Long-term
regulation
of four renal
aquaporins
in rats
JAMES TERRIS,
CAROLYN
A. ECELBARGER,
WREN
NIELSEN,
AND MARK A. KNEPPER
Laboratory
of Kidney and Electrolyte
Metabolism,
National Heart, Lung, and Blood Institute,
Bethesda 20892; Department
of Physiology,
Uniformed
Services University
of the Health Sciences,
Bethesda, Maryland
20814; and Department
of Cell Biology, Institute
of Anatomy,
University
of Aarhus,
DK-8000 Aarhus,
Denmark
loop of Henle; collecting
duct; water permeability;
water
channels; vasopressin;
urinary concentrating
mechanism
OF OSMOTIC WATER transport across the
renal collecting duct epithelium depends on at least two
independent mechanisms that manifest themselves
over different time scales. 1) Short-term regulation of
collecting duct water permeability is a consequence of
the rapid action of arginine vasopressin (AVP) to increase the water permeability of the apical plasma
membrane of collecting duct cells by triggering the
insertion of aquaporin-2 water channels from an intracellular vesicular pool (23, 24, 29, 36). This short-term
response to AVP is complete within 30-40 min of
exposure to the peptide (26, 35). The short-term effect
of AVP may also be partially dependent on adenosine
3’,5’-cyclic monophosphate (CAMP)-dependent
phosphorylation of aquaporin-2 (19). 2) Long-term regulation of collecting duct water permeability is manifested
as a stable increase in collecting duct water permeability after a restriction in water intake for 24 h or more
(8, 10,20).
The primary goal of the present study is to investigate long-term regulation of expression of renal aquapo-
REGULATION
F414
rin water channels as a possible factor in the long-term
regulation of collecting duct water permeability. Previous studies using immunoblotting
revealed that the
level of aquaporin-2 expression was markedly increased in the renal inner medulla in response to fluid
restriction of rats (25). Immunocytochemical
observations suggested that the increase in aquaporin-2 abundance occurred in both the plasma membrane and in
intracellular vesicles. Studies using Northern blotting
have demonstrated an associated increase in aquaporin-2 mRNA in the inner medullas of water-restricted
rats (22, 37). These studies, therefore, provided strong
evidence that the long-term upregulation of water
permeability in the rat inner medullary collecting duct
in response to water restriction was due, at least in
part, to increased expression of the aquaporin-2 water
channel. Subsequently, DiGiovanni et al. (4) demonstrated in the Brattleboro rat that infusion ofAVP for 5
days induced a threefold increase in inner medullary
aquaporin-2 expression associated with a proportionate threefold increase in osmotic water permeability of
isolated perfused inner medullary collecting ducts.
These results further supported the view that longterm regulation of collecting duct water permeability is
due to changes in aquaporin-2 expression. Based on
these studies, two possible mechanisms for upregulation of aquaporin-2 expression in response to increased
circulating levels of AVP were proposed as follows. 1)
AVP may act directly on the collecting duct cells to
increase aquaporin-2 expression, possibly as a result of
CAMP-induced increases in transcription of the aquaporin-2 gene. 2) The increase in aquaporin-2 expression
could be a result of the high osmolality or ionic strength
of the interstitial
fluid during antidiuresis and thus
could be an indirect response to high AVP levels.
Obviously, these two possibilities are not mutually
exclusive.
In addition to aquaporin-2, three additional members of the aquaporin family of water channels have
been found to be expressed in the kidney, namely,
aquaporin-1 (3, 27, 30), aquaporin-3 (5, 6, 14, 21), and
aquaporin-4 (11,16,33). Among these, aquaporin-3 and
aquaporin-4 have both been found to be present in the
basolateral plasma membrane of collecting duct cells
(5,9,14,21,33)
and thus could conceivably be involved
in the long-term regulation of collecting duct water
permeability. In fact, we have recently demonstrated
that the inner medullary abundance of aquaporin-3 (5),
but not aquaporin-4 (33), increases in response to 48-h
fluid restriction in rats. The remaining renal aquaporin, aquaporin-1 (or CHIP28), is expressed in the
descending limb of Henle’s loop and in the proximal
tubule (3,27,30) but not in the collecting duct (27).
Downloaded from http://ajprenal.physiology.org/ by 10.220.33.4 on June 18, 2017
Terris, James, Carolyn
A. Ecelbarger,
Sgren Nielsen,
and Mark A. Knepper.
Long-term
regulation
of four renal
aquaporins
in rats. Am. J. Physiol. 271 (Renal FZuid Electrolyte PhysioZ. 40): F414-F422,
1996.-The
aquaporins
are
molecular
water channels expressed in the kidney and other
organs. To investigate
long-term
regulation
of renal expression of these water channels, we carried out immunoblotting
studies using membrane
fractions from rat renal cortex and
medulla. Both 48-h water restriction
in Sprague-Dawley
rats
and &day arginine vasopressin (AVP) infusion in Brattleboro
rats caused significant
increases in the expression
levels of
two aquaporins,
aquaporin-2
and aquaporin-3,
while the
levels of aquaporin-1
and aquaporin-4
were unchanged.
The
increases in aquaporin-2
and aquaporin-3
expression
were
seen in inner and outer medulla as well as cortex. Ablation of
the corticomedullary
interstitial
osmotic gradient
with an
infusion of furosemide
did not eliminate
the upregulatory
response to AVP infusion in Brattleboro
rats. Furthermore,
&day furosemide
infusion to Sprague-Dawley
rats did not
decrease expression levels of the collecting duct aquaporins,
but rather increased them. We conclude that the expression of
aquaporin-2
and aquaporin-3,
but not aquaporin-1
or aquaporin-4, is increased
in response to elevated circulating
AVP.
Because regulation
of aquaporin-2
and aquaporin-3
levels
was observed in the cortex and because osmotic gradient
ablation
did not abrogate
the increase, we conclude that
changes in interstitial
osmolality
are not necessary for the
AVP-induced
upregulation
of aquaporin-2
and aquaporin-3
expression.
