Full text of DOI:10.1038/sj.bjp.0704528

British Journal of Pharmacology (2002) 135, 891 ± 900
ã
2002 Nature Publishing Group All rights reserved 0007 ± 1188/02 $25.00
www.nature.com/bjp
Cyclooxygenase-1 derived prostaglandins are involved in the
maintenance of renal function in rats with cirrhosis and ascites
1
5
2
,1
1
1
pez-Parra, * Joan Claria, Anna Planaguma, Esther Titos, Jaime L. Masferrer,
 Á Á
5
Marta Lo
B. Mark Woerner, Alane T. Koki, Wladimiro Jime
5
2
4
nez, Rosario Altuna, Vicente Arroyo,
3
Â
3
Francisca Rivera & Joan Rodes
Â
1
DNA Unit, Hospital Clõ
Â
nic, Institut d'Investigacions Biome
Á
diques August Pi i Sunyer (IDIBAPS), Universitat de Barcelona,
2
Barcelona 08036, Spain; Hormonal Laboratory, Hospital Clõ
IDIBAPS), Universitat de Barcelona, Barcelona 08036, Spain; Liver Unit, Hospital Clõ
Biome
Â
nic, Institut d'Investigacions Biome
Á
diques August Pi i Sunyer
nic, Institut d'Investigacions
3
(
Â
4
Á
diques August Pi i Sunyer (IDIBAPS), Universitat de Barcelona, Barcelona 08036, Spain; Pharmacia Spain, Barcelona
5
0
8190, Spain and Pharmacia Research and Development, St Louis, Missouri, MO 63167, U.S.A.
1
The maintenance of renal function in decompensated cirrhosis is highly dependent on
prostaglandins (PGs). Since PG synthesis is mediated by cyclooxygenase-1 and -2 (COX-1 and
COX-2), the present study was designed to examine which COX isoform is involved in this
phenomenon.
2
Renal COX-1 and COX-2 protein expression and distribution were analysed by Western blot and
immunohistochemistry in nine rats with carbon tetrachloride-induced cirrhosis and ascites and 10
control animals. The e€ects of placebo and selective COX-1 (SC-560) and COX-2 (celecoxib)
inhibitors on urine ¯ow (V), urinary excretion of sodium (UNaV) and PGE
ltration rate (GFR), renal plasma ¯ow (RPF), the diuretic and natriuretic responses to furosemide
and renal water metabolism were assessed in 88 rats with cirrhosis and ascites.
COX-1 protein levels were found to be unchanged in kidneys from cirrhotic rats. In contrast,
2
(UPGE2V), glomerular
®
3
these animals showed enhanced renal COX-2 protein expression which was focally increased in the
corticomedullary region. Although UPGE2V was equally reduced by SC-560 and celecoxib, only SC-
5
60 produced a signi®cant decrease in UNaV, GFR and RPF and a pronounced impairment in the
diuretic and natriuretic responses to furosemide in rats with cirrhosis and ascites. Neither SC-560
nor celecoxib a€ected renal water metabolism in cirrhotic rats.
4 These results indicate that despite abundant renal COX-2 protein expression, the maintenance of
renal function in cirrhotic rats is mainly dependent on COX-1-derived prostaglandins.
British Journal of Pharmacology (2002) 135, 891 ± 900
Keywords: Experimental cirrhosis; renal COX expression; selective COX inhibition; renal function
Abbreviations: Celecoxib, 4-[5-(4-methylphenyl)-3-(tri¯uoromethyl)-1H-pyrazol-1-yl]; GFR, glomerular ®ltration rate; NSAIDs,
nonsteroidal anti-in¯ammatory drugs; PG, prostaglandin; RPF, renal plasma ¯ow; SC-560, [5-(4-chlorophenyl)-
1-(4-methoxyphenyl)-3-tri¯uoromethylpyrazole); V, urine ¯ow; UPGE2V, urinary PGE
sodium excretion
2
excretion; UNaV, urinary
Introduction
Non-steroidal anti-in¯ammatory drugs (NSAIDs) are among
the most widely prescribed class of pharmaceutical agents
world-wide, having broad clinical utility in treating pain,
fever and in¯ammation (Payan & Katzung, 1995). Despite a
relatively low incidence of renal side e€ects in healthy
subjects, administration of NSAIDs to patients with
unbalanced e€ective arterial blood volume, such as decom-
pensated cirrhosis, represents a major clinical problem since
renal function in these patients is critically dependent on
prostaglandins (PGs) (Arroyo et al., 1983; 1986; Dunn,
Arroyo et al., 1983; 1986; Planas et al., 1983; Mirouze et al.,
1983; Perez-Ayuso et al., 1984; Dunn, 1984). Thus, in
clinical practice, patients with decompensated cirrhosis
cannot be treated with NSAIDs on a long term basis
because of the high risk of developing renal failure and
refractory ascites.
Cyclooxygenase (COX) is the key enzyme in the
formation of PGs from arachidonate and is the major
therapeutic target for NSAIDs (Vane, 1971). Two isoforms
of COX, designated COX-1 and COX-2, have been
identi®ed (Kujubu et al., 1991, Masferrer et al., 1992; Hla
& Neilson, 1992). COX-1 is ubiquitous and has been
previously linked to the cytoprotective e€ects of PGs in
the gut as well as to the integrity of platelet function.
Conversely, COX-2 is undetectable in most tissues, but its
expression can be induced by a variety of stimuli related to
in¯ammatory response (Herschman, 1996). Conventional
NSAIDs inhibit COX-1 and COX-2 and this feature
Â
1
984). In fact, acute PG inhibition with NSAIDs in patients
with cirrhosis and ascites is associated with a signi®cant
impairment in renal hemodynamics, sodium excretion, free
water clearance and renal response to furosemide and
spironolactone (Boyer et al., 1979; Zipser et al., 1979;
*
Author for correspondence; E-mail: jclaria@clinic.ub.es

892
M. L o pez-Parra et al
Selective cyclooxygenase inhibition in cirrhosis
accounts for both therapeutic and side e€ects (Warner et al.,
999). Recently, selective COX-2 inhibitors have been
developed to e€ectively inhibit COX-2-dependent in¯amma-
inhibitors were provided by Pharmacia Research and
Development (St Louis, MO, U.S.A.).
1
tion while sparing physiologic PG production (Seibert et al.,
1
Induction of cirrhosis in rats
994; Marnett & Kalgutkar, 1999). These novel compounds
could potentially be the NSAIDs of choice in patients in
whom, as occurs in cirrhosis with ascites, renal function is
critically dependent on PGs.
To address the renal-damaging e€ects of NSAIDs in liver
disease, we have previously used rats with carbon tetra-
The study was performed in 97 rats with cirrhosis and ascites.
