1 Department of Pharmacology, the Panum Institute, University of Copenhagen, DK-2200 Copenhagen; and 2 Department of Cell Biology, Institute of Anatomy, University of Århus, DK-8000 Århus, Denmark
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ABSTRACT |
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Previous studies have suggested that
mineralocorticoids are needed for a normal action of vasopressin on
collecting duct osmotic water permeability. However, the mechanisms
behind this are unknown. To investigate if aldosterone-receptor
blockade influences vasopressin type 2 receptor
(V2)-mediated renal water
reabsorption and the renal expression of the vasopressin-regulated
water channel aquaporin-2 (AQP2), rats were treated with the
aldosterone-receptor antagonist canrenoate (20 mg/day iv) for 4 wk.
Daily urine flow was increased significantly by 44%, and urine
osmolality was decreased by 27% in canrenoate-treated rats. Acute
V2-receptor blockade (OPC-31260, 800 µg · kg1 · h
1)
was performed under conditions in which volume depletion was prevented.
In control rats, OPC-31260 induced a significant increase in urine flow
rate (V, +25%) and free water clearance
(CH2O,
29%). In canrenoate-treated rats, the effect of OPC-31260 was
significantly reduced, and semiquantiative immunoblotting demonstrated
a significant reduction (45%) in AQP2 expression. Because rats with
common bile duct ligation (CBL) have a reduced vasopressin-mediated
water reabsorption compared with normal rats (V:
24%;
CH2O:
28%, and 86% downregulation of AQP2), the effect of canrenoate
combined with OPC-31260 was tested. Canrenoate treatment of CBL rats
significantly increased daily urine flow, decreased urine osmolality,
and impaired the aquaretic response to OPC-31260 (V:
23%;
CH2O:
31%) with maintained suppression of the renal AQP2 expression.
Thus canrenoate treatment of normal and CBL rats showed
1) increased urine production, 2) reduced aquaretic effect of acute
V2-receptor blockade, and 3) a marked reduction in AQP2
expression. This strongly supports the view that aldosterone plays a
significant role for vasopressin-mediated water reabsorption.
aquaporin-2; V2-receptor; OPC-31260; collecting ducts; canrenoate; cirrhosis
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INTRODUCTION |
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CHRONIC ADRENAL insufficiency is characterized by an inability to generate a maximally concentrated urine as shown in patients with Addison's disease (49) or in adrenalectomized animals (42). Treatment with aldosterone-receptor antagonists like spironolactone increases dilute urine production (48). However, the mechanism behind the impaired renal concentrating ability during adrenal insufficiency or aldosterone-receptor blockade is unknown.
Studies in isolated collecting ducts (CD) suggest that the presence of mineralocorticoids is needed for a normal action of vasopressin on transepithelial osmotic water permeability (5, 37, 42). However, the mechanism behind this action of mineralocorticoids on CD water permeability is unknown. Furthermore, in vivo microperfusion studies of Henle's loop and in vitro studies on isolated segments of the thick ascending limb of Henle's (TAL) have demonstrated that aldosterone stimulates sodium transport in rat medullary TAL (44, 51). This suggests that aldosterone is involved in the regulation of the interstitial hyperosmolality in the renal medulla, which is the driving force for the transepithelial water transport across the CD epithelium. Therefore, the lack of stimulation with mineralocorticoids on both TAL and CD segments may be involved in the impaired concentrating ability during adrenal insufficiency.
Recently, a number of studies have shown that vasopressin stimulates water reabsorption in the CD principal cells by vasopressin type 2 (V2 receptor)-mediated stimulation of aquaporin-2 (AQP2) water channels (30). AQP2 is localized in the apical plasma membrane and in cytoplasmic vesicles, and acute increases in the plasma vasopressin concentration are associated with insertion of AQP2 from cytoplasmic vesicles into the apical plasma membrane (28, 30, 38, 53). During prolonged increases in plasma vasopressin levels, the AQP2 expression is markedly increased (30). However, mechanisms other than vasopressin seem to be involved in the long-term regulation of AQP2 expression. AQP2 expression is increased in pregnant rats with normal plasma vasopressin levels (24). Moreover, downregulation of renal AQP2 protein levels has been reported in a number of conditions with normal or increased plasma vasopressin levels like nephrotic syndrome (1, 14), hypokalemia (27), hypercalcemia (10, 39), ureteral obstruction (16), and compensated liver cirrhosis (18).
