Upregulation of Na+ transporter abundances in
response to chronic thiazide or loop diuretic treatment in
rats
Ki Young
Na1,
Yoon Kyu
Oh2,
Jin Suk
Han1,
Kwon Wook
Joo1,
Jung Sang
Lee1,
Jae-Ho
Earm3,
Mark A.
Knepper4, and
Gheun-Ho
Kim5
1 Department of Internal Medicine, Seoul National
University, Clinical Research Institute of Seoul National University
Hospital, 2 Department of Internal Medicine, Eulji Medical
College, 5 Department of Internal Medicine, Hallym University
Hangang Sacred Heart Hospital, Seoul, 110-744; 3 Department
of Internal Medicine, Chungbuk National University, Cheongju
361-711, South Korea; 4 and Laboratory of Kidney and
Electrolyte Metabolism, National Institutes of Health, Bethesda,
Maryland 20892
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ABSTRACT |
Furosemide
and hydrochlorothiazide (HCTZ) exert their diuretic actions by binding
to apical Na+ transporters, viz., the
Na+-K+-2Cl
cotransporter in
the thick ascending limb and the Na+-Cl
cotransporter in the distal convoluted tubule, respectively. We carried
out semiquantitative immunoblotting and immunohistochemistry of rat
kidneys to investigate whether chronic administration of furosemide or
HCTZ is associated with compensatory changes in the abundance of
Na+ transporters downstream from the primary site of
action. Osmotic minipumps were implanted into Sprague-Dawley rats to
deliver furosemide (12 mg/day) or HCTZ (3.75 mg/day) for 7 days. To
prevent volume depletion, all animals were offered tap water and a
solution containing 0.8% NaCl and 0.1% KCl as drinking fluid. The
diuretic/natriuretic response was quantified in response to both agents
by using quantitative urine collections. Semiquantitative
immunoblotting revealed that the abundances of thick ascending limb
Na+-K+-2Cl
cotransporter and all
three subunits of the epithelial Na+ channel (ENaC) were
increased by furosemide infusion. HCTZ infusion increased the
abundances of thiazide-sensitive Na+-Cl
cotransporter and
-ENaC in the cortex and
- and
-ENaC in the outer medulla. Consistent with these results,
-ENaC
immunohistochemistry showed a remarkable increase in immunoreactivity
in the principal cells of collecting ducts with either diuretic
treatment. These increases in the abundance of Na+
transporters in response to chronic diuretic treatment may account for
the generation of diuretic tolerance associated with long-term diuretic use.
collecting duct; epithelial sodium channel; distal convoluted
tubule; thiazide-sensitive sodium-chloride cotransporter
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INTRODUCTION |
DIURETICS ARE
FREQUENTLY PRESCRIBED for treatment of hypertension and edematous
disorders. Although they are clinically useful to induce negative
Na+ balance, diuretic resistance is often encountered to
limit their further use. Diuretic resistance may be explained in part
by increased Na+ absorption in downstream renal tubule
segments related to increased Na+ delivery
(10).
Furosemide and hydrochlorothiazide (HCTZ) are diuretics commonly used
in clinical practice. Furosemide exerts its diuretic action by binding
to the Na+-K+-2Cl
cotransporter
(NKCC2) in the thick ascending limb and blocking ion transport
(34). HCTZ exerts its diuretic action by binding to the
Na+-Cl
cotransporter (NCC) in the distal
convoluted tubule (4). Increased Na+ delivery
to the distal nephron may result in enhanced Na+ absorption
downstream from the distal site of diuretic action. Recent research in
the pathophysiology of other disorders of water and electrolyte balance
suggests that long-term adaptive mechanisms may be important in these
disorders and may involve altered expression of transporter proteins,
such as aquaporin water channels and Na+ transporters
(13, 19, 21, 22, 30). In this context, it appears possible
that diuretic resistance produced by long-term diuretic administration
is in part due to adaptive increases in the abundances of
Na+ transporters in the distal nephron and collecting ducts.