REGULATION
OF RENAL
The objectives of the present study were I) to determine which of the three collecting duct water channels
display increases
in expression
level in response
to
long-term increases in circulating
AVP and 2) to determine whether
the effect of AVP on water
channel
expression
is dependent on increased interstitial
tonicity.
METHODS
IN RATS
F415
needed for maximal stimulation
of osmotic water permeability in isolated perfused collecting ducts, i.e., 100 pM (32).
The third study was designed to evaluate the effects of a
5-day infusion of AVP in the setting of chronic furosemide
treatment,
which was utilized
to decrease the corticomedullary interstitial
osmotic gradient.
All 12 Brattleboro
rats
(150-160
g) were implanted
with osmotic minipumps
(model
2MLl; Alza) that delivered furosemide (Sigma Chemical) at a
rate of 7.2 mg. 100 g body wt-l-day-l
following
a procedure
originally
reported by Kaissling
and Stanton (17). The furosemide was dissolved in 0.9% sodium chloride and adjusted to
pH 9-10 with 10 N sodium hydroxide.
In addition
to the
furosemide,
six rats were infused with 2.52 ug AVP/day by
separate minipumps.
Volume depletion
was prevented
by
giving the rats a drinking
solution containing
0.8% sodium
chloride and 0.1% potassium chloride.
The fourth study examined the effects of a 5-day infusion of
furosemide to Sprague-Dawley
rats (140-160
g) in the setting
of high levels of circulating
AVP. All rats were infused with
AVP (2.52 ug AVP/day by minipump)
as described above. Six
of these rats were also infused with furosemide
(7.2 mg. 100
g-l-day-l
by minipump)
as described
above. As with the
previous study using furosemide,
0.8% sodium chloride and
0.1% potassium chloride was given as drinking
fluid.
In all studies, rats were maintained
in metabolism
cages to
allow urine collections. Urine osmolality was measured in the
24-h urine collected preceding
death of the rats using a
vapor-pressure
osmometer
(model 5lOOC; Wescor, Logan,
UT). Animals
were killed by decapitation,
and trunk blood
was collected into heparinized
beakers for determination
of
AVP levels and serum sodium concentration.
The kidneys
were rapidly removed into an ice-cold isolation
buffer solution. This solution contained
250 mM sucrose plus IO mM
triethanolamine
(Calbiochem,
La Jolla, CA) and was adjusted
to pH 7.6 with 1 N NaOH.
It also contained
protease
inhibitors
[ 1 ug/ml leupeptin
(Bachem California,
Torrance,
CA) and 0.1 mg/ml phenylmethylsulfonyl
fluoride (United
States Biochemical,
Toledo, OH)]. The inner medulla, outer
medulla, and cortex were separated using sharp, curved, iris
scissors. AVP levels were determined
in serum samples by
radioimmunoassay
(Incstar, Stillwater,
MN).
Membrane
preparation
and immunoblotting.
Membrane
fractions from each of the three renal regions were prepared
as previously
described
(5, 23, 33). Briefly, tissues were
homogenized
in ice-cold isolation
solution (composition
described above). Homogenates
were centrifuged
at 1,000 g for
10 min. Supernatants
were saved, and the pellets were
rehomogenized
and centrifuged
again at 1,000 g. For aquaporin-1, -2, and -3, supernatants
from the two centrifugations
were combined and centrifuged
at 200,000 g. The resulting
pellet contained plasma membranes
and intracellular
vesicles
(5,23). For aquaporin-4
determinations
in the inner medulla,
a sample of the 1,000 g supernatant
was spun at 4,000 g, and
this pellet was used for blots. Previous studies had established that virtually
all of the aquaporin-4
in the inner
medulla
is present in this fraction
(33). Inner and outer
medullary
pellets were resuspended
in 100 ul of isolation
solution, and cortical pellets were resuspended
in 1,000 ul.
Total protein concentration
was determined
with a Pierce
bicinchoninic
acid protein assay reagent kit. A quantity of 5 X
Laemmli
buffer [7.5% sodium dodecyl sulfate, 30% glycerol,
50 mM tris(hydroxymethyl)aminomethane,
pH 6.8, bromopheno1 blue] with 30 mg/ml dithiothreitol
was added to the
samples in a ratio of 1:4, which were then heated to 60°C for
15 min. Electrophoresis
was performed
on minigels
of 12%
polyacrylamide,
and the proteins were transferred
electrophoretically
to nitrocellulose
membranes.
After blocking
with
Downloaded from http://ajprenal.physiology.org/ by 10.220.33.4 on June 18, 2017
Animals. The animals used for these studies were pathogenfree male Sprague-Dawley
rats (Taconic Farms, Germantown, NY) or male Brattleboro
homozygous
(di/di; HarlanSprague-Dawley,
Indianapolis,
IN) as indicated.
Weights of
control and experimental
animals were carefully matched in
each protocol, and all were equilibrated
and maintained
on an
ad libitum
intake of a diet containing
a moderate
level of
sodium chloride (NIH 31-autoclavable
rodent diet, 130 meq
Na+/kg; Ziegler Bros., Gardner,
PA). The rats were kept in
filter-top microisolators
with autoclaved feed and bedding, to
maintain
a pathogen-free
state, or metabolic
cages, as required.
Antibodies.
Antibodies
to aquaporin-2
(4), aquaporin-3
(5),
and aquaporin-4
(33) were characterized
previously. All polyclonal antibodies
were raised to synthetic peptides (18-24
amino acids) corresponding
to a region in the carboxyl termini
of the respective water channels and were affinity purified
using columns
on which the immunizing
peptides
were
immobilized
(Immunopure
Ag/Ab immobilization
kit no. 2;
Pierce, Rockford,
IL). For this study, we prepared
a new
antibody against aquaporin1 using the carboxyl terminal
sequence for rat aquaporin-l(2),
HZN-CGQVEEYDLDADDINSRVEMKPK-COOH.
