Vol. 275, Issue 2, F216-F225, August 1998
Decreased vasopressin-mediated renal water reabsorption in
rats with compensated liver cirrhosis
Thomas E. N.
Jonassen1,
Søren
Nielsen2,
Sten
Christensen1, and
Jørgen
Søberg
Petersen1
1 Department of Pharmacology,
the Panum Institute, University of Copenhagen, DK-2200 Copenhagen
N; and 2 Department of Cell
Biology, Institute of Anatomy, University of Aarhus, DK-8000
Aarhus, Denmark
 |
ABSTRACT |
Experiments were performed to investigate vasopressin type 2 receptor (V2)-mediated renal
water reabsorption and the renal expression of the
vasopressin-regulated water channel aquaporin-2 (AQP-2) in
cirrhotic rats with sodium retention but without ascites. In addition,
the expression of the furosemide-sensitive type 1 Na-K-2Cl
cotransporter (BSC-1) and the natriuretic response to an
intravenous test dose furosemide (7.5 mg/kg) during acute
V2-receptor blockade was measured.
Acute V2-receptor blockade with
the selective nonpeptide antagonist OPC-31260 (800 µg · kg
1 · h
1)
was performed during conditions in which volume depletion was prevented
by computer-driven, servo-controlled intravenous volume replacement
with 150 mM glucose. OPC-31260 produced a significantly smaller
increase in urine flow rate (
26%) and free water clearance (
18%) in cirrhotic rats than in control rats. The natriuretic response to an intravenous test dose furosemide (7.5 mg/kg) was significantly increased in cirrhotic rats (+52%), but pretreatment with OPC-31260 did not affect the natriuretic response to furosemide in
neither cirrhotic nor in control rats. Semiquantitative immunoblotting showed a significant downregulation of AQP-2 in the renal cortex (
72%) and in the outer medulla (
44%). The relative
expression of BSC-1 in the outer medulla was unchanged in cirrhotic
rats. The corticopapillary gradient of Na was significantly increased in cirrhotic rats. Since daily urine flow rate was similar in cirrhotic
and sham-operated rats, we suggest that non-vasopressin-mediated water
reabsorption is increased in cirrhotic rats probably as a result of an
increased corticomedullary gradient due to exaggerated NaCl
reabsorption in the thick ascending limb of Henle's loop.
vasopressin V2 receptor; OPC-31260; aquaporin-2; thick ascending limb; furosemide; BSC-1
 |
INTRODUCTION |
LIVER CIRRHOSIS is a chronic disease with marked
progressive changes in systemic and renal hemodynamics. Initially,
patients with cirrhotic liver disease have peripheral vasodilation and increased cardiac output but without clinical signs of fluid retention (the compensated state). During the late decompensated state, liver
cirrhosis is associated with sodium retention, edema, and ascites. The
renal mechanisms that initiate sodium retention during the early
compensated stage of liver cirrhosis are still unknown. Experimental
studies suggest that the sodium retention that initiates edema and
ascites formation in cirrhosis begins 1-2 wk before ascites become
detectable (13, 19). The early sodium retention seems to be mediated by
an increased tubular NaCl reabsorption, since the glomerular filtration
rate (GFR) is unaltered at this stage of the disease (18, 41, 42). In
rats with secondary biliary cirrhosis induced by common bile duct
ligation (CBL), we recently reported that rats with sodium retention
but without ascites, i.e., compensated liver cirrhosis, had an
exaggerated natriuretic response to furosemide and an increased volume
of the thick ascending limb of Henle's loop (TAL) epithelium in the inner stripe of the outer medulla (14). These functional and structural
changes suggest that increased NaCl reabsorption in the TAL may be
involved in the early sodium retention observed during liver cirrhosis.
Furthermore, plasma vasopressin levels were unchanged in CBL rats, but
since both the functional and the structural changes in the TAL were
absent in CBL rats with hereditary vasopressin deficiency, these
findings suggested that vasopressin plays a permissive role for the
adaptive changes in the TAL in cirrhotic rats.
Vasopressin plays a central role in the kidneys concentration ability
by stimulation of water reabsorption in the collecting duct. In the
collecting duct principal cell, vasopressin binds to vasopressin type 2 receptors (V2 receptors) in the
basolateral plasma membrane and increases intracellular cAMP
concentration, which in turn increases the water permeability (26).
Several studies have demonstrated that the vasopressin-sensitive
aquaporin-2 (AQP-2) water channel plays a central role in the
regulation of collecting duct water permeability. AQP-2 is localized in
the luminal plasma membrane and in cytoplasmic vesicles (26). Acute increases in the plasma vasopressin concentration are associated with
insertion of AQP-2 from cytoplasmic vesicles into the luminal plasma
membrane (24, 25, 32, 44), whereas prolonged increases in plasma
vasopressin levels are associated with marked increases in AQP-2
expression (26).
In addition, in vitro studies on isolated segments of medullary TAL
from rats and mice have shown that vasopressin increases adenylate
cyclase activity and stimulates transepithelial sodium transport in
this nephron segment (34, 40). Like the effect of vasopressin on the
collecting duct, the effect of vasopressin on the medullary TAL is
mediated by V2 receptors (3).