Therefore, this study was undertaken to elucidate the molecular basis
of the adaptive mechanisms in long-term use of diuretics. We
hypothesized that a secondary increase in distal delivery of Na+ may induce compensatory changes in the abundance of
Na+ transporters downstream from the primary site of the
diuretic action. To test this hypothesis, we administered either
furosemide or HCTZ for 7 days to rats and investigated the effects on
the expression of Na+ transporter proteins by using
semiquantitative immunoblotting and immunohistochemistry on tissue from
rat kidneys.
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METHODS |
Animals and experimental protocols.
Specific pathogen-free male Sprague-Dawley rats (170-230 g; SLC,
Shizuoka, Japan) were placed in metabolism cages 3 days before the
beginning of the study. Control and treated rats were designated randomly, and all were provided with a daily, fixed amount of finely
ground regular rat chow (18 g · 200 g body
wt
1 · day
1) and two separate
bottles of drinking water, one containing 0.8% NaCl and 0.1% KCl and
the other containing tap water. All the rats ate all of the offered rat
chow and showed a steady increase in body weight throughout the study
period. The chronic studies were carried out as follows.
Chronic furosemide infusion.
Rats were anesthetized with enflurane (Choongwae Pharma, Seoul, Korea),
and osmotic minipumps (model 2ML1, Alzet, Palo Alto, CA) were
subcutaneously implanted to deliver 12 mg/day of furosemide (Handok,
Seoul, Korea). Furosemide was dissolved in a 1.7% ethanolamine solution. Control rats were implanted with the minipumps containing vehicle (ethanolamine) alone.
Chronic HCTZ infusion.
Rats were infused with either 3.75 mg/day of HCTZ (YuHan, Seoul, Korea)
or vehicle for 7 days. These infusions were achieved by using the same
vehicle solution (1.7% ethanolamine) and osmotic minipumps as
described above.
Physiological measurements.
During the course of the studies, daily urine was collected to evaluate
responses to the diuretics. Urine osmolality was measured with a
cryoscopic osmometer (Osmomat 030-D-M, Gonotec, Berlin, Germany), and
urine electrolytes and creatinine were measured with an ion-selective
method (System E4A, Beckman Coulter, Fullerton, CA). Serum samples were
collected at the time each rat was killed for determination of the
serum aldosterone concentration by radioimmunoassay (SPAC-S aldosterone
kit, Daiichi Pharmaceutical, Tokyo, Japan).
Semiquantitative immunoblotting.
After 7 days of infusion, the rats were killed by decapitation,
and kidneys were rapidly removed and placed in chilled isolation solution containing 250 mM sucrose, 10 mM triethanolamine (Sigma, St.
Louis, MO), 1 µg/ml leupeptin (Sigma), and 0.1 mg/ml
phenylmethylsulfonyl fluoride (Sigma) titrated to pH 7.6. Next, the
kidneys were dissected to obtain cortex and inner stripe of the outer
medulla. Each region was separately homogenized in 10 (cortex) or 1 ml
(outer medulla) of ice-cold isolation solution at 15,000 rpm with three
strokes for 15 s with a tissue homogenizer (PowerGun 125, Fisher
Scientific, Pittsburgh, PA). After homogenization, total protein
concentration was measured by using the bicinchoninic acid protein
assay reagent kit (Sigma) and adjusted to 2 µg/µl with isolation
solution. The samples were then stabilized by the addition of 1 vol 5×
Laemmli sample buffer/4 vol sample, heated to 60°C for 15 min.
