Departments of Internal Medicine (A.J., B.P.) and
Pharmacology and Therapeutics (B.P., D.D.S.), Faculty of
Medicine, St. Boniface Research Centre (A.J., L.C.), University of
Manitoba, Winnipeg, Manitoba, Canada
Aquaporin-2 (AQP-2), the major water channel responsible for water
balance, has been shown to be regulated by the binding of vasopressin
to V2 vasopressin receptors in the medullary collecting duct. 
2-Adrenoceptor agonists such as clonidine have
been associated with an increase in free water clearance that was
secondary to an inhibition of the ability of vasopressin to increase
cAMP levels in the collecting ducts. This investigation focused on the
possibility that this increase in free water clearance following
administration of an 2-adrenoceptor agonist was
associated with a reduction in medullary AQP-2 expression. In the
anesthetized rat, clonidine increased urine flow rate (32 ± 5 versus 137 ± 16 µl/min, p < .05) and free
water clearance (
58 ± 6 versus 3 ± 8 µl/min,
p < .05) compared with the group receiving the
saline vehicle infusion. The increase in free water clearance with
clonidine administration was associated with a reduction in whole
kidney AQP-2 mRNA levels (282 ± 25 versus 216 ± 11 A units, p < .05). This
decrease in water reabsorption was associated with a redistribution of
AQP-2 away from the luminal membrane of the medullary collecting duct to the cytosol. These effects were not secondary to changes in serum
vasopressin levels, as these were similar in the vehicle control and
clonidine groups (59 ± 5 pg/ml versus 64 ± 7 pg/ml, p = NS). The rapid redistribution of AQP-2 and the
reduction in AQP-2 mRNA following clonidine administration are
consistent with the hypothesis that the 2 adrenoceptor
regulates water excretion at least in part by effects on AQP-2.
 |
Introduction |
The
regulation of water excretion has implications for a number of clinical
situations. The interaction of vasopressin and 2-adrenoceptors has been well documented. In
isolated collecting tubules, vasopressin increased cAMP accumulation
(Chabardes et al., 1984
; Umemura et al., 1985) and water permeability
(Krothapalli and Suki, 1984). This vasopressin-induced increase in
water permeability, as well as the increase in cAMP, was found to be
attenuated in the presence of an
2-adrenoceptor agonist in both isolated
tubular segments (Chabardes et al., 1984; Krothapalli and Suki,
1984; Umemura et al., 1985) and isolated perfused rat kidney (Smyth et
al., 1985). In vivo studies have confirmed the ability of
2-adrenoceptor stimulation to increase free
water clearance secondary to the inhibition of the renal actions of
vasopressin (Strandhoy et al., 1982; Gellai and Ruffolo, 1987;
Blandford and Smyth, 1990).
More recently, the effects of vasopressin on water permeability in the
kidney have been found to be mediated through the regulation of
aquaporin-2 (AQP-2) water channels (Deen et al., 1994; Knepper, 1997).
cAMP has been found to be a second messenger in AQP-2 gene transcription and translocation into the luminal membrane after stimulation of V2 receptors by vasopressin
(Hozawa et al., 1996; Deen et al., 1997; Matsumura et al., 1997). As
2-adrenoceptor stimulation has been shown to
decrease the ability of vasopressin to increase cAMP (Chabardes et al.,
1984; Umemura et al., 1985), we determined the effects of clonidine, an
2-adrenoceptor agonist, on AQP-2 expression
and localization. The present study reports on the ability of
2-adrenoceptor stimulation to decrease the level of whole kidney AQP-2 mRNA levels as early as 1 h after administration. In addition, AQP-2 redistributes into the cytosol away
from the luminal membrane of medullary collecting duct cells. This
effect of clonidine failed to alter plasma vasopressin levels, suggesting an effect at the level of the nephron. These findings may
have implications for the potential treatment of disorders associated
with impaired regulation of water balance.
| |
Materials and Methods |
Animal Preparation and Functional Studies.