An increased plasma aldosterone level is considered among the most important mechanisms involved in the avid sodium and water retention in patients with severe congestive heart failure or decompensated liver cirrhosis. This provides the rationale for the use of aldosterone-receptor antagonists in the management of edema, ascites, and hyponatremia in these clinically important conditions (2, 13, 47). However, our understanding of the mechanism by which aldosterone-receptor antagonists affect renal water handling in such conditions is still incomplete.
In the present study, V2-receptor-mediated water reabsorption in the CD was examined in chronically instrumented rats treated by continous intravenous infusion (20 mg/day) for 4 wk with the aldosterone-receptor antagonist canrenoate. Experiments were performed in normal Wistar rats and in rats with liver cirrhosis induced by common bile duct ligation (CBL). Untreated rats were used as controls. Acute V2-receptor blockade was induced by intravenous administration of the selective V2-receptor antagonist OPC-31260. V2-receptor blockade was achieved in the absence of changes in fluid balance, by use of a computer-driven, servo-controlled intravenous volume replacement system that replaced urinary losses momentarily by intravenous infusion of 150 mM glucose. In an additional group of animals, the expression of the vasopressin-sensitive water channel AQP2 was determined by semiquantitative immunoblotting.
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METHODS |
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Materials. Barrier-bred and specific pathogen-free female Wistar rats (210-230 g) were obtained from the Department of Experimental Medicine, Panum Institute, University of Copenhagen (Copenhagen, Denmark). The animals were housed in a temperature (22-24°C)- and moisture (40-70%)-controlled room with a 12:12-h light-dark cycle (light on from 6:00 AM to 6:00 PM). All animals were given free access to tap water and pelleted rat diet containing ~140 mmol/kg sodium, 275 mmol/kg potassium, and 23% protein (Altromin catalog no. 1310; Altromin International, Lage, Germany).
Animal preparation. During halothane-nitrous oxide anesthesia, CBL was performed as described by Kountouras et al. (21). Normal rats were subjected to sham operation. One week later, during halothane-nitrous oxide anesthesia, a Silastic catheter was implanted in the left external jugular vein in the rats subjected to chronic aldosterone-receptor blockade. The venous catheter was connected to an osmotic minipump (Alzet model 2ML4; pumping rate 2.5 µl/h; Alza, Palo Alto, CA) that was filled with potassium canrenoate (Searle Scandinavia, Malmø, Sweden) at a concentration that produced an infusion rate of 20 mg base/24 h. Three weeks after CBL or sham-CBL, all rats were anesthetized with halothane-nitrous oxide, and permanent medical-grade Tygon catheters were implanted in the abdominal aorta and in the inferior caval vein via a femoral artery and vein. A permanent suprapubic bladder catheter was implanted in the urinary bladder and was sealed with a silicone-coated stainless steel pin after the bladder was flushed with 0.6 mg/ml ampicillin (Anhypen; Nycomed Pharma, Oslo, Norway). Catheters were produced, fixed, and sealed as described previously (36). After instrumentation, the animals were housed individually. All surgical procedures were performed during aseptic conditions. To relieve postoperative pain, rats were treated with 0.2 mg/kg body wt ip buprenorfin (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).
Efficacy of aldosterone-receptor
blockade. To investigate the degree of
aldosterone-receptor blockade, we performed a dose-response experiment
in which the antinatriuretic and antilithiuretic responses to acute
administration of the mineralocorticoid DOCA were investigated. Experiments were performed in chronically instrumented rats treated with intravenous canrenoate. Untreated rats were used as controls. After 2-wk treatment with canrenoate, the animals were transferred to a
restraining cage, and intravenous infusion (150 mM glucose, 13 mM
sodium chloride, and 3 mM lithium chloride; 2.5 ml/h) with [3H]inulin (batch nos.
145 and 147; specific activity, 48.5 and 42.5 Gbq/mmol, respectively;
infusion rate 3.5 µCi/h; Amersham, Buckinghamshire, UK) was started.
After a 90-min equilibration period, urine was collected during 30-min
control periods. Next, intravenous infusion of the mineralocorticoid
DOCA was started (prime: 4 µg/kg body wt; 8 µg · kg1 · h
1
iv). Collections were made in one 60-min period followed by four 30-min periods.
Experimental groups. The following groups of animals were studied: sham (n = 6), sham-operated rats; sham-CAN (n = 6), sham-operated rats chronically treated with canrenoate (20 mg/24 h); CBL (n = 6), CBL rats; and CBL-CAN (n = 6), CBL rats chronically treated with canrenoate (20 mg/24 h). Within each group, an additional six to eight rats were used for immunoblotting analysis to determine AQP2 expression levels (see Membrane fractionation for immunoblotting).
