From the Department of Cell Physiology, Nijmegen
Center for Molecular Life Sciences, UMC St. Radboud, Nijmegen, 6500 HB,
The Netherlands, the § Program in Membrane Biology
and Renal Unit, Department of Medicine, Massachusetts General Hospital
and Harvard Medical School, Charlestown, Massachusetts 02129, and the
** Department of Cell Biology, University of Utrecht, 3584 CX
Utrecht, The Netherlands
Received for publication, July 22, 2002, and in revised form, September 13, 2002
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ABSTRACT |
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In renal collecting ducts, vasopressin
increases the expression of and redistributes aquaporin-2 (AQP2) water
channels from intracellular vesicles to the apical membrane, leading to
urine concentration. However, basolateral membrane expression of AQP2, in addition to AQP3 and AQP4, is often detected in inner medullary principal cells in vivo. Here, potential mechanisms that
regulate apical versus basolateral targeting of AQP2 were
examined. The lack of AQP2-4 association into heterotetramers and the
complete apical expression of AQP2 when highly expressed in Madin-Darby canine kidney cells indicated that neither heterotetramerization of AQP2 with AQP3 and/or AQP4, nor high expression levels of AQP2 explained the basolateral AQP2 localization. However, long term hypertonicity, a feature of the inner medullary interstitium, resulted
in an insertion of AQP2 into the basolateral membrane of Madin-Darby
canine kidney cells after acute forskolin stimulation. Similarly, a
marked insertion of AQP2 into the basolateral membrane of principal
cells was observed in the distal inner medulla from normal rats and
Brattleboro rats after acute vasopressin treatment of tissue slices
that had been chronically treated with vasopressin to increase
interstitial osmolality in the medulla, but not in tissues from
vasopressin-deficient Brattleboro rats. These data reveal for the first
time that chronic hypertonicity can program cells in vitro
and in vivo to change the insertion of a protein into the
basolateral membrane instead of the apical membrane.
The renal collecting duct is involved in urine concentration via a
process that is regulated by the antidiuretic hormone arginine vasopressin (AVP).1 After
binding to its receptor on target cells in the kidney collecting duct,
AVP initiates an intracellular signaling cascade that increases cytosolic cAMP and calcium levels (1-3). Upon activation of protein kinase A, aquaporin-2 (AQP2) is phosphorylated and is rapidly redistributed from intracellular vesicles to the apical membrane of
collecting duct principal cells. Driven by an osmotic gradient, water
then moves into the cell apically via AQP2, and exits across the
basolateral membrane via AQP3 and/or AQP4 (4, 5). In addition to this
short term effect, increased circulating AVP levels also lead to an
increased expression of AQP2 protein, which is mediated via a cAMP
responsive element in the AQP2 gene promoter (6-8). Additionally, the
expression of AQP3 is increased, but the level of AQP4 remains
unchanged (9, 10).
Although the majority of AQP2 is located in the apical plasma membrane
under "steady-state" conditions in normally hydrated animals,
immunocytochemical studies have shown that AQP2 antigenicity can also
be detected in the basolateral plasma membrane of collecting duct
principal cells in these rats. This basolateral staining pattern
becomes more prominent with increased AVP levels or water deprivation
in rats, and is especially prominent in the principal cells of the
inner medulla (11).
The factors and mechanisms that determine the partitioning of AQP2
between the apical and basolateral membrane of principal cells in the
kidney are unknown, and the goal of the present study was to
investigate this process further. Three hypotheses that might explain
basolateral AQP2 targeting were tested using renal tissue, as well as
oocytes and cultured renal epithelial cells heterologously expressing
AQP2, AQP3, and/or AQP4. We considered (a) that
heterotetramer formation among differentially targeted aquaporins might
be involved, (b) that higher levels of AQP2 expression both
in vivo and in vitro might cause AQP2 to traffic
to the membrane in both apical and basolateral pathways, and
(c) that a hypertonic environment such as that found in the
renal medullary interstitium could play a role. Our data indicate that
long term exposure of cells to hypertonicity primes epithelial cells to
insert AQP2 into the basolateral membrane upon acute stimulation with
AVP and/or forskolin.
Plasmids--
To stably express AQP2 in MDCK cells in high
amounts, the pBS-AQP2 (12) construct was digested with XbaI,
blunted, and cut with HindIII. Subsequently, the full-length
human AQP2 cDNA was isolated and ligated into the blunted
BglII site and HindIII site of the eucaryotic
expression vector pCB6 (13), thereby generating pCB6-AQP2. To generate
the oocyte expression construct pT7Ts-AQP4, a pBluescript vector
containing the entire cDNA of human AQP4a (pBS-AQP4a (14)) was
digested with EcoRV and XbaI, full-length AQP4a
cDNA was isolated and cloned into the EcoRV and
SpeI sites of pT7Ts. The pSPORT-AQP3 construct, encoding
full-length rat AQP3 (15), was kindly donated by M. Echevarria,
Sevilla, Spain.
AQP Expression in Oocytes--
Xenopus
laevis oocytes were isolated and cultured as described (16).