Male
Sprague-Dawley rats (200-225 g) had their right kidneys removed under
ether anesthesia 7 to 10 days before the day of the experiment, as
previously described (Blandford and Smyth, 1990). On the day of the
experiment, the animals were anesthetized with 50 mg/kg pentobarbitone
(BDH Chemicals Ltd., Poole, England) and placed on a thermostatically
controlled heating blanket that maintained body temperature at
37.5°C. The left jugular vein was cannulated (PE-50) for the infusion
of saline (97 µl/min). The carotid artery was cannulated with PE-160
tubing for the measurement of blood pressure with a Statham
pressure transducer (model P23Dc; Farmingdale, NY) connected to a Grass
polygraph model V (Astro-Med, Inc., Grass Div., West Warwick,
RI). The left kidney was exposed by a flank incision, and the
ureter was cannulated (PE-10). A 31-gauge needle was advanced through
the aorta into the renal artery and secured with glue for the infusion
of the clonidine directly into the kidney.
The preparation was allowed to stabilize for 45 min, after which a
30-min control urine collection was obtained. Immediately following the
first urine collection, the intrarenal infusion (3.4 µl/min) of
vehicle (saline) or clonidine (3 nmol/kg/min) was begun and maintained
for the duration of the experiment. During this collection two
additional urine collections of 30 min each were obtained. At the end
of the experiment, a blood sample was obtained for measurement of
vasopressin, and the kidney was removed for the studies described below.
Vasopressin Assay.
Cervical blood was collected in
prechilled tubes containing EDTA (1 mg/ml of blood) and aprotinin (500 kallikrein inhibitor units/ml of blood). Plasma was separated by
centrifugation at 1600g for 15 min at 0°C. Plasma
vasopressin concentrations were determined with a kit (Peninsula
Laboratories, Inc., Belmont, CA) according to the manufacturer's instructions.
RNA Extraction and Northern Hybridization.
RNA isolation was
performed by the method of Chomczynski and Sacchi (1987). Following
extraction, total RNA was dissolved in sterile water, and the
concentration was determined by absorbency readings at 260 nm. Twenty
micrograms per lane were loaded onto a 1% agarose gel containing 20 mM
3-(N-morpholino)propanesulfonic acid, 1 mM EDTA, 5 mM
sodium acetate (pH 7.0), and 2.2 M formaldehyde. After separation by
electrophoresis, RNA was transferred to nylon membranes (Duralon UV;
Stratagene, La Jolla, CA) and fixed by UV cross-linking (Stratalinker;
Stratagene). Prehybridization was performed at 60°C for 4 h in
5× standard saline citrate (SSC), 5× Denhardt's reagent, 50 mM
Tris-hydrochloride (pH 7.5), 0.1% sodium pyrophosphate, 0.2% SDS, 200 µg/ml denatured salmon testes DNA, and 100 µg/ml yeast tRNA.
cDNA probe for rat AQP-2 (985-base pair fragment corresponding to major
coding region of rat AQP-2) was generously provided by Dr. K. Fushimi
(Fushimi et al., 1993). This probe was labeled with
32P by the random priming method (Promega Corp.,
Madison, WI.). After hybridization at 42°C for 16 to 18 h in
buffer containing 50% deionized formamide, 5× SSC, 1× Denhardt's
reagent, 50 mM Tris-hydrochloride, pH 7.5, 0.1% sodium pyrophosphate,
1% SDS, 100 µg/ml salmon testes DNA, and 100 µg/ml yeast tRNA and
washing in 2× SSC and 0.1% SDS, membranes were subjected to
autoradiography (Kodak XAR-5 film), stripped, and reprobed for
glyceraldehyde-3-phosphate dehydrogenase. The signal for hybridized
AQP-2 and GAPDH mRNA was quantified with the National Institutes of
Health Image Program from a scan of the radiograph. All values are
reported in absorbance (A) factored for variations in
loading of total RNA as determined by the density of GAPDH.
Western Analysis.
Total protein was isolated from medullary
tissue with Trizol (Life Technologies, Inc.) according to the
manufacturer's instructions. Tissue was homogenized in Trizol reagent
(1 ml/100 g) with a tissue homogenizer. After separation of RNA and
DNA, proteins were precipitated from the phenol-ethanol supernatant
with isopropanol. The precipitated protein was pelleted by
centrifugation, washed three times with 0.3 M guanidine hydrochloride
in 95% ethanol, and given a final wash in ethanol. The protein
pellet was vacuum dried and resuspended in 1% SDS in preparation for electrophoresis.