Cirrhosis was induced by CCl4 inhalation in adult male
Wistar rats (Charles-River, Saint Aubin les Elseuf, France)
initially weighing 160 ± 180 g, following a method described
elsewhere (Claria & Jimenez, 1999). Cirrhotic rats were
Á Â
chloride (CCl
that closely reproduces the systemic and renal abnormalities
seen in human cirrhosis (Claria & Jimenez, 1999). In fact,
CCl -induced cirrhotic rats also show increased renal PG
synthesis and develop renal failure after receiving conven-
tional NSAIDs such as aspirin (Ros et al., 1995) and
Â
ketorolac (Bosch-Marce et al., 1999). Interestingly, we have
recently shown that renal function in these animals remains
una€ected by the administration of SC-236 (a research pre-
clinical compound with high selectivity for COX-2 (Bosch-
4
)-induced cirrhosis, an experimental model
obtained from a group of 136 animals submitted to the
cirrhosis induction protocol. Thirty-nine of these animals
could not be included in the study for several reasons: 24 rats
died before the development of ascites and/or impairment of
renal water excretion, seven rats died before completing the
experimental procedures and eight rats did not develop
ascites and/or impairment to renal water excretion. Animals
were maintained at the University of Barcelona animal
facility and were fed ad libitum with standard chow and
Á
Â
4
71
distilled water containing phenobarbital (0.3 g l ) as drink-
ing ¯uid. Cirrhotic rats were studied after the development of
Marce et al., 1999)), suggesting that the renal side-e€ects of
Â
conventional NSAIDs in cirrhosis are secondary to COX-1
inhibition. Given that a selective COX-1 inhibitor was not
available in that study, the current investigation was aimed
to unequivocally establish which COX isoform is responsible
for the synthesis of PGs involved in the maintenance of
renal function in decompensated cirrhosis. To this end, we
ascites, usually between 12 and 18 weeks of CCl
tration.
4
adminis-
Western blot analysis of COX-1 and COX-2 protein
expression in renal tissue
®
rst analysed COX-1 and COX-2 protein expression and
distribution by Western blot and immunohistochemistry,
respectively, in kidneys from rats with CCl -induced
Protein expression for COX-1 and COX-2 was assessed in
renal tissue samples isolated from six cirrhotic rats with
ascites and from seven control animals. Animals were
4
71
cirrhosis and ascites. In these animals, we next compared
the e€ects of a selective COX-1 inhibitor (SC-560) with
those of a selective COX-2 inhibitor (celecoxib) on renal
hemodynamics and renal sodium handling. Subsequently, we
assessed the e€ects of these COX inhibitors on the diuretic
and natriuretic response to furosemide, and ®nally char-
acterized which COX isoform is involved in the regulation
of renal water metabolism in cirrhotic rats.
anaesthetized with an i.m. injection of ketamine (50 mg kg )
and the kidneys were rapidly dissected and stored in liquid
nitrogen. Frozen tissues (150 ± 180 mg) were homogenized in
30 mM Tris/HCl pH 7.4, 100 mM phenylmethylsulphonyl
71
¯uoride, containing protease inhibitors (1 mg ml
leupeptin, pepstatin A and aprotinin. The homogenate was
each):
centrifuged at 10006g for 10 min and the supernatant was
taken and subjected to a ®nal centrifugation step at
100,0006g for 70 min. Total protein concentration was
determined by the Bradford Protein Assay. Aliquots from
each sample containing equal amounts of protein (30 ± 50 mg)
were resuspended in SDS-containing Laemmli sample bu€er
and heated at 1008C for 5 min, electrophoresed on 10 ± 12%
SDS-polyacrylamide gels and transferred overnight to PVDF
membranes. The eciency of the transfer was visualized by
Ponceau staining. The blots were subsequently blocked for
6 h with 50 mM Tris/HCl pH 7.4 and 100 mM NaCl (TBS)
containing 5% nonfat dry milk and 0.5% Tween 20, followed
by incubation (1 : 1000 dilution) for 16 h with antisera speci®c
for either COX-1 or COX-2. After washing three times for
5 min each with 0.1% Tween 20 in TBS, the blots were
incubated for 1 h at room temperature with 1 : 2000 dilution
of sheep anti-mouse or donkey anti-rabbit secondary
antibodies conjugated to horseradish peroxidase. Bands were
visualized by an enhanced chemiluminescence (ECL) detec-
tion system.
Methods
Materials
1
25
I-Iothalamate, speci®c PGE
2
EIA and the ECL system
were from Amersham Pharmacia (Buckinghamshire, U.K.).
PAH was obtained from Merck & Co (West Point, PA,
U.S.A.). Sep-Pack C18 cartridges and HPLC columns were
from Waters (Milford, MA, U.S.A.). Furosemide was from
1
Hoechst Marion Roussel (Seguril , Barcelona, Spain). MBF
was from Strecks Laboratories (Omaha, NE, U.S.A.).
Immobilon-P PVDF membranes were from Millipore (Bed-
ford, MA, U.S.A.). Tween 80, aprotinin, leupeptin, pepstatin
A and phenylmethylsulphonyl ¯uoride were purchased from
Sigma Chemical Co (St Louis, MO, U.S.A.). Ketorolac,
COX-1 and COX-2 protein standards, COX-1 (murine)
monoclonal antibody and COX-2 (murine) polyclonal anti-
body (Western blot) were from Cayman Chemical Company
Immunohistochemical analysis of COX-1 and COX-2
protein distribution in renal tissue
(
Ann Arbor, MI, U.S.A.). Polyclonal rabbit anti-human
COX-2 serum and anti-ovine COX-1 monoclonal antibody
were from Oxford Biomedical Research (Oxford, MI,
U.S.A.). Selective COX-1 (SC-560) and COX-2 (celecoxib)
Localization of COX-1 and COX-2 expression was assessed
in renal tissue isolated from three cirrhotic rats with ascites
British Journal of Pharmacology vol 135 (4)

M. L o pez-Parra et al
Selective cyclooxygenase inhibition in cirrhosis
893
7
1
and three control rats. Animals were anaesthetized with an
(n=10): Selective COX-1 inhibitor, SC-560 (20 mg kg , i.v.);
Group (n=10): Selective COX-2 inhibitor, celecoxib
71
i.m. injection of ketamine (50 mg kg ) and sacri®ced. The
kidneys were rapidly resected and cut to the appropriate size
to facilitate ecient ®xative penetration of the molecular
biology ®xative (MBF). MBF is an aqueous bu€ered solution
containing organic and inorganic salts, supplemented with a
bacteriostatic and fungistatic agent and provides excellent
preservation of tissue structure and COX antigenicity. Tissues
were ®xed for 24 h at 48C, transferred to 70% ethanol,
embedded in paran, sectioned at 5 mm onto Fisher Gold
SuperFrost Plus glass slides and ®nally, deparanized in
xylene and re-hydrated in descending alcohols. Tissues were
3
(20 mg kg , i.v.).
71
The dose of SC-560 was selected from previous studies
demonstrating inhibition of constitutive PG synthesis in
euvolemic rats (Smith et al., 1998; Wallace et al., 2000;
Gretzer et al., 2001). The dose of celecoxib was selected from
previous studies reporting complete inhibition of PG
synthesis in the rat carrageenan footpad and airpouch models
of in¯ammation (Smith et al., 1998; Wallace et al., 2000).
Moreover, by using the zymosan-induced in¯ammation
model in rats, Niederberger et al. (2001) have recently
reported that celecoxib retains its anti-in¯ammatory ecacy
then blocked for endogenous peroxidase (3% H O2 in
2
71
MeOH) and avidin/biotin (Avidin Biotin Blocking Kit SP-
at doses of up to 100 mg kg .