Metabolism studies. During the last 5 days before the renal function study, rats were housed in metabolic cages (Techniplast, model 1700; Scandbur, Lellinge, Denmark) that allowed accurate determination of 24-h urine volume and food and water intake. Daily sodium balance was calculated as sodium intake minus urinary sodium excretion. To optimize urinary recovery of sodium, the metabolic cage was rinsed with 40-50 ml of demineralized water after every urine collection. During housing in metabolic cages, the diet was changed to a granulated standard diet (Altromin catalogue no. 1310; Altromin International) to which lithium citrate was added (12 mmol lithium/kg dry diet). This dose of lithium given in the diet produced plasma lithium concentrations in the range 0.1-0.2 mmol/l without influencing renal function (22). After 2 days of adaptation, daily sodium balance was measured during the last 3 days before the renal function study.
Renal clearance study. Renal function
was examined by clearance techniques 5 wk after CBL or sham-CBL. Before
the renal clearance experiments, all rats were adapted to the
restaining cage used for these experiments by training them for two
periods of 2 h each. To examine the rats at the same level of
hydration, all experiments were started at 9:00 AM. The animal was
transferred to a restraining cage, and intravenous infusion (150 mM
glucose, 13 mM sodium chloride, 3 mM lithium chloride; 2.5 ml/h) with
[3H]inulin (batch nos.
145 and 147; specific activity, 48.5 and 42.5 Gbq/mmol, respectively;
infusion rate 3.5 µCi/h; Amersham) and
[14C]tetraethylammonium
bromide (lot no. 2957-517, specific activity 0.10 Gbq/mmol;
infusion rate 1.5 µCi/h; New England Nuclear, Boston, MA) 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 was started (prime: 400 µg/kg body wt; 800 µg · kg1 · h
1;
Otsuka America Pharmaceuticals; see Ref. 54). This dose of OPC-31260
was chosen based on dose-response experiments that 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. Total body water content was kept constant during
V2-receptor blockade by
intravenous replacement of urine losses with 150 mM glucose. Volume
replacement was performed as described earlier by use of a
computer-driven servo-control system written in LabView (National
Instruments, Austin, TX) and was developed in collaboration with Bie
Data (Copenhagen, Denmark; see Refs. 3 and 18). Urine collections were
made in one 60-min period followed by three 30-min periods. A
steady-state diuresis was achieved 45-60 min after the onset of
the OPC-31260 infusion. Arterial blood samples of 300 µl each were
collected in ammonium-heparinized capillary tubes at the end of the
equilibration period, at the end of the control period, 1 h after
OPC-31260 administration was started, and at the end of the experiment.
At the beginning of the equilibration period (i.e., at 9:00 AM), a
0.2-ml blood sample was collected for measurement of plasma sodium and
potassium concentrations and plasma osmolality, and, for measurement of
the plasma concentration of vasopressin, a 1.0-ml blood sample was
collected in a prechilled test tube with 20 µl of 0.5 M EDTA, pH 7.4, and 10 µl of 20 × 106
IE/ml aprotinin. After centrifugation at 4°C, plasma was
transferred to a prechilled test tube and was stored at
20°C
for later determination. 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 HR couplers (Hugo Sachs, Hugstetten, Germany) and were sampled
on-line using a data-acquisition program written in LabView (National
Instruments) and developed in collaboration with Bie Data. 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. Two days later, an additional 800-µl blood sample
was drawn in a prechilled test tube for measurements of the plasma
aldosterone concentration. The blood sample was centrifuged immediately
at 4°C, and plasma was transferred to a prechilled test tube and
stored at 20°C until analysis. An additional 0.1-ml arterial
blood sample was drawn for analysis of plasma bilirubin and alanine
aminotransaminase (ALAT), and then the rats were killed.
Analytic 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 ALAT were measured by reflometry using a Reflotron (Boehringer Mannheim, Mannheim, Germany). The plasma concentration of aldosterone was measured by RIA using a commercial kit (Coat-A-Count Aldosterone; DPC, Los Angeles, CA). Vasopressin was extracted from plasma on C18 Sep-Pak cartridges and was measured by RIA, as described earlier (20).
