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Methyl-_-cyclodextrin induces vasopressin-independent apical accumulation
2011-05-22 08:40:35Methyl-_-cyclodextrin induces vasopressin-independent apical accumulation
of aquaporin-2 in the isolated, perfused rat kidney
Leileata M. Russo, Mary McKee, and Dennis Brown
Program in Membrane Biology and Division of Nephrology, Department of Medicine,
Submitted 2 November 2005; accepted in final form 26 January 2006
Russo, Leileata M., Mary McKee, and Dennis Brown. Methyl-
_-cyclodextrin induces vasopressin-independent apical accumulation
of aquaporin-2 in the isolated, perfused rat kidney. Am J
Physiol Renal Physiol 291: F246 –F253, 2006. First published
January 31, 2006; doi:10.1152/ajprenal.00437.2005.—Vasopressin
increases urine concentration by stimulating plasma membrane accumulation
of aquaporin-2 (AQP2) in collecting duct principal cells,
allowing bulk water flow across the collecting duct from lumen to
interstitium down an osmotic gradient. Mutations in the vasopressin
type 2 receptor (V2R) cause hereditary X-linked nephrogenic diabetes
insipidus (NDI), a disease characterized by excessive urination and
dehydration. Recently, we showed that inhibition of endocytosis by
the cholesterol-depleting drug methyl-_-cyclodextrin (m_CD) induces
plasma membrane accumulation of AQP2 in transfected renal
epithelial cells overexpressing epitope-tagged AQP2. Here, we asked
whether m_CD could induce membrane accumulation of AQP2 in
situ using the isolated, perfused kidney (IPK). By immunofluorescence
and electron microscopy, we show that AQP2 was shifted from
a predominantly intracellular localization to the apical membrane of
principal cells following 1-h perfusion of Sprague-Dawley rat kidneys
with 5 mM m_CD. Quantification of staining revealed that the
intensity of AQP2 was increased from 647 _ 114 (control) to 1,968 _
299 units (m_CD; P _ 0.001), an effect similar to that seen after
perfusion with 4 nM dDAVP (1,860 _ 298, P _ 0.001). Similar
changes were observed following m_CD perfusion of kidneys from
vasopressin-deficient
on the basolateral distribution of AQP3 and AQP4 was detected.
These data indicate that AQP2 constitutively recycles between the
apical membrane and intracellular vesicles in principal cells in situ
and that inducing apical AQP2 accumulation by inhibiting AQP2
endocytosis is a feasible goal for bypassing the defective V2R signaling
pathway in X-linked NDI.
diabetes insipidus; collecting duct; endocytosis; immunocytochemistry;
aquaporin recycling
AQUAPORIN-2 (AQP2) is a hydrophobic _29-kDa protein that is
composed of six transmembrane segments and cytoplasmically
located NH2 and COOH termini (2). Expressed in principal
cells of the kidney collecting duct (CD), AQP2 plays a major
role in whole body water homeostasis through water reabsorption
that results in urine concentration (6, 8). This function is
achieved by the insertion of AQP2 into the apical membrane of
principal cells, where it acts as a hydrophilic pore allowing the
osmotic flow of water into the cell. Water then exits basolaterally
into the interstitium via aquaporins-3 and/or -4 (12, 35)
and perhaps also AQP2 in some kidney regions (9, 17a, 25).
Dysfunction of the AQP2 membrane accumulation process
results in whole body deregulation of water homeostasis,
contributing to excessive urination and dehydration, a disease
known as diabetes insipidus (DI) (10, 24, 26, 31). AQP2
distribution and function are chiefly regulated by the antidiuretic
hormone arginine vasopressin (AVP), which is released
by the posterior pituitary gland in response to high-serum
osmolarity and/or blood volume reduction (17). Circulating
AVP binds to the vasopressin type 2 receptor (V2R), located
on the basolateral side of principal cells of the CD, causing
intracellular cAMP levels to increase. This activates protein
kinase A (PKA) (27), resulting in the phosphorylation of AQP2
at residue S256. This phosphorylation is necessary for the
AVP-induced redistribution of AQP2 from a vesicular to a
membrane localization (13, 18), allowing water reabsorption to
occur in the CD. AQP2 membrane accumulation can also occur
after an increase in cytoplasmic cGMP levels; protein kinase G
(PKG) is also capable of phosphorylating AQP2 on residue
S256 (4).