2
001, Vector Laboratories, Burlingame, CA, U.S.A.). Sec-
tions were permeabilized in TNB-BB (0.1 M Tris pH 7.5,
.015 M NaCl, 0.5% blocking agent, 0.3% Triton-X, 0.2%
saponin), and incubated overnight at 48C with polyclonal
Subsequently, four 20-min urine collection periods were
performed and changes in MAP, HR, urine volume (V),
urinary sodium excretion (UNaV), glomerular ®ltration rate
(GFR) and renal plasma ¯ow (RPF) were recorded during
the entire period. At the midpoint of each clearance period, a
0
71
rabbit anti-human COX-2 serum diluted to 2.5 mg ml
Oxford Biomedical Research). COX-1 was immunolocalized
with the anti-ovine monoclonal antibody (diluted to
(
0.5 ml blood sample was obtained. Urinary PGE excretion
2
(UPGE2V) was measured under baseline conditions and
during the fourth 20-min urine collection period following
the administration of COX inhibitors.
71
1
mg ml ) (Oxford Biomedical Research). Immunoreactive
complexes were detected using tyramide signal ampli®cation
TSA-indirect, NEN Life Science), and were visualized with
(
Sodium and potassium concentrations in plasma and urine
samples were measured by ¯ame photometry (IL 943,
Instrumentation Laboratory, Lexington, MA, U.S.A.) and
serum osmolality by the osmometric depression of the
freezing point in an Advanced Instruments Osmometer
(model 3MO, Needham, MA, U.S.A.). GFR and RPF were
the peroxidase substrates AEC or DAB. Control sections
were treated with isotype-matched controls, or were pre-
incubated with 100 fold excess of human recombinant COX-1
or COX-2 protein. To ensure rigid inter-slide consistency all
slides were stained simultaneously on an autoimmunostainer.
Pathological changes were described by a licensed pathologist
following preparation of standard hematoxylin and eosin
staining.
1
25
estimated from I-Iothalamate and PAH clearance respec-
125
tively, using the standard formula: CIothalamate=urinary
I
1
25
counts6V/plasma
I counts and CPAH=urinary PAH
concentration6V/plasma PAH concentration. The radio-
1
25
Drug administration studies
activity of
I-Iothalamate samples was counted in an
automatic scintillation counter (Wallac, Turku, Finland).
PAH was determined by RP ± HPLC as described (Decosterd
et al., 1997) with modi®cations. Brie¯y, 2 ml of plasma or
urine in MeOH (®nal volume 25 ml) were injected into a RP ±
HPLC system which consisted of a Waters integrated system
controller (model 600E) equipped with a 996 Photodiode
Array Detector (DAD) and Millennium HPLC analysis
software (Waters, Milford, MA, U.S.A.). For analysis of
PAH, a HPLC column (either a Kromasil 100 C18 (5 mm,
Study 1 The goal of this study was to investigate the e€ects
of selective COX-1 (SC-560) and COX-2 (celecoxib)
inhibitors on renal function in conscious rats with cirrhosis
and ascites. For this purpose, 28 rats with cirrhosis and
ascites were anaesthetized with an i.m. injection of ketamine
71
(
50 mg kg ) and prepared with a PVC-50 catheter in the left
femoral artery for hemodynamic measurements and blood
collection, a double-lumen PVC-100 catheter in the right
jugular vein for the infusion of substances, and a PVC-50
tube in the bladder for urine collection. The femoral artery
catheter was connected to a highly sensitive transducer and a
multichannel recorder (MX4P and MT4, Lectromed Ltd,
Jersey, Channel Islands, U.K.) to register pulsatile mean
arterial pressure (MAP) and heart rate (HR), while the
jugular vein catheter was continuously perfused with Ringer
4.66250 mm) or
a
Tracer Extrasil ODS2 (5 mm,
4.66250 mm) was eluted with a linear gradient of MeOH:ci-
71
trate-citric acid pH 3.9 (from 1 : 99 to 15 : 85, v v
over
15 min) at a ¯ow rate of 1.0 ml min . The spectrophoto-
metric UV-VIS DAD was set at 273 nm. PGE levels in urine
71
2
were measured by speci®c EIA after extraction of samples on
Sep-Pack C18 cartridges.
71
solution (0.5 ml h ). Catheters were connected to a swivel
Harvard Apparatus, South Natick, MA, U.S.A.) and
animals were placed in rectangular cages with no restriction
(
Study 2 The aim of this study was to investigate the e€ect
of SC-560 and celecoxib on the renal diuretic and natriuretic
responses to furosemide in rats with cirrhosis and ascites.
Twenty-six rats with cirrhosis and ascites were prepared as
described in Study 1 and received placebo (n=6), the selective
1
25
of movement. Twenty-four hours later, a priming dose of I-
Iothalamate (0.37 mCi) and para-aminohippurate (PAH)
(
constant infusion (2 ml h ) of a Ringer solution containing
3 mg) was given through the jugular vein, followed by a
71
71
COX-1 inhibitor SC-560 (20 mg kg , i.v.) (n=10) or the
71
1
25
71
71
I-Iothalamate (0.37 mCi ml
)
Animals were equilibrated for 1 h and after two baseline
and PAH (3 mg ml ).
selective COX-2 inhibitor celecoxib (20 mg kg
,
i.v.)
(n=10). Sixty minutes later, they received an i.v. injection
71
71
2
Tween 80 in Dulbecco's Phosphate Bu€ered Saline (DPBS)
0-min urine collection periods received 1 ml kg
of 2%
of furosemide (5 mg kg ) and changes in V, UNaV, GFR
and RPF were recorded during four 20-min urine collection
periods. In an additional group of 13 rats with cirrhosis and
containing the following: Group 1 (n=8): Placebo; Group 2
British Journal of Pharmacology vol 135 (4)

894
M. L o pez-Parra et al
Selective cyclooxygenase inhibition in cirrhosis
ascites, the e€ects of increasing doses of SC-560 (10 and
71
71
3
0 mg kg , i.v) and celecoxib (10 and 30 mg kg , i.v,) on
the diuretic and natriuretic response to furosemide were also
evaluated.
Study 3 The aim of this study was to investigate the e€ect of
SC-560 and celecoxib on renal water metabolism in rats with
cirrhosis and ascites. Renal water metabolism was estimated in
cirrhotic rats resting in metabolic cages, as follows: two hours
71
after removing water and food, a water load (50 ml kg ) was
administered via a gastric tube inserted under light ether
anaesthesia. Immediately afterwards, the animals were re-
introduced into their metabolic cages where each spontaneous
urine void was individually collected for a total period of 3 h.