(Note the addition of a cysteine residue
at the amino terminal
end of the peptide to facilitate conjugation). The peptide was purified by high-performance
liquid
chromatography
and conjugated to a carrier protein (keyhole
limpet hemocyanin)
via the added amino-terminal
cysteine,
and rabbits were immunized
with the conjugate. Antiserum
from a rabbit (LL266) with an enzyme-linked
immunosorbent
assay titer of greater
than 1:32,000 was utilized
for all
experiments
reported here. Labeling
with the anti-aquaporin-1 antibody on immunoblots
(see RESULTS) and immunocytochemistry
(not shown) was identical
to that seen in rat
tissues with a previously characterized
antibody raised against
purified human aquaporin-I
(27).
AnimaZ treatments. Four studies were performed to examine the long-term regulation
of expression of aquaporins-1,
-2,
-3, and -4 in rat kidney. The first study, modified slightly from
that of Lankford et al. (20), was designed to assess the effect
of a 48-h water restriction
on male, Sprague-Dawley
rats
(320-370
g). Initially,
all 12 rats received sweetened water
(600 mM sucrose) as the sole drinking
fluid for a 48-h
equilibration
period. A moderate water diuresis developed,
which was the control state for all animals. Following
this
equilibration
period, six rats were thirsted for 48 h and six
rats (hydrated
controls) continued
to receive the sweetened
water for the same 48-h period.
The second study addressed the effects of a &day infusion
of AVP (Sigma Chemical,
St. Louis, MO) to Brattleboro
homozygous (di/di) (150-160
g) rats. Under light methoxyflurane anesthesia,
the rats were implanted
with osmotic
minipumps
(model 2002; Alza, Palo Alto, CA). Six rats
received AVP (2.52 pg/day) dissolved in 5% dextrose-0.05%
acetic acid vehicle solution,
and the remaining
six rats
received the vehicle only. This dose rate was chosen to yield
plasma AVP levels approximately
equivalent
to the level
AQUAPORINS
F416
REGULATION
OF RENAL
IN RATS
THIRSTED
HYDRATED
Inner
Medulla
Outer
Medulla
-
35
-
28
-
35
-
28
-
35
-
28
Cortex
RESULTS
Effect of thirsting on aquaporin levels in kidney
regions. In the first experiment,
we compared
waterloaded rats with rats that were thirsted for 48 h. Urine
osmolality in the hydrated rats averaged 642 -t 161
compared with 1,903 + 478 mosmol/kgH20 in the
thirsted rats (P < 0.0001). As expected, the plasma AVP
levels increased in response to thirsting (hydrated,
2.9 -t 1.8; thirsted, 16 i- 1.8 pg/ml; P < 0.001).
Figure 1 shows Western blotting results for aquaporin-2 in inner medulla, outer medulla, and cortex. In all
three regions, two bands were observed as demonstrated previously (251, i.e., one sharp band at 29 kDa
representing the nonglycosylated protein and one broad
band at -35 kDa representing the mature glycosylated
protein. As we have demonstrated before (25), there
was a striking upregulation of aquaporin-2 expression
in inner medmlain response to thirsting (Fig. 1, top 1.In
THIRSTED
Inner
Medulla
Outer
Medulla
HYDRATED
-
35
-
29
-
35
-
29
-
35
-
29
Cortex
Fig. 1. Immunoblots
of aquaporin-2
in inner medulla,
outer medulla,
and cortex of hydrated
and thirsted
Sprague-Dawley
rats. Quantities
of 1,3, and 10 pg of total protein
were loaded onto the gel per lane for
inner medulla,
outer medulla,
and cortex, respectively.
Initially
all 12
rats were given 600 mM sucrose as the sole drinking
fluid for 48 h.
Following
this equilibration
period,
one-half
of the animals
were
deprived
of drinking
water for the next 48 h while the remaining
6
continued
to receive the sucrose . water.. In_. all 3 . regions
band
--.- a mature
appears
at 35 kDa and a nonglycosylated
band at 29 kUa.
Fig. 2. Immunoblot
of aquaporin-1
in inner medulla,
outer medulla,
and cortex of hydrated
and thirsted
Sprague-Dawley
rats. Quantities
of 3, 2, and 1 pg total protein
were loaded onto the gel per lane for
inner medulla,
outer medulla,
and cortex,
respectively.
Membrane
samples were obtained
from the same rats as those used for Fig. 1.
addition, there was also a marked increase in aquaporin-2 expression in outer medulla and cortex (Fig. 1,
middle and bottom). Because the response was seen in
the cortex where interstitial
osmolality varies very
little, we conclude that the increase in aquaporin-2
expression is not dependent on a large increase in local
interstitial osmolality.
Figure 2 shows results in the same animals using the
anti-aquaporin-1 antibody. Again, as with aquaporin-2,
there are two bands, one at 28 kDa and a second broad
band at -35 kDa, as shown previously with another
antibody (27). In contrast to aquaporin-2, there was no
detectable increase in aquaporin-1 expression in any of
the three regions with thirsting.
Figure 3 shows results in the same animals using the
anti-aquaporin-3 antibody. As we have previously reported (5), thirsting increased the expression level of
aquaporin-3 in the rat inner medulla (Fig. 3, top).
Furthermore, thirsting also significantly increased the
expression of aquaporin-3 in outer medulla and cortex
(Fig. 3, middle and bottom), just as was seen for
aquaporin-2. The fractional increase in aquaporin-3
expression appeared to be most pronounced in the
outer medulla and cortex. Finally, aquaporin-4, which
is detectable only in the inner medulla with this
technique, was not changed in the thirsted rats (Fig. 4).
Figure
5 presents
quantitation
of the
changes
in
expression levels seen in response to thirsting, as
determined by densitometry. Thirsting significantly
increased the expression of aquaporin-2 and -3, but not
that of aquaporin-1 or -4. Subsequent results will be
given in the format shown in Fig. 5, rather than
showing individual immunoblots.