These findings suggest that vasopressin not only increases the renal concentration ability by stimulation of collecting duct water permeability, but also by increasing the corticomedullary osmotic gradient through
V2-receptor-mediated stimulation
of NaCl reabsorption in the TAL.
In the present study,
V2-receptor-mediated water
reabsorption in the collecting ducts was examined in chronically
instrumented rats with compensated liver cirrhosis. Liver cirrhosis was
induced by CBL, and sham-operated rats (sham-CBL) were used as
controls. V2-receptor blockade was
induced by intravenous treatment with the selective
V2-receptor antagonist, OPC-31260.
V2-receptor blockade was achieved
in absence of changes in fluid balance, by use of a computer-driven,
servo-controlled intravenous volume replacement system, which replaced
urinary losses momentarily by intravenous infusion of 150 mM glucose.
In an additional group of animals with CBL or sham-CBL, the expression
of the vasopressin-sensitive water channel AQP-2 was determined in
cortex, outer, and inner medulla by semiquantitative immunoblotting.
The role of V2-receptor-mediated
NaCl reabsorption in the TAL was examined by studying the natriuretic
response to a test dose of furosemide during acute
V2-receptor blockade with
OPC-31260. Furthermore, to examine the expression of the
furosemide-sensitive Na-K-2Cl cotransporter (BSC-1) in cirrhotic rats,
semiquantitative immunoblotting was performed on renal outer medulla
from CBL and sham-CBL rats. Finally, to examine whether the increased
furosemide-sensitive NaCl reabsorption in the medullary TAL in
cirrhotic rats was associated with an increased corticopapillary
gradient, interstitial solute concentrations were measured in the
cortex, in the inner stripe of the outer medulla, and in the papilla in
CBL and sham-CBL rats.
 |
METHODS |
Materials. Barrier-bred and specific
pathogen-free female Wistar rats (220-240 g) were obtained from
the Department of Experimental Medicine, the Panum Institute,
University of Copenhagen, Denmark. The animals were housed in a
temperature-controlled (22-24°C) and moisture-controlled
(40-70%) room with a 12-h light-dark cycle (light on from 6:00
A.M. to 6:00 P.M.). All animals were given free access to tap water and
pelleted rat diet containing ~140 mmol/kg of sodium, 275 mmol/kg
potassium, and 23% protein (catalog no. 1314; Altromin International,
Lage, Germany). Three days before the clearance experiments, the rats
diet was changed to a similar diet to which lithium citrate (12 mmol
Li/kg dry diet) was added. When Li is given by this mode of
administration and in this dose, it does not influence renal function
(20).
Animal preparation. During
halothane-nitrous oxide anesthesia, CBL or sham-CBL was performed as
previously described by Kountouras et al. (16). Three weeks later, rats
used for renal clearance experiments were anesthetized with
halothane-nitrous oxide, and permanent medical grade Tygon catheters
were implanted into the abdominal aorta and into the inferior caval
vein via a femoral artery and vein. A permanent suprapubic bladder
catheter was implanted into the urinary bladder, which was sealed with
a silicone-coated stainless steel pin after flushing the bladder with
ampicillin, 0.6 mg/ml (Anhypen; Nycomed Pharma, Oslo, Norway).
Catheters were produced, fixed, and sealed as described previously
(31). After instrumentation, the animals were housed individually. All
surgical procedures were performed during aseptic conditions. To
relieve postoperative pain, rats were treated postoperatively with
buprenorfin, 0.2 mg/kg body wt ip (Anorfin; GEA, Copenhagen, Denmark),
and to accelerate postoperative recovery, animals were given access to
1.5% sodium chloride in addition to tap water until they reached preoperative weight (3-4 days later).
Renal clearance study. Renal function
was examined by clearance techniques 5 wk after CBL or sham-CBL. On the
day of the experiment, the animal was transferred to a restraining
cage, and intravenous infusion (150 mM glucose, 13 mM sodium chloride,
3 mM lithium chloride; 2.0 ml/h) with
[3H]inulin (Amersham,
Buckinghamshire, UK; batch no. 145 and 147 specific
activity, 48.5 and 42.5 GBq/mmol respectively; infusion rate 3.5 µCi/h) and
[14C]tetraethylammonium
bromide (New England Nuclear, Boston, MA; lot no. 2957-517,
specific activity 0.10 GBq/mmol, infusion rate 1.5 µCi/h) was
started. After a 90-min equilibration period, urine was collected
during two 30-min control periods. Next, intravenous infusion of the
selective V2-receptor antagonist
OPC-31260 (prime, 400 µg/kg body wt; 800 µg · kg
1 · h
1;
Otsuka America Pharmaceuticals) (43) or vehicle (150 mM glucose) was
started. This dose of OPC-31260 was chosen based on dose-response experiments which demonstrated that 800 µg · kg
1 · h
1
produced a diuretic response that was ~90% of the maximal response to OPC-31260, and since higher doses caused sedation, this dose was
used. Extracellular fluid volume was kept constant during V2-receptor blockade by
intravenous replacement of urine losses with 150 mM glucose. Volume
replacement was performed by a computer-driven servo-control system in
which urine output was monitored continuously by collecting urine in
test tubes placed on an electronic balance (type MC 1; Sartorius,
Gottingen, Germany) that was connected to an IBM-compatible computer,
which in turn controlled the infusion rate of an infusion pump
(Perfusor Secura; B. Braun, Melsungen, Germany) (2). Urine collections
were made in one 30-min period followed by four 15-min periods. A
steady-state diuresis was achieved 45-60 min after onset of the
OPC-31260 infusion. After 2 h of OPC-31260 infusion, a test dose of
furosemide (7.5 mg/kg body wt; Dumex, Copenhagen, Denmark) was given as
an intravenous bolus injection (37.5 µg furosemide/s) and urine was
collected in additional four periods of 10 min each. After furosemide
administration, the total intravenous infusion rate was fixed at the
rate used during the preceding urine collection period. Arterial blood
samples of 300 µl each were collected into ammonium-heparinized
capillary tubes at the end of the equilibration period, at the end of
the control period, before furosemide administration, and at the end of
the experiment. During the equilibration period, a 1.0-ml blood sample
was collected in a prechilled test tube with 20 µl 0.5 M EDTA, pH
7.4, and 10 µl 20 × 106
IU/ml aprotinin. After centrifugation at 4°C, plasma
was transferred to a prechilled test tube and stored at
20°C
for later determination of plasma vasopressin concentration. All blood
samples were replaced immediately with heparinized blood from a normal
donor rat.
