
female (F) rats were separated using SDS-PAGE. The NKCC2 was
identified using polyclonal antibodies. b Male levels were set at
100%. Each column represents the mean B SEM from experiments
carried out in triplicate on three different homogenates for each
experimental groups. # p ! 0.05.
is actively pumped out of the cell into the interstitium by
the basolateral Na-K-ATPase. The Cl ions exit via a baso-
lateral chloride channel and/or a KCl cotransporter. Elec-
troneutrality is maintained by paracellular Na transport
driven by a lumen-positive electrical potential across the
epithelium. When FUR exerts its action, important incre-
ments in the urinary excretion of Na, H2O, K, phosphate,
Ca, and Mg are observed, the increment of K excretion
being in part a function of the delivery of Na and H2O to
the K-secretory sites in the distal nephron.
In this study, it was observed that FE% Na,, FE% K,
and FE% H2O were similar in both sexes in the absence of
FUR, as was previously demonstrated by us [4]. The
present study shows that female rats have an increased
Furosemide Effects in Male and Female
Rats
natriuretic, kaliuretic, and diuretic response to FUR in the
presence of identical excreted load of FUR. When the
effects of FUR were expressed as the efficiency of the drug
(defined as the relationship between diuretic delivery and
response, measured as FE% Na, FE% K, and FE% H2O),
the increased efficiencies of FUR in female rats were still
present. This might be explained by the lower abundance
of NKCC2 observed in homogenates from renal medullae
of female rats. The amount of FUR reaching the co-
transporters is probably sufficient to inhibit all of them in
female but not in male rats. The abundance of the NKCC2
in the TAL is regulated in response to changes in circulat-
ing cyclic adenosine monophosphate (cAMP) levels, since
the 5) flanking region of the NKCC2 gene contains a
cAMP-regulatory element [19]. It has been described that
nitric oxide (NO) and prostaglandin E2 (PGE2) decrease
the cAMP levels [20, 21]. In this connection, it has been
shown in humans and animals that the NO release is great-
er in females than in males [22, 23]. This is probably due to
an increased availability of NO, because estrogens not
only stimulate the NO production [23], but also decrease
the inactivation of NO by oxygen radicals [24]. Neugarten
et al. [25] have described that steady state levels of mRNA
for inducible NO synthase were found to be higher in the
inner medulla of female rats as compared with male rats.
Moreover, Verhagen et al. [22] observed an increased
renal medullary endothelial NO synthase activity in the
kidneys of female rats. PGE2, a major product of the
cyclooxygenase pathway in the kidney, also decreases the
cAMP levels in the TAL in vitro. In this connection, the
medulla of female rats has a high basal level of vasodilator
prostaglandins [26]. In addition, it has been described that
the presence of testosterone increases the cAMP levels
[27]. So, the lower levels of testosterone and the higher lev-
els of NO and PGE2 described in females might account
for lower levels of cAMP in the TAL, leading to the lower
abundance of NKCC2 that we observed in renal medullae
of female as compared with male rats.
Female rats showed a lower GFR as compared with
male rats which is in accordance with results of our pre-
vious studies [4] and with those of other authors [28, 29].
The administration of FUR caused a reduction in the
GFR, due to the predominantly renal vasoconstrictor
effects of the diuretic which have been previously reported
in rats [9, 30–34]. The lower efficiency of FUR in decreas-
ing the GFR in female rats might be due to the higher circu-
lating levels of PGE2 previously reported in females [26].
The present study, demonstrating a gender difference
in the NKCC2 abundance, has possible implications in
the development of hypertension. It is an established epi-
Pharmacology 2004;70:107–112 111
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demiological observation that the incidence of hyperten-
sion is lower in women (before the menopause) than in
men. In this connection, it has been recently demon-
strated that the development of genetic hypertension in
the Milano strain of genetic hypertensive rats is associated
with an upregulation of NKCC2 mRNA and protein
abundance along the TAL of Henle’s loop [35]. So, it is
possible that the lower incidence of hypertension in
females might be associated with the lower abundance of
NKCC2 observed in females as compared with males.
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