Effect of AVP infusion on aquaporin levels in kidney
regions. In the next experiment, we used Brattleboro
rats, which manifest central diabetes insipidus. One
group was infused with AVP for 5 days via osmotic
Downloaded from http://ajprenal.physiology.org/ by 10.220.33.4 on June 18, 2017
5 g/d1 nonfat dry milk for 30 min, the membranes were probed
with affinity-purified
antibodies to the respective aquaporins
for 24 h at 4”C, washed, and exposed to secondary antibody
(donkey anti-rabbit
immunoglobulin
G conjugated with horseradish peroxidase, Pierce no. 31458, diluted 1:5,000) for 1 h at
room temperature.
Sites of antibody-antigen
reaction were
visualized using luminol-based
enhanced chemiluminescence
(LumiGLO;
Kirkegaard
and Perry Laboratories,
Gaithersburg, MD). The blots were quantitated
by densitometry
(Molecular
Dynamics
model PDSl-P90,
ImageQuaNT
~4.2
software).
For all experiments,
control minigels
were run prior to
Western blotting
and were Coomassie
stained to confirm
equality of loading in each lane. Several representative
bands
were quantified by densitometry
to assure equality of loading.
Statistics.
The data are reported as means ? SE. Statistical comparisons
were made by use of unpaired
Student’s
t-test.
AQUAPORINS
REGULATION
THIRSTED
OF RENAL
HYDRATED
Inner
Medulla
-
33
-
26
-
33
-
26
-
33
-
26
minipumps. Controls received a vehicle infusion. The
efficacy of the infusion was documented with urinary
osmolality measurements (vehicle, 362 & 99; AVP
infused, 2,400 -+ 278 mosmolkg; P < O.OOOl>,as well as
with measurements of plasma AVP levels (vehicle,
1.15 t 0.32;AVP infused, 252 +- 78 pg/ml; P < 0.01).
Figure 6 shows the percent change in expression in
response to AVP infusion for each aquaporin in each
renal region. Figure 6A shows results for aquaporin-2.
As we have demonstrated previously (4), a 5-day AVP
infusion in Brattleboro rats resulted in a marked
increase in aquaporin-2 levels in inner medulla. Furthermore, similar increases in aquaporin-2 expression
were seen in the outer medulla and cortex. These
findings parallel observations with thirsting (compare
with Fig. 5).
Figure 6B shows findings for aquaporin-3. As with
aquaporin-2, there was a significant increase in the
inner medullary and outer medullary regions in response to AVP infusion, although a statistically significant increase was not seen in the cortex. Finally, as
shown in Fig. 6 (C and D) AVP infusion did not
significantly
affect either aquaporin-4 (Fig. 6C) or
aquaporin-1 (Fig. 6D) expression in any region.
Effect ofAVP infusion on aquaporin levels in a setting
of chronic furosemide infusion. In the third experiment,
THIRSTED
Inner
Medulla
HYDRATED
IN RATS
F417
we investigated the response to AVP in Brattleboro rats
that were simultaneously infused with furosemide to
prevent the maintenance of a normal interstitial
osmotic gradient. All rats were infused with furosemide
by minipump. Half of the furosemide-infused
rats
received AVP by minipump, and the other half received
vehicle. Furosemide infusion prevented the large increase in urinary osmolality normally seen with AVP,
although the difference was still significant (vehicle,
285 t 19; AVP, 559 + 20 mosmolkg; P < 0.0001). AVP
levels in the AVP-infused animals averaged 8.14 +- 1.9
pg/ml, and AVP was undetectable in controls.
Figure 7 shows the percent changes in the levels of
each of the four renal aquaporins in response to AVP
infusion. As shown in Fig. 7A, furosemide infusion did
not prevent the increase in aquaporin-2 expression
caused by AVP infusion. Large increases in aquaporin-2
levels were seen in all three regions, as well as in inner
medulla and outer medulla for aquaporin-3 (Fig. 7B).
Furthermore, as was seen in the absence of furosemide
in the second study, AVP infusion did not alter the
expression of either aquaporin-4 (Fig. 7C) or aquaporin-1 (Fig. 70).
Effect of long-term furosemide infusion on aquaporin
levels in a setting of chronic AVP infusion. The last
experiment was done to test whether furosemide infusion decreases the expression of any of the aquaporins.
If any of the aquaporins are upregulated by interstitial
hypertonicity,
we would expect furosemide infusion to
decrease the expression in the renal medulla. In these
experiments, Sprague-Dawley rats received an infusion of AVP, and one-half of this group received in
addition a 5-day infusion of furosemide. As expected,
the urinary osmolality was markedly reduced by furosemide (AVP alone, 1,707 +- 118; AVP plus furosemide,
606 t 47 mosmol/kg; P < 0.0001). AVP levels were not
significantly different (AVP alone, 246 5 20; AVP plus
furosemide, 378 + 51 pg/ml).
As illustrated in Fig. 8, instead of decreasing in
response to furosemide, the expression level of all three
of the collecting duct aquaporins (aquaporin-2, -3, and
-4) increased in response to furosemide. We speculate
that a portion of this increase could have been due to
the general hypertrophic effect of furosemide on the
collecting ducts and connecting tubules, as demonstrated by others (17), which would be expected to
increase the levels of collecting duct-specific proteins
relative to total protein levels. Consistent with this, the
expression level of aquaporin-1, which is expressed upstream from the site of action of furosemide, was either
decreased or was unchanged in the three regions.
DISCUSSION
-32
Fig. 4. Immunoblot
of aquaporin-4
in inner medulla
of hydrated
thirsted
Sprague-Dawley
rats. Aquantity
of 10 pg of total protein
loaded onto the gel per lane. Membrane
samples were obtained
the same rats as those used for Fig. 1.
and
was
from
The studies reported here have demonstrated that
the expression levels of aquaporin-2 and aquaporin-3
increase in the kidney in response to elevated circulating AVP. In contrast, the levels of aquaporin-4 and
aquaporin-1 were unaffected, indicating that the adaptation was selective for aquaporin-2 and -3. Because
the upregulation of aquaporin-2 and aquaporin-3 expression occurred in the cortex as well as the medulla in
Downloaded from http://ajprenal.physiology.org/ by 10.220.33.4 on June 18, 2017
Fig. 3. Immunoblot
of aquaporin-3
in inner medulla,
outer medulla,
and cortex of hydrated
and thirsted
Sprague-Dawley
rats. Quantities
of 5,5, and 10 pg of total protein
were loaded onto the gel per lane for
inner medulla,
outer medulla,
and cortex,
respectively.