Membrane fractionation for
immunoblotting. An additional series of rats was
prepared for immunocytochemical examination
(n = 7-8 in all groups). The rats
were anesthetized with halothane-nitrous oxide, and the right kidney
was removed and immediately frozen in liquid nitrogen and stored at
80°C before analysis. The kidneys were homogenized
[0.3 M sucrose, 25 mM imidazole, 1 mM EDTA (pH 7.2), 8.5 µM
leupeptin, and 1 mM phenylmethylsulfonyl fluoride], and the
homogenates were centrifuged at 4,000 g for 15 min. Next, the supernatant
was centrifuged at 200,000 µg for 1 h to produce a pellet containing
both plasma membrane and intracellular vesicle fractions (26, 28). Gel
samples were prepared using Laemmli sample buffer containing 2% SDS.
Electrophoresis and immunoblotting. Samples of membrane fractions (~2 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 (45). The other gel was subjected to immunoblotting. Blots were blocked with 5% milk in 80 mM Na2HPO4, 20 mM Na2HPO4, 100 mM NaCl, and 0.1% Tween 20, pH 7.5, for 1 h and were incubated with affinity-purified anti-AQP2 (40 ng IgG/µl IgG; see Refs. 9 and 28-30). The labeling was visualized with horseradish peroxidase-conjugated secondary antibody (diluted 1:3,000; P448; Dako) using an enhanced chemiluminescence (ECL) system (Amersham). Controls were prepared with replacement of the primary antibody with an antibody preabsorbed with immunizing peptide IgG or with nonimmune IgG.
Quantitation of AQP2 expression. ECL films with bands within the linear range were scanned (28) using a Hewlett-Packard ScanJet scanner. For AQP2, both the 29-kDa and the 35- to 50-kDa bands corresponding to the nonglycosylated and the glycosylated species (40) were scanned as described earlier (16, 26, 28, 45). The labeling density was quantitated (26, 28) from blots from canrenoate-treated rats and untreated CBL rats run on a gel along with control material taken from untreated sham-operated animals. AQP2 labeling in samples from the canrenoate-treated rats and the untreated cirrhotic rats was expressed relative to the mean expression in the corresponding control material run on the same gel.
Preparation of RNA samples and Northern blotting. Total RNA was extracted from whole kidney from untreated and canrenoate-treated sham rats (n = 6 in both groups) using the acid guanidium-isothiocyanate-phenol-chloroform method (6). Quantification of AQP2 message was performed using a digoxigenin-labeled AQP2 RNA probe (7). The synthesis and digoxigenin labeling of AQP2 RNA probe were performed by in vitro transcription using a Maxiscript in vitro transcription kit (Ambion, Austin, TX). RNA samples (7 µg) were denatured and separated by electrophoresis on a gel agarose (1.2%) containing 0.6 M formaldehyde. Equal RNA loading was verified by visual inspection after coloration with ethidium bromide. The RNA were transferred overnight from gel to nylon membranes (Hybond-N; Amersham Life Science) that were then baked in a vacuum oven (2 h at 80°C). Blots were placed in a glass hybridization tube containing 5× saline sodium citrate (SSC), 50% formamide, 0.1% sarcosyl, 0.02% SDS, and 2% blocking solution (blocking reagent in maleic acid; Boehringer). Prehybridization was performed at 55°C for 30 min in a hybridization oven. The digoxigenin-labeled AQP2 RNA probe was then added to prehybridization medium, and membranes were incubated overnight at 55°C. The blots were washed two times at 25°C in 2× SSC and 0.1% SDS for 5 min and two times at 68°C in 0.1× SSC and 0.1% SDS for 15 min. Blots were then equilibrated for 1 min in maleic acid solution containing 0.3% Tween 20 and were blocked for 30 min. After incubation for 30 min with anti-digoxigenin-alkalinephosphatase conjugate (Boehringer), blots were washed two times for 15 min in maleic acid solution containing 0.3% Tween 20 and were equilibrated for 5 min in 0.1 M Tris · HCl and 0.1 M NaCl. The bands were visualized using a chemiluminescent substrate (CSPD, Boehringer). ECL films with bands within the linear range were scanned using an AGFA ARCUS-II scanner and Corel Photo-Paint software. The labeling density was quantitated using specially written software. The band of ~1.6 kb corresponding to the AQP2 mRNA (7) was scanned. Values were corrected for potential differences in loading of total RNA in the specific lines by densitometry of 18S and 28S bands visualized by ethidium bromide on the same gel. AQP2 mRNA levels in the experimental animals were calculated as a fraction of control levels, which were normalized to 100%.