The renal ability of these animals to excrete free water was
established by measuring the minimum urinary osmolality
(
mUOsm) from the osmometric depression of the freezing
Figure 1 Renal COX-1 and COX-2 protein levels. Upper panels:
representative Western blot analysis of COX-1 (M
COX-2 (M , 72 kDa) in renal tissue samples from two control rats
CT1 and CT2) and two cirrhotic rats with ascites (CH1 and CH2).
r
, 70 kDa) and
point of each spontaneously voided sample and by gravime-
tically measuring the percentage (%) of the water load
excreted during the 3-h urine collection period. After a 24-h
period, animals received 1 ml kg of 2% Tween 80 in 0.5%
carboxymethylcellulose containing the following treatments
and doses selected from Smith et al. (1998), Wallace et al.
r
(
Renal protein extracts were electrophoresed and probed with speci®c
anti-COX-1 and anti-COX-2 antibodies. Lower panels: COX-1 and
COX-2 band intensities were determined by scanning densitometry.
Results show the mean+s.e.m. of six cirrhotic and seven control rats.
71
(2000), Gretzer et al. (2001) and Niederberger et al. (2001).
Group 1 (n=6): Placebo; Group 2 (n=7): SC-560 (30 mg
7
1
71
kg , p.o.); Group 3 (n=8): celecoxib (30 mg kg , p.o.).
Twenty minutes after receiving the drug, the animals were
vasculature, and papillary interstitial cells, being its expres-
sion intense in the papillary collecting ducts and low-to-
moderate in the cortical collecting ducts (Figure 2). On the
other hand, positive COX-2 immunoreactive protein was
localized in the macula densa and outer medulla collecting
ducts (Figure 3).
7
1
submitted to a second oral water load of 50 ml kg and the
percentage of water load excreted and the mUOsm were
again determined during the 3-h period following the water
administration (see above).
At the end of each study, tissue specimens were obtained
from the middle liver lobe of each animal, ®xed in 10%
bu€ered formalin and stained with hematoxylin-eosin,
reticulin and Masson's trichrome for histological examina-
tion.
Study 1: Effect of selective COX inhibitors on renal
hemodynamics and renal excretory function in rats with
cirrhosis and ascites
Statistical analysis of the results was performed by one-way
ANOVA, Newman-Keuls and paired and unpaired Student's
t-tests, as appropriate. Data are expressed as mean+s.e.m.
and were considered signi®cant at a P level of 0.05 or less.
All animal studies were conducted in accordance with the
criteria of the Investigation and Ethics Committee of the
No statistically signi®cant di€erences were detected in the
three groups of rats receiving placebo, SC-560 or celecoxib
with respect to baseline body weight, MAP, HR, serum
sodium and potassium concentrations, serum osmolality, V,
U
NaV, UPGE2V, GFR and RPF (Table 1).
Figure 4 shows the e€ects of placebo, SC-560 or celecoxib
Â
Hospital Clõnic and the European Community laws govern-
ing the use of experimental animals.
on V, UNaV, GFR and RPF in cirrhotic rats with ascites.
The i.v. administration of the selective COX-1 inhibitor SC-
5
60 signi®cantly decreased V, UNaV, GFR and RPF in
cirrhotic rats. In these animals, renal function was impaired
as early as 20 min following SC-560 administration and
experienced a progressive decline throughout the entire study
period, except for UNaV which reached a maximum plateau
at 40 min after the administration of the drug (Figure 4). In
contrast, the renal e€ects associated with selective COX-2
inhibition with celecoxib were similar to those of placebo
except that celecoxib induced a reduction in V which reached
statistical signi®cance at 60 min after administration of the
drug (Figure 4). This e€ect, however, was transient and
rapidly reversed and after 80 min of receiving celecoxib, the
V values in this group of animals did not di€er from those of
the placebo group. No changes in MAP and HR were
observed throughout the entire course of the study in any
group of cirrhotic animals (data not shown).
Results
Animals treated with CCl showed the characteristic features
4
of micronodular cirrhosis (i.e. a stage of well-established
cirrhosis with a combination of nodular regeneration of liver
cell plates surrounded by thick connective tissue septa and
proliferating bile ducts). The volume of ascites of cirrhotic
rats ranged between 12 and 40 ml.
Constitutive COX-1 and COX-2 protein expression was
detected by Western blot and immunohistochemistry analysis
in renal tissue collected from rats with cirrhosis and ascites
(Figures 1 ± 3). However, whereas COX-1 protein expression
was found to be unchanged as compared to controls, COX-2
expression was consistently up-regulated in kidneys from
cirrhotic rats (Figure 1). In these animals, COX-1 immunor-
eactivity was observed in the collecting ducts, renal
The administration of SC-560 and celecoxib to cirrhotic
a 48% (from 312+82 to
rats was associated with
British Journal of Pharmacology vol 135 (4)

M. L o pez-Parra et al
Selective cyclooxygenase inhibition in cirrhosis
895
Figure 2 Localization of COX-1 protein expression in control rats (A) and rats with cirrhosis and ascites (B). COX-1 is observed
in the collecting ducts, renal vasculature, and papillary interstitial cells. Intense COX-1 immunoreactivity is detected in the papillary
collecting ducts, and low-to-moderate expression detected in the cortical collecting ducts.
A
B
C
D
Figure 3 Localization of immunoreactive COX-2 protein expression in kidneys from rats with cirrhosis and ascites. Low-
magni®cation micrograph showing a longitudinal section of the kidney stained with hematoxylin-eosin (A) or reacted with a
polyclonal rabbit antimurine COX-2 (B). In the cortex, COX-2 staining was observed in the macula densa (C), whereas in the
medulla, strong immunoreactivity for COX-2 was localized in tubular epithelial cells (D).
7
1
1
2
63+54 pg min , P50.05) and 47% (from 389+93 to
e€ect was rapid (reached a maximum after 20 min) and
transient (disappeared in most cases 60 ± 80 min after the
administration of furosemide). Parallel changes in GFR and
RPF were also observed in this group of animals following
the administration of furosemide, although the duration of
7
1
03+111 pg min ), inhibition in UPGE2V, respectively.
Study 2: Effect of selective COX inhibitors on the renal
response to furosemide in rats with cirrhosis and ascites
these changes was shorter than that elicited on V and UNa
V
Figure 5 shows the results obtained in the three groups of
animals included in this study. As expected, in cirrhotic rats
(data not shown). The injection of furosemide to cirrhotic
rats treated with the selective COX-1 inhibitor SC-560 was
associated with reduced diuretic and natriuretic response to
this drug (Figure 5). In sharp contrast, the diuretic and
natriuretic ecacy of furosemide in cirrhotic rats was not
71
receiving placebo, the i.v. injection of 5 mg kg
furosemide was followed by a pronounced increase in V
diuretic e€ect) and UNaV (natriuretic e€ect) (Figure 5). This
of
(
British Journal of Pharmacology vol 135 (4)

896
M. L o pez-Parra et al
Selective cyclooxygenase inhibition in cirrhosis
Table 1 Baseline values for the three groups of cirrhotic rats receiving placebo, SC-560 or celecoxib included in Study 1.