To generate AQP2, AQP3, and AQP4 cRNAs, pT7Ts-AQP2 (17) was linearized
with SalI, whereas pSPORT-AQP3 and pT7Ts-AQP4 were
linearized with XbaI. Synthesis of G-capped cRNA transcripts and determination of their integrity and concentration were done as
described (17). Two days after injection, oocytes were subjected to
assays described below.
Culturing and Transfection of MDCK Cells--
All cells used in
this study were derived from MDCK type I cells (18) and were grown in
Dulbecco's modified Eagle's medium supplemented with 5% (v/v) fetal
calf serum at 37 °C in 5% CO2. Transfected cells used
were those stably expressing human AQP2 (Wt10 cells (19)). To obtain
MDCK cells expressing high levels of AQP2, MDCK cells stably
transfected with the pCB6-AQP2 construct were generated as described
(20).
To test the effect of hypertonicity on the steady state localization of
AQP2, cells were seeded on 1.13-cm2 filters at 3.0 × 105 cells/cm2 and grown in medium for 8 h.
Subsequently, the osmolarity of the medium was increased from 297 to
672 mosmol/kg of H2O in three steps of 125 mosmol/kg of
H2O at t = 8, 24, and 32 h using NaCl, sucrose, raffinose, or mannitol as osmolytes. The MDCK cells were analyzed at 3 days after seeding, which meant that the cells were exposed to hypertonicity for about 64 h (starting 8 h after
seeding) and to a full hypertonicity (672 mosmol/kg) for 40 h.
Control cells were seeded at 1.5 × 105 cells per
cm2. Three days after seeding, the cells were directly
prepared for confocal laser scanning microscopy analysis or first
incubated for 45 min in hypertonic medium containing 1 × 10 Isolation of Membranes--
Total membranes of oocytes were
isolated as described previously (16). For membranes of renal cells,
kidneys were removed from control or 24-h water-deprived rats and
homogenized in 5 ml of HbA per 350 mg of wet tissue. After removing
nuclei and unbroken cells by centrifugation at 1000 × g at 4 °C for 10 min, each supernatant was centrifuged at
100,000 × g for 1 h to pellet the membranes.
Subsequently, oocyte (20 µl/oocyte) and kidney (5 ml/sample)
membranes were incubated for 30 min at 37 °C in solubilization buffer (4% Na-desoxycholate, 20 mM Tris (pH 8.0), 5 mM EDTA, 10% glycerol, 1 mM
phenylmethylsulfonyl fluoride, 5 µg/ml leupeptin and pepstatin) to
dissolve the membranes. Next, undissolved membranes were removed with a
centrifugation step at 100,000 × g at 4 °C for
1 h.
Antibodies--
For analysis of AQP2, rabbit (17) and guinea pig
(21) antibodies, raised against a synthetic peptide corresponding to the last 15 amino acids of rat AQP2 were used. AQP3 antibodies were
raised against a peptide based on the predicted 15 COOH-terminal amino
acids of rat AQP3, which was conjugated to keyhole limpet hemocyanin
(17). By passing whole serum over a bovine serum albumin-AQP3-coupled
Affi-Gel 15 column, affinity purified antibodies were isolated
(Amersham Biosciences, Uppsala, Sweden). Antibodies were eluted
with 0.1 M glycine (pH 2.8) and directly neutralized.
To generate AQP4 antibodies, a fragment of 389 nucleotides encoding the
entire C-terminal tail of AQP4a was isolated from pBS-AQP4a, by
digestion with EcoRI, and cloned into the EcoRI site of the pGEX1 vector (Amersham Biosciences). After transformation of DH5 Immunoprecipitations and Sucrose Gradient
Analysis--
Immunoprecipitations were performed as previously
described (16). Samples (300 µl) of solubilized renal and oocyte
membranes were loaded onto a 3.2-ml 5-17.5% linear sucrose gradient
(in 20 mM Tris (pH 8.0), 5 mM EDTA, 0.1%
Triton X-100, 1 mM phenylmethylsulfonyl fluoride, 5 µg/ml
leupeptin and pepstatin). Gradients were centrifuged at 100,000 × g in a Beckman SW-60 rotor for 16 h at 8 °C. After centrifugation, 200-µl fractions, annotated A-Q, were taken from the
top and 15-µl samples were analyzed by immunoblotting. As size
markers, bovine serum albumin (68 kDa), phosphorylase b (97 kDa), yeast alcohol dehydrogenase (150 kDa), and Immunoblotting--
To prepare Wt10 cell lysates, cells from a
1.13-cm2 filter were incubated in 170 µl of Laemmli
buffer for 30 min, after which the DNA was sheared by pulling the
sample through a 30-gauge needle 3 times. When indicated,
N-linked sugar groups were removed with peptide
N-glycosidase F according to the manufacturer (New
England Biolabs, Beverly, MA). Samples were denatured for 30 min at
37 °C in Laemmli buffer and subjected to 13% SDS-PAGE
electrophoresis. Proteins were transferred to polyvinylidene difluoride
membranes (Millipore Corp., Bedford, MA) as described (17). For
immunoblot analysis, AQP3 antibodies were biotinylated with Sulfo-NHS
biotin (Pierce) according the manufacturer. Membranes were incubated with 1:4000 diluted guinea pig AQP2 antibodies in TBST (20 mM Tris, 140 mM NaCl, 0.2% Tween 20 (pH 7.6)),
1:3000 diluted rabbit AQP2 antibodies, 1:1000 biotinylated rabbit AQP3
antibodies or guinea pig AQP4 antibodies, or 1:1000 mouse monoclonal
AQP1 antibodies (22), which were all diluted in TBST with 1% nonfat
dried milk. As secondary antibodies, goat anti-rabbit (1:5000 in TBST;
Sigma), sheep anti-mouse (1:2000 in TBST; Sigma), goat anti-guinea pig (1:10,000 in TBST; Sigma), or streptavidin (1:8000 in TBST with 1%
nonfat dried milk; Jackson ImmunoResearch, West Grove, PA), all coupled
to horseradish peroxidase, were used. Proteins were visualized using
enhanced chemiluminescence (Pierce).