Calculations. Renal clearances (C) and
fractional excretions (FE) were calculated by the standard formula
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The effective filtration fraction (EFF) was calculated as
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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 the fractional excretion of lithium (FELi) in excess of 2-5% can be attributed to changes in proximal tubular sodium reabsorption (15, 43). 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 (15, 35).
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 30-min periods during OPC-31260-induced diuresis. Within-group comparisons were analyzed with Student's paired t-test. Between-group comparisons were performed by one-way ANOVA followed by Fisher's least-significant difference test. Differences were considered significant at the 0.05 level.
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RESULTS |
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Efficacy of aldosterone-receptor
blockade. In untreated rats, acute DOCA infusion
induced a significant fall in fractional sodium and lithium excretion
without changes in GFR. This antinatriuresis and antilithiuresis were
completely absent in rats treated with 20 mg canrenoate/day. Because
higher doses of canrenoate caused hyperkalemia and decreased the daily
weight gain in normal rats, we chose the dose of 20 mg canrenoate/day
(Fig. 1).
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Effect of canrenoate on plasma biochemistry and plasma
osmolality. Chronic aldosterone-receptor blockade had
no effect on the plasma level of vasopressin, which was significantly
increased in the cirrhotic rats. The plasma levels of aldosterone,
sodium, and potassium and the plasma osmolality were similar in the
four experimental groups (Table 1).
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Effect of canrenoate on renal water excretion and
sodium balance. In normal rats, chronic
aldosterone-receptor blockade significantly increased the daily urine
flow rate by 44% (11.1 ± 0.9 vs. 7.7 ± 0.9 ml · day1 · 100 g body wt
1;
P < 0.05) and decreased urine
osmolality by 27% (919 ± 94 vs. 1,261 ± 107 mosmol/kgH2O;
P < 0.05). An even more pronounced
effect of chronic aldosterone-receptor blockade on daily water handling was observed in the cirrhotic rats. The untreated cirrhotic rats had a
urine output similar to normal rats, but, as shown in Table 2 and Fig. 2,
canrenoate treatment increased the daily urine flow rate by 94% (15.3 ± 2.8 vs. 7.9 ± 0.7 ml · day
1 · 100 g body wt
1;
P < 0.05) and decreased the urine
osmolality by 39% (807 ± 137 vs. 1,314 ± 96 mosmol/kgH2O;
P < 0.05). Thus chronic
aldosterone-receptor blockade induced a significant increase in
solute-free urine.
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In accordance with previous studies (19), daily sodium intake was similar in all groups, but daily sodium excretion was significantly decreased in untreated cirrhotic rats, which caused sodium retention relative to control animals. Despite the well-described effect of aldosterone-receptor blockade on the CD reabsorption, canrenoate treatment had no significant effect on the daily sodium excretion in normal rats when investigated during the fourth week of canrenoate treatment. This indicates that a new steady state in sodium balance was reached after a initial sodium loss in the first week of treatment. However, in cirrhotic rats, canrenoate prevented sodium retention due to a significant 22% increase in the daily sodium excretion (Table 2 and Fig. 2). This indicates that canrenoate significantly inhibited an increased tubular sodium reabsorption in cirrhotic rats.
Effect of canrenoate on systemic and renal
hemodynamics and fractional lithium excretion. Table
3 shows baseline levels of systemic and
renal hemodynamics and renal lithium handling during the clearance
experiments performed after 4 wk intravenous treatment with canrenoate.
Canrenoate had no effect on MAP (which was significantly decreased in
the cirrhotic rats), GFR, or FELi.
However, canrenoate treatment normalized the increased renal plasma
flow (ERPF) and the decreased filtration fraction (EFF) found in the
cirrhotic rats. Acute V2-receptor
blockade had, as previously shown (18), no effect on MAP, ERPF, GFR,
EFF, or FELi (data not shown).
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Effect of V2-receptor blockade on renal
water handling.
1
Acute intravenous treatment with the
V2-receptor antagonist OPC-31260
significantly increased urine flow rate, free water clearance
(CH2O), and
V/CLi in all four groups (Fig.