Placebo (n=8)
SC-560 (n=10)
Celecoxib (n=10)
Body weight (g)
MAP (mmHg)
595+28
90+7
439+2
138+1
3.9+0.2
294+2
31+5
1.9+0.2
212+92
3.6+0.5
13.7+1.6
569+21
91+6
436+17
130+7
3.7+0.2
292+3
28+4
1.8+0.3
312+82
3.3+0.3
12.9+2.1
569+30
91+6
401+13
133+3
3.8+0.2
290+2
28+4
1.8+0.3
389+93
3.2+0.2
14.2+1.7
7
1
HR (beats min
Serum sodium (mEq l
Serum potassium (mEq l
Serum osmolality (mOsm kg
)
71
)
7
1
)
71
)
71
V (ml min
)
7
1
U
NaV (mEq min
)
7
1
U
GFR (ml min
PGE2V (pg min
)
71
)
RPF (ml min7
1
)
Figure 4 E€ect of selective COX inhibitors on renal function in rats
with cirrhosis and ascites. Urine ¯ow (V), urinary sodium excretion
(
UNaV), glomerular ®ltration rate (GFR) and renal plasma ¯ow
(
RPF) were measured before (720 and 0 min) and up to 80 min after
the i.v. administration of placebo (n=8), the selective COX-1
7
1
inhibitor (SC-560, 20 mg kg
, n=10) or the selective COX-2
1
7
inhibitor (celecoxib, 20 mg kg , n=10) to rats with cirrhosis and
ascites. Results are given as mean+s.e.m. Data are compared with
the Student's t-test for paired data. *P50.05; **P50.025 and
*
**P50.001 vs values at 0 min.
a€ected by celecoxib (Figure 5). Consequently, rats treated
with SC-560 showed a pronounced reduction in overall urine
output and cumulative sodium excretion over the 80 min
period after furosemide injection, whereas these parameters
remained unaltered in those rats treated with celecoxib
Figure 5 E€ect of selective COX inhibitors on the renal response to
furosemide in rats with cirrhosis and ascites. Animals received i.v.
71
placebo (n=6), the selective COX-1 inhibitor (SC-560, 20 mg kg
n=10) or the selective COX-2 inhibitor (celecoxib, 20 mg kg
,
,
71
(Figure 6). The decrease in the response to furosemide in
rats receiving SC-560 was dose-dependent and reached
n=10) and 1 h later they were challenged with an i.v. injection of
furosemide (5 mg kg7 ) (time 0 min). Changes in urine ¯ow (V) and
urinary sodium excretion (UNaV) were recorded for four 20-min
urine collection periods after the injection of furosemide. Results are
given as mean+s.e.m. Data are compared by the Student's t-test for
unpaired data. *P50.05; **P50.025 and ***P50.005 vs placebo-
treated group.
1
71
statistical signi®cance at doses of 20 and 30 mg kg (Figure
).
6
Study 3: Effects of COX inhibitors on renal water
metabolism in rats with cirrhosis and ascites.
Under baseline conditions, all three treatment groups exhibited
similar renal ability to excrete free water, as estimated by the
percentage of water load excreted (placebo: 82+11%; SC-560:
of water load excreted and mUOsm under baseline conditions
and following the oral administration of the drugs to rats
with cirrhosis and ascites. In these animals, neither placebo,
SC-560 or celecoxib produced any signi®cant change in
either the percentage of water load excreted or mUOsm
(Figure 7).
9
2+6% and celecoxib: 86+7%) and mUOsm (Placebo:
71
71
1
03+15 mOsm kg ; SC-560: 69+5 mOsm kg and celecox-
71
ib: 88+17 mOsm kg ). Figure 7 shows the percentage
British Journal of Pharmacology vol 135 (4)

M. L o pez-Parra et al
Selective cyclooxygenase inhibition in cirrhosis
897
Figure 6 Diuretic and natriuretic responses to furosemide in rats with cirrhosis and ascites treated with increasing doses of
selective COX inhibitors. Graph bars show the overall urine output and the cumulative sodium excretion over the 80 min after the
administration of furosemide (5 mg kg ) to di€erent groups of rats treated with placebo (0 mg kg ), SC-560 (10, 20 or
71
71
71
71
3
unpaired data and P values denote statistically signi®cant di€erences with respect to the placebo-treated group.
0 mg kg ) or celecoxib (10, 20 or 30 mg kg ). Results are given as mean+s.e.m. Data are compared by the Student's t-test for
Discussion
Renal PG synthesis is markedly increased in patients with
cirrhosis and ascites as compared to patients with compen-
sated cirrhosis (no ascites) and normal subjects (Arroyo et al.,
1
986; Dunn, 1984; Arroyo et al., 1983; Planas et al., 1983). In
compensated cirrhosis, this increase is a homeostatic response
to antagonize the exacerbated activity of the endogenous
vasoconstrictor systems (i.e. the renin-angiotensin system, the
sympathetic nervous system and the antidiuretic hormone
(
ADH)). Two lines of evidence support this contention.
Firstly, acute PG inhibition with NSAIDs (indomethacin,
ibuprofen, naproxen, sulindac or aspirin) in patients with
cirrhosis and ascites is associated with a signi®cant decrease
in V, UNaV, GFR and RPF (Dunn, 1984; Arroyo et al.,
1983; Boyer et al., 1979; Mirouze et al., 1983; Zipser et al.,
1979). Patients with high plasma renin activity and plasma
norepinephrine concentrations are particularly sensitive to
these adverse e€ects. NSAIDs do not impair renal function in
patients with compensated cirrhosis who do not show
increased activity of the renin-angiotensin and sympathetic
nervous systems. Secondly, acute PG inhibition with aspirin
in patients with cirrhosis, ascites and increased plasma ADH
levels is associated with a signi®cant reduction in the renal
ability to excrete free water (Perez-Ayuso et al., 1984). In
Â
addition, PGs are also important in the renal response to
diuretics since the administration of aspirin, naproxen,
indomethacin and sulindac suppresses the renal hemody-
namic e€ect and reduces the natriuretic ecacy of furosemide
in cirrhotic patients with ascites (Planas et al., 1983).
Although the role of PGs in the maintenance of renal
function in cirrhosis is well established, at present it is
unknown which COX isoform (COX-1 or COX-2) is
responsible for the synthesis of PGs involved in renal
homeostasis in this disease. Under normal conditions, both
Figure 7 E€ects of selective COX inhibitors on renal water
metabolism in rats with cirrhosis and ascites. The percentage (%)
water load excreted (A) and the mUOsm (B) were measured before
71
and after the administration of placebo (n=6), SC-560 (30 mg kg
,
71
n=7) or celecoxib (30 mg kg , n=8) to cirrhotic rats with ascites.
Results represent the mean+s.e.m. and data are compared by the
Student's t-test for paired data.