During the clearance experiment, mean arterial pressure (MAP) and heart
rate (HR) were measured continuously using Baxter Uniflow pressure
transducers (Bentley Laboratories, Uden, Holland) connected to pressure
and heart rate couplers (Hugo Sachs, Hugstetten, Germany). Signals were
displayed on a model WR 3101 Linearcorder Mark VII (Watanabe
Instruments, Tokyo, Japan) and sampled on-line using a data acquisition
program written in LabView (National Instruments, Austin, TX) and
developed in collaboration with Bie Data, Copenhagen, Denmark. After
the clearance experiment, all catheters were sealed, the bladder was
flushed with ampicillin (0.6 mg/ml), and the animals were returned to
their home cages. To replace furosemide-induced sodium losses, rats
were given free access to 1.5% sodium chloride in addition to tap
water for 24 h after the renal function study.
Experimental groups. For renal
clearance experiments, 9 sham-operated rats (sham-CBL) were used for
control experiments and 13 rats with CBL were used to study responses
in cirrhotic rats. Renal function studies were performed in the
following four groups: sham-CBL/vehicle
(n = 8), sham-CBL/OPC-31260
(n = 9), CBL/vehicle (n = 8), and CBL/OPC-31260
(n = 9). Rats that were used for both control and OPC-31260 experiments were allowed a 3-day recovery period
between experiments.
Analytical procedures. Urine volume
was determined gravimetrically. Concentrations of sodium, potassium,
and lithium in plasma and urine were determined by atomic absorption
spectrophotometry using a Perkin-Elmer (Allerød, Denmark) model
2380 atomic absorption spectrophotometer. Urine and plasma osmolality
were determined by use of a cryomatic osmometer (model 3 CII; Advanced
Instruments, Needham Heights, MA).
[3H]inulin and
[14C]tetraethylammonium
bromide in plasma and urine were determined by dual-label liquid
scintillation counting on a Packard Tri-Carb liquid scintillation
analyzer (model 2250CA; Packard Instruments, Greve, Denmark). Plasma
concentrations of bilirubin and alanine aminotransaminase (ALAT) were
measured by reflometry using a Reflotron (Boehringer,
Mannheim, Germany). Vasopressin was extracted from plasma on
C18 Sep-Pak cartridges and
measured by a radioimmunoassay as described earlier (15).
Metabolism studies. An additional
series of rats was prepared with either CBL
(n = 12) or sham-CBL
(n = 8). Four to five weeks after CBL
or sham-CBL, the rats were transferred to metabolic cages (Techniplast,
model 1700; Scandbur, Lellinge, Denmark), which allowed accurate
determination of urine flow and food and water intake. During housing
in metabolic cages, the diet was changed to a granulated standard diet
(catalog no. 1310; Altromin International). After 2 days of adaptation,
determination of sodium and fluid balance was performed daily during
the following 3 days.
Membrane fractionation for
immunoblotting. Rats used in the metabolism study were
later used for immunocytochemical examination. The rats were
anesthetized with halothane-nitrous oxide, and the right kidney was
removed and immediately frozen in liquid nitrogen. The kidney was
stored at
80°C prior to analysis. The cortex, the outer
medulla, and the inner medulla were homogenized separately (0.3 M
sucrose/25 mM imidazole/1 mM EDTA, pH 7.2/8.5 µM leupeptin/1 mM
phenylmethylsulfonyl fluoride), and the homogenates were centrifuged in
a Beckman L8M centrifuge at 4,000 g
for 15 min. Next, the supernatant was centrifuged at 200,000 g for 1 h to produce a pellet
containing both plasma membrane and intracellular vesicle fractions
(22, 24). Gel samples were prepared using Laemmli sample buffer
containing 2% SDS.