3). However, the aquaretic effect of
OPC-31260 was significantly attenuated in normal rats with chronic
aldosterone-receptor blockade: V,
29% (57 ± 8 vs. 81 ± 4 µl/min/100 g; P < 0.01);
CH2O,
29% (59 ± 8 vs. 83 ± 5 µl/min/100 g;
P < 0.01); and
V/CLi,
26% (22 ± 2 vs. 29 ± 3%; P < 0.01). Thus
there is a marked reduction in vasopressin-dependent water reabsorption
in response to canrenoate treatment. To test if this also was the case
in rats with CBL-induced cirrhosis, cirrhotic rats were subjected to
combined canrenoate and OPC-3160 treatment. Cirrhotic rats had, as
previously demonstrated (18), a significantly decreased aquaretic
response to acute V2-receptor
blockade:
V,
29% (58 ± 4 vs. 81 ± 4 µl · min
1 · 100 g
1;
P < 0.01);
CH2O,
28% (59 ± 3 vs. 83 ± 5 µl · min
1 · 100 g
1;
P < 0.01); and
V/CLi,
34% (29 ± 3 vs. 19 ± 1%; P < 0.01). In
canrenoate-treated cirrhotic rats, the aquaretic response to V2-receptor blockade was further
impaired:
V,
21% (45 ± 3 vs. 58 ± 4 µl · min
1 · 100 g
1;
P < 0.05);
CH2O,
32% (40 ± 5 vs. 59 ± 3 µl · min
1 · 100 g
1;
P < 0.05); and
V/CLi,
32% (13 ± 2 vs. 19 ± 1%; P < 0.05).
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DISCUSSION |
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The present results demonstrate that chronic treatment with the aldosterone receptor-antagonist canrenoate (20 mg/day iv for 4 wk) significantly 1) increases urine production and decreases urine osmolality, 2) decreases the aquaretic effect of selective vasopressin V2-receptor blockade, and 3) decreases AQP2 protein abundance, in the absence of changes in plasma vasopressin levels. Together these results suggest that chronic treatment with the aldosterone antagonist canrenoate decreases vasopressin-mediated renal water reabsorption and increases the daily production of solute-free urine and that this involves downregulation of the CD water channel AQP2.
Changes in CD function during chronic aldosterone-receptor blockade. Conditions with adrenal insufficiency are characterized by an inability to generate a maximally concentrated urine (49). Similarly, adrenalectomized rabbits have an impaired urinary concentration ability that can be normalized by administration of either gluco- or mineralocorticoids (42). In vitro studies on isolated cortical CD from adrenalectomized rabbits showed that the vasopressin-stimulated increase in osmotic water permeability was impaired in adrenalectomized animals but could be restored by treatment with mineralocorticoids (42). Furthermore, acute or 4-14 days treatment with mineralocorticoid increased the vasopressin-mediated osmotic water permeability in isolated cortical CD in normal rabbits (5). In rats, this synergistic action of mineralocorticoid and vasopressin on osmotic water permeability was absent in isolated cortical CD from normal rats treated with mineralocorticoid for 4-8 days (5). However, Ray et al. (37) showed that the vasopressin-mediated osmotic water permeability was significantly impaired in papillary segments of CD from adrenalectomized rats 3 wk after adrenalectomy. Together, these observations strongly suggests that mineralocorticoids are involved in the regulation of the CD water permeability. The present results demonstrate that chronic aldosterone-receptor blockade inhibits the vasopressin-mediated water reabsorption in the absence of changes in the plasma vasopressin level in normal rats and in rats with liver cirrhosis. Moreover, the results suggest that downregulation of AQP2 plays a significant role in this. Theoretically, this effect could be due to a primary polydipsic effect of canrenoate causing secondary polyuria, but the lack of changes in plasma vasopressin strongly indicates that this is not the case. Therefore, the results suggests that mineralocorticoid receptor blockade downregulates AQP2 abundance in the CD and thereby the CD water permeability. The mechanisms behind this effect are unknown, but the lack of a significant downregulation of the AQP2 mRNA level (Fig. 6) could suggest that an increased degradation of the AQP2 protein was involved. Because plasma vasopressin levels are normal, vasopressin-independent mechanisms may play a role for this reduction in AQP2 expression. This will be discussed below.