British Journal of Pharmacology vol 135 (4)

898
M. L o pez-Parra et al
Selective cyclooxygenase inhibition in cirrhosis
COX-1 and COX-2 are constitutively expressed in the
kidneys (O'Neill & Ford-Hutchinson, 1993; Harris et al.,
and that selective COX-2 inhibition spares furosemide-
induced renal salt transport in cirrhosis. It is noteworthy to
point out that in cirrhotic rats the e€ects of selective COX-1
inhibition with SC-560 on furosemide-induced diuretic and
natriuretic response were dose-dependent. However, contrary
to what was previously reported by Katayama et al. (1984),
who found that indomethacin can either enhance or inhibit
the e€ect of furosemide-induced natriuresis depending on the
dose of this conventional NSAID used, we found that at low
1
1
994; Vio et al., 1997; Jensen & Kurtz, 1997; Khan et al.,
998; Bosch-Marce et al., 1999). Speci®cally, in normal rats,
Â
COX-1 is mainly expressed in cells of the collecting duct and
renal vasculature and in a small number of papillary
interstitial cells, while COX-2 expression is consistently focal
and limited to the macula densa of the juxtaglomerular
apparatus, epithelial cells of the thick ascending limb and
papillary interstitial cells (Khan et al., 1998; Vio et al., 1997;
Harris et al., 1994; Jensen & Kurtz, 1997). Our results show
that both COX-1 and COX-2 are also present in kidneys
from rats with cirrhosis and ascites. Moreover, whereas the
expression of COX-1 was found to be unchanged, the
expression of COX-2 was up-regulated in kidneys of cirrhotic
rats. These ®ndings are consistent with the notion that COX-
71
doses (10 mg kg ), SC-560 induced no changes whereas
71
higher doses (20 and 30 mg kg ) signi®cantly inhibited the
response to furosemide.
PGs are also involved in renal water transport by
modulating the tubular actions of ADH (Mattix & Badr,
2000). Speci®cally, PGE
ADH whereas PG inhibition with NSAIDs enhances the
tubular e€ect of this hormone (Mattix & Badr, 2000; Hebert et
2
inhibits the hydroosmotic e€ect of
2
, but not COX-1, is regulated in a cell-speci®c fashion in
Â
response to altered volume status. Indeed, increased COX-2
expression has been reported in the macula densa and peri-
macula densa region of rats and dogs with chronic salt
depletion and in the renal medulla of rats with a high-salt
diet (Khan et al., 1998; Harris et al., 1994; Jensen & Kurtz,
al., 1990). In our study, neither SC-560 nor celecoxib modi®ed
renal water metabolism in rats with cirrhosis and ascites.
These results are in sharp contrast with those previously
obtained with the NSAID ketorolac which signi®cantly
impaired the ability to excrete free water in cirrhotic rats
1
997; Yang et al., 1998). Furthermore, increased COX-2
(Bosch-Marce et al., 1999). Given that ketorolac is a mixed
Â
expression has been reported in the renal medulla of water-
deprived rats (Yang et al., 1999). Collectively, these results
suggest that renal COX-2 up-regulation in cirrhosis is likely
to be a consequence of changes in volume status, although
the exact mechanism for this phenomenon is, at present, not
understood.
COX-1/COX-2 inhibitor, these ®ndings suggest that renal
water homeostasis in cirrhosis is dependent on PGs derived
from both COX isoforms. Thus, a compensatory mechanism
appears to be present in cirrhotic rat kidneys, in such a way
that when only one isoform is inhibited, water metabolism can
be maintained by PGs produced by the other isoform.
Despite cirrhotic rats showing increased COX-2 protein
immunoreactivity in the renal area, renal function in these
animals appears to be mainly dependent on PGs derived from
COX-1. In fact, in Study 1 of this investigation, we observed a
Controlled clinical studies have demonstrated that selective
COX-2 inhibitors have equal therapeutic ecacy but with a
lower incidence of gastrointestinal ulcers and erosions than
conventional NSAIDs (Emery et al., 1999; Bombardier et al.,
2000). The marketed selective COX-2 inhibitors rofecoxib
and celecoxib have also been evaluated in randomized
controlled trials in terms of their e€ects on renal PGs, renal
function and occurrence of adverse renal events. Whereas in
elderly subjects, rofecoxib did not produce any signi®cant
alteration in GFR after 2 weeks of treatment, it produced a
reduction in urinary sodium excretion similar to that with
indomethacin during the ®rst 3 days of therapy (Catella-
Lawson et al., 1999). In contrast, in elderly patients with mild
renal impairment and stabilized on a low-sodium diet,
rofecoxib produced decreases in GFR of similar magnitude
to those observed in indomethacin-treated subjects, without
a€ecting urinary sodium excretion (Swan et al., 2000). In
elderly patients, celecoxib, like rofecoxib, failed to decrease
GFR, but did produce transient reductions in urinary sodium
excretion similar to those of naproxen (Whelton et al., 2000).
Finally, in a trial in salt-depleted subjects, celecoxib not only
promoted sodium retention but also produced signi®cant
dose-related decreases in GFR (Rossat et al., 1999). There-
fore, the unwanted renal-side e€ects of selective COX-2
inhibitors still remain, at present, uncharacterized. This
subject is particularly important in circumstances more
susceptible to NSAID-induced renal failure, such as advanced
liver disease, congestive heart failure and nephrotic syn-
drome. For this reason, our ®ndings in the experimental
model of cirrhosis warrant further studies in humans
investigating the feasibility of selective COX-2 inhibitors
possibly being the anti-in¯ammatory option of choice in
patients with chronic liver disease.
2
signi®cant inhibition in renal PGE synthesis concomitant with
marked impairment in renal haemodynamics and renal sodium
handling following the administration of a selective COX-1
4
inhibitor (SC-560) to CCl -induced cirrhotic rats. In contrast,
the selective COX-2 inhibitor celecoxib did not compromise
renal function in cirrhotic rats. Interestingly, and although
celecoxib administration was not associated with severe renal
e€ects in cirrhotic rats, this compound reduced UPGE2V to
levels equivalent to SC-560. McAdam et al. (1999) also found a
similar suppression in urinary PG excretion in both healthy
subjects treated with celecoxib and in those receiving ibuprofen.
Taken together, these results suggest that urinary PGs are
derived from both COX-1 and COX-2, yet only those from
COX-1 are involved in the maintenance of renal function in
cirrhosis. The physiological signi®cance of PGs derived from
COX-2 in the kidneys, not only in disease states, but also in
healthy conditions, remains to be determined.
PGs are clearly involved in the renal natriuretic response to
loop diuretics (Katayama et al., 1984). Loop diuretics, such
as furosemide, are the most powerful diuretics currently
available. These drugs inhibit sodium reabsorption in the
ascending limb of the loop of Henle by acting on a speci®c
+
7
+
co-transport system, the Na -2Cl K carrier (Puschett,
981). Loop diuretics also increase GFR and renal blood
ow and the renal production of PGE whereas NSAIDs can
1
¯
2
modulate the renal vasodilatory e€ect and the natriuretic
eciency of these drugs (Katayama et al., 1984). The results
obtained in Study 2 demonstrate that response to furosemide
in cirrhotic rats is mainly dependent on COX-1 derived PGs
British Journal of Pharmacology vol 135 (4)

M. L o pez-Parra et al
Selective cyclooxygenase inhibition in cirrhosis
Consumo (BEFI 98/9314), respectively. A. Planaguma
899
These studies were supported in part by Ministerio de Ciencia y
Á
received a
Tecnologõ
ias (FIS 00/0616). M. Lo
Ministerio de Ciencia y Tecnologõ
Â
a (SAF 00/0043) and Fondo de Investigaciones Sanitar-
pez-Parra and E. Titos were supported by
a and Ministerio de Sanidad y
fellowship from IDIBAPS. The authors are indebted to M. Borrajo
for expert technical assistance.