Membrane
samples were obtained
from the same rats as those used for Fig. 1.
AQUAF’ORINS
F418
REGULATION
OF RENAL
A
IN RATS
*
400
AQP3
%
x
350
SO-
0
ai?
500
soo-
La
QC
3#
.E
cn a>
>e
Fig. 5. Change in aquaporin-2
(A), aquaporin-3
(B),
aquaporin-4
(C), and aquaporin-1
(D) water channel
expression
in inner
medulla
(IM),
outer
medulla
(OM),
and cortex
(COR)
of hydrated
and thirsted
Sprague-Dawley
rats, as determined
by densitometry of Western
blots. Values
are expressed
as percent change (means
+ SE) of thirsted
animals
relative
to the mean
for the
hydrated
controls.
* Statistically
significant
(P < 0.05). Expression
levels of aquaporin-4
in outer medulla
and cortex were
too low to be quantitated
(NQ).
AQUAPORINS
250
250-
200
200-
150
150-
zg
100
loo-
E
.-
60
5
I
so0
O
I
-50 ’
C
I
I
IM
OM
COR
0
300
I
OM
I
COR
4007
AQP4
AQPI
350
300i
1
260
1
-50
’
I
I
IM
response to thirsting, we conclude that a large increase
in interstitial
tonicity is not a prerequisite for the
response to AVP. Furthermore, the failure of furosemide infusion to prevent the increase in aquaporin-2
and -3 expression in response to AVP also supports the
A
Fig. 6. Change
in aquaporin-2
(A), aquaporin-3
(B), aquaporin-4
(C), and aquaporin-1
(D) water
channel
expression
in inner medulla
(IM), outer
medulla
(OM),
and cortex
(COR)
in response
to
arginine
vasopressin
(AVP) infusion
in Brattleboro
rats, as determined
by densitometry
of Western
blots. Values
are expressed
as percent
change
(means t SE) relative
to mean for vehicle-infused
controls.
* Statistically
significant
(P < 0.05).Expression
levels of aquaporin-4
in outer medulla
and cortex were too low to be quantitated
(NQ).
1
-50
’
250-
>a”
a-
I
I
I
IM
OM
COR
B
300
AQP2
I
‘““1
I
I
OM
COR
-50
AQP3
’
I
I
I
IM
OM
COR
D 300-
C 300-
>5
n 2
’
notion that the AVP-induced increase in expression
levels of these two water channels does not require a
large increase in local interstitial osmolality. Thus we
conclude that the upregulatory action of AVP on aquaporin-2 and aquaporin-3 expression mav be a direct
IM
al
==
rF
ala
$tj
-50
I
COR
OM
AQP4
AQPI
250-
200-
200-
l50-
150-
loo-
loo-
50n .
-50
NQ
NQ
I
I
IM
I
OM
I
COR
-50
’
I
I
I
IM
OM
COR
Downloaded from http://ajprenal.physiology.org/ by 10.220.33.4 on June 18, 2017
3SO
I
IM
D
400
-8
-3
-50 ’
I
REGULATION
B 550
OF RENAL
I
Ii&lOM
COR
IM
OM
COR
AQPl
450(
Fig. 7. Change
in aquaporin-2
(A), aquaporin-3
(B), aquaporin-4
(C), and aquaporin-1
(D) water
channel
expression
in inner medulla
(IM),
outer
medulla
(OM),
and cortex (COR)
in response
to
AVP infusion
in furosemide-infused
Brattleboro
rats. Values
are expressed
as percent
change
(means
+ SE) relative
to mean for control
rats
infused
with furosemide
alone. * Statistically
significant
(P < 0.05). Expression
levels of aquaporin-4
in outer medulla
and cortex were too low
to be quantitated
(NQ).
350
uj+
>5
n,g
>g
Qz-
250-
250
150-
150
IM OMCOR
IM
OM
effect of the hormone on collecting duct cells rather
than an indirect effect of medullary hypertonicity.
In
the remainder of the discussion, we discuss these
observations in greater detail in the context of the
foregoing literature.
COR
It has been recognized for many years that long-term
restriction of fluid intake in humans results in an
increase in the maximal urinary osmolality attainable
in response to injections of vasopressin (7,X). Based on
studies in rats (4,20), we have proposed that this long-
250
200
150
100
50
0
-50
-100
COR
C
D
AQP4
300-
250
250-
3
E
200
zoo-
O-
300
*
09
SO-
150
92
a)0
loo-
100
eg
5
g
II
50
soNQ
42
2
0
0
-50
-5o-100
I
IM
I
OM
I
COR
-100
IM
OM
COR
Fig. 8. Change
in aquaporin-2
(A), aquaporin-3
(B), aquaporin-4
(C), and aquaporin-1
(D) water
channel
expression
in inner medulla
(IM), outer
medulla
(OM),
and cortex (COR)
in response
to
chronic furosemide
infusion
in AVP-infused
Sprague-Dawley
rats. Values are expressed
as percent
change (means + SE) relative
to mean for control
rats infused with AVP alone. * Statistically
significant (P < 0.05). Expression
levels of aquaporin-4
in outer medulla
and cortex were too low to be
quantitated
(NQ).
Downloaded from http://ajprenal.physiology.org/ by 10.220.33.4 on June 18, 2017
AQP4
F419
*
250
Ii4
IN RATS
AQP3
1
350
250
AQUAPORINS
F420
REGULATION
OF RENAL
IN RATS
osmolality
could have an independent
effect on water
channel expression.