Initially, "loading gels" were done on each sample set to allow
fine adjustment of loading amount to guarantee equal loading on
subsequent immunoblots. Five micrograms of protein from each sample
were loaded into each individual lane and electrophoresed on 12%
polyacrylamide-SDS minigels by using Mini-PROTEAN III electophoresis apparatus (Bio-Rad, Hercules, CA) and then stained with Coomassie blue
dye (0.025% solution made in 4.5% methanol and 1% acetic acid;
G-250, Bio-Rad). Selected bands from these gels were scanned (GS-700
Imaging Densitometry, Bio-Rad) to determine density (Molecular Analyst
version 1.5, Bio-Rad) and relative amounts of protein loaded in each
lane. Finally, protein concentrations were "corrected" to reflect
these measurements. For immunoblotting, the proteins were transferred
electrophoretically from unstained gels to nitrocellulose membrane
(Bio-Rad). After being blocked with 5% skim milk in PBS-T (80 mM
Na2HPO4, 20 mM NaH2PO4,
100 mM NaCl, and 0.1% Tween-20, pH 7.5) for 30 min, membranes were
probed overnight at 4°C with the respective primary antibodies. For
probing blots, all antibodies were diluted into a solution containing
150 mM NaCl, 50 mM sodium phosphate, 10 mg/dl sodium azide, 50 mg/dl
Tween 20, and 0.1 g/dl bovine serum albumin (pH 7.5). The secondary
antibody was goat anti-rabbit IgG conjugated to horseradish peroxidase
(31458, Pierce, Rockford, IL) diluted to 1:3,000. Sites of
antibody-antigen reaction were viewed by using enhanced
chemiluminescence substrate (ECL RPN 2106, Amersham Pharmacia Biotech,
Buckinghamshire, UK) before exposure to X-ray film (Hyperfilm, Amersham
Pharmacia Biotech).
Immunohistochemistry.
The kidneys were perfused by retrograde perfusion via the abdominal
aorta with 1% PBS to remove blood and then with
periodate-lysine-paraformaldehyde (0.01 M NaIO4, 0.075 M
lysine, and 2% paraformaldehyde in 0.0375 M
Na2HPO4 buffer, pH 6.2) for kidney fixation for
3 min. After completion of perfusion, each kidney was sliced into
5-mm-thick pieces and immersed in 2% periodate-lysine-paraformaldehyde
solution overnight at 4°C. Each slice was dehydrated with a graded
series of ethanol and embedded in polyester wax. The embedded pieces of
kidney were sectioned at 5-µm thickness on a microtome (RM 2145, Leica Instruments, Nussloch, Germany).
The sections were dewaxed with a graded series of ethanol and treated
with 3% H2O2 for 30 min to eliminate
endogenous peroxidase activity. They were blocked with 6% normal goat
serum (S-1000, Vector Laboratory, Burlington, CA) for 15 min. They were
incubated overnight at 4°C with the respective primary antibodies
diluted in PBS. After incubation, they were washed with PBS and
incubated for 30 min in biotinylated goat anti-rabbit IgG (BA-1000,
Vector Laboratory) at room temperature. Next, peroxidase standard
vectastatin ABC kit (PK-4000, Vector Laboratory) was added for
30-60 min at room temperature. The sections were washed with PBS
and incubated in 3,3'-diaminobenzidine substrate kit (SK-4100, Vector
Laboratory). Hematoxylin staining was used as a counterstain. The
slides were mounted with Canadian balsam.
Primary antibodies.
For semiquantitative immunoblotting and immunohistochemistry, we used
previously characterized polyclonal antibodies. Affinity-purified polyclonal antibodies against the thick ascending limb NKCC2
(9), thiazide-sensitive NCC (20), and
-,
-, and
-subunits of the epithelial Na+ channel (ENaC)
(26) were used. In addition, the present study utilized
polyclonal antibodies against aquaporin-1 (AQP1) (40), AQP2 (32), and AQP3 (8).
Statistics.
Values were presented as means ± SE. Quantitative comparisons
between the groups were made by Mann-Whitney U-test
(Statview software, Abacus Concepts, Berkeley, CA). To facilitate
comparisons in the semiquantitative immunoblotting, we normalized the
band density values by dividing by the mean value for the control
group. Thus the mean for the control group is defined as 100%. P
< 0.05 was considered as indicative of statistical significance.
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RESULTS |
Chronic furosemide infusion.