Electrophoresis and immunoblotting.
Samples of membrane fractions from each kidney zone (1-10
µg/lane) were run on 12% polyacrylamide minigels (Bio-Rad Mini
Protean II). For each gel, an identical gel was run in parallel and
subjected to Coomassie staining to assure identical loading (36). The
other gel was subjected to immunoblotting. Blots were blocked with 5%
milk in PBS-T (80 mM Na2HPO4/20
mM
Na2HPO4/100
mM NaCl/0.1% Tween 20, pH 7.5) for 1 h, and incubated with
affinity-purified anti-AQP-2 (40 ng IgG/µl IgG; see Refs. 5, 24, 25,
26) or anti-BSC-1 (6). The labeling was visualized with horseradish
peroxidase-conjugated secondary antibody (P448; Dako; diluted 1:3,000)
using an enhanced chemiluminescence system (ECL, Amersham). Controls
were prepared with replacement of the primary antibody with an antibody
preabsorbed with immunizing peptide IgG, or with nonimmune IgG.
Quantitation of AQP-2 and BSC-1
expression. ECL films with bands within the linear
range were scanned (24) using a Hewlett-Packard ScanJet scanner. For
AQP-2 both the 29-kDa and the 35- to 50-kDa band (corresponding to the
nonglycosylated and the glycosylated species; Ref. 33) and for BSC-1
the 161-kDa band were scanned as described earlier (6, 10, 22, 23, 36).
The labeling density was quantitated (22, 23) from blots where samples from each renal zone from CBL rats were run on a gel along with control
material taken from the same renal zones from sham-CBL animals. AQP-2
and BSC-1 labeling in samples from the cirrhotic rats was expressed
relative to the mean expression in the corresponding control material
run on the same gel.
Measurement of interstitial concentrations of
solutes. A third group of animals was prepared with
either CBL (n = 8) or sham-CBL (n = 8) 5 wk earlier. After 24-h
thirsting, the rats were anesthetized with halothane-nitrous oxide, and
the right kidney was removed. Samples from the cortex, the inner stripe
of the outer medulla, and the whole papilla were rapidly dissected and
frozen by clamping with aluminum tongs precooled by immersion in liquid
nitrogen. The tissue samples were placed in prechilled test tubes of
known weight, and the wet weight was determined. Dry weight was
determined after an overnight lyophilization. Lyophilized dry tissue
was extracted for determination of solutes as previously described (4).
Water content in samples was expressed as percentage of wet weight. Na
and K were determined by atomic absorption spectrophotometry, and urea
was determined after hydrolysis with urease as originally described by
Fawcett and Scott (8).
Calculations. Renal clearances (C) and
fractional excretions (FE) were calculated by the standard formulas;
i.e., C = U · V/P; FE = C/GFR; where U is
concentration in urine, V is urine flow rate, and P is plasma
concentration. Inulin clearance was used as a marker for GFR, and
tetraethylammonium clearance was used as a marker for the effective
renal plasma flow (ERPF) (29).
The effective filtration fraction (EFF) was calculated as EFF = GFR/ERPF. Assuming that the renal venous pressure was 5 mmHg throughout
the experiment, the effective renal vascular resistance (ERVR) was
calculated as ERVR = (MAP
5) · (1
Hct)/ERPF, where Hct is hematocrit. Lithium clearance
(CLi) was used as a marker for
the outflow of tubular fluid from the proximal tubules (37). Thus
CLi/GFR is an estimate of the
fractional delivery of fluid and sodium from the proximal tubules,
CNa/CLi
is an estimate of the fractional excretion of sodium from the distal
nephron, i.e., nephron segments beyond the proximal tubules, and
V/CLi represents the fractional
distal water excretion.
Micropuncture studies on the effect of furosemide on tubular lithium
handling suggest that during control conditions, 2-5% of filtered
lithium may be reabsorbed in the TAL, and therefore only changes of
FELi in excess of 2-5% can
be attributed to changes in proximal tubular sodium reabsorption (9,
35). However, when comparisons are performed between groups in which
all animals are treated with furosemide, any difference among groups
can be ascribed to changes in proximal tubular sodium reabsorption,
since there is no evidence for lithium reabsorption beyond the early distal convoluted tubules in sodium replete rats (9, 30). Like most
other clearance markers, the validity of the use of lithium clearance
as a marker for proximal tubular sodium transport has not been examined
in cirrhotic animals, but in animal models with normal plasma levels of
vasopressin there is no evidence for increased lithium reabsorption in
the TAL, distal convoluted tubules, or in the collecting ducts (39).
Statistics. Data are presented are
means ± SE. To evaluate the effects of
V2-receptor blockade, the average
value during the two 30-min control periods was compared with the
average value during the last two 15-min periods during
OPC-31260-induced diuresis. The response during the period with
furosemide-induced peak diuresis was used to evaluate the effect of
furosemide. Within-group comparisons were analyzed with Student's
paired t-test. Between-group
comparisons were performed by one way analysis of variance followed by
Fisher's least significant difference test. Differences were
considered significant at the 0.05 level.
 |
RESULTS |
Organ weights, sodium balance, diuresis, and plasma
biochemistry. Body weights at the end of the study were
similar in CBL and sham-CBL rats, and the average weight gain during
the 5-wk experimental period was 1.0 ± 0.1 g/day in both groups.