Although it is conceivable that regulation
of aquaporin-2 and -3 levels can occur by a direct effect on
translation
or degradation
of these proteins, it is most
likely that the effects observed here are a result of
changes in aquaporin-2
and -3 mRNA levels. In fact,
thirsting
has been reported to increase mRNA levels for
aquaporin-2
(22, 37) in rats. Whether
the thirstinginduced increases in aquaporin-2
and -3 mRNA levels
are due to increased transcription
rates or to increased
mRNA stability
has not been investigated.
Cloning of
the Y-flanking
region of the aquaporin-2
gene has
revealed the presence
of a CAMP-response
element
(CRE), which could play a role in the AVP-induced
increase in aquaporin-2
expression
(34). In contrast, no
CRE has been identified
in the upstream
regulatory
region of the aquaporin-3
gene (13). However,
the
aquaporin-3
gene does contain
Spl and AP2 cisregulatory
elements, which have been associated with
CAMP-mediated
transcriptional
regulation
(1, 12, 28,
31) .
One byproduct of these investigations
is the observation that the degree of glycosylation
of aquaporin-2
differs in the medulla and the cortex (Fig. 1). That is, a
much greater fraction
of the total amount of aquaporin-2 appears to be glycosylated
in the inner medulla
than in the cortex. In addition, as shown in Fig. 2, a
large difference in glycosylation
of aquaporin-1
is seen
between cortex and medulla. However, with this water
channel, the band corresponding
to the glycosylated
form is most abundant
in the cortex. Neither
the
mechanistic
basis nor the functional
significance
of
these differences
are ascertainable
from the present
studies.
An additional finding of this study was that furosemide infusion for 5 days caused a striking
increase in
abundance of all three of the collecting duct aquaporins
(aquaporin-2,
-3, and -4) (Fig. 8). This finding is probably attributable
to the fact that chronic furosemide
infusion causes marked hypertrophy
of the renal collecting duct as well as other segments downstream
from
the site of furosemide action in the thick ascending limb
(17). In contrast,
chronic furosemide
infusion did not
increase the abundance
of aquaporin-1,
which is expressed in the proximal tubule and in the descending
limb, i.e., at a site upstream
from the thick ascending
limb, which does not hypertrophy
in response to chronic
furosemide
infusion.
Based on these studies, we suggest that the longterm regulation of aquaporin-2
and -3 expression
in the
renal collecting duct may play a role in the overall
regulation
in concentrating
ability in the mammalian
kidney. We have also observed that a long-term
deficiency in aquaporin-2
expression
as seen in the
Brattleboro
rat is associated with a marked downregulation of collecting duct water permeability
(4). Consequently, clinical situations
manifesting
chronically
low
levels of AVP, such as compulsive
water drinking
and
Downloaded from http://ajprenal.physiology.org/ by 10.220.33.4 on June 18, 2017
term enhancement
of urinary
concentrating
ability is
due in part to a “conditioned”
increase in intrinsic water
permeability
of the renal collecting ducts. These studies demonstrated
that fluid restriction
for 24 h or more
in normal rats (20) or long-term
infusion
of AVP in
Brattleboro
rats (4) increases the intrinsic water permeability of the renal collecting
ducts even after the
collecting ducts are removed from the short-term
influence of AVP. In previous studies (4, 25), we have demonstrated
that the increase in water permeability
is
associated with a marked increase in the abundance of
aquaporin-2
in the collecting duct cells. In Brattleboro
rats, the maximum
stimulated
water permeability
of
perfused inner medullary
collecting duct segments was
markedly
enhanced after long-term AVP infusion in the
rats, suggesting
that these collecting ducts can achieve
a higher water permeability
in vivo (4). Presumably,
the larger number of water channels in the collecting
duct cells allows more water channels to be translocated to the apical plasma membrane in response to an
acute rise in intracellular
CAMP, thus accounting
for
the higher water permeability.
More recently, we have
demonstrated
that the expression
level of aquaporin-3
is upregulated
in the renal inner medulla in response to
restriction
of fluid intake in rats (5).
In the present
study, we have confirmed
and extended the prior results.
Restriction
of fluid intake
resulted
in an increase
in the abundance
of aquaporin-2 and aquaporin-3
in all three regions of the
kidney (Figs. 1, 3, and 5). The abundance of these two
water
channels
also increased
in response
to AVP
infusion in Brattleboro
rats (Fig. 6). In contrast,
the
expression
levels of neither aquaporin-1
nor -4 were
affected by fluid restriction
or AVP infusion. Thus the
effects of water restriction
and increased AVP levels to
increase water channel expression
appear to be selective for aquaporin-2
and -3.
We investigated
the stimulus
for the increase
in
aquaporin-2
and -3 water channel expression.
First,
since the upregulatory
response to thirsting
was seen
not only in the medulla but also in the cortex, where
interstitial
osmolality is maintained
close to that of the
general circulation
(18), we conclude that the upregulatory response does not require a large increase in local
interstitial
osmolality.
Furthermore,
the increase in
aquaporin-2
and -3 expression
in response to AVP was
not prevented
by washout
of the corticomedullary
osmolality
gradient by infusion of furosemide
(Fig. 7).
This observation
adds further support to the view that
the effect of AVP on aquaporin expression
is not exclusively dependent
on its effect to increase medullary
interstitial
osmolality. Based on these observations,
we
believe that the effect of water restriction
and AVP
infusion to increase aquaporin-2
and -3 expression
in
these studies is probably mediated predominantly
by a
direct effect of AVP on the collecting duct cells rather
than through
an alteration
in medullary
osmolality.
However,
it must be pointed out that these studies do
not rule out the possibility
that changes in interstitial
AQUAPORINS
REGULATION
OF RENAL
central diabetes insipidus,
can be expected to be accompanied by a “physiological
nephrogenic
diabetes insipidus” due to downregulation
of water channel expression, which delays correction
of the associated
water
balance disorder until the downregulation
is corrected.
Further studies will be required to definitively
test this
possibility.
AQUAPORINS
15.