DI has several distinct causes. Hereditary forms may be
“nephrogenic (NDI)” (the result of mutations in the AQP2 or
V2R genes) or “central (CDI)” (the result of mutations in the
AVP gene). Several mutations in the AQP2 gene have been
identified and they commonly lead to the mistargeting of AQP2
to subcellular organelles rather than the plasma membrane,
resulting in AQP2 degradation (23). X-linked NDI is caused by
mutations in the V2R and is much more common than autosomal
NDI in which AQP2 is defective. The AQP2 protein
expressed in X-linked NDI patients as well as in patients with
CDI is probably functional, but the inability of the kidney to
respond to AVP or the lack of active AVP hormone precludes
AQP2 accumulation on the plasma membrane of principal
cells.
In cases where AVP itself is dysfunctional, the symptoms of
CDI may be treated by administration of 1-desamino(8-Darginine)
vasopressin or dDAVP, a synthetic structural analog
of AVP that has a high specificity for the V2R (1). Treatment
of CDI with dDAVP is generally successful; however, dDAVP
in combination with some drugs may cause dilutional hyponatremia
(12a). Of course, dDAVP is not effective in most cases
where the V2R receptor is mutated or unresponsive to AVP,
although high doses of AVP may be effective in a few
instances where the V2R has a lower, but not absent, affinity
for the hormone. To date, therefore, the treatment of NDI
resulting from V2R mutations is limited and relies largely on
reducing urine flow by (seemingly paradoxical) treatment with
thiazide diuretics with Address for reprint requests and other correspondence: L. M. Russo, concomitant sodium restriction.
Program in Membrane Biology, Division of Nephrology, Massachusetts General
Hospital/Harvard Medical School,
receptor.mgh.harvard.edu).
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.
Am J Physiol Renal Physiol 291: F246–F253, 2006.
First published January 31, 2006; doi:10.1152/ajprenal.00437.2005.
F246 0363-6127/06 $8.00 Copyright © 2006 the American Physiological Society http://www.ajprenal.org
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However, some recent studies are emerging that suggest
alternative strategies for therapeutic intervention in NDI. These
are based on our increasing understanding of the signal transduction
and trafficking pathways that result in the membrane
accumulation of AQP2. Previous work in vitro showed that
AQP2 is constitutively recycled between the plasma membrane
and intracellular vesicles, even in the absence of AVP (22, 32).
The relative rates of endo- and exocytosis of AQP2 were
proposed to be critical in determining the amount of AQP2 in
the plasma membrane (14, 25). After reaching the cell surface,
AQP2 is subsequently internalized by clathrin-mediated endocytosis
(32). The depletion of plasma membrane cholesterol by
methyl-_-cyclodextrin (m_CD) prevents the formation and
budding of clathrin-coated pits, resulting in the acute blockade
of endocytosis (29). With the use of this strategy, a very rapid
plasma membrane accumulation of AQP2 was demonstrated in
AQP2-transfected LLC-PK1 and inner medullary CD (IMCD)
cells in culture. Furthermore, inhibiting endocytosis with dominant-
negative (K44A) dynamin also resulted in membrane
accumulation of AQP2 in cultured cells (32).
The aim of our present study was to determine whether the
endocytosis inhibitor m_CD would cause vasopressin-independent
AQP2 accumulation in principal cells of the functioning,
intact rat kidney. This would indicate that AQP2 is
constitutively recycling not only in transfected cell models in
vitro but also in CD principal cells in situ. However, m_CD
Fig. 1. Methyl-_-cyclodextrin (m_CD) treatment
increases apical aquaporin-2 (AQP2) expression in
the isolated, perfused kidney (IPK): immunofluorescence
microscopy. Sprague-Dawley kidneys were
perfused under control conditions, in the presence of
5 mM m_CD, or with 4 nM dDAVP for 60 min.