There were no signs of ascites in rats with CBL when the abdomen
was exposed. Plasma concentrations of vasopressin,
bilirubin, ALAT, Na, and K in rats used for the renal clearance
experiments are shown in Table 1. Plasma
vasopressin concentrations were similar in CBL and sham-CBL animals.
Plasma concentrations of bilirubin and ALAT were significantly
increased in CBL rats. Plasma concentrations of Na and K were unchanged
in cirrhotic rats. Table 2 shows daily urine flow, urine osmolality, and Na balance in CBL and sham-CBL animals. Cirrhotic rats had Na retention relative to sham-operated animals, whereas 24-h urine flow and urine osmolality were similar in
cirrhotic and sham-operated control animals.
Systemic and renal hemodynamics before and during
V2-receptor blockade.
Table 3 shows systemic and renal
hemodynamics before and during treatment with OPC-31260 or vehicle.
During the control period, HR were similar in all groups (not shown).
CBL rats treated with vehicle had a slightly lower ERPF and GFR than
CBL rats treated with OPC-31260. The reason for these differences in
systemic and renal hemodynamics is unclear, but it is probably a
reflection of the vehicle group being closer to the stage of
decompensation (decreased ERPF and decreased GFR) than the
OPC-treated group of CBL animals. However, none of the CBL rats had any
signs of ascites, decreased ERPF, or decreased GFR, and therefore all
CBL animals were considered to be in the clinically compensated stage of cirrhosis. Overall, ERVR was significantly decreased in CBL rats
compared with sham-operated control animals (13.5 ± 1.0 vs. 19.8 ± 1.6 mmHg · ml
1 · min
1 · 100 g body wt
1;
P < 0.01), suggesting peripheral
vasodilation. All systemic and renal hemodynamic parameters were
unchanged during treatment with OPC-31260 and vehicle.
Renal tubular electrolyte handling before and
during V2-receptor
blockade.
Data on the renal handling of Na, K, and Li before and during treatment
with OPC-31260 or vehicle are shown in Table
4. During baseline conditions, urinary
sodium excretion rate (UNaV),
FENa, FEK, and
FELi were similar in CBL and
sham-CBL rats.
Urine flow rate, free water clearance
(CH2O), and fractional excretion of
water before and during V2-receptor
blockade.
Plasma osmolality was similar in all groups and unchanged throughout
the renal clearance experiment, which suggests that the servo-controlled intravenous volume replacement was effective. The
renal handling of water during baseline conditions and during OPC-31260
treatment is shown in Fig. 1. During
baseline conditions, urine flow rate,
CH2O, and the
fractional excretion of water (V/GFR), as well as the fractional distal
excretion of water (V/CLi), were similar in cirrhotic and sham-operated animals.
V2-receptor blockade significantly
increased these parameters, but compared with sham-operated control
animals, the increases were significantly attenuated in the cirrhotic
rats, as follows: V,
26% (64 ± 5 vs. 86 ± 4 µl · min
1 · 100 g
1;
P < 0.001);
CH2O,
18% (69 ± 5 vs.
84 ± 3 µl · min
1 · 100 g
1;
P < 0.01); V/GFR,
19% (6.66 ± 0.46 vs. 8.19 ± 0.33%; P < 0.01); and V/CLi,
26%
(22.4 ± 2.1 vs. 27.4 ± 1.6%;
P < 0.05).

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Fig. 1.
Urine flow rate (V, A), free water
clearance (CH2O,
B), fractional excretion of water
[V vs. glomerular filtration rate (GFR);
C], and fractional distal water
clearance (V/CLi,
D) during control conditions and
during V2-receptor blockade with
OPC-31260. OPC-31260 was administered as an intravenous infusion (800 µg · kg 1 · h 1)
to conscious, chronically instrumented, female Wistar rats subjected to
common bile duct ligation (CBL; solid bars) or sham operation (open
bars) 5 wk earlier. Volume depletion during
V2-receptor blockade was prevented
by computer-driven, servo-controlled intravenous volume replacement
with 150 mM glucose. Values are means ± SE;
n = 8-9 animals per group.
* P < 0.01 vs. sham-CBL within
period.
|
|
Effect of furosemide. MAP, HR, ERPF,
GFR, EFF, and ERVR were unchanged in all groups during
furosemide-induced peak diuresis. Furosemide significantly increased
urine flow rate and fractional sodium excretion in all groups (Fig.
2). However, the diuretic and natriuretic
responses to furosemide were significantly increased in the cirrhotic
animals compared with the sham-operated control animals. In relative
terms, the diuretic response to furosemide was increased by 41% (169.7 ± 4.6 vs. 120.4 ± 8.8 µl · min
1 · 100 g body wt
1;
P < 0.001) and the natriuretic
response was increased by 52% (19.4 ± 0.7 vs. 12.8 ± 1.4 µmol · min
1 · 100 g body wt
1;
P < 0.001) in cirrhotic rats. The
change in fractional sodium excretion was 75% higher in CBL than in
sham-CBL rats (15.4 ± 1.2 vs. 8.8 ± 0.9%;
P < 0.001).