16
’
17.
Address
for reprint
requests:
M. A. Knepper,
Health,
Bldg. 10, Rm. 6N307,
10 Center
Drive
MD 20892-1598.
Received
22 December
1995;
accepted
in final
National
Institutes
of
MSC 1598, Bethesda,
form
15 March
1996.
REFERENCES
19.
20.
21.
22.
23.
24
25.
26.
27.
28.
29.
30.
31.
32.
F421
Jones,
R. V. H., and H. E. de Wardener.
Urine
concentration
after fluid deprivation
or pitressin
tannate
in oil. Br. Med. J. 1:
271-274,1956.
Jung,
J. S., R. V. Bhat,
G. M. Preston,
W. B. Guggino,
J. M.
Baraban,
and P. Agre.
Molecular
characterization
of an aquaporin cDNA from brain: candidate
osmoreceptor
and regulator
of
water
balance.
Proc. NatZ. Acad. Sci. USA 91: 13052-13056,
1994.
Kaissling,
B., and B. A. Stanton.
Adaptation
of distal tubule
and collecting
duct to increased
sodium
delivery.
I. Ultrastructure. Am. J. Physiol.
255 (Renal Fluid Electrolyte
Physiol.
24):
F1256-F1268,1988.
Knepper,
M., and M. Burg.
Organization
of nephron
function.
Am. J. Physiol.
244 (Renal Fluid Electrolyte
Physiol.
13): F579F589,1983.
Kuwahara,
M., K. Fushimi,
Y. Terada,
L. Bai, F. Marumo,
and S. Sasaki.
CAMP-dependent
phosphorylation
stimulates
water
permeability
of aquaporin-collecting
duct water
channel
protein
expressed
in Xenopus
oocytes.
J. BioZ. Chem.
270:
10384-10387,1995.
Lankford,
S. P., C.-L.
Chou,
Y. Terada,
S. M. Wall,
J. B.
Wade, and M. A. Knepper.
Regulation
of collecting
duct water
permeability
independent
of CAMP-mediated
AVP response.
Am. J. Physiol.
261 (Renal Fluid EZectroZyte
PhysioZ.
30): F554F566,1991.
Ma, T., A. Frigeri,
H. Hasegawa,
and A. S. Verkman.
Cloning
of a water channel
homolog
expressed
in brain meningeal
cells
and kidney
collecting
duct that functions
as a stilbene-sensitive
glycerol transporter.
J. BioZ. Chem. 269: 21845-21849,1994.
Ma, T., H. Hasegawa,
W. R. Skach,
A. Frigeri,
and A. S.
Verkman.
Expression,
functional
analysis,
and in situ hybridization of a cloned rat kidney
collecting
duct water channel.
Am. J.
Physiol.
266 (CeZZ Physiol.
35): C189-C197,
1994.
Marples,
D., M. A. Knepper,
E. I. Christensen,
and S.
Nielsen.
Redistribution
of aquaporin-2
water channels
induced
by vasopressin
in rat kidney
inner medullary
collecting
duct.
Am. J. Physiol.
269 (CeZZ Physiol.
38): C655-C664,
1995.
Nielsen,
S., C.-L. Chou,
D. Marples,
E. I. Christensen,
B. K.
Kishore,
and M. A. Knepper.
Vasopressin
increases
water
permeability
of kidney
collecting
duct by inducing
translocation
of aquaporin-CD
water
channels
to plasma
membrane.
Proc.
NatZ. Acad. Sci. USA 92: 1013-1017,1995.
Nielsen,
S., S. R. DiGiovanni,
E. I. Christensen,
M. A.
Knepper,
and H. W. Harris.
Cellular
and subcellular
immunolocalization
of vasopressin-regulated
water
channel
in rat kidney. Proc. NatZ. Acad. Sci. USA 90: 11663-11667,1993.
Nielsen,
S., and M. A. Knepper.
Vasopressin
activates
collecting duct urea
transporters
and water
channels
by distinct
physical
processes. Am. J. Physiol.
265 (Renal Fluid Electrolyte
Physiol.
34): F204-F213,1993.
Nielsen,
S., B. L. Smith,
E. I. Christensen,
M. A. Knepper,
and P. Agre.
CHIP28
water
channels
are localized
in constitutively
water-permeable
segments
of the nephron.
J. Cell BioZ.
120: 371-383,1993.
Roesler,
W. J., G. R. Vandenbark,
and R. W. Hanson.
Cyclic
AMP and the induction
of eukaryotic
gene transcription.
J. BioZ.
Chem. 263: 9063-9066,1988.
Sabolic,
I., T. Katsura,
J. M. Verbabatz,
and D. Brown.
The
AQP2 water
channel:
effect of vasopressin
treatment,
microtubule disruption,
and distribution
in neonatal
rats. J. Membr.
BioZ. 143: 165-177,
1995.
Sabolic,
I., G. Valenti,
J.-M. Verbabatz,
A. N. Van Hoek,
A. S.
Verkman,
D. A. Ausiello,
and D. Brown.
Localization
of the
CHIP28
water
channel
in rat kidney.
Am. J. Physiol.
263 (CeZZ
Physiol.
32): C1225-C1233,
1992.
Shiotani,
A., and J. L. Merchant.
CAMP regulates
gastrin
gene
expression.
Am. J. Physiol.
269 (Gastrointest.
Liver PhysioZ.
32):
G458-G464,1995.
Star, R. A., H. Nonoguchi,
R. Balaban,
and M. A. Knepper.
Calcium
and cyclic adenosine
monophosphate
as second messengers for vasopressin
in the rat inner medullary
collecting
duct. J.
CZin. Invest. 81: 1879-1888,1988.
Downloaded from http://ajprenal.physiology.org/ by 10.220.33.4 on June 18, 2017
1. Chang,
C.-Y., Huang,
I.-C. Guo, H.-M.
Tsai, D.-A. Wu, and B.
Chung.