Kidneys were then fixed, sectioned, and immunostained
using anti-AQP2 antibodies. For all treatments,
examples of tubules sectioned transversely
from the inner stripe of the outer medulla (A, C, E)
and longitudinally in the inner medulla/papilla (B,
D, F) are illustrated. Under control conditions (A,
B), AQP2 has a cytosolic distribution in principal
cells of the collecting duct. In the inner stripe
sections (A, C, E), AQP2-negative intercalated cells
appear as dark, unstained “holes” in the epithelial
layer. After perfusion with 5 mM m_CD (C, D),
AQP2 shows an increased apical localization in
principal cells of the inner stripe and inner medulla.
After perfusion with 4 nM dDAVP, a similar and
expected increased apical localization of AQP2 in
medullary collecting ducts is seen in the IPK preparation.
However, dDAVP also induced a clear
basolateral localization of AQP2 in the inner medulla
(F and arrows, inset) but not in the inner stripe
(E) principal cells. Bar _ 40 _m.
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targets and binds cholesterol, and its use in vivo is limited due
to its potential toxicity and its ability to lyse erythrocytes.
Therefore, we examined the effect of m_CD on AQP2 localization
using the ex vivo isolated, perfused kidney technique
(IPK). This technique allows the kidney to function on an
artificial closed circuit, hence removing extra renal influences,
making the IPK an invaluable tool for the investigation of
drugs or compounds that may be toxic or display complex
interactions in vivo (33). The results obtained confirm previous
in vitro studies and demonstrate that AQP2 constitutively
recycles to the apical membrane, a process that is
halted by the depletion of cholesterol using m_CD and
which results in the accumulation of AQP2 on the plasma
membrane (22). This provides proof-of-principle evidence
that specific targeting of m_CD (or other agents that inhibit
endocytosis) to CDs may provide a novel form of treatment
for DI resulting from dysfunctional V2R or AVP or as an
alternative to dDAVP treatment in cases where its use is
contraindicated.
MATERIALS AND METHODS
IPK preparation. All animal experiments carried out were approved
by the Institutional Committee on Research Animal Care, in
accordance with National Institutes of Health (NIH) Guide for the
Care and Use of Laboratory Animals. Male Sprague-Dawley rats or
male
interference of oxytocin release on AQP2 distribution during surgery
(19),
overnight before the experiment. Rats were anesthetized by an intraperitoneal
injection of 45 mg/kg body wt of pentobarbital sodium
(Nembutal; Abbott Laboratories,
mannitol and 200 IU heparin (final volume 1 ml; Sigma,
MO) was injected into the femoral vein. To prevent dehydration, 1.5
ml of 0.9% NaCl were also injected into the femoral vein immediately
before surgery. A laparotomy was performed and the right ureter was
catheterized with polyethylene tubing PE/1 (Scientific Commodities).
The right renal artery was cannulated via the superior mesenteric
artery and the kidney was removed by en bloc dissection. The whole
procedure was completed within 10 min. The kidney perfusion pressure
was maintained at 95–100 mmHg with a peristaltic pump monitored
using a calibrated aneroid manometer. The kidney was perfused
with a recirculating Krebs-Henseleit buffer containing 5% BSA,
Fig. 2. Apical accumulation of AQP2 in cortical collecting ducts (CCDs) is
induced by m_CD and dDAVP. Sprague-Dawley kidneys were perfused under
control conditions (CON), in the presence of 5 mM m_CD or with 4 nM
dDAVP for 60 min. Kidneys were then fixed, sectioned, and immunostained
using anti-AQP2 antibodies. Under control conditions (A), AQP2 has a
predominantly cytosolic distribution but some cells have an apical distribution
in the cortical collecting duct. After perfusion with 5 mM m_CD (B), AQP2
has an increased apical localization in the collecting duct. After perfusion with
4 nM dDAVP (C), a similar and expected increased apical localization of
AQP2 in the collecting ducts is seen in the IPK preparation. G, glomerulus.