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Fig. 2.
Furosemide-induced changes in urine flow rate ( V,
A) and fractional sodium excretion
( FENa,
B). Furosemide was administered as a
bolus injection (7.5 mg/kg body wt) to conscious, chronically
instrumented, female Wistar rats subjected to CBL (solid bars) or sham
operation (open bars) 5 wk earlier. Sham-Veh
(n = 8), sham-operated rats pretreated
with vehicle (150 mM glucose); Sham-OPC
(n = 9), sham-operated rats with acute
V2-receptor blockade with the
V2-receptor antagonist OPC-31260
(800 µg · h 1 · kg
body wt 1 iv); CBL-Veh
(n = 8), cirrhotic rats pretreated
with vehicle; CBL-OPC (n = 9), cirrhotic rats with acute
V2-receptor blockade with
OPC-31260. Values are means ± SE.
* P < 0.01 vs. sham-CBL within
period.
|
|
Pretreatment with OPC-31260 did not affect the natriuretic response to
furosemide in cirrhotic or in sham-operated rats. Although V2-receptor blockade did not
affect the diuretic response to furosemide in CBL rats, the diuretic
response to furosemide was significantly attenuated in sham-operated
control rats pretreated with OPC-31260 (90.7 ± 7.5 vs. 120.4 ± 8.8 µl · min
1 · 100 g body wt
1;
P < 0.01).
The furosemide-induced increases in
CLi and
FELi were similar in all four
groups. However, the furosemide-induced increase in
CNa/CLi
was significantly increased in CBL compared with sham-CBL rats (26.2 ± 1.8% vs. 15.5 ± 1.7%; P < 0.001). The furosemide-induced increase in
FEK was similar in all four
groups.
Renal expression of aquaporins.
Figures 3 and 4 show immunoblots of
membrane fractions (20 µg/lane) from the renal cortex (Fig. 3) and
the outer medulla (Fig. 4). As previously
shown, the affinity-purified anti-AQP-2 antibody recognizes the 29-kDa and the 35- to 50-kDa band (23, 36), corresponding to nonglycosylated and glycosylated AQP-2, respectively (33).

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Fig. 3.
Immunoblots of membrane fractions (20 µg/lane) from renal cortex
prepared from female Wistar rats subjected to CBL (solid bars) or sham
operation (open bars) 5 wk earlier. A:
immunoblot was reacted with affinity-purified anti-aquaporin-2
(anti-AQP-2) and revealed 29-kDa and 35- to 50-kDa AQP-2 bands.
B: densitometry was performed on all
sham-operated (n = 5) and all
cirrhotic (Cir.) rats (n = 8). Values
are means ± SE. * P < 0.01 vs. sham-CBL.
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Fig. 4.
Immunoblots of membrane fractions (20 µg/lane) from renal outer
medulla prepared from female Wistar rats subjected to CBL (solid bars)
or sham operation (open bars) 5 wk earlier.
A: immunoblot was reacted with
affinity-purified anti-AQP-2 and revealed 29-kDa and 35- to 50-kDa
AQP-2 bands. B: densitometry was
performed on all sham-operated (n = 8)
and all cirrhotic rats (n = 12).
Values are means ± SE. * P < 0.01 vs. sham-CBL.
|
|
As shown in Fig. 3A and
4A, a significant decrease of both the
29-kDa and the 35- to 50-kDa AQP-2 bands was observed in the cortex as
well as in the outer medulla from cirrhotic rats, whereas inner
medullary tissue from cirrhotic and control rats produced bands of
similar density (not shown). Relative to kidneys from sham-operated
control rats, densitometry revealed a marked decrease in AQP-2
expression in cirrhotic rats in the renal cortex (100 ± 28% vs. 28 ± 7%; P < 0.01) and in the
outer medulla (100 ± 14% vs. 56 ± 10%;
P < 0.01), whereas the expression
was similar to control rats in the inner medulla (100 ± 14% vs. 99 ± 17%; not significant). Recently reported data from our
laboratory have shown selective hypertrophy of the inner stripe of the
outer medulla in CBL rats with increased volume of the TAL as well as
the collecting duct epithelium. However, there was sign of cortical or
inner medulla hypertrophy in Wistar CBL rats (14). Together these results suggest that the AQP-2 downregulation is most pronounced in the
renal cortex.
Renal expression of BSC-1. Figure
5 shows immunoblots of membrane
fractions (20 µg/lane) from the outer medulla. As shown by Ecelbarger
et al. (6), the affinity-purified anti-BSC-1 antibody recognizes the
161-kDa band. The expression of BSC-1 per microgram membrane protein
was similar in CBL and sham-CBL, but since the volume of the TAL
epithelium is significantly increased in the outer medulla in CBL rats
(14), the results suggest that the total amount of BSC-1 is
significantly increased in rats with compensated cirrhosis induced by
CBL.

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|
Fig. 5.