Vasopressin regulates water permeability in the renal CD by short-term and long-term regulation. CD water permeability increases within a few minutes in response to an acute increase in plasma vasopressin concentration, and this is mediated by shuttling of AQP2 from intracellular vesicles into the apical plasma membrane via exocytosis (29, 30, 38, 54). For long-term regulation of body water, the total amount of AQP2 protein in the principal cells is increased (30) along with increased AQP2 mRNA levels (23) due, at least in part, to increased AQP2 gene transcription (32). Conversely, in the absence of vasopressin, e.g., in vasopressin-deficient Brattleboro rats, AQP2 expression is suppressed (9). From several studies, it has become clear that both vasopressin-dependent and vasopressin-independent mechanisms operate to modulate AQP2 expression levels (for recent review see Ref. 31). Long-term treatment of vasopressin-deficient Brattleboro rats with vasopressin resulted in 1) a marked increase in AQP2 expression levels, 2) increased osmotic water permeability of inner medullary CD, and 3) complete restoration of the urinary concentration defect (9). This directly demonstrated that vasopressin regulates AQP2 expression levels. The identification of a cAMP-response element in the 5'-flanking region of the AQP2 gene (47) is consistent with an important role of vasopressin V2 receptor-mediated increases in cAMP and cAMP-dependent protein kinase activity on AQP2 expression. The first indication that vasopressin-independent regulation may also be involved came from a study with rats having extremely severe nephrogenic diabetes insipidus due to chronic lithium treatment. Thirsting of such rats for 48 h produced a much greater increase in AQP2 expression than did 7 days of 1-desamino-8-D-arginine vasopressin (DDAVP) treatment (26). Subsequently, Ecelbarger and colleagues (11, 12) demonstrated that water loading of rats that had clamped high levels of plasma DDAVP levels (which prior to water loading increased AQP2 expression) escapes from the effect of DDAVP and produces a significant reduction in AQP2 levels. Recently, it was also demonstrated that thirsting of rats in the continued presence of chronic V2-receptor blockade (OPC-31260) markedly increased AQP2 expression levels (25). These studies together support the view that vasopressin-independent mechanisms may play a significant role in modulating AQP2 expression levels, and several studies suggest that this pathway may be involved in several water balance disorders.
Dysregulation of AQP2 expression has been shown to be associated with
several diseases or conditions with severe disturbances in renal water
and salt handling. Deen et al. (8) demonstrated that mutant,
nonfunctional AQP2 was the cause of very severe non-X-linked inherited
nephrogenic diabetes insipidus in humans, making it clear that AQP2 was
essential for renal water conservation. Subsequently, it was
demonstrated that downregulation of AQP2 expression and reduced
targeting of AQP2 was associated with several forms of acquired
nephrogenic diabetes insipidus, such as lithium treatment (26),
hypokalemia (27), hypercalcemia (10, 39), and ureteral obstruction
(16). Conversely, it was found that AQP2 expression is increased in
rats with severe congestive heart failure associated with hyponatremia
and increased plasma vasopressin levels (33, 52), and also pregnant
rats with water retention (24) have been shown to have increased AQP2
expression levels. Thus dysregulation of AQP2 appears to be involved in
many water balance disorders. The present study demonstrates that
chronic aldosterone-receptor blockade is associated with a 45%
reduction in AQP2 expression levels (Fig. 4) and a significant polyuria
(44% increase, Table 2 and Fig.
2) in the absence of changes in plasma vasopressin levels. Thus this condition shares similarities with other forms of
acquired nephrogenic diabetes insipidus with moderate polyuria and
urinary concentrating defects such as hypokalemia, hypercalcemia, and
postobstructive polyuria (as described above). All of these conditions
are also associated with a 50-200% increase in urine production
and 40-70% reduction in AQP2 expression levels. The demonstration
of a significant attenuation of the aqauaretic response to acute
vasopressin V2-receptor blockade
(with OPC-31260) in canrenoate-treated animals (Fig. 3) is consistent
with the view that downregulation of AQP2 expression is likely to play
a significant role in producing the polyuria (i.e., the defect in the
CD; aldosterone-receptor blockade is also likely to induce effects in
other tubule segments, see below). This reduction in AQP2 expression
occurred in the absence of changes in plasma vasopressin levels. Thus
this is also similar to other forms of acquired nephrogenic diabetes
insipidus in which plasma vasopressin levels are unchanged or perhaps
even increased, indicating that vasopressin-independent regulation (or
dysregulation) of AQP2 may be involved in these conditions. The present
study raises the possibility that modulation of aldosterone receptor
activation may play a significant role in regulating (or maintaining)
AQP2 expression. However, further studies are required to fully define
the general importance of this in the physiology of water balance and
pathophysiology of water balance disorders.