Â
Â
References
ARROYO, V., PLANAS, R., GAYA, J., DEULOFEU, R., RIMOLA, A.,
Â
JENSEN, B.L. & KURTZ, A. (1997). Di€erential regulation of renal
cyclooxygenase mRNA by dietary salt intake. Kidney Int., 52,
1242 ± 1249.
Â
PEREZ-AYUSO, R.M., RIVERA, F. & RODES, J. (1983). Sympa-
thetic nervous activity, renin-angiotensin system and renal
excretion of prostaglandin E
functional renal failure and sodium and water excretion. Eur. J.
2
in cirrhosis. Relationship to
KATAYAMA, S., ATTALLAH, A.A., STAHL, R.A., BLOCH, D.L. & LEE,
J.B. (1984). Mechanism of furosemide-induced natriuresis by
Clin. Invest., 13, 271 ± 278.
Â
direct stimulation of renal prostaglandin E
F555 ± F561.
2
. Am. J. Physiol., 247,
ARROYO, V., GINES, P., RIMOLA, A. & GAYA, J. (1986). Renal
function abnormalities, prostaglandins, and e€ects of nonster-
oidal anti-in¯ammatory drugs in cirrhosis with ascites. An
overview with emphasis on pathogenesis. Am. J. Med., 81,
KHAN, K., VENTURINI, C.M., BUNH, R.T., BRASSARD, J.A., KOKI,
A.T., MORRIS, D.L., TRUMP, B.F., MAZIASZ, T.J. & ALDEN, C.L.
(1998). Interspecies di€erences in renal localization of cycloox-
ygenase isoforms: implications in nonsteroidal antiin¯ammatory
drug-related nephrotoxicity. Toxic. Pathol., 26, 612 ± 620.
104 ± 122.
BOMBARDIER, C., LAINE, L., REICIN, A., SHAPIRO, D., BURGOS-
VARGAS, R., DAVIS, B., DAY, R., FERRAZ, M.B., HAWKEY, C.J.,
KUJUBU, D.A., FLETCHER, B.S., VARNUM, B.C., LIM, R.W.
&
HOCHBERG, M.C., KVIEN, T.K.
Comparison of upper gastrointestinal toxicity of rofecoxib and
naproxen in patients with rheumatoid arthritis. N. Engl. J. Med.,
&
SCHNITZER, T.J. (2000).
HERSCHMAN, H.R. (1991). TIS10, phorbol ester tumor
a
promoter-inducible mRNA from Swiss 3T3 cells, encodes a
novel prostaglandin synthase/cyclooxygenase homologue. J.
Biol. Chem., 266, 12866 ± 12872.
3
BOSCH-MARCE, M., CLARIA, J., TITOS, E., MASFERRER, J.L.,
43, 1520 ± 1528.
Â
Á
MARNETT, L.J. & KALGUTKAR, A.S. (1999). Cyclooxygenase 2
inhibitors: discovery, selectivity and the future. TIPS, 20, 465 ±
469.
MASFERRER, J.L., SEIBERT, K., ZWEIFEL, B.S. & NEEDLEMAN, P.
(1992). Endogenous glucocorticoids regulate an inducible
cyclooxygenase enzyme. Proc. Natl. Acad. Sci. U.S.A., 89,
3917 ± 3921.
MATTIX, H.J. & BADR, K.F. (2000). Arachidonic acid metabolites
and the kidney, In The Kidney. ed. Brenner, B.M., Rector, F.C.
pp. 756 ± 792, Philadelphia: Saunders.
MCADAM, B.F., CATELLA-LAWSON, F., MARDINI, I.A., KAPOOR, S.,
LAWSON, J.A. & FITZGERALD, G.A. (1999). Systemic biosynth-
esis of prostacyclin by cyclooxygenase (COX)-2: the human
pharmacology of a selective inhibitor of COX-2. Proc. Natl.
Acad. Sci. U.S.A., 96, 272 ± 277.
Â
ALTUNA, R., POO, J.L., JIMENEZ, W., ARROYO, V., RIVERA, F.
Â
RODES, J. (1999). Selective inhibition of cyclooxygenase-2
&
spares renal function and prostaglandin synthesis in cirrhotic rats
with ascites. Gastroenterology, 116, 1167 ± 1175.
BOYER, T.D., ZIA, P.
indomethacin and prostaglandin A
plasma renin activity in alcoholic liver disease. Gastroenterology,
7, 215 ± 222.
&
REYNOLDS, T.B. (1979). E€ect of
1
on renal function and
7
CATELLA-LAWSON, F., MCADAM, B., MORRISON, B.W., KAPOOR,
S., KUJUBU, D., ANTES, L., LASSETER, K.C., QUAN, H., GERTZ,
B.J. & FILTZGERALD, G.A. (1999). E€ects of speci®c inhibition
on cyclooxygenase-2 on sodium balance, hemodynamics and
vasoactive eicosanoids. J. Pharm. Exp. Ther., 289, 735 ± 741.
Á
Â
CLARIA, J. & JIMENEZ, W. (1999). Renal dysfunction and ascites in
carbon tetrachloride-induced cirrhosis in rats, In The Liver and
MIROUZE, D., ZIPSER, R.D. & REYNOLDS, T.D. (1983). E€ects of
inhibitors of prostaglandin synthesis on induced diuresis in
cirrhosis. Hepatology, 3, 50 ± 55.
the Kidney. ed. Arroyo, V., Schrier, R.W., Rode
79 ± 396, Boston: Blackwell Science.
DECOSTERD, L.A., KARAGIANNIS, A., ROULET, J.M., BELAZ, N.,
APPENZELLER, M., BUCLIN, T., VOGEL, P. & BIOLLAZ, J. (1997).
High-performance liquid chromatography of the renal blood
s, J., Gines, P. pp.
 Á
3
Â
NIEDERBERGER, E., TEGEDER, I., VETTER, G., SCHMIDTKO, A.,
È
È
SCHMIDT, H., EUCHENHOFER, C., BRAUTIGAM, L., GROSCH, S.
& GEISSLINGER, G. (2001). Celecoxib loses its anti-in¯ammatory
ecacy at high doses through activation of NF-kB. FASEB J.,
15, 1622 ± 1624.
¯
ow marker p-aminohippuric acid (PAH) and its metabolite N-
acetyl PAH improves PAH clearance measurements. J. Chroma-
tog., 703, 25 ± 36.
O'NEILL, G.P. & FORD-HUTCHINSON, A.W. (1993). Expression of
mRNA for cyclooxygenase-1 and cyclooxygenase-2 in human
tissues. FEBS Lett., 330, 156 ± 160.
DUNN, M.J. (1984). Role of eicosanoids in the control of renal
function in severe hepatic disease. Gastroenterology, 87, 1392 ±
1
395.
PAYAN, D.G. & KATZUNG, B.G. (1995). Nonsteroidal anti-in¯am-
matory drugs; nonopioid analgesics; drugs used in gout, In Basic
& Clinical Pharmacology. ed. Katzung, B.G. pp. 536 ± 559
Norwalk: Appleton & Lange.