Immunoblots of membrane fractions (20 µg/lane) from renal outer
medulla prepared from female Wistar rats subjected to CBL or sham
operation 5 wk earlier. Immunoblot was reacted with affinity-purified
anti-BSC-1 and revealed a 161-kDa BSC-1 band.
|
|
Interstitial concentrations of Na, K, and
urea. Data on interstitial water content and
concentrations of Na, K, and urea in the cortex, the inner stripe of
the outer medulla, and in the papilla from CBL and sham-CBL rats
subjected to 24-h thirsting are shown in Table
5. The interstitial concentration of Na was significantly increased in the inner stripe of the outer medulla and in
the papilla in CBL rats. There were no significant changes in water
content or K and urea concentration in any of the renal zones.
 |
DISCUSSION |
The present results demonstrate that rats with compensated liver
cirrhosis and normal plasma concentrations of vasopressin have
decreased expression of the vasopressin-regulated water channel AQP-2
along with a decreased diuretic response to selective
V2-receptor blockade with the
V2-receptor antagonist, OPC-31260.
These results suggest that vasopressin-mediated renal water
reabsorption is decreased in rats with compensated liver cirrhosis.
Furthermore, as we have shown previously (14), cirrhotic rats had an
increased diuretic and natriuretic response to a test dose of
furosemide along with an increased corticopapillary Na gradient as
reflected by an increased interstitial Na concentration in the inner
stripe of the outer medulla and in the papilla. The expression of BSC-1 per microgram membrane protein was unchanged in cirrhotic rats, but
since the volume of the TAL epithelium is significantly increased in
the outer medulla in rats with compensated cirrhosis (14), these
results suggest the total amount of BSC-1 is increased in rats with
compensated liver cirrhosis. These results are compatible with our
observation that the furosemide-sensitive tubular Na reabsorption in
the TAL is increased in compensated cirrhosis. Since daily urine flow
rate and proximal tubular Na handling were similar in cirrhotic and
sham-operated rats, the blunted diuretic response to OPC-31260 in
cirrhotic rats suggests that non-vasopressin-mediated renal water
reabsorption is increased in rats with compensated liver cirrhosis. The
increased corticopapillary interstitial gradient due to an exaggerated
NaCl reabsorption in the TAL may explain the increased driving force
for non-vasopressin-mediated water reabsorption in compensated
cirrhosis.
Vasopressin regulates water permeability in the renal collecting duct
by different mechanisms in short-term and in long-term regulation.
Collecting duct water permeability increases within a few minutes after
an acute increase in plasma vasopressin concentration, and it is
mediated by shuttling of AQP-2 from intracellular vesicles into the
apical plasma membrane via exocytosis (24, 25, 32, 44). Mechanisms
involved in the effects of vasopressin on long-term regulation of water
permeability are activated during prolonged (>24 h) increases in the
plasma vasopressin concentration. During prolonged elevations of the
plasma vasopressin level, the density of AQP-2 in the principal cells
is increased (26) along with increased AQP-2 mRNA levels (21) due to
increased AQP-2 gene transcription (27).
Defects in the long-term regulation of AQP-2 expression have been
described in a number of experimental conditions with impaired renal
ability to handle water. Conditions with impaired concentration ability, like central diabetes insipidus in the homozygous Brattleboro rat (5), acquired diabetes insipidus due to lithium intoxication (22),
prolonged hypokalemia (23), bilateral ureteral obstruction (10), and
nephrotic syndrome due to puromycin aminonucleotide toxicity (1), have
a decreased renal expression of AQP-2. Recently, it was demonstrated
that AQP-2 expression is increased in conditions associated with avid
water retention and hyponatremia, as in rats with incompensated
congestive heart failure (28, 43), rats with
incompensated liver cirrhosis (11), and in a model of syndrome of
inappropriate antidiuretic hormone secretion (11). During conditions
with water retention and hyponatremia, the development of hyponatremia
is limited by "vasopressin escape," which is the term used to
describe the situation where the water-retaining action of vasopressin
is impaired (17). With the onset of vasopressin escape, water excretion
increases despite high levels of vasopressin, thereby allowing a new
steady-state water balance during conditions with low plasma sodium
concentration. During water loading of rats treated with high doses of
the selective V2-receptor agonist desmopressin, Ecelbarger et al. (7) recently found that
the renal AQP-2 expression as well as AQP-2 mRNA levels significantly decreased from day 2 of water loading,
whereas AQP-2 trafficking was intact. These results suggest that
vasopressin-independent mechanisms downregulate AQP-2 levels and
decrease collecting duct water permeability during vasopressin escape.
Similar to these findings, Apostol et al. (1) found that AQP-2
expression was significantly decreased in rats with puromycin
aminonucleoside-induced nephrotic syndrome, despite an increased plasma
vasopressin concentration.
In the present study, cirrhotic rats with sodium retention, but a
normal plasma vasopressin concentration, had an impaired long-term
regulation of renal collecting duct water permeability with a
significant downregulation of AQP-2 expression in the renal cortex and
in the outer medulla. Thus it may be speculated that this
downregulation occurs as a physiological compensatory response like in
vasopressin escape and in puromycin aminonucleoside-induced nephrotic
syndrome. The mechanisms behind the altered long-term regulation of
AQP-2 in the models of vasopressin escape, puromycin aminonucleoside-induced nephrotic syndrome, as well as in the present
model of compensated liver cirrhosis, are still unknown. The AQP-2 gene
contains a putative cAMP regulatory element (38), suggesting that
intracellular cAMP concentrations may be involved in the long-term
regulation of AQP-2 expression.