|
Changes in TAL function during chronic aldosterone-receptor blockade. The TAL plays a major role in the renal concentration mechanism. A number of hormones, including vasopressin, stimulate sodium reabsorption in the TAL (41, 50). In addition to the well-known stimulatory effect of aldosterone on sodium reabsorption in the CD, studies using in vivo perfusion of Henle's loop of superficial nephrons (44) and in vitro perfusion of isolated TAL (51) have shown that aldosterone-replacement therapy normalizes the decreased TAL sodium reabsorption in adrenalectomized rats. We recently demonstrated that rats with compensated liver cirrhosis have increased furosemide-sensitive sodium chloride reabsorption and tubular hypertrophy of the TAL. These functional and structural changes are associated with sodium retention and an increased interstitial sodium concentration in the renal medulla (17, 18). As a consequence of the increased corticopapillary interstitial osmotic gradient, the driving force for non-vasopressin-mediated water reabsorption is increased in cirrhotic rats. This likely explains why cirrhotic rats, despite a significant downregulation of AQP2 expression and an attenuated diuretic response to selective V2-receptor blockade, had a normal daily urine production. Thus the downregulation of AQP2 may be compensatory to avoid water retention, in similarity to the downregulation of AQP2 seen in water-loaded, DDAVP-treated rats (11, 12), thereby preventing water intoxication. In the present study, chronic aldosterone-receptor blockade increased the daily urine production by 92% in cirrhotic rats compared with 44% in normal rats. The response to acute V2-receptor blockade was blunted to the same extent as in the normal rats, but the immunoblotting did not show any further significant downregulation of AQP2 in the canrenoate-treated cirrhotic rats. We have recently shown that canrenoate inhibits the increased furosemide-sensitive sodium reabsorption in the TAL in cirrhotic rats (19). These data suggest that canrenoate, at least in cirrhotic rats, decreases sodium chloride reabsorption in the TAL and thereby impairs the corticopapillary interstitial gradient. Thus, in cirrhotic rats, canrenoate also decreases the driving force for transepithelial water reabsorption across the CD and increases urine flow rate. Therefore, inhibition of an increased sodium chloride reabsorption in the TAL in cirrhotic rats may explain why canrenoate produced a significantly greater increase in 24-h urine production in cirrhotic rats than in normal animals.
In summary, chronic treatment with the aldosterone-receptor antagonist canrenoate (20 mg/day iv for 4 wk) significantly 1) increases urine production and decreases urine osmolality, 2) decreases the aquaretic effect of selective V2-receptor blockade, and 3) decreases the AQP2 protein abundance, and this occurs in the absence of changes in plasma vasopressin levels. Together these results suggest that chronic treatment with the aldosterone antagonist canrenoate decreases vasopressin-mediated renal water reabsorption, decreases CD AQP2 expression, and increases the daily production of solute-free urine. These findings support the view that aldosterone-receptor antagonists may be particularly effective drugs during conditions with avid vasopressin-mediated water retention and hyponatremia.
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ACKNOWLEDGEMENTS |
---|
The technical assistance of Anette Francker, Lisette Knoth-Nielsen, Anette Nielsen, Iben Nielsen, Mette Vistisen, and Annette Blak Rasmussen is acknowledged. We gratefully acknowledge Dr. J. Warberg for performing the plasma vasopressin analyses.
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FOOTNOTES |
---|
This work was supported by 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-Jrgensen Foundation, The Helen and Ejnar
Bjørnow Foundation, The Karen Elise Jensen Foundation, the Aage
Thuesen Bruun Foundation, the University of Aarhus, and the European Union (EU) Commission (EU-Biotech Programme and
EU-Training Mobility Research programme).
Part of this study was presented in preliminary form at the annual meeting of the American Societies of Nephrology, San Antonio, TX, on November 2-5, 1997.
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.
1 Renal clearance experiments were performed during the inactive period of the rat (i.e., during day time) in which sodium- and water-retaining mechanisms are maximally activated. To get a stable urine production during these conditions, all rats were slightly water loaded by infusion of a hypotonic glucose solution, 2.5 ml/h, as previously described (18). Therefore, baseline levels of urine flow rate (V), free water clearance (CH2O), and the fractional distal excretion of water (V/CLi) were similar (i.e., clamped) in all four groups (data not shown), as previously demonstrated (18). Furthermore, in accordance with previous studies (18, 19), the renal sodium and potassium handling were similar in all four groups and were unchanged during V2-receptor blockade (data not shown). Plasma osmolality was similar in all groups and was unchanged throughout the renal clearance experiment, which indicates that the servo-controlled intravenous volume replacement was effective.
Address for reprint requests and other correspondence: T. E. N. Jonassen, Dept. of Pharmacology, The Panum Institute, Univ. of Copenhagen, 3 Blegdamsvej, Bldg. 18.6, DK-2200 Copenhagen N, Denmark (E-mail: fitj{at}farmakol.ku.dk).
Received 30 November 1998; accepted in final form 31 August 1999.
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