EMERY, P., ZEIDLER, H., KVIEN, T.K., GUSLANDI, M., NAUDIN, R.,
STEAD, H., VERBURG, K.M., ISAKSON, P.C., HUBBARD, R.C. &
GEIS, G.S. (1999). Celecoxib versus diclofenac in long-term
management of rheumatoid arthritis: randomised double-blind
comparison. Lancet, 354, 2106 ± 2111.
GRETZER, B., MARICIC, N., RESPONDEK, M., SCHULIGOI, R. &
PESKAR, B.M. (2001). E€ects of speci®c inhibition of cyclo-
oxygenase-1 and cyclo-oxygenase-2 in the rat stomach with
normal mucosa and after acid challenge. Br. J. Pharmacol., 132,
Â
PEREZ-AYUSO, R.M., ARROYO, V., CAMPS, J., RIMOLA, A., GAYA,
Â
J., COSTA, J., RIVERA, F. & RODES, J. (1984). Evidence that renal
prostaglandins are involved in renal water metabolism in
cirrhosis. Kidney Int., 26, 72 ± 80.
PLANAS, R., ARROYO, V., RIMOLA, A., PEREZ-AYUSO, R.M. &
Â
Â
RODES, J. (1983). Acetylsalicylic acid suppresses the renal
1
565 ± 1573.
hemodynamic e€ect and reduces the diuretic action of furosemide
in cirrhosis with ascites. Gastroenterology, 84, 247 ± 252.
PUSCHETT, J.B. (1981). Sites and mechanisms of diuretics in the
kidney. J. Clin. Pharmacol., 21, 564 ± 574.
HARRIS, R.C., MCKANNA, J.A., AKAI, Y., JACOBSON, H.R., DUBOIS,
R.N. & BREYER, M.D. (1994). Cyclooxygenase-2 is associated
with the macula densa of rat kidney and increases with salt
restriction. J. Clin. Invest, 94, 2504 ± 2510.
HEBERT, R.L., JACOBSON, H.R. & BREYER, M.D. (1990). PGE
Á
Â
Â
ROS, J., CLARIA, J., JIMENEZ, W., BOSCH-MARCE, M., ANGELI, P.,
Â
Â
2
ARROYO, V., RIVERA, F. & RODES, J. (1995). Role of nitric oxide
inhibits AVP-induced water ¯ow in cortical collecting ducts by
protein kinase C activation. Am. J. Physiol., 259, F318 ± F325.
HERSCHMAN, H.R. (1996). Prostaglandin synthase 2. Biochim.
Biophys. Acta., 1299, 125 ± 140.
HLA, T. & NEILSON, K. (1992). Human cyclooxygenase-2 cDNA.
Proc. Natl. Acad. Sci. U.S.A., 89, 7384 ± 7388.
and prostacyclin in the control of renal perfusion in experimental
cirrhosis. Hepatology, 22, 915 ± 920.
ROSSAT, J., MAILLARD, M., NUSSBERGER, J., BRUNNER, H.R. &
BURNIER, M. (1999). Renal e€ects of selective cyclooxygenase-2
inhibition in normotensive salt-depleted subjects. Clin. Pharma-
col. Ther., 66, 76 ± 84.
British Journal of Pharmacology vol 135 (4)

900
M. L o pez-Parra et al
Selective cyclooxygenase inhibition in cirrhosis
SWAN, S., RUDY, D.W., LASSETER, K.C., RYAN, C.F., BUECHEL,
K.L., LAMBRECHT, L.J., PINTO, M.B., DILZER, S.C., OBRDA, O.,
SUNDBLAD, K.J., GUMBS, C.P., EBEL, D.L., QUAN, H., LARSON,
P.J., SCHWARTZ, J.I., MUSLINER, T.A., GERTZ, B.J., BRATER, C.
WARNER, T.D., GIULIANO, F., VOJNOVIC, I., BUKASA, A., MITCH-
ELL, J.A. & VANE, J.R. (1999). Nonsteroid drug selectivities for
cyclooxygenase-1 rather than cyclooxygenase-2 are associated
with human gastrointestinal toxicity: a full in vitro analysis. Proc.
Natl. Acad. Sci. U.S.A., 96, 7563 ± 7568.
&
YAO, S.-L. (2000). E€ect of cyclooxygenase-2 inhibition on
renal function in elderly persons receiving a low-salt diet. Ann.
Intern. Med., 133, 1 ± 9.
SEIBERT, K., ZHANG, Y., LEAHY, K., HAUSER, S., MASFERRER, J.,
PERKINS, W., LEE, L. & ISAKSON, P. (1994). Pharmacological and
biochemical demonstration of the role of cyclooxygenase 2 in
in¯ammation and pain. Proc. Natl. Acad. Sci. U.S.A., 91, 12013 ±
WHELTON, A., SCHULMAN, G., WALLEMARK, C., DROWER, E.J.,
ISAKSON, P.C., VERBURG, K.M. & GEIS, G.S. (2000). E€ects of
celecoxib and naproxen on renal function in the elderly. Arch.
Intern. Med., 160, 1465 ± 1470.
YANG, T., SCHNERMANN, J.B. & BRIGGS, J.P. (1999). Regulation of
cyclooxygenase-2 expression in renal medulla by tonicity in vivo
and in vitro. Am. J. Physiol., 277, F1 ± F9.
1
2017.
SMITH, C.J., ZHANG, Y., KOBOLDT, C.M., MUHAMMAD, J.,
ZWEIFEL, B.S., SHAFFER, A., TALLEY, J.J., MASFERRER, J.L.,
SEIBERT, K. & ISAKSON, P.C. (1998). Pharmacological analysis of
cyclooxygenase-1 in in¯ammation. Proc. Natl. Acad. Sci. U.S.A.,
YANG, T., SINGH, I., PHAM, H., SUN, D., SMART, A., SCHNER-
MANN, J.B. & BRIGGS, J.P. (1998). Regulation of cyclooxygenase
expression by dietary salt intake. Am. J. Physiol., 274, F481 ±
F489.
9
VANE, J.R. (1971). Inhibition of prostaglandin synthesis as a
5, 13313 ± 13318.
ZIPSER, R., HOEFS, J.C., SPECKART, P.F., ZIA, P.K. & HORTON, R.
(1979). Prostaglandins: modulators of renal function and pressor
resistance in chronic liver disease. J. Clin. Endocrinol. Metab., 48,
895 ± 900.
mechanism of action for the aspirin-like drugs. Nature, 231,
2
32 ± 235.
VIO, C.P., CESPEDES, C., GALLARDO, P. & MASFERRER, J.L. (1997).
Renal identi®cation of cyclooxygenase-2 in a subset of thick
ascending limb cells. Hypertension, 30, 687 ± 692.
(Received November 1, 2001
Accepted November 23, 2001)
WALLACE, J.L., McKNIGHT, W., REUTER, B.K. & VERGNOLLE, N.
(
2000). NSAID-induced gastric damage in rats: requirement for
inhibition of both cyclooxygenase 1 and 2. Gastroenterology, 199,
06 ± 714.
7
British Journal of Pharmacology vol 135 (4)