Results from the present renal clearance studies showed that
V2-receptor blockade with the
highly selective V2-receptor
antagonist OPC-31260 significantly increased the urine flow rate,
CH2O, fractional water excretion
(V/GFR), and fractional distal water excretion (V/CLi) without any changes in
systemic or renal hemodynamics or renal tubular sodium or lithium
handling. These results suggest that OPC-31260 is a highly selective
aquaretic agent. The attenuated aquaretic response in CBL
rats is in accordance with the downregulation of AQP-2 in this model.
Together these results strongly suggest that vasopressin-mediated renal
water reabsorption is decreased in rats with compensated liver
cirrhosis induced by CBL.
Stimulation of vasopressin
V2-receptors in medullary TAL of
rats and mice causes activation of the adenylate cyclase, which results
in stimulation of tubular sodium transport and increased transepithelial voltage (34, 40). We recently demonstrated that the
functional and structural changes in TAL found in rats with compensated
liver cirrhosis and normal plasma concentration of vasopressin were
completely absent in homozygous Brattleboro rats with CBL (14).
Therefore, we suggested that vasopressin has a permissive role for the
observed changes in this nephron segment; i.e., the presence of
vasopressin is required for the expression of increased tubular NaCl
reabsorption and structural changes in the TAL in cirrhotic rats. To
examine the effects of acute changes in the vasopressin tone on
furosemide-sensitive NaCl reabsorption in rats with compensated liver
cirrhosis, a test dose of furosemide (7.5 mg/kg body wt iv) was given
during steady-state V2-receptor
blockade. This experiment showed that furosemide produced similar
hemodynamic, diuretic, natriuretic, and lithiuretic responses in CBL
rats pretreated with vehicle and OPC-31260. This suggests that acute
V2-receptor blockade does not
modify the exaggerated natriuretic response to furosemide in rats with
compensated liver cirrhosis.
Sodium chloride transport across the apical plasma membrane in the TAL
is mediated by an Na-K-2Cl cotransporter (alternatively termed BSC-1)
that is directly inhibited by furosemide as shown by Greger (12). The
outer medulla is the renal zone with the highest density of TAL
segments, and to examine whether the exaggerated natriuretic response
in CBL rats was associated with changes in the renal expression of
BSC-1, we performed semiquantitative immunoblotting on renal outer
medulla in CBL and sham-operated control rats. The expression of BSC-1
in this renal zone was found to be unchanged in the cirrhotic rats.
Recent morphometric examinations of in vivo perfused kidneys
demonstrated a 47% increase in the volume of the inner stripe of the
outer medulla, with a 55% increase in the volume of TAL epithelium in
cirrhotic rats relative to controls (14). Thus, along with our previous
findings, the immunoblotting data suggest that the total amount of
BSC-1 is increased in rats with compensated cirrhosis. These findings
suggest that the increased non-vasopressin-mediated renal water
reabsorption in CBL rats is due to an increased corticopapillary
interstitial gradient due to an exaggerated sodium chloride
reabsorption in the TAL.
In summary, the aquaretic response to a near-maximal dose of the
selective V2-receptor antagonist
OPC-31260 was significantly attenuated in rats with compensated liver
cirrhosis compared with sham-operated control rats. This was paralleled
by a significant decrease in AQP-2 expression in the renal cortex and
outer medulla of cirrhotic rats. These results suggest that
vasopressin-mediated renal water transport is decreased in rats with
compensated liver cirrhosis. Furthermore, acute
V2-receptor blockade did not
modify the natriuretic response to furosemide, neither in cirrhotic
rats nor in sham-operated control rats. These findings suggests that acute changes in V2-receptor
stimulation do not influence furosemide-sensitive NaCl reabsorption in
the TAL.
 |
ACKNOWLEDGEMENTS |
We gratefully acknowledge Dr. J. Warberg for performing the plasma
vasopressin analyses. We also acknowledge the technical assistance of
Anette Francker, Lisette Knoth-Nielsen, Iben Nielsen, Mette Vistisen,
Trine Møller, and Gitte Christensen.
 |
FOOTNOTES |
This work received financial support from The Danish Medical Research
Council, The Novo Nordic Foundation, The P. Carl Petersen Foundation,
The Eva and Robert Voss Hansen Foundation, The Ruth Kønig-Petersen
Foundation, The Knud Øster-Jørgensen Foundation, and The Helen
and Ejnar Bjørnow Foundation.
This study was presented in preliminary form at the annual meeting of
the Federation of American Societies for Experimental Biology,
Experimental Biology '97, New Orleans, LA, April 6-9, 1997.
Address for reprint requests: T. E. N. Jonassen, Dept. of Pharmacology,
The Panum Institute, Univ. of Copenhagen, 3 Blegdamsvej, Bldg. 18.6, DK-2200 Copenhagen N, Denmark.
Received 1 December 1997; accepted in final form 23 April 1998.
 |
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