Physiological data from urine collections confirmed the effects
of furosemide. Table 1 shows urinary
data on the final day of chronic furosemide infusion. Urine output was
markedly increased and urine osmolality was decreased by furosemide
infusion compared with vehicle-infused controls. In furosemide-infused
rats, urinary Na+ and Cl
excretion was
remarkably increased, but there was no significant change in urinary
K+ excretion. Creatinine clearance was also not
significantly affected by furosemide infusion.
Despite persistent osmotic diuresis for 7 days, body weight measured
before euthanasia was not different between the two groups (furosemide-infused rats, 198 ± 4 g, vs. vehicle-infused
rats, 204 ± 4 g). Furthermore, serum aldosterone level was
not altered by furosemide infusion (furosemide-infused rats, 0.40 ± 0.15 nM, vs. vehicle-infused rats, 0.38 ± 0.14 nM).
Analysis of the renal cortical homogenates prepared for immunoblotting
revealed a significant increase in total amount of protein in
furosemide-infused rat cortices compared with vehicle-infused controls
(80.8 ± 3.7 vs. 66.0 ± 3.5 mg/100 g body wt,
P < 0.05). This finding is consistent with the
previously demonstrated furosemide-induced cortical hypertrophy
(17, 18). On the other hand, there was no difference in
total amount of protein in the outer medullary homogenates between the
two groups.
Figure 1A shows an immunoblot
of the thick ascending limb NKCC2 from cortical homogenates.
Furosemide infusion for 7 days resulted in a significant increase in
NKCC2 abundance in the cortex. Normalized band densities for
furosemide-infused and vehicle-infused rats were 151 ± 10 and
100 ± 10, respectively (means ± SE, P < 0.05). In Fig. 1C, the abundance of NKCC2 in the outer
medulla was also increased by furosemide infusion (122 ± 5 for
furosemide-infused rats vs. 100 ± 3 for vehicle-infused rats,
P < 0.01). Parallel Coomassie blue-stained
SDS-polyacrylamide gels demonstrated uniform loading among all samples
(Fig. 1, B and D), ruling out the possibility that the increase in band density in cortex or outer medulla could be
due to differences in loading.

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Fig. 1.
Effects of furosemide infusion on abundance of thick
ascending limb Na+-K+-2Cl
cotransporter (NKCC2). Immunoblots of cortical homogenates
(A) and outer medullary homogenates (C) from
Sprague-Dawley rats receiving a subcutaneous infusion of furosemide (12 mg/day) or vehicle via osmotic minipump are shown. Each lane was loaded
with protein sample from a different rat. Blots were probed with rabbit
polyclonal anti-NKCC2 antibody L224. A: blot of cortex was
loaded with 10 µg total protein/lane. Band density for NKCC2 protein
was significantly increased by furosemide infusion. C: blot
of outer medulla was loaded with 5 µg total protein/lane. Band
density for NKCC2 protein was significantly increased by furosemide
infusion. Twelve percent Coomassie blue-stained SDS-polyacrylamide gels
from cortical homogenates (B) and outer medullary
homogenates (D) confirm equal loading among lanes.
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The abundance of thiazide-sensitive NCC in the cortex was not altered
by furosemide infusion (Fig. 2).
Normalized band densities for furosemide-infused rats vs.
vehicle-infused rats were 101 ± 11 vs. 100 ± 19, respectively. Figure 3 shows NCC
immunohistochemistry from the cortex of furosemide-infused and
vehicle-infused rats. Consistent with previous studies (12,
17), distal tubular hypertrophy was noted in furosemide-infused
rats. However, no difference in NCC immunoreactivity was found between
furosemide-infused and vehicle-infused rats.

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Fig. 2.
Effect of furosemide infusion on abundance of
thiazide-sensitive Na+-Cl cotransporter (NCC)
in cortex. Immunoblot of the same cortical homogenates from
Sprague-Dawley rats as used for Fig. 1 is shown. Each lane was
loaded with a protein sample from a different rat. The blot was loaded
with 10 µg total protein/lane and was probed with rabbit polyclonal
anti-NCC antibody L573. Band density for NCC protein was not
significantly altered by furosemide infusion.
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Fig. 3.
Immunohistochemistry of thiazide-sensitive NCC in cortex from
Sprague-Dawley rats infused with vehicle or furosemide. Top,
×100; bottom, ×400.
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Figure 4 shows the immunoblots carried
out for the three ENaC subunits in cortical homogenates. Significant
increases in all three subunits of ENaC protein abundance were found in
response to chronic furosemide infusion. Normalized band densities of
each subunit protein were as follows:
-subunit (Fig. 4A),
187 ± 25 for furosemide vs. 100 ± 22 for vehicle,
P < 0.05;
-subunit (Fig. 4B), 155 ± 8 for furosemide vs. 100 ± 15 for vehicle, P < 0.05; and
-subunit (Fig. 4C) 168 ± 16 for
furosemide vs. 100 ± 9 for vehicle, P < 0.05. Responses of the ENaC subunits in the outer medulla of the same rats
were qualitatively the same as those seen in the cortex (Fig.
5). Band density of each subunit protein was as follows:
-subunit (Fig. 5A), 171 ± 27 for
furosemide vs. 100 ± 17 for vehicle, P < 0.05;
-subunit (Fig. 5B), 986 ± 91 for furosemide vs.
100 ± 33% for vehicle, P < 0.01; and
-subunit (Fig. 5C), 242 ± 24 for furosemide
vs. 100 ± 22 for vehicle, P < 0.01.

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Fig. 4.
Effects of furosemide infusion on abundance of epithelial
Na+ channel (ENaC) in cortex. Each lane was loaded with
cortical homogenate from a different rat. A: blot was loaded
with 30 µg total protein/lane and was probed with rabbit polyclonal
anti- -ENaC antibody L766. B: blot was loaded with 30 µg
total protein/lane and was probed with rabbit polyclonal anti- -ENaC
antibody L558. C: blot was loaded with 30 µg total
protein/lane and was probed with rabbit polyclonal anti- -ENaC
antibody L550. Band density for each subunit protein was significantly
increased by furosemide infusion.
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Fig. 5.
Effects of furosemide infusion on abundance of ENaC in
outer medulla. Each lane was loaded with outer medullary homogenate
from a different rat. A: blot was loaded with 30 µg total
protein/lane and was probed with rabbit polyclonal anti- -ENaC
antibody L766. B: blot was loaded with 30 µg total
protein/lane and was probed with rabbit polyclonal anti- -ENaC
antibody L558. C: blot was loaded with 30 µg total
protein/lane and was probed with rabbit polyclonal anti- -ENaC
antibody L550. Band density for each subunit protein was significantly
increased by furosemide infusion.
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Figure 6 demonstrates ENaC
immunohistochemistry from the renal cortex of furosemide-infused and
vehicle-infused rats by using the
-ENaC antibody. Figure 6,
top, shows low-power images demonstrating a marked increase
in immunostaining in furosemide-infused vs. vehicle-infused rat. In
higher-power images (Fig. 6, bottom), the size of the
tubular epithelial cells appeared larger in a furosemide-infused rat
compared with a vehicle-infused rat. Staining conditions and exposure
settings on the microscope were identical for the two images. Similar
observations were made in three pairs of rats.

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Fig. 6.
Immunohistochemistry of -ENaC in cortex from
Sprague-Dawley rats infused with vehicle or furosemide. Each panel
shows a marked increase in immunostaining in furosemide-infused vs.
vehicle-infused rat. Top, ×100; bottom,
×400.
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We also carried out immunoblotting for AQP1-3 in the cortex and
outer medulla (not shown). In the cortex, there were no significant changes in the band densities of AQP1 (105 ± 7 for furosemide vs.
100 ± 7 for vehicle), AQP2 (149 ± 18 for furosemide vs.
100 ± 17 for vehicle), and AQP3 (126 ± 17 for furosemide
vs. 100 ± 12 for vehