Vasopressin regulates apical targeting of aquaporin-2 but not
of UT1 urea transporter in renal collecting duct
Takeaki
Inoue1,
James
Terris1,2,
Carolyn A.
Ecelbarger1,
Chung-Lin
Chou1,
Soren
Nielsen3, and
Mark A.
Knepper1
1 Laboratory of Kidney and
Electrolyte Metabolism, National Heart, Lung and Blood Institute,
National Institutes of Health, Bethesda 20892;
2 Department of Physiology,
Uniformed Services University of Health Sciences, Bethesda, Maryland
20814; and 3 Department of
Cell Biology, University of Aarhus, DK-8000 Aarhus,
Denmark
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ABSTRACT |
In the renal inner
medullary collecting duct (IMCD), vasopressin regulates
two key transporters, namely aquaporin-2 (AQP2) and the
vasopressin-regulated urea transporter (VRUT). Both are present in
intracellular vesicles as well as the apical plasma membrane.
Short-term regulation of AQP2 has been demonstrated to occur by
vasopressin-induced trafficking of AQP2-containing vesicles to the
apical plasma membrane. Here, we have carried out studies to determine
whether short-term regulation of VRUT occurs by a similar process. Cell
surface labeling with NHS-LC-biotin in rat IMCD suspensions revealed
that vasopressin causes a dose-dependent increase in the amount of AQP2
labeled at the cell surface, whereas VRUT labeled at the cell surface
did not increase in response to vasopressin. Immunoperoxidase labeling
of inner medullary thin sections from Brattleboro rats treated with
1-desamino-8-D-arginine vasopressin (DDAVP)
for 20 min revealed dramatic translocation of AQP2 to the apical region
of the cell, with no change in the cellular distribution of VRUT. In
addition, differential centrifugation of inner medullary homogenates
from Brattleboro rats treated with DDAVP for 60 min revealed a marked
depletion of AQP2 from the low-density membrane fraction (enriched in
intracellular vesicles) but did not alter the quantity of VRUT in this
fraction. Finally, AQP2-containing vesicles immunoisolated from a
low-density membrane fraction from renal inner medulla did not contain
immunoreactive VRUT. Thus vasopressin-mediated regulation of AQP2, but
not of VRUT, depends on regulated vesicular trafficking to the plasma membrane.
surface biotinylation; immunocytochemistry; differential
centrifugation; urinary concentrating mechanism
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INTRODUCTION |
THE INNER MEDULLARY collecting duct of the mammalian
kidney responds to vasopressin with a marked increase in water
permeability and in urea permeability (14, 17, 22). Both of these
processes are critical to the overall regulation of water excretion.
Both processes are mediated by cAMP (10, 27) and are initiated within
40 s of exposure of the collecting duct to increased levels of
vasopressin (29). Studies of aquaporin-2 (AQP2), the
vasopressin-regulated water channel, have demonstrated that the
short-term effect of vasopressin to raise water permeability is a
result of vasopressin-stimulated trafficking of AQP2-bearing vesicles
to the apical plasma membrane (13, 15, 20, 30). It has been
hypothesized that vasopressin may increase the urea permeability by a
similar mechanism (18). However, this hypothesis has not yet been
directly addressed.
Urea transport across the apical plasma membrane of the inner medullary
collecting duct (IMCD) cells is facilitated by a specialized phloretin-sensitive urea transporter initially identified by
physiological techniques (4, 21, 26) and eventually cloned by Shayakul et al. (25). The apical plasma membrane is the rate-limiting membrane
for overall transcellular urea transport and is the site at which
vasopressin regulates urea transport (4, 26). Therefore, the apical
urea transporter is referred to as the vasopressin-regulated urea
transporter (VRUT) of the IMCD (12). More recently, the cloned VRUT has
been referred to as either "UT1" (25) or "UT-A1" (23).
Immunocytochemical studies, using a polyclonal antibody raised to a
synthetic peptide corresponding to the carboxy-terminal tail of the 929 amino acid protein predicted from the open reading frame of the UT1
cDNA clone, have confirmed apical localization of the urea transporter
protein in the IMCD (18). The chief aim of the present study was to
determine whether vasopressin activates VRUT by the same mechanism
involved in activation of the AQP2 water channel, i.e., by stimulation
of VRUT trafficking to the plasma membrane of IMCD cells.
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METHODS |
Antibodies. Antibodies recognizing the
carboxy-terminal region of AQP2 (L127) (5), the carboxy-terminal region
of VRUT (L194) (18), and the middle loop of VRUT (L448) (28)
have been previously characterized. They were affinity
purified as described (5, 18, 28).
IMCD suspensions. IMCDs were prepared
from rat whole inner medulla suspensions as described by Chou et al.
(3). Inner medullas were dissected from rat kidneys and digested at
37°C with collagenase B (3 mg/ml; Boehringer-Mannheim,
Indianapolis, IN) and hyaluronidase (600 U/ml; Worthington
Biochemicals, Freehold, NJ) in HEPES-buffered solution (in mM: 118 NaCl, 5 KCl, 25 NaHCO3, 4 Na2HPO4,
2 CaCl2, 1.2 MgSO4, 5 CH3COONa, and 5.5 glucose)
containing 0.1% (wt/vol) bovine serum albumin (ICN Biomedicals,
Aurora, OH) under continuous supplement of 95% air-5%
CO2 until IMCDs were free from
adherent thin limbs. DNase I (Boehringer-Mannheim) at a final
concentration of 0.001% (wt/vol) was added to the digesting solution
to reduce aggregation of separated tubule segments. After incubation
for another 15 min in this solution, the suspension was separated into
IMCD-enriched and IMCD-depleted fractions by low-speed centrifugation (50 g) (Sorvall RT-6000B). The
pellet was resuspended in HEPES, and the 50 g centrifugation was repeated three
times. The final pellet contained mostly IMCD fragments, whereas the
supernatant contained thin limbs and vascular elements. Aliquots were
taken for measurement of protein concentration (Pierce BCA protein
assay reagent kit; Pierce Chemical, Rockford, IL).
Surface biotinylation of IMCD
suspension. Biotinylation of IMCD suspensions was
carried out as described by Gottardi et al. (9) with slight
modification. Briefly, after prewarming suspensions in HEPES buffer
solution containing 0.1% (wt/vol) BSA for 60 min at 37°C,
suspensions were incubated in the same solution without vasopressin
(AVP) or with 10
10,
10
8, and
10
6 M AVP for 20 min. At
the end of the incubation, suspensions were put into ice
to inhibit endocytosis. After centrifugation at 50 g for 10 s at 4°C, the supernatant
was discarded and the pellet was washed three times at 4°C with
ice-cold biotinylation buffer solution (in mM: 215 NaCl, 4 KCl, 2.5 Na2HPO4,
2 CaCl2, 1.2 MgSO4, 5.5 glucose, and 10 triethanolamine, pH 7.4) without biotin. Final pellet was resuspended
in 1 ml ice-cold biotinylation solution containing 3.0 mg/ml of
NHS-LC-biotin (ImmunoPure no. 21335, Pierce) and incubated for 60 min
at 4°C with gentle horizontal motion to ensure mixing. The
suspensions were then washed three times with ice-cold biotin quenching
solution (0.1 mM CaCl2, 1 mM
MgCl2, and 260 mM glycine in PBS,
pH 7.4) followed by gentle mixing for 20 min at 4°C with the same
solution. After washing with ice-cold lysis buffer (150 mM NaCl, 5 mM
EDTA, and 50 mM Tris, pH 7.4) containing protease inhibitors (1 µg/ml
of leupeptin and 0.1 mg/ml of phenylmethylsulfonyl fluoride) without
detergent three times, the suspension was solubilized in 1 ml of lysis
buffer (which included 1% NP-40) for 60 min at 4°C with gentle
motion. The samples were centrifuged at 14,000 g for 10 min at 4°C, and 900 µl
of the supernatant was added to 200 µl of streptavidin beads
(ImmunoPure no. 20349, Pierce). The bead suspension was incubated
overnight (16 h) at 4°C with continuous agitation. After washing
the beads three times with lysis buffer containing 1% NP-40, twice
with high-salt wash buffer (500 mM NaCl, 5 mM EDTA, and 50 mM Tris, pH
7.5), and once with no-salt wash buffer (10 mM Tris, pH 7.5) at
4°C, biotinylated proteins were eluted from the beads with 100 µl
of 1.5% SDS in Laemmli sample buffer incubated at 60°C for 15 min.
Experiments were done to test whether biotinylation is restricted to
surface proteins on the plasma membranes. An IMCD tubule suspension
pooled from five rats was divided into five tubes and biotinylated with
0, 0.02, 0.2, 2, and 10 mg of NHS-LC-biotin in 1 ml of biotinylation
solution. After quenching of excess free biotin as above, the
suspensions were pelleted by low-speed centrifugation and resuspended
in ice-cold isolation solution [10 mM triethanolamine (pH 7.6),
250 mM sucrose]. The suspensions were homogenized in ice-cold
isolation solution with protease inhibitors using a tissue homogenizer
(Omni 1000 fitted with a micro-sawtooth generator). Differential
centrifugation was carried out as described below, yielding a
high-density membrane fraction and a low-density membrane fraction.
Both fractions were solubilized in 1 ml of 1% NP-40 lysis buffer with
protease inhibitors for 60 min at 4°C with gentle motion. These
samples were incubated with streptavidin beads, which were subsequently
eluted with Laemmli sample buffer to obtain biotinylated proteins as
described above. The resulting samples were subjected to
immunoblotting. The high-density fraction is enriched in plasma
membranes and should contain biotinylated proteins. Conversely, since
the low-density fraction is virtually devoid of plasma membranes (6,
13), it should not contain biotinylated proteins if biotinylation is
limited to plasma membrane proteins.
Differential centrifugation.
Differential centrifugation was carried out as previously described (6,
13). Inner medullary homogenates were initially centrifuged at 4,000 g for 10 min at 4°C (Tomy,
MTX-150) to remove incompletely homogenized fragments and nuclei. The
pellets were resuspended in ice-cold isolation solution with protease
inhibitors and centrifuged again at 4,000 g for 10 min. The supernatants were
collected and centrifuged at 17,000 g
for 20 min (Sorvall RC2-B centrifuge with SS34 rotor). The pellets
("high-density membrane fraction") were retained, and the
supernatants were then pelleted by centrifugation at 200,000 g for 1 h (Beckman ultracentrifuge
with Ti-80 rotor) ("low-density membrane fraction"). As
previously documented (6, 13), the low-density membrane fraction from
this protocol is virtually devoid of plasma membranes.
Immunoisolation of AQP2-bearing intracellular
vesicles. AQP2-bearing intracellular vesicles were
immunoisolated using affinity-purified AQP2 polyclonal antibody
covalently coupled to magnetic beads (Dynabeads no. M-280 with
covalently attached sheep anti-rabbit IgG; Dynal, Lake Success, NY)
according to the manufacturer's instructions. Either 5.1 µg of AQP2
antibody or 5.1 µg of preimmune IgG from the same rabbit was
incubated with 1.7 mg magnetic beads in 100 µl of washing solution
(PBS pH 7.4, 0.1% BSA) with 0.02% azide overnight at 4°C with
gentle mixing. After washing four times in same solution with
bidirectional mixing for 30 min each, the beads were resuspended in 1 ml of 0.2 M triethanolamine (pH 8.2) and were washed two times with the
same solution. Then, 20 mM dimethylpimelimidate (DMP), a
homobifunctional cross-linker, in 10 ml of 0.2 M triethanolamine
solution was added to the beads and incubated for 45 min at room
temperature with bidirectional mixing. After incubation in 10 ml of
cross-linking solution without DMP for a further 2 h, beads
were resuspended and incubated in 10 ml of 1% NP-40 containing 0.2 M
triethanolamine for another 10 min to completely eliminate
noncovalently bound IgG.
A low-density membrane fraction containing intracellular vesicles but
not plasma membranes (6, 13) was prepared from inner medullas by
differential centrifugation as described above, except that the
low-density membrane fraction (200,000 g pellet) was resuspended in washing
buffer. After washing the antibody-bearing beads in 1 ml of washing
buffer three times, 166 µl of the low-density membrane fraction
(~0.3 µg/µl total protein) was incubated with the beads for 5 h
at 4°C with gentle mixing. After washing in 1 ml of washing buffer
three times, the proteins were eluted by 100 µl of Laemmli sample
buffer and were solubilized at 60°C for 10 min.
Electrophoresis and immunoblotting.
Samples prewarmed at 37°C for 20 min were loaded on precast 12%
SDS-PAGE minigels (Novex) and electrophoresed using an X-Cell II
minicell (Novex, San Diego, CA). The proteins on the gel were
transferred electrophoretically to nitrocellulose membranes using a
Bio-Rad Mini Trans-Blot cell (Bio-Rad, Hercules, CA). After blocking
with 5% (wt/vol) nonfat dry milk in blot wash buffer [150 mM
NaCl, 50 mM
NaH2PO4,
and 0.05% (vol/vol) Tween 20, pH 7.5] for 30 min at room
temperature, the membranes were incubated with either anti-VRUT
antibody (L194) or anti-AQP2 antibody (L127) in antibody dilution
buffer solution (blot wash buffer with 0.1% BSA and 0.02%
NaN3) overnight at 4°C. After washing with blot wash buffer, the nitrocellulose membranes were
incubated with 0.16 µg/ml of donkey anti-rabbit IgG conjugated to
horseradish peroxidase (Pierce no. 31458) in blot wash buffer containing 5% (wt/vol) nonfat dry milk at room temperature for 1 h.
Sites of antibody-antigen reaction were visualized by chemiluminescence using SuperSignal Substrate (Pierce) and exposure to light-sensitive imaging film (Kodak no. 165-1579 Scientific Imaging Film).
Dot blotting. One microliter of each
sample was applied directly onto nitrocellulose membranes. Subsequent
procedure after air drying of membranes for 30 min at room temperature
was the same as for immunoblotting as described above. The dot density was analyzed by scanning densitometry (Molecular Dynamics, Sunnyvale, CA) and normalized by the amount (in µg) applied onto the membrane. Data are presented as mean ± SE. Comparisons between groups were made by unpaired Student's t-test.
Experimental animals. For in vivo
studies of short-term regulation of AQP2 and VRUT by vasopressin, we
used pathogen-free male Brattleboro homozygous (di/di; Harlan Sprague
Dawley, Indianapolis, IN) rats. All rats were fed a standard rat chow
ad libitum containing 18% protein and 130 meq
Na+ per kg (NIH-31 autoclavable
rodent diet; Zeigler Bros., Gardner, PA) and had free access to water.
The rats were maintained at all times under pathogen-free conditions.
Animal protocol: Differential centrifugation
studies. Body weights were carefully matched (control,
240 ± 6 g; DDAVP, 253 ± 3 g). The rats were maintained in
metabolic cages to allow urine collections. Urine osmolalities were
measured using a vapor-pressure osmometer (model 5100C; Wescor, Logan,
UT). Six rats received an intramuscular injection of 2 nmol DDAVP
(Sigma Chemical, St. Louis, MO) dissolved in water, and six rats were
injected with the same volume of 0.9% NaCl. One hour after injection,
the rats were killed by decapitation. The kidneys were rapidly removed into ice-cold isolation buffer (composition described above), and the
inner medullas were separated from the outer medulla and cortex using
sharp, curved, iris scissors. Homogenization and differential
centrifugation were carried out as described above to yield
high-density and low-density membrane fractions.
Animal protocol: Immunocytochemical
studies. Brattleboro rats, weighing 250-300 g,
were anesthetized with intraperitoneal pentobarbital sodium (75 mg/kg)
and injected via the femoral vein with DDAVP, a stable
V2-selective analog (1 ng/animal),
or with vehicle. After 20 min, the kidneys were perfusion fixed by
retrograde perfusion through the abdominal aorta with 8%
paraformaldehyde in 0.1 M sodium cacodylate buffer, pH 7.2. Tissue
blocks were postfixed in the same fixative for 2 h, infiltrated for 30 min with 2.3 M sucrose containing 2% paraformaldehyde, mounted on holders, and rapidly frozen in liquid nitrogen. Semi-thin cryosections (0.85 µm) were obtained with a cryoultramicrotome (Reichert Jung, Vienna, Austria), and the sections were placed on gelatin-coated glass
slides. After preincubation with PBS containing 1% BSA and 0.05% M
glycine, the sections were incubated with 1 µg IgG/ml of either
affinity-purified anti-AQP2 (L127) or anti-VRUT antibody (L194) in PBS
containing 1% BSA and 0.3% Triton X-100. The labeling was visualized
using anti-rabbit IgG conjugated to horseradish peroxidase (P448,
1:100; DAKO, Glostrup, Denmark). Sections were counterstained with
Meier counterstain. The following controls were performed:
1) incubation with protein
A-purified rabbit IgG instead of primary antibody;
2) adsorption controls, made by
preincubating the affinity-purified antibody with an excess of peptide
conjugated to BSA; 3) and incubation
without use of primary antibody or without primary and secondary
antibody. All controls revealed absence of labeling.
 |
RESULTS |
Surface biotinylation of plasma membrane proteins in
IMCD suspension. We used surface biotinylation to assay
for protein trafficking to the plasma membrane in IMCD cells. This
technique employs a membrane-impermeant reagent that reacts with
primary amine groups to label all plasma membrane proteins with
externally accessible lysines. Biotinylated proteins were collected by
binding to streptavidin beads and were eluted from the beads with
SDS-containing sample buffer for immunoblotting. A preliminary
experiment to test whether biotinylation was restricted to plasma
membrane proteins is summarized in Fig. 1.
Here, high-density (17,000 g) and
low-density (200,000 g pellet from
17,000 g supernatant) membrane
fractions were prepared from IMCD suspensions after incubation with
different amounts of NHS-LC-biotin as described in
METHODS and probed with anti-AQP2 antibody. As previously demonstrated, the 17,000 g fraction from this differential
centrifugation protocol is enriched in plasma membranes, whereas the
200,000 g fraction is virtually free
of plasma membrane proteins from collecting ducts, with AQP2 being abundant in both fractions (6, 13). As shown in Fig. 1,
differential centrifugation of the derivatized samples revealed that
only the 17,000 g fraction contained
biotinylated AQP2. AQP2 in the 200,000 g fraction, which contains diverse
intracellular vesicles, was not biotinylated, supporting the idea that
only the cell surface is biotinylated. Biotinylation of AQP2 was nearly
maximal at 2 mg/ml NHS-LC-biotin. Based on this result, all subsequent
biotinylation experiments were carried out using 3 mg/ml of
NHS-LC-biotin for IMCD suspensions from one rat.

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Fig. 1.
Surface biotinylation of rat inner medullary collecting duct (IMCD).
IMCD suspensions were divided into equal aliquots and subjected to
surface biotinylation with 0, 0.02, 0.2, 2, and 10 mg of NHS-LC-biotin.
High-density membrane fraction (17,000 g) and low-density membrane fraction
(200,000 g) were prepared by a
series of centrifugations as described in
METHODS. Immunoblot was probed with
affinity-purified anti-aquaporin-2 (anti-AQP2) antibody.
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Figure 2 shows a representative surface
biotinylation experiment done to test whether the amount of AQP2 and
VRUT in the plasma membrane is increased by incubation of the IMCD
suspension with AVP. Figure 2A shows
the results for AQP2 using immunoblotting (top) and dot blotting
(bottom). As expected from previous
studies demonstrating that vasopressin stimulates translocation of AQP2 to the plasma membrane of collecting duct cells, there was a marked dose-dependent increase in AQP2 labeling by surface biotinylation in
response to AVP. These observations, therefore, provide an appropriate
positive control for VRUT observations. Figure
2B shows the immunoblotting and dot
blotting results for VRUT using the same samples. In contrast to AQP2,
VRUT showed no perceptible change in labeling by surface biotinylation
in response to increasing concentrations of AVP. As we have previously
demonstrated (1), solubilization of membranes using a nonionic
detergent results in stabilization of a VRUT complex (most likely a
VRUT dimer) represented by a broad band centered at around 206 kDa.

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Fig. 2.
Effect of arginine vasopressin (AVP) on extent of biotinylation of AQP2
and vasopressin-regulated urea transporter (VRUT) at IMCD cell surface.
Immunoblots (top) and immuno-dot
blots (bottom) of biotinylated
proteins from IMCD suspension were probed with either affinity-purified
anti-AQP2 (A) or anti-VRUT antibody
(B). This is an example that is
typical of 6 experiments. For immunoblots, 10 µl was loaded in each
lane. For dot blots, 1 µl of each sample was directly applied to the
nitrocellulose membrane.
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Figure 3 summarizes quantification of six
such studies using laser densitometry to assess dot density on AQP2 and
VRUT dot blots. There was a significant increase in cell surface
labeling of AQP2 to a maximum of 350% of the AVP-independent value,
whereas there was no change in biotinylation of VRUT at the cell
surface. Thus we find no evidence for vasopressin-stimulated
trafficking of the urea transporter VRUT.

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Fig. 3.
Quantification of dot density in 6 biotinylation experiments. Solid
bars, results from AQP2 dot blots; hatched bars, results from VRUT dot
blots. Dot density was analyzed by scanning densitometry, and values
were normalized to show percent change.
* P < 0.05, significantly
different based on ANOVA with Bonferroni's method employed for
individual contrasts vs. the unstimulated control.
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Immunocytochemical localization of AQP2 and VRUT:
Effect of vasopressin. To address further whether
vasopressin stimulates redistribution of VRUT in IMCD cells,
immunocytochemical localization was carried out. Brattleboro rats were
injected with either DDAVP (n = 3) or
with vehicle (n = 3), and the kidneys
were perfusion fixed with paraformaldehyde fixative as described in
METHODS. Thin sections were prepared,
and horseradish-peroxidase immunocytochemistry was carried out in the
inner medulla using antibodies against AQP2 and VRUT. Figure
4, top,
shows the localization of AQP2 in the IMCD after vehicle injection
(Fig. 4A) and after DDAVP injection (Fig. 4B). In the vehicle-treated
control IMCDs, AQP2 was distributed both to the apical region of the
cell and throughout the cytoplasm (Fig.
4A). The AQP2 in the cytoplasm is
known to be due to the presence of this water channel in
intracytoplasmic vesicles (16). In contrast, after DDAVP, the labeling
was almost exclusively in the extreme apical region of the IMCD cells
(Fig. 4B), consistent with the
conclusion that DDAVP stimulates translocation of AQP2 vesicles to the
apical plasma membrane. Figure 4,
bottom, shows VRUT labeling in the
IMCD. In the IMCDs from vehicle-treated rats, the VRUT labeling of
collecting duct cells was distributed both to the apex of the cells and
intracytoplasmic vesicles (Fig. 4C), as previously demonstrated (16). In contrast to AQP2, VRUT labeling in
the IMCD cells was not affected by DDAVP administration to the rats
(Fig. 4D). Similar observations were
made in two other pairs of rats (not shown). Labeling was absent in
parallel sections when an IgG fraction of the preimmune serum was
substituted for the primary antibody (not shown). These
immunocytochemical results are consistent with the observations made in
surface biotinylation experiments (above) and further support the
conclusion that vasopressin does not stimulate trafficking of VRUT to
the apical plasma membranes of IMCD cells.

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Fig. 4.
Immunocytochemical localization of AQP2 and VRUT. Immunoperoxidase
labeling of thin sections from inner medullas of Brattleboro rats
treated with DDAVP (B and
D) or vehicle
(A and
C).
A and
B were labeled with anti-AQP2
antibody; C and
D were labeled with anti-VRUT
antibody. Note that DDAVP stimulated a redistribution of AQP2, but not
VRUT, toward the apical plasma membrane. Observations shown were
confirmed in two other pairs of rats (not shown).
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Differential centrifugation.
Differential centrifugation techniques have previously been employed to
study regulated trafficking of AQP2 (6, 13). We used the same protocol
to investigate whether VRUT is regulated by trafficking in the IMCD.
Inner medullas from Brattleboro rats injected intravenously with either
2 nmol of DDAVP (n = 4) or with
vehicle (n = 4) were subjected to
differential centrifugation as previously described (13). The
DDAVP-injected rats showed an increase in urinary osmolality from 240 ± 9 to 682 ± 78 mosmol/kgH2O 60 min after
injection. The vehicle-injected control rats showed urinary
osmolalities of 278 ± 26 mosmol/kgH2O prior to injection
and 356 ± 18 mosmol/kgH2O 60 min after vehicle injection. Figure
5A shows
immunoblots run with the low-density membrane fraction (pellet from
200,000 g spin using supernatant from
17,000 g spin), which contains
predominantly intracellular vesicles. As shown, there was a marked
reduction in AQP2 band density in the low-density membrane fraction
from DDAVP-treated rats relative to vehicle-treated rats (Fig.
5A), indicating a depletion of AQP2
from intracellular vesicles. In contrast, when the same samples were
run for VRUT, there was no evident decrease (Fig.
5B). Figure
6 shows a summary of quantification of the
blots from the differential centrifugation experiments expressed as the
ratio of band density of the high-density fraction (17,000 g) to the band density of the
low-density fraction (200,000 g). As
previously demonstrated (13), this ratio for the AQP2 blots was
markedly increased by DDAVP administration, consistent with vasopressin-induced trafficking to the plasma membrane. However, the
corresponding ratio for VRUT was unchanged. This combines with the
evidence from the surface biotinylation studies and immunocytochemical studies (above) to show that although vasopressin stimulates
trafficking of AQP2 in the IMCD, parallel trafficking of VRUT cannot be
observed.

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Fig. 5.
Differential centrifugation of rat inner medullary membranes showing
effect of DDAVP on abundance of AQP2 and VRUT in low-density membrane
fraction. Immunoblots were carried out using low-density membrane
fraction from vehicle- or DDAVP-treated Brattleboro rats and were
probed with either affinity-purified anti-AQP2
(A) or anti-VRUT antibody
(B). Two micrograms of protein was
loaded in each lane for AQP2 blots, and 15 µg protein per lane was
loaded for VRUT blots.
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Fig. 6.
Quantification of effect of DDAVP on the ratio of band density of the
high-density fraction to the band density of the low-density fraction
(HD:LD). Low-density membrane fraction and high-density membrane
fraction from inner medullas of vehicle-treated
(n = 4) or DDAVP-treated
(n = 4) Brattleboro rats were prepared
by a series of centrifugations as described in
METHODS. Immunoblots were probed with
either affinity-purified anti-AQP2
(A) or anti-VRUT antibody
(B). Band density was analyzed by
scanning densitometry. Ratio of band density of the high-density
fraction (HD) to the band density of low-density fraction (LD) was
significantly increased by DDAVP for AQP2
(P < 0.03, t-test), but there was no significant
(NS) change in VRUT.
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VRUT is not present in immunoisolated AQP2
vesicles. We immunoisolated IMCD vesicles using the
anti-AQP2 antibody to test whether these vesicles contain VRUT. The
starting material was a low-density membrane fraction (200,000 g pellet from the 17,000 g supernatant) from rat inner medullas
that was previously demonstrated to be devoid of plasma membrane
markers (6, 13). Typical results are shown in Fig.
7. In Fig. 7,
left half, is an immunoblot probed
with anti-AQP2. AQP2 normally runs as two bands at 29 kDa (nonglycosylated form) and 38 kDa (glycosylated form) (16). As can be
seen, when vesicles were immunoisolated using anti-AQP2-bearing beads
(left lane), there was a substantial
yield of AQP2. In contrast, when purified preimmune IgG from the same
rabbit was substituted on the beads
(middle lane), there was little or no
yield of AQP2, consistent with lack of substantial nonspecific binding
of AQP2-bearing vesicles to the beads. The
third lane shows the AQP2 present in the initial low-density membrane fraction. Figure 7,
right half, shows an immunoblot run
with the same samples, but probed instead with the anti-VRUT antibody.
VRUT normally runs at 97 kDa when solubilized in SDS as done here and
as seen in the right lane loaded
with the initial low-density membrane fraction prior to the immunoisolation. As can be seen in Fig. 7,
right half, little or no VRUT was
detectable in the eluates from the anti-AQP2 beads (left lane) or the control beads
(middle lane), despite
the fact that much more of the eluates was loaded on this blot than for the blot probed with anti-AQP2. The bands seen just above the 48-kDa
marker are due to the presence of small amounts of reduced IgG heavy
chain that detached from the immunoisolation beads. From this
experiment and three others of identical design (not shown), we
conclude that AQP2-containing intracellular vesicles in the IMCD do not
contain appreciable amounts of VRUT.

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Fig. 7.
Absence of VRUT in immunoisolated AQP2 vesicles. Low-density membrane
fraction from inner medulla was incubated with beads with covalently
bound affinity-purified anti-AQP2 or IgG from preimmune serum purified
by protein A column as described in
METHODS. Proteins were eluted with
Laemmli sample buffer. Immunoblots were probed with either
affinity-purified anti-AQP2 (left)
or anti-VRUT antibody (right). Two
and 10 µl of eluate from beads were loaded for AQP2 and VRUT,
respectively. Third lane of each panel was loaded with low-density
membrane fraction used as starting material for the immunoisolation.
|
|
 |
DISCUSSION |
It is well established that vasopressin dramatically increases the
water and urea permeability of the IMCD epithelium (14, 17, 22) and
does so via increases in intracellular cAMP (27). It is also well
established that the rapid increase in water permeability seen with
vasopressin stimulation (seen within minutes of vasopressin exposure)
is a result of redistribution of AQP2-containing membrane domains
within the collecting duct cell to increase the number of AQP2 water
channels in the apical plasma membrane at the expense of AQP2 in
intracellular vesicles (13, 15, 20, 30). The finding of a substantial
degree of labeling of intracellular vesicles with a rabbit polyclonal
antibody to VRUT (18) raised the possibility that
vasopressin-stimulated increases in urea transport in the IMCD are due
to stimulation of trafficking of VRUT to the plasma membrane, either in
parallel to the trafficking of AQP2 vesicles or perhaps via
translocation of vesicles containing both VRUT and AQP2. In this study,
we have provided several types of evidence that refute this hypothesis.
First, surface biotinylation studies, although demonstrating a marked
increase in AQP2 at the surface of IMCD cells in response to
vasopressin, failed to show any increase in surface labeling of VRUT.
Second, immunocytochemical localization experiments revealed that,
although vasopressin induces a major redistribution of AQP2 labeling in
IMCD cells to the apex of the cells, there was no demonstrable
redistribution of VRUT labeling. Third, differential centrifugation
studies, although revealing a shift of AQP2 out of the low-density
membrane fraction, demonstrated no such shift in the distribution of
VRUT. Finally, VRUT was not present in intracellular vesicles
immunoisolated from the IMCD using a polyclonal antibody to AQP2.
Therefore, we conclude from these studies that the ability of
vasopressin to activate VRUT does not depend on stimulation of
trafficking of VRUT to the cell surface. This conclusion is in
agreement with conclusions from physiological studies in isolated
perfused IMCD segments demonstrating that the time course of urea
permeability changes and water permeability changes in response to
addition or washout of vasopressin can be dissociated under certain
conditions, indicating that different physical processes account for
short-term regulation of urea and water permeability (17). Similar
conclusions could be drawn from the observed action of atrial
natriuretic peptide in the IMCD, which decreases osmotic water
permeability without affecting urea permeability (19). Furthermore, a
similar dissociation of regulation of urea and water transport was
previously demonstrated in the toad bladder (2), a
vasopressin-responsive epithelium that has served as a reliable model
of the mammalian collecting duct.
If the short-term regulation of VRUT by vasopressin does not depend on
VRUT trafficking to the plasma membrane, then what is the mechanism of
the response? One possibility is direct phosphorylation of the urea
transporter which may alter its transport characteristics. The VRUT
protein has been shown to possess several serines and threonines that
are potential targets for phosphorylation by protein kinase A (25).
Preliminary reports have provided evidence suggesting that VRUT is
phosphorylated in response to physiological manipulations (11) and that
serine-499 found in the middle loop of the 929 amino acid VRUT protein
is crucial for activation of VRUT by cAMP (24). However, a functional
role for such phosphorylation in the regulation of VRUT has not been
reported. Another possibility is that regulatory proteins may exist
that may exert vasopressin-mediated regulatory influences on VRUT,
perhaps similar to regulatory proteins involved in cAMP-mediated
regulation of the type 3 Na/H exchanger, NHE3 (31). Such a possibility
has not been explored in detail for VRUT. Chemical cross-linking
studies, however, have demonstrated that VRUT exists in the plasma
membrane of IMCD cells as a protein complex of ~206 kDa (1). Although
this complex is believed to be a VRUT homodimer, the possibility that
additional proteins may exist in the complex has not been ruled out.
The present observations, although providing evidence that
vasopressin-mediated regulation of VRUT does not occur via changes in
VRUT trafficking to the plasma membrane, do not rule out the possibility that other modes of VRUT regulation involve regulated trafficking. For example, urea transport in isolated perfused IMCD
segments is markedly increased by increases in the osmolality of the
fluid bathing the tubule, a process at least in part independent of the
vasopressin-mediated regulatory process (7, 8). Further studies are
needed to address the possible role of VRUT trafficking in this response.
 |
ACKNOWLEDGEMENTS |
We thank Wilford Saul and Annette Rasmussen for expert technical assistance.
 |
FOOTNOTES |
Funding for this study was derived from the intramural budget of the
National Heart, Lung, and Blood Institute (National Institutes of
Health Project Z01-HL-01282-KE to M. A. Knepper), the Karen Elise
Jensen Foundation (to S. Nielsen), and the Novo Nordic Foundation (to
S. Nielsen).
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.
Address for reprint requests and other correspondence: M. A. Knepper,
National Institutes of Health, Bldg. 10, Rm. 6N260, 10 Center Drive MSC
1603, Bethesda, MD 20892-1603 (E-mail:
knep{at}helix.nih.gov).
Received 5 October 1998; accepted in final form 3 December 1998.
 |
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