Protein samples were electrophoresed on a denaturing, 10%
polyacrylamide gel, transferred onto a 0.45 µ polyvinylidene
difluoride membrane (Millipore Corp., Bedford, MA), and
subjected to Western blotting as follows. After blocking with 5%
nonfat dry milk in Tris-buffered saline with Tween 20 at room
temperature for 1 h, the membrane was washed with three changes of
Tris-buffered saline before incubation with anti-AQP-2 antibody (1:500
dilution, kindly provided by P. Yves-Martin and R.W. Schrier,
University of Colorado) for 1 h (Xu et al., 1997). Following
another three washes with Tris-buffered saline with Tween 20, the
membrane was incubated with horseradish peroxidase-conjugated, donkey,
anti-rabbit IgG (1:1000 dilution, Amersham Pharmacia Biotech,
Piscataway, NJ) at room temperature for 1 h. Antibody-bound
proteins were detected by enhanced chemiluminescence (Amersham
Pharmacia Biotech). Equality of protein loading was confirmed by
ponceau S staining.
Immunofluorescence.
On removal of kidneys, medullary tissue
was dissected away from the renal cortex, embedded in optimal cutting
temperature medium, and immediately immersed in liquid nitrogen
and stored at 70°C until sectioning. Embedded medullary tissue was
sectioned into 5-µm sections on a cryostat (Cryo-Cut; American
Optical Scientific Instruments, Buffalo, NY) and placed on
silanized slides. Sections were permeabilized by incubating with 0.1%
Triton X-100 in phosphate-buffered saline. After blocking with
universal blocking agent (DAKO Corp., Carpinteria, CA), sections were
incubated with anti-AQP-2 antibody for 1 h at 37°C. Specific
labeling was visualized by incubation for 1 h at 37°C with
fluorescein-conjugated, goat, anti-rabbit antibody (1:100 dilution;
Jackson ImmunoResearch Laboratories, Inc., West Grove, PA) and
photographed on an Olympus BH-2 fluorescence microscope (Olympus
Optical Corp. Ltd., Tokyo, Japan) at a magnification of 300× using
Kodak Ektrachrome 400 print film.
Statistical Analysis.
All results are expressed as mean ± S.E. The unpaired Student's t test with the
Bonferroni correction for multiple comparisons was used for the
statistical comparisons between groups, and results were considered
significant at the p < .05 level.
| |
Results |
Renal Function.
The values obtained during the first urine
collection were similar in both groups (data not shown). For
presentation, data from the third collection period have been shown
(Table 1). Intrarenal infusion of
clonidine at 3 nmol/kg/min failed to significantly alter blood
pressure, heart rate, and creatinine clearance. Clonidine at this
infusion rate increased urine flow rate and sodium excretion. This
increase in urine flow rate was secondary to an increase in free water
clearance and osmolar clearance. The plasma vasopressin levels were
similar following the saline vehicle infusion and the clonidine
infusion (59 ± 5 versus 64 ± 7 pg/ml; NS;
n = 6).
AQP-2 Gene Expression, Protein Level and Distribution.
Compared with the group receiving the saline vehicle infusion, there
was a reduction in whole kidney AQP-2 mRNA following only 1 h of
clonidine administration (Fig. 1;
282 ± 25 versus 216 ± 11 A units;
p < .05, n = 4). This
suggested that the expression of this water channel was rapidly
regulated. At this early time point, this difference in mRNA levels was
not reflected by a decrease in AQP-2 protein as detected by Western
blot analysis (Fig. 2).
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Fig. 1.
Representative lanes from hybridization of 30 µg of total RNA with cDNA probe for rat AQP-2 and GAPDH.
Clonidine-infused rats (lanes 1, 3, 5, and 7) and saline-infused rats
(lanes 2, 4, 6, and 8).
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Fig. 2.
Western blot analysis of total protein probed with
anti-AQP-2 antibody in kidneys from saline-infused (lanes 1, 3, 5, and
7) and clonidine-infused (lanes 2, 4, 6, and 8) rats. When factored for
variations in loading, as determined by ponceau S staining, there was
no significant difference between groups. The 29-kD band represents
AQP-2 and the 36- to 45-kD band represents AQP-2 in glycated form.
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Localization of AQP-2 in Medullary Tissues.
Representative
immunoflouresence profiles of saline- or clonidine-treated rats are
shown in Figs. 3, A and B, respectively. AQP-2 shifted away from the luminal membrane to the cytoplasm in
inner medullary collecting duct cells. This redistribution would lessen
the ability of this region to abstract water from the glomerular
ultrafiltrate and likely accounts for the increase in free water
clearance that was seen with 2-adrenoceptor agonist administration.
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Fig. 3.
Representative immunofluoresence profiles for the
localization of AQP-2 in medullary tissue from saline- (A) and
clonidine-treated (B) rats. Relocation of aquaporin-2 from the luminal
membrane to the cytoplasm of inner medullary collecting duct cells in
clonidine-treated rats.
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Discussion |
The ability of vasopressin to enhance water transport by renal
epithelial cells has been previously shown to be inhibited by
stimulation of 2-adrenoceptors (Krothapalli
and Suki, 1984). In vivo studies by our laboratory and others
(Strandhoy et al., 1982; Gellai and Ruffolo, 1987; Blandford and Smyth,
1990) have documented an increase in free water clearance at doses of
2-adrenoceptor agonists that do not alter
plasma vasopressin levels (Leander et al., 1985). The importance of
vasopressin in the response to 2-adrenoceptor
agonists has been previously documented by the failure of
2-adrenoceptor agonists to increase free water
clearance in Brattleboro rats (Gellai, 1990) and in normal rats
pretreated with a specific V2 vasopressin
receptor antagonist (Blandford and Smyth, 1990). In this study, the
intrarenal administration of the
2-adrenoceptor agonist clonidine increased
free water clearance. This occurred despite lack of significant
differences in plasma vasopressin levels, systemic blood pressure, or
glomerular filtration rate, as measured by creatinine clearance.
Micropuncture studies have suggested that
2-adrenoceptor stimulation increases water and
sodium excretion in the collecting tubule (Stanton et al.,
1987). Vasopressin alters water excretion at this site (Krothapalli and
Suki, 1984). This site is consistent with an interference with the
effects of vasopressin on AQP-2 expression on the luminal surface of
collecting duct cells. AQP-2 has recently been cloned and localized to
the principal cells in the medullary collecting duct (Fushimi et al.,
1993). It has been shown that expression of AQP-2 is increased in
animal models of congestive heart failure (Nielsen et al., 1997) and
decreased in rats during lithium administration (Marples et al., 1995),
low protein diet (Sands et al., 1996), bilateral ureteric obstruction
(Frokiaer et al., 1996), purine aminonucleoside nephrosis
(Apostol et al., 1997), and hypokalemia (Marples et al., 1996).
The mechanism by which 2-adrenoceptor
stimulation altered AQP-2 expression in the rat was most conceivably
secondary to changes in cAMP levels. It has been well documented that
in the rat, 2-adrenoceptor stimulation
decreases vasopressin-mediated increases in cAMP levels (Chabardes et al., 1984; Krothapalli and Suki, 1984; Umemura et al.,
1985; Pettinger et al., 1987). However, this effect of
2-adrenoceptor stimulation has not been
observed in dogs or humans (Brooks et al., 1991; Edwards et al., 1992).
cAMP has been documented as the second messenger that participates in
the trafficking of AQP-2 to the luminal membrane (Deen et al., 1997).
Recently, it has been shown that the promoter region of AQP-2 contains
a cAMP response element (Yasui et al., 1997). Therefore, the observed
decrease in whole kidney mRNA for AQP-2 in clonidine-treated rats may
well be related to decreased cAMP levels in principal cells of the
medullary collecting duct. However, a stabilization rather than an
increase in the rate of transcription of the AQP-2 gene may also
explain these findings. The levels of AQP-2 mRNA appear to be acutely
regulated to parallel the cellular location of this water channel and
reflect a tight regulation of water-handling requirements as suggested
by the decrease in AQP-2 message as early as 1 h after stimulation
with clonidine. The lack of a decrease in AQP-2 protein at this early
time point is not surprising, because it is likely that sufficient time
had not passed for the change in mRNA to be translated into a change in
protein abundance. In other studies, 24 h after a stimulus for
AQP-2 expression, a parallel increase in AQP-2 protein levels has been
observed. This decrease in AQP-2 mRNA in the present study occurred
despite similar levels of plasma vasopressin with and without clonidine
administration. Hence the ability of clonidine to influence
AQP-2 shuttling and mRNA expression was unlikely to be secondary to a
change in vasopressin binding to the V2
vasopressin receptor.
In summary, the present study has demonstrated for the first time the
ability of 2-adrenoceptor agonists to alter
the expression and translocation of AQP-2 in the rat kidney. This
alteration in AQP-2 expression occurred independently of changes in
vasopressin activity and may represent an alternative modality for the
treatment of disorders associated with impaired regulation of water balance.
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2-Adrenoceptor Agonist Clonidine in the Rat1
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Abstract
Introduction