Bar _ 50 _m.
Fig. 3. Apical accumulation of AQP2 in outer medullary collecting ducts is
induced by m_CD and dDAVP. Sprague-Dawley kidneys were perfused under
control conditions, in the presence of 5 mM m_CD, or with 4 nM dDAVP for
60 min. Kidneys were then fixed, sectioned, and immunostained using anti-
AQP2 antibodies. Under control conditions (A), AQP2 has a predominantly
cytosolic distribution. After perfusion with 5 mM m_CD (B), AQP2 shows an
increased apical localization in the collecting duct. After perfusion with 4 nM
dDAVP (C), the expected increased apical localization of AQP2 in the
collecting ducts is seen in the IPK preparation. Bar _ 40 _m.
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glucose, essential amino acids, and oxygen radical scavengers as
previously described (34). The perfusate was filtered using a 0.45-_m
filter and was oxygenated with 95% O2-5% CO2, pH 7.4, and
maintained at 37°C throughout the entire procedure. The kidney was
first equilibrated on the apparatus for 10 min and was then perfused
for a 60-min time period. Urine and plasma samples were collected at
40 and 60 min. Urine osmolality was measured using a Wescor Vapor
Pressure Osmometer 5520 (Wescor,
m_CD and dDAVP experiments were carried out using exactly the
same procedure except that the perfusate contained 5 mM m_CD
(Sigma) or 4 nM dDAVP (Sigma).
Tissue fixation and immunostaining. Following perfusion on the
IPK apparatus, the kidney was immediately flushed with paraformaldehyde
lysine periodate (PLP) fixative and then immersion-fixed
overnight in PLP, as described previously (3). Tissue was then
extensively washed in PBS and cryoprotected in 30% sucrose. Tissue
was embedded in OCT and 5-_m-thick cryosections were cut. Sections
were treated with 1% SDS in PBS, an antigen retrieval technique
(7), and then blocked using a solution of 1% BSA in PBS. Previously
characterized primary antibodies used were against the COOH terminus
of AQP2 (30), AQP3, or AQP4 (15). Secondary antibodies used
were goat anti-rabbit IgG coupled to CY3 or FITC (Jackson Immuno-
Research Laboratories,
Staining was analyzed using a Nikon E800 fluorescence microscope
equipped with a Hamamatsu Orca CCD camera and IP Lab
Spectrum acquisition and image analysis software, or with a Zeiss
Axioplan microscope equipped with a Radiance 2000 confocal laserscanning
system (Bio-Rad). For quantification, digital images were
acquired using the Nikon microscope with a _40 objective. All
images from different slides were collected with the same microscope
setting. Perpendicularly sectioned cells from the inner stripe/inner
medulla boundary region were chosen at random and a line was drawn
through the cell from the basolateral to apical pole, passing through
the region of the nucleus. The line was analyzed for intensity and the
intensity at the level of the apical pole was recorded. Twenty-five
individual cells were quantified in each of three to four kidneys from
each group (75–100 points in total for each group). Analysis was
performed on a Macintosh computer using the public domain NIH
Image program (developed at the U.S. NIH and available on the
Internet at http://rsb.info.nih.gov/nih-image/).
Electron microscopy and immunogold staining. For electron microscopy
and immunogold staining, small pieces of PLP-perfused rat kidney
(_1 mm3) were dehydrated through a graded series of ethanol to 100%
ethanol. They were then infiltrated with LR White resin (Electron Microscopy
Sciences,
White for a few hours and embedded in gelatin capsules at 50°C
overnight. For the immunostaining, thin sections were cut on a Reichert
Ultracut E ultramicrotome and were collected on formvar-coated nickel
grids. The grids were incubated on drops of 1% BSA/PBS _ 5% normal
goat serum (Sigma) for 10 min at room temperature as a blocking step.
They were then placed on drops of either DAKO antibody diluent
(negative control, DAKO,
antibody diluted in DAKO diluent for 1 h, at room temperature. Following
several rinses on drops of PBS, the grids were incubated on drops of
gold-conjugated secondary anti-rabbit IgG (Ted Pella,
1 h at room temperature. Finally, the grids were rinsed several times on
drops of distilled water and stained for 5 min on drops of aqueous uranyl
acetate (
examined in a JEOL 1011 transmission electron microscope at 80 kV,
and imaged digitally.
Statistical analysis. All data are expressed as means _ SE where n
is the number of measurements. Statistical analysis was performed
using Student’s t-test where P _ 0.05 was taken as statistically
significant. Statistical analysis of apical intensity was carried out by
performing a one-way ANOVA followed by Student’s t-test.
RESULTS AND DISCUSSION
Following perfusion of intact, isolated kidneys with 5 mM
m_CD for 60 min, immunocytochemical analysis of AQP2 revealed
a striking redistribution of AQP2. It relocated from a
predominantly cytoplasmic location in outer medullary (inner
stripe) principal cells of the nontreated IPK (Fig. 1A) to an apical
distribution in the m_CD-treated kidney (Fig. 1C), an effect
similar to that of dDAVP (Fig. 1E). This marked apical redistribution
of AQP2 induced by both m_CD and dDAVP was also
evident in CDs of the cortex and the outer stripe (Figs. 2 and 3).
In the middle portion of the papilla, AQP2 was also relocated to
the apical pole of principal cells following m_CD treatment,
whereas dDAVP induced not only apical accumulation, but also
basolateral localization of AQP2 in this portion of the CD (Fig. 1,
B, D, F), as previously described in vivo (36). In addition,
basolateral AQP2 was also seen in a population of cortical
connecting segments (data not shown), a phenomenon that has
also been described earlier (9). The AQP2 redistribution was also
evident by electron microscopy (Fig. 4, A-C), where AQP2 gold
labeling showed an increased apical plasma membrane localization
following m_CD (Fig. 4B) and dDAVP (Fig. 4C) treatment.
However, a marked basolateral accumulation of AQP2 was noted
after dDAVP, but not m_CD treatment in the middle portion of
Fig. 4. Immunogold electron microscopy showing that m_CD increases apical
membrane expression of AQP2 in the IPK. Sprague-Dawley kidneys were
perfused in control conditions, in the presence of 5 mM m_CD, or with 4 nM
dDAVP for 60 min as in Fig. 1. Kidneys were then fixed and processed in LR
White embedding resin for electron microscopy analysis using immunogold
labeling for AQP2. A: AQP2 is found in a mainly subapical location (some
indicted by arrowheads) in the control IPK, although some apical AQP2 (some
indicated by arrows) is also detectable. B: apical plasma membrane AQP2
expression is greatly increased in the m_CD-treated IPK (some indicated by
arrows). C: apical AQP2 distribution is also increased in the dDAVP-treated
IPK (some indicated by arrows). Bar _ 0.5 _m.
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the inner medulla (not shown). Morphologically, the CDs from all
IPK groups looked similarly intact, and epithelial cells were well
preserved when analyzed by electron microscopy, demonstrating
that the change in AQP2 distribution using m_CD treatment was
not accompanied by gross cellular alterations. In support of this,
both AQP3 and AQP4 maintained their usual tight basolateral
localization in both the nontreated control and m_CD-treated IPK
(Fig. 5, A-D).
Analysis of AQP2 distribution in the apical pole of principal
cells from CDs along the inner stripe/inner medullary border
(where the effect was the most pronounced) using NIH Image
revealed the apical AQP2 redistribution to be statistically
significant in both m_CD-treated group (P _ 0.001) and
dDAVP-treated group (P _ 0.001) compared with the nontreated
control IPK (Fig. 6). Both treatments resulted in an
almost threefold increase in apical membrane staining intensity
in this system. However, despite this increase in apical AQP2,
no significant change in urine concentrating ability was observed
[330 _ 11 (n _ 4) control IPK vs. 362 _ 6.8 (n _ 3)
dDAVP-treated IPK]. This is consistent with the results of
Lieberthal et al. (21), who demonstrated that the addition of
dDAVP resulted in the partial production of concentrated urine
by the IPK only when erythrocytes (40–45% hematocrit) were
included in the circulating medium (20). This maneuver was
not possible in the present set of experiments, because m_CD
efficiently lyses erythrocytes, which can lead to tubular necrosis
due to the release of heme proteins (5).
To determine whether the same effects of m_CD could be
obtained in a vasopressin-deficient model in which AQP2
levels are considerably lower than in normal rats (28),
rats which lack functional AVP were used in a parallel
study to determine the effect of m_CD on AQP2 distribution.
Our data revealed a similar redistribution of AQP2 from a
cytoplasmic localization to an apical localization following
5 mM m_CD perfusion in these animals (Fig. 7, A and B).
Similar to the results from Sprague-Dawley rats, AQP3 distri-
Fig. 5. m_CD has no effect on AQP3 and AQP4
localization in the IPK: confocal immunofluorescence
microscopy. Sprague-Dawley kidneys were
perfused under control conditions or in the presence
of 5 mM m_CD for 60 min. Kidneys were then
fixed and immunostained using anti-AQP3 and anti-
AQP4 antibodies. A and C: AQP3 distribution under
control IPK conditions demonstrates the expected
basolateral distribution of AQP3 (A) and AQP4 (C)
in principal cells of the inner medullary collecting
duct. B and D: AQP3 and AQP4 distribution in 5
mM m_CD IPK also demonstrates basolateral staining
for AQP3 (B) and AQP4 (D) comparable to that
observed in the control IPK. This result demonstrates
that m_CD did not interfere with the normal
basolateral expression of AQP3 or AQP4. Bar _
10 _m.
Fig. 6. Effect of m_CD on the apical intensity of AQP2 immunostaining in the
IPK. Quantitation of AQP2 distribution in the IPK was analyzed using images
similar to those shown in Fig. 1, in control conditions, after 5 mM m_CD
treatment, and after perfusion with 4 nM dDAVP (see MATERIALS AND METHODS).
Collecting ducts from the inner stripe/inner medulla boundary were
selected for quantification. The data revealed significant and comparable
increases in AQP2 apical intensity following 60-min perfusion with either
5 mM m_CD perfusion (P _ 0.001) or 4 nM dDAVP (P _ 0.001) compared
with the control IPK. Data are expressed as arbitrary units of fluorescence
intensity. *P _ 0.001, n _ 3 for control and dDAVP perfusions and n _ 4 for
m_CD perfusion.
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bution (Fig. 7, C and D) and AQP4 distribution (not shown)
were also unaffected by the perfusion with m_CD in the
the absence of preexposure to AVP in vivo, AQP2 can be
sequestered to the apical membrane of principal cells by
exposure to m_CD.
The Sprague-Dawley-derived IPK perfused with 5 mM
m_CD displayed a decrease in kidney flow rate and urine
production (Table 1), although perfusion pressure was not
significantly different from nontreated control IPKs (Table 1).
Urine osmolality showed a tendency to be higher in the
m_CD-treated rat kidneys (as also seen for the dDAVP-treated
rats), although this was not statistically significant (Table 1).
As mentioned above, this finding is in agreement with previous
data suggesting that the IPK has a compromised concentrating
ability (20, 21). Although attempts were also made to analyze
the effect of m_CD on endocytosis through the injection of a
concentrated bolus of FITC dextran or horseradish peroxidase
(HRP) into the renal artery, we were unable to visualize the
endocytosis of these markers in any of the three groups studied.
Previous reports indicate that the IPK has disrupted sodium
handling as well as progressive ischemic injury of the medullary
thick ascending limb contributing to a decreased concentrating
ability (20, 21). In the present study, this decreased
concentrating ability also hampered efforts to analyze endocytosis
of the fluid phase markers FITC-dextran and HRP as they
did not achieve a high enough concentration in the renal tubule
to permit ready visualization.
Our data demonstrate for the first time the ability of m_CD
to sequester AQP2 in the apical membrane of CD principal
cells in the intact ex vivo functioning kidney. The addition of
m_CD to the IPK perfusate was able to significantly increase
the level of apical AQP2 in the CD in all kidney regions,
without affecting the expression and localization of AQP3 or
AQP4. However, the effect was most pronounced in the inner
stripe and the initial two-thirds of the inner medulla. A similar
effect was also found in kidneys from the vasopressin-deficient
Fig. 7. m_CD increases apical AQP2 expression in
IPK from vasopressin-deficient
conditions and in the presence of 5 mM m_CD for
a total of 60 min. Kidneys were then fixed and
stained using anti-AQP2 antibodies. A: AQP2 in the
control IPK has a subapical distribution in principal
cells of the inner stripe collecting duct. B: after
perfusion with 5 mM m_CD, AQP2 has an increased
apical localization, similar to that seen in
Sprague-Dawley rat kidneys (see Fig. 1). C: AQP3
displayed a basolateral distribution in both control
IPK collecting ducts (C) and in the presence of
5 mM m_CD (D). Bar _ 10 _m.
Table 1. Physiological parameters for IPK perfusion of Sprague-Dawley rat kidneys
Time
Pressure, mmHg Flow Rate, ml/min Urine Volume, ml Plasma Osmolarity, mosM Urine Osmolarity, mosM
C M C M C M C M C M
40 min 94_2.5 99_0.5 21.8_2.3 7.0_1.5* 0.92_0.3 0.19_0.1† 321_5 326_2.5 320_19 354_6.5
20 min 96_2.5 100_1 20.7_2.4 6.7_1.6* 0.65_0.2 0.23_0.1† 322_5 327_1.5 330_11 365_13.5
Values are means _ SE, n _ 4 for each group. Where time is perfusion time, C is control isolated, perfused kidney (IPK) and M is methyl-_-cyclodextrin
IPK. *P _ 0.001, †P _ 0.05 compared with control IPK.
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AQP2 constitutively recycles between cytoplasmic vesicles
and the apical plasma membrane of principal cells of the
kidney in situ, in the absence of vasopressin, a process that is
regulated at least in part by clathrin-mediated endocytosis (22,
32). However, the observation that dDAVP but not m_CD can
also induce basolateral accumulation of AQP2 in some regions
of the CD implies that while they are similar, the mechanisms
of action of the hormone and the drug may not completely
overlap.
Although we previously reported that AQP2 constitutively
recycles in LLC-PK1 and IMCD cells in culture (22, 32), the
fact that this constitutive recycling process occurs in situ is an
important finding for the development of future therapies for
NDI. The ability of AQP2 to be sequestered in the apical
membrane is paramount for the CD to exert its water reabsorption
function in the kidney. Although m_CD is toxic in vivo,
our present findings provide solid support for the idea that
specifically blocking apical endocytosis in the CD of nephrogenic
DI patients may eventually provide a plausible form of
therapy for NDI.
ACKNOWLEDGMENTS
We thank G. Hawthorn for excellent technical support. We also thank Dr.
W. D. Comper (
apparatus that was used in these studies.
GRANTS
This work was supported by National Institutes of Health Grant DK-38452.
Dr. L. M. Russo is the recipient of a Juvenile Diabetes Research Foundation
Postdoctoral Fellowship. The Microscopy Core facility of the MGH Program
in Membrane Biology receives additional support from the Boston Area
Diabetes and
the Study of Inflammatory Bowel Disease (DK-43341).
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