Confocal Laser Scanning Microscopy on Cell
Cultures--
Preparation of MDCK cells for CLSM analysis was done as
described (20). The filters were incubated overnight with 1:100 diluted
affinity purified rabbit anti-AQP2, followed by an incubation with
1:100 diluted Alexa-594-coupled goat anti-rabbit antibodies (Molecular
Probes, Eugene, OR). When co-stained for AQP3, 1:100 dilutions of
affinity purified rabbit anti-AQP3 and guinea pig anti-AQP2 antibodies
were used, followed by 1:100 dilutions of affinity purified goat
anti-rabbit or guinea pig IgGs, coupled to Alexa-488 or Alexa-594
(Molecular Probes, Leiden, The Netherlands), respectively. Using Adobe
Photoshop, all signals were maximally expanded over the intensity
range. All figures shown are representative images of at least three
independent experiments.
Whole Animal Studies--
Animal experiments were approved
by the Institutional Committee on Research Animal Care of the
Massachusetts General Hospital, in accordance with the NIH guide
for the Care and Use of Laboratory Animals. Male adult Sprague-Dawley
and homozygous, vasopressin-deficient Brattleboro rats were purchased
from Harlan Sprague-Dawley (Indianapolis, IN).
Chronic Vasopressin Treatment of Brattleboro Rats--
Adult
male Brattleboro homozygous rats weighing 300-360 g were used to study
the effects of chronic AVP pretreatment on the polarity of AQP2
membrane insertion after acute AVP treatment in the tissue slice
preparation. The Brattleboro rats were divided into two groups (3 animals/group). One group of animals was not treated (control) and the
other group received the vasopressin analogue
1-desamino-8-D-arginine vasopressin (dDAVP) at a rate of 5 µl/h via osmotic minipumps as described (23). This dose has been
shown to produce comparable plasma vasopressin levels to those
achieved in normal rats during water restriction (24). All Brattleboro
rats had free access to food and water for the duration of the studies.
A 5500 Wescor vapor pressure osmometer (Wescor, Logan, UT) was used to
measure the urine osmolarity. Urine samples were collected by "clean
catch" before and after the implantation of the minipumps, and at
various times during the 11-day treatment period.
Acute Effects of Vasopressin in Control Rat Kidney
Slices--
Adult Brattleboro rats, pretreated for 11 days (see
above), and male Sprague-Dawley rats were anesthetized with an
injection of sodium pentobarbital (65 mg/kg; intraperitoneally). Both
kidneys were removed from the rats, cut into ~2-3 mm thick slices
using a razor blade, and quickly placed in Hanks' balanced salt
solution (pH 7.4) at 37 °C equilibrated with 5%
CO2, 95% O2. Slices of 0.5 mm were then
cut as described (25). The thin slices were first incubated at 37 °C
for 15 min in equilibrated Hanks' balanced salt solution only (vial A)
to wash out endogenous AVP and cause internalization of cell-surface
AQP2. These slices were then simultaneously transferred to fresh vials
containing either arginine AVP (10 nM) plus forskolin (10 µM) (Sigma) in Hanks' balanced salt solution (vial B) or
to vials containing Hanks' balanced salt solution alone (vial C) for
15 min at 37 °C. Next, all slices were fixed by immersion in
periodate-lysine containing 4% paraformaldehyde as described (25).
Slices were then rinsed several times in phosphate-buffered saline and
stored in phosphate-buffered saline containing 0.02% NaN3
at 4 °C. To determine the localization of AQP2 prior to treatment
with AVP plus forskolin, some slices from vial A were fixed
immediately. Additionally, some kidney slices from Brattleboro rats
were fixed in paraformaldehyde immediately after preparation
(i.e. before (dD)AVP washout).
Immunocytochemistry on Tissue Slices--
Kidney cryosections (4 µm thick) were prepared as described (26). After rehydration in
phosphate-buffered saline for 15 min, sections were treated with 1%
SDS for antigen retrieval (27). Blocking and immunostaining of the
sections was done as described (26), except that affinity purified
antiserum raised against the second extracellular loop of AQP2 (28) and
goat anti-rabbit IgG conjugated to indocarbocyanine (CY3) (2 µg/ml;
Jackson ImmunoResearch) were used as primary and secondary antibodies,
respectively. Sections were mounted in Vectashield diluted 1:1 in 1.5 M Tris-HCl (pH 8.9). Sections were examined using a Bio-Rad
Radiance 2000 confocal laser scanning microscope (Bio-Rad Microscience
Ltd., Hemel Hempstead, United Kingdom) or a Nikon 800 epifluorescence
microscope coupled to a Hamamatsu Orca CCD camera and IP Lab Spectrum
software (Scanalytics, Vienna, VA).
Image Quantification Analysis--
To create an objective index
for basolateral versus apical expression of AQP2, the
integrated optical density (IOD) of equal basolateral or apical
membrane segments within a fixed square area was determined using
Image-Pro Plus analysis software (Media Cybernetics, Silver Spring,
MD). Background IOD values, determined within the nucleus area of the
particular cell, were subtracted from the obtained basolateral and
apical IOD values. The B/Asorting index is defined
as the IOD of the basolateral membrane segment divided by the IOD of
the apical membrane segment. Of 8 or 15 independent cells (indicated)
of representative images, and 3 segments of the basolateral and apical
membrane per cell, the mean B/Asorting index ± S.E.
was determined. The significance of a change in sorting index between
two experimental settings was determined with an independent two
population t test.
AQP2, AQP3, and AQP4 Do Not Form Heterotetramers
Some membrane proteins that are mostly expressed as homomultimeric
proteins can also form heteromultimeric complexes consisting of
related, but distinct, subunits (29, 30). The function, trafficking,
and regulation of such heteromeric complexes, such as the heterodimeric
GABAB1-2 receptor (31),
Aquaporin-3 Is Expressed as Tetramers--
To be able to address
this hypothesis, it is essential that AQP2, AQP3, and AQP4 are all
expressed as tetramers and that the tetramers are not disrupted by the
membrane isolation and extraction procedure. Whereas it has been shown
that AQP2 and AQP4 form homotetramers (16, 37), this has not been
reported for AQP3. To allow these analyses, antibodies were raised
against AQP3 and AQP4, affinity purified, and tested for their AQP
specificity. Immunoblotting of total membranes from AQP2-, AQP3-, or
AQP4-expressing oocytes revealed that each of these antibodies
specifically recognized the AQP against which it was raised (not
shown). Next, membranes of oocytes expressing AQP2, AQP3, or AQP4 were
isolated, solubilized, and sedimented through a sucrose gradient.
Immunoblotting of fractions taken from these gradients revealed that
AQP3 peaked in fraction J (Fig. 1)
between the 97- and 150-kDa marker proteins that were run in parallel.
Because an AQP3 monomer has a calculated molecular mass of 31.4 kDa,
the observed sedimentation is consistent with the presence of an AQP3
homotetramer. AQP4 bands of 32 and 34 kDa were obtained, which are, as
shown before (38), derived from the use of alternative translational
starting methionines (M1 and M23), both of which are contained in the
AQP4 cDNA construct used here. AQP2 and AQP4, with monomeric
molecular masses of 29 and 32/34 kDa, respectively, also peaked in
fraction J, which also supports the presence of homotetramers. These
data revealed that, besides AQP2 and AQP4, AQP3 is also expressed as
homotetramers and that the tetrameric structure remains intact upon
solubilization of membranes with desoxycholate.
Do Aquaporins Form Heterotetramers?--
To investigate whether
AQP2, AQP3, and AQP4 form heterotetramers in vivo, rats
either received water ad libitum, or were water deprived for
24 h to increase the expression of AQP2 and AQP3, and to maximize
basolateral expression of AQP2 (11). Total kidney membranes were
isolated, solubilized, and subjected to AQP-specific immunoprecipitation. As shown in Fig.
2A, none of the other AQPs co-precipitated with the immunoprecipitated AQP. However, AQP2, AQP3
and, to a lesser extent, AQP4 were readily detectable in the lanes
containing total kidney membranes. These results indicate that none of
the AQPs form heterotetramers. Note that only the 32-kDa isoform of
AQP4 was detected in these experiments, which is more abundant in the
kidney (37).
In collecting duct cells of water-deprived rats, the majority of AQP2
is still located in the apical membrane and a relative small number of
AQP2/AQP3 or AQP2/AQP4 heterotetramers might have been overlooked.
Therefore, to examine this further, the three aquaporins were
co-expressed in oocytes, where they are all located in the plasma
membrane (15, 17, 38). Immunoblotting of the immunoprecipitates for the
different AQPs clearly demonstrated that the AQPs did not
co-precipitate, even though each AQP was expressed at a high level in
this system (Fig. 2B, TM). These data show that AQP2, AQP3,
and AQP4 are not able to form heteroligomers and, therefore, strongly
suggest that the basolateral localization of AQP2 is not a result of
heterotetramerization of AQP2 with AQP3 or AQP4. The ~32-kDa AQP3
band on blots disappeared upon digestion with peptide
N-glycosidase F, indicating that this band corresponded to
glycosylated AQP3 (not shown).
Increased Expression of AQP2 Does Not Lead to Its Basolateral
Localization
Although most AQP2 is still found in the apical membrane,
basolateral expression of AQP2 is especially prominent in antidiuresis. Because in this condition AQP2 expression is increased (6, 11), we
speculated that this might lead to expression in the basolateral
membrane because of saturation of the apical sorting pathway, and
"overflow" of AQP2 into the basolateral pathway. This hypothesis
was tested using MDCK cells that express AQP2 at moderate levels,
driven by an SV40 promoter (Wt10 cells), or at high levels driven by a
strong cytomegalovirus promoter. As previously reported (19), confocal
microscopy showed that cells with a moderate expression level inserted
AQP2 into the apical membrane upon stimulation with forskolin (Fig.
3A). Similarly, cells with
high AQP2 expression also inserted AQP2 apically (Fig. 3B).
Semiquantification of the AQP2 expression in the basolateral versus apical membrane in Wt10 cells resulted in a
B/Asorting index of 0.07 ± 0.00 (n = 8), which indicated that at these detection levels nearly 20 times more
AQP2 was present in the apical than basolateral membrane. In the cells
in which AQP2 expression was derived from the cytomegalovirus promoter,
no basolateral membrane AQP2 expression was detected, whereas the AQP2
expression in the apical membrane was saturated. These results indicate
that basolateral localization of AQP2 is probably not because of
saturation of the apical sorting pathway, at least in this culture
system.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
5 M forskolin to induce translocation of
AQP2 to the plasma membrane (19).
bacteria with this construct and induction of protein expression with isopropyl-1-thio-
-D-galactopyranoside,
the soluble glutathione S-transferase-AQP4 fusion protein
was isolated using glutathione-Sepharose 4B (Amersham Biosciences).
Antibodies raised in rabbit and guinea pig were affinity purified as
described above.
-amylase (200 kDa)
were centrifuged in a parallel tube. To determine the peak fractions of
marker proteins, fractions were analyzed by SDS-PAGE, after which the
proteins were visualized using Coomassie Brilliant Blue staining.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-amino-3-hydroxy-5-methyl-4-isoxazole propionate receptor
(32), and the opioid receptor (33) are different from that of homomeric
complexes. Within the AQP family of proteins, altered trafficking of
wt-AQP2 has been found upon formation of heterotetramers with the AQP2
mutants AQP2-E258K or AQP2-delG, and this provided the explanation for
the occurrence of a dominant form of NDI (16, 34). Whereas all AQPs
tested up to now (AQP0-AQP2 and AQP4) are expressed as homotetramers (16, 35-37), the possibility existed that some AQP2 might be targeted
to the basolateral membrane because of the formation of heterotetramers
with AQP3 and/or AQP4, which are both basolateral membrane proteins.
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Fig. 1.
AQP3 is expressed as a homotetramer. Two
days after injection, total membranes of oocytes expressing AQP2, AQP3,
or AQP4 were isolated, solubilized in desoxycholate, and subjected to
sucrose gradient centrifugation. Fractions of 200 µl were taken, of
which fractions C to P (indicated) were immunoblotted for AQP2, AQP3,
or AQP4. AQP3 fractions were treated with peptide
N-glycosidase-F before loading. To estimate the mass of the
AQP complexes, sedimentation marker proteins, bovine serum albumin (68 kDa), phosphorylase b (97 kDa), yeast alcohol dehydrogenase
(150 kDa), and -amylase (200 kDa) were sedimented in a parallel
tube. Their peak fractions are indicated at the bottom. The
mass of a marker protein (in kDa) is given on the left. All
aquaporins sedimented between the 97- and 150-kDa markers, indicating a
tetrameric assembly, which was maintained throughout the extraction and
centrifugation procedure.
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Fig. 2.
AQP2, AQP3, and AQP4 do not form
heterotetramers in vivo and in vitro.
A, in vivo. Membranes of renal medulla from
control (C) or water-deprived (D) rats were
isolated, solubilized in desoxycholate, and subjected to
immunoprecipitation (IP) with AQP2-, AQP3-, or AQP4-specific
antibodies. The proteins of the precipitates were separated by SDS-PAGE
and immunoblotted for AQP2 (upper panel), AQP3 (middle
panel), or AQP4 (lower panel). Proteins from total
membranes (TM) of renal medullas were taken as controls.
B, in vitro. Oocytes were injected with a
mixture of cRNAs encoding AQP2, AQP3, and AQP4. Two days after
injection, membranes were isolated, solubilized in desoxycholate, and
analyzed as under A. Proteins from total membranes of
noninjected (C) or AQP2-4 expressing oocytes were taken as
negative and positive controls, respectively. In addition to AQP2 and
AQP4, unglycosylated (uAQP3) and glycosylated AQP3
(gAQP3) are indicated. The mass of a marker protein (in kDa)
is given on the left. These data indicate that AQP2, AQP3,
and AQP4 do not form heterotetramers in renal medullas and in oocytes
expressing all three aquaporins, because only the aquaporin that was
specifically immunoprecipitated is detectable in the respective
lanes.
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Fig. 3.
Intracellular localization of AQP2 with
overexpression. X-Z images of AQP2 localization in MDCK cells with
moderate expression of AQP2 (Wt10 cells; panel A) and pooled
colonies of MDCK expressing high levels of AQP2 (derived from pCB6-AQP2
construct; panel B). The cells were grown to confluence in
normal medium, treated with forskolin, fixed, and subjected to AQP2
immunocytochemistry and confocal laser scanning microscopy. For both
figures, identical CLSM settings were used.
Hypertonicity Results in the Basolateral Localization of AQP2 in MDCK Cells
Because in antidiuresis, the tonicity of the inner medullary
collecting duct is increased (39), we tested whether basolateral targeting of AQP2 could be induced by hypertonicity. The osmolarity of
the medium of Wt10 cells was gradually increased with NaCl from 297 to
672 mosmol/kg of H2O over a 3-day period. Cells were either
untreated or exposed to forskolin, fixed, and examined by
immunocytochemistry. Confocal analysis revealed that without forskolin,
AQP2 was mainly localized in vesicles (Fig.
4, C1 and H1). With
forskolin stimulation, however, hypertonicity indeed resulted in a
pronounced basolateral localization of AQP2 (Fig. 4, H2),
which was underscored by its co-localization with AQP3 (Fig. 4,
H, AQP3). As reported (40), AQP3 appeared to be
induced in its expression in MDCK cells by hypertonicity (not shown)
and has shown to be localized in the basolateral membrane (40, 41). The
B/Asorting index was 1.02 ± 0.04 (n = 8; Fig. 4, H2), which indicated that with hypertonicity the
basolateral versus apical expression of AQP2 was about
14-fold increased compared with Wt10 cells grown under isotonic
conditions (0.07 ± 0.00; n = 8; p < 0.001; Fig. 4, C2). To further test whether this was
osmolyte-dependent, Wt10 cells were treated as above, using
different sugars as osmolytes. Confocal analysis of these cells and
subsequent determination of B/Asorting indexes revealed
that compared with cells grown under isotonic conditions, basolateral
versus apical expression of AQP2 was 15-17-fold increased
for mannitol (index of 1.20 ± 0.14; n = 8),
sucrose (1.08 ± 0.04; n = 8), or raffinose
(1.22 ± 0.05; n = 8) upon forskolin treatment
(p < 0.001; shown for mannitol in Fig. 4,
H3).
|
Basolateral Localization of AQP2 in Kidney Principal Cells in Situ
Previous studies have demonstrated that AQP2 can be detected on the basolateral plasma membrane of collecting duct principal cells in situ, and that this basolateral location is (a) more prominent in the inner medulla and (b) increased after vasopressin treatment or dehydration. To examine this further, experiments on normal rats and vasopressin-deficient Brattleboro rats were performed.
Effect of Acute Vasopressin Treatment on AQP2 Distribution in
Normal Rat Kidney Slices--
Kidney slices were prepared from
normally hydrated Sprague-Dawley rats, and were incubated in
vitro with and without vasopressin/forskolin. Confocal analysis
revealed a heterogeneous pattern of AQP2 distribution in collecting
duct principal cells that was different in various regions of the
kidney. After removal from the animal and incubation in
vitro in buffer alone to wash out endogenous vasopressin, plasma membrane staining was considerably reduced (Figs.
5, A and C) compared with tissues that had been exposed to vasopressin/forskolin (Fig. 5, B and D). However, a remarkable
difference in the polarity of AQP2 insertion was seen between the
proximal third and the distal portion of the inner medullary collecting
duct. As seen in Fig. 5B, a strong apical staining was
induced after 15 min treatment in the initial portion of the inner
medulla, although basolateral staining was also detectable
(B/Asorting index of 0.39 ± 0.01; n = 15). In more distal regions of the collecting duct, however, the
basolateral over apical staining was more than 2-fold higher than in
the initial portion (0.87 ± 0.08; n = 15; p < 0.001; Fig. 5D). In the inner stripe of
the outer medulla, AQP2 staining was predominantly apical after
AVP/forskolin treatment (not shown), as we have previously described
(26). These data support the hypothesis that hypertonicity could be
involved in determining the polarity of AQP2 distribution, because the
most intense basolateral staining was seen in the region of the inner medulla in which interstitial osmolarity is the highest.
|
Effect of Acute AVP Stimulation on AQP2 Distribution in Kidney
Slices from Brattleboro Rats--
To test this hypothesis further, we
used vasopressin-deficient homozygous Brattleboro rats, which are
unable to concentrate their urine, and in which the interstitial
osmolarity is reduced compared with normal rats (42). The acute AQP2
insertion response to AVP/forskolin was compared in tissue slices from
control, diuretic Brattleboro rats and from Brattleboro rats whose
concentration defect had been "corrected" by administration of
dDAVP by osmotic minipump for 11 days. As shown in Fig.
6A, AQP2 was located at the
apical plasma membrane of principal cells from the inner medulla of
diuretic Brattleboro rats, although a granular staining was also
detected in the basolateral pole of some cells. After acute stimulation
of tissue slices from the group of rats chronically treated with dDAVP
prior to the in vitro experiment, a different AQP2 staining
pattern was found. In addition to some apical staining, a very marked
basolateral staining was induced, with the staining being especially
pronounced along the lateral aspects of principal cells (Fig.
6C). This lateral staining was especially evident in
tangential sections of tubules in which a bright honeycomb staining
pattern, indicative of basolateral AQP2 insertion, was seen in the
dDAVP-treated rats (Fig. 6D), but not the untreated rats
(Fig. 6B). These data also indicate that the differential polarized insertion of AQP2 between the two groups resulted from an
effect of chronic dDAVP treatment, and did not result from an acute
effect of osmolarity, because the osmolarity of the buffer in which the
slices were incubated was the same (isotonic) for all experimental
groups.
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DISCUSSION |
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Basolateral Localization of AQP2 in MDCK Cells-- The present study was designed to investigate potential factors or mechanisms that might be involved in determining basolateral AQP2 localization. We clearly showed that, besides AQP2 and AQP4, AQP3 is also expressed as a homotetramer. However, while co-expressed in the same cells, AQP2, AQP3, and AQP4 appeared not to co-precipitate, even when overexpressed in Xenopus oocytes. Also, immunoprecipitation of AQP2 from Wt10 cells treated with hypertonic medium, a condition that AQP2 and AQP3 co-localize and at which endogenous AQP3 expression is induced, did not result in co-precipitation of AQP3 (not shown). No AQP4 expression was detected in Wt10 cells treated with hypertonic medium. Although we cannot rule out the possibility that in contrast to AQP2-4 homotetramers, AQP2/3 or AQP2/4 heterotetramers disintegrate in 4% desoxycholate, our data strongly indicate that the basolateral routing of AQP2 in principal cells is probably not because of the formation of heterotetramers of AQP2 with basolaterally targeted AQP3 or AQP4. In addition, analysis of MDCK cells expressing high levels of AQP2 driven by the cytomegalovirus promoter did not reveal any detectable staining of the basolateral membrane, which indicates that overexpression of AQP2 is also not a likely cause for its basolateral targeting. This finding is consistent with the fact that basolateral AQP2 expression in the kidney is segment-specific, and is not readily seen in the outer medulla, despite an increase in AQP2 expression levels in all collecting duct segments during dehydration or chronic vasopressin treatment.
Hypertonicity, however, appeared to alter the trafficking of AQP2 in epithelial cells, because AQP2-expressing MDCK cells grown in hypertonic medium for nearly 3 days showed a 14-18 times increase in basolateral over apical membrane insertion of AQP2 after acute forskolin stimulation compared with control cells grown in isotonic medium. In fact, this change in ratio is even underestimated, because to obtain a detectable apical membrane signal for AQP2 in hypertonic Wt10 cells, the AQP2 signal in the basolateral membrane was often saturated. In contrast to AQP2, the distribution of the basolateral protein AQP3 was not altered by growth in hypertonic media.
Basolateral Localization of AQP2 in the Kidney-- The role of hypertonicity in AQP2 trafficking was also evaluated using an established in vitro kidney slice model in which AVP-induced AQP2 membrane insertion has been previously demonstrated (25, 26). In kidney slices from normally hydrated Sprague-Dawley rats, treatment with a AVP/forskolin mixture resulted a more than 2-fold increase of the AQP2 B/Asorting index of principal cells of the distal inner medulla compared with the proximal inner medulla of the same tissue slice. The first segment is the kidney region, which, in vivo, is exposed to the highest interstitial osmolarity (up to 1200 mosmol/kg). We have previously shown that in the outer medulla, regulated AQP2 insertion is almost exclusively apical (26). Thus, the polarity of acute insertion of AQP2 in medullary collecting ducts is segment specific, and can be correlated with the level of hypertonicity to which the tubule segments had been exposed in vivo.
The data from dDAVP-treated and nontreated Brattleboro rats support this contention. When tissue slices from control, vasopressin-deficient Brattleboro rats were challenged in vitro with an AVP/forskolin mixture, apical insertion was seen, although some staining remained at the basolateral pole of the cells. This situation resembled that found in the proximal inner medulla of normal rats. However, the striking basolateral AQP2 insertion detected in the distal inner medulla of slices from Brattleboro rats after chronic pretreatment with dDAVP supports the idea that a high interstitial osmolarity is necessary for this process to occur. Because only tubules from the distal inner medulla showed this marked basolateral staining, a direct effect of dDAVP on this process is unlikely, because principal cells in all kidney regions were exposed to circulating dDAVP.
In addition, even after ~45 min of bathing in an isotonic incubation medium, the in vivo environment modulates the subsequent response of collecting ducts in excised tissue slices. This clearly indicated that the effect is not a direct and rapid effect of exposure of cells to an increased osmolarity, but probably reflects a longer term adaptation of the cell to the high interstitial osmolarity that occurs in vivo (or nearly 3 days adaptation of MDCK cells to hypertonicity in vitro).
Cellular Changes with Hypertonicity-- In the short term, a hypertonicity induced cell volume decrease is followed by a regulated volume increase, which is achieved by an influx of inorganic solutes. Over the long term, cells adapt to increased extracellular osmolarity by an intracellular accumulation of organic osmolytes, such as myo-inositol, glycerophosphorylcholine, taurine, betaine, and sorbitol (43-45), which is brought about by an increased expression of their transporters. This slow process of adaptation protects the cells from growth retardation and apoptosis, which has been reported to occur with acute high salt challenge in inner medullary collecting duct cells (46). Indeed, acute treatment of our MDCK cells with 375 mosmol of extra NaCl resulted in a significant loss of cells, which precluded the analysis of the short term effect of a hyperosmotic/tonic agent on the localization of AQP2. In addition to the accumulation of inorganic or organic solutes, the process of adaptation to hypertonicity also affects basic cellular functions, such as alterations in cell metabolism, cell growth and differentiation, transcriptional activation, or repression of specific genes and reorganization of cellular structure (via the cytoskeleton; Ref. 47).
These different forms of adaptation to a hypertonic environment are likely to play a role in the redistribution of AQP2 from the apical to the basolateral membrane. In Wt10 cells, the basolateral translocation was observed with membrane-impermeant solutes (NaCl, sucrose, and raffinose), which indicated that hypertonicity (osmotic gradient) and not simply hyperosmolarity (increased solute content) induced the effect. The requirement for hypertonic, rather than hyperosmolar, conditioning provides some insight into upstream events, as it suggests that perturbation of the cell membrane or cytoskeleton may be necessary for AQP2 translocation to occur. Indeed, several studies have reported that osmomechanical stress can activate numerous membrane-associated events including activation of plasma membrane ion channels, calcium signaling events, and phosphatidylinositol turnover (48), which are known to play critical roles in membrane trafficking and cytoskeleton reorganization (49). Of particular relevance to our present observations is that in renal proximal tubule cells, hypoosmotic stress induces exocytosis followed by endocytosis of vesicles at the basolateral membrane and a basolateral-to-apical translocation of vesicles and ion channels (48). Possibly, hyperosmotic stress has the opposite effect of inducing an apical to basolateral translocation of vesicles and associated membrane proteins.
Physiological Relevance of Basolateral AQP2 in the Renal Medulla-- In mammals, only cells of the renal medulla are subject to substantial fluctuations in extracellular solute concentrations, because in antidiuresis, medullary cells are confronted with high extracellular NaCl and urea concentrations, which then fall rapidly during the onset of water loading (50). Our present model does not, therefore, explain the basolateral localization of AQP2 in connecting tubules, which are not exposed to significant hypertonicity.
Whereas other factors may be involved in the targeting process, our study suggests that an increased hypertonicity of the renal medulla might be fundamental to the pronounced basolateral localization of AQP2 in principal cells in antidiuresis. The physiological relevance of this redistribution remains unclear, but three scenarios are possible. First, basolateral AQP2 insertion might be required to increase the water permeability of the basolateral membrane under high flow conditions, despite the presence of AQP3 and AQP4. Second, diversion of AQP2 to the basolateral membrane may be a protective mechanism to limit the apical flow of water and to prevent hypervolemia during prolonged antidiuresis and/or hypernatremia. Third, basolateral AQP2 insertion may represent a transient part of an indirect trafficking pathway in which AQP2 is first delivered basolaterally, followed by internalization and re-routing to the apical membrane by transcytosis. Such an indirect pathway of apical membrane protein insertion has been described for several membrane proteins in other cell types (51).
It is now clear that the same protein, including AQP2, can be inserted
into different membrane domains when expressed in different cell types,
implying that targeting signals on proteins are not interpreted
identically by the sorting machineries of all cells (20, 52-54).
However, it is unusual for the polarity of any given protein to be
modified under normal physiological, nonpathological conditions. The
kidney is largely responsible for maintaining body fluid, electrolyte
and acid/base homeostasis, and the remarkable plasticity of epithelial
cells in some kidney regions may reflect a continual need to monitor
prevailing physiological conditions, and adapt to them by modulating
vectorial transport processes across the epithelium. In this organ,
systemic acid-base alterations can lead to altered polarity of the
H+-ATPase in collecting duct intercalated cells (55, 56),
and we now report that hypertonicity can modify the polarity of AQP2 insertion in principal cells of some regions of the collecting duct.
Thus, hypertonicity represents a novel regulatory factor involved in
modifying the polarity of membrane protein insertion in the kidney, and
possibly in other cell types that are exposed to alterations in their
extracellular osmotic environment. Whereas these findings might explain
the basolateral localization of AQP2 in the inner medullary collecting
duct cells of the kidney, factors involved in basolateral AQP2
expression in the cortex, as well as the physiological/cell biological
role of basolateral AQP2 insertion remain to be determined.
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ACKNOWLEDGEMENT |
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We thank Dr. W. J. H. Koopman, Department of Cell Physiology, UMC St. Radboud, Nijmegen, for help with setting up the image quantification analysis.
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FOOTNOTES |
---|
* This work was supported in part by Dutch Organization of Scientific Research Grant NWO-MW 902-18-092 (to P. M. T. D. and P. v. d. S.) and European Union Grant QLRT-2000-00778 (to P. M. T. D.).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. Section 1734 solely to indicate this fact.
¶ Supported by National Research Service Award, National Institutes of Health Grant HD08684.
Supported by a grant from the National Kidney Foundation and
National Institutes of Health Grant DK38452.
Supported by National Institutes of Health Grant DK38452.
§§ To whom correspondence should be addressed: 160, Dept. of Cell Physiology, UMC St. Radboud Nijmegen, P. O. Box 9101, 6500 HB Nijmegen, The Netherlands. Tel.: 31-24-3617347; Fax: 31-24-3616413; E-mail: p.deen@ncmls.kun.nl.
Published, JBC Papers in Press, October 8, 2002, DOI 10.1074/jbc.M207339200
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ABBREVIATIONS |
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The abbreviations used are: AVP, arginine vasopressin; AQP2, aquaporin-2; MDCK, Madin-Darby canine kidney; dDAVP, 1-desamino-8-D-arginine vasopressin; IOD, integrated optical density.
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