Transcription
of the human
ferredoxin
gene through
a
single promoter
which contains
the 3’,5’-cyclic
adenosine
monophosphate-responsive
sequence and Spl-binding
site. MoZ. Endocrinol. 92: 1362-1370,1992.
2. Deen,
P M,, J. A. Dempster,
B. Wieringa,
and C. H. van OS.
Isolation
of a cDNA for rat CHIP28
water
channel:
high mRNA
expression
in kidney
cortex
and inner medulla.
Biochem.
Biophys. Res. Commun.
188: 1267-1273,1992.
3. Denker,
B. M., B. L. Smith,
F. P. Kuhajda,
and P. Agre.
Identification,
purification,
and partial
characterization
of a
novel M, 28,000 integral
membrane
protein
from erythrocytes
and renal tubules.
J. Biol. Chem. 263: 15634-15642,1988.
4. DiGiovanni,
S. R., S. Nielsen,
E. I. Christensen,
and M. A.
Knepper.
Regulation
of collecting
duct water
channel
expression by vasopressin
in Brattleboro
rat. Proc. Natl. Acad. Sci. USA
91: 8984-8988,1994.
5. Ecelbarger,
C. A., J. Terris,
G. Frindt,
M. Echevarria,
D.
Marples,
S. Nielsen,
and M. A. Knepper.
Aquaporin-3
water
channel
localization
and regulation
in rat kidney. Am. J. Physiol.
269 (Renal Fluid Electrolyte
Physiol.
38): F663-F672,
1995.
6. Echevarria,
M., E. E. Windhager,
S. S. Tate, and G. Frindt.
Cloning
and expression
of AQP3,
a water
channel
from the
medullary
collecting
duct of rat kidney.
Proc. NatZ. Acad. Sci.
USA 91: 10997-11001,1994.
7. Epstein,
F. H., C. R. Kleeman,
and A. Hendrikx.
The influence of bodily hydration
on the renal concentrating
process. J.
CZin. Invest. 36: 629-634,
1957.
8. Flamion,
B., K. R. Spring,
and M. Abramow.
Adaptation
of
inner medullary
collecting
duct to dehydration
involves
a paracellular
pathway.
Am. J. Physiol.
268 (Renal
FZuid
Electrolyte
Physiol.
37): F53-F63,1995.
9. Frigeri,
A., M. A. Gropper,
C. W. Turck,
and A. S. Verkman.
Immunolocalization
of the mercurial-insensitive
water
channel
and glycerol
intrinsic
protein
in epithelial
cell plasma
membranes. Proc. NatZ. Acad. Sci. USA 92: 4328-4331,1995.
10. Han,
J. S., Y. Maeda,
C. Ecelbarger,
and M. A. Knepper.
Vasopressin-independent
regulation
of collecting
duct water
permeability.
Am. J. Physiol.
266 (Renal Fluid Electrolyte
Physiol.
35): F139-F146,1994.
11. Hasegawa,
H., T. Ma, W. Skach,
M. A. Matthay,
and A. S.
Verkman.
Molecular
cloning
of a mercurial-insensitive
water
channel expressed
in selected water-transporting
tissues. J. BioZ.
Chem. 269: 5497-5500,1994.
12. Imagawa,
M., R. Chiu,
and M. Karin.
Transcription
factor
AP-2 mediates
induction
by two different
signal-transduction
pathways:
protein
kinase C and CAMP. Cell 51: 251-260,1987.
13. Inase,
N., K. Fushimi,
K. Ishibashi,
S. Uchida,
M. Ichioka,
S. Sasaki,
and F. Marumo.
Isolation
of human
aquaporin
3
gene. J. BioZ. Chem. 270: 17913-17916,1995.
14. Ishibashi,
K., S. Sasaki,
K. Fushimi,
S. Uchida,
M. Kuwahara,
H. Saito, T. Furukawa,
K. Nakajima,
Y. Yamaguchi,
T.
Gojobori,
and F. Marumo.
Molecular
cloning
and expression
of
a member
of the aquaporin
family
with permeability
to glycerol
and urea in addition
to water
expressed
at the basolateral
membrane
of kidney
collecting
duct cells. Proc. NatZ. Acad. Sci.
USA 91: 6269-6273,1994.
18*
IN RATS
F422
REGULATION
OF RENAL
33. Terris,
J., C.A. Ecelbarger,
D. Marples,
M.A. Knepper,
and
S. Nielsen.
Distribution
of aquaporin-4
water
channel
expression within
rat kidney. Am. J. Physiol.
269 (Renal Fluid Electrolyte Physiol.
38): F775-F785,
1995.
34. Uchida,
S., S. Sasaki,
K. Fushimi,
and F. Marumo.
Isolation
of human
aquaporin-CD
gene. J. Biol. Chem. 269: 23451-23455,
1994.
35. Wall,
S. RI., J. S. Han,
C.-L.
Chou,
and M. A. Knepper.
Kinetics
of urea and water permeability
activation
by vasopressin in rat terminal
IMCD.
Am. J. Physiol.
262 (Renal
Fluid
Electrolyte
Physiol.
3 1): F989-F998,
1992.
AQUAPORINS
IN RATS
36. Yamamoto,
N., S. Sasaki,
K. Fushimi,
K. Ishibashi,
E.
Yaiota,
K. Kawasaki,
F. Marumo,
and I. Kihara.
Vasopressin
increases
A&P-CD
water
channel
in the apical
membrane
of
collecting
duct cells without
affecting
AQP3
distribution
in
Brattleboro
rat. Am. J. Physiol.
268 (Cell Physiol.
37): C1546c1551,1995.
37. Yamamoto,
T., S. Sasaki,
K. Fushimi,
K. Kawasaki,
E.
Yaoita,
K. Oota,
K. Hirata,
F. Marumo,
and I. Kihara.
Localization
and expression
of a collecting
duct water
channel,
aquaporin,
in hydrated
and dehydrated
rats. Exp. Nephrol.
3:
193-201,1995.
Downloaded from http://ajprenal.physiology.org/ by 10.220.33.4 on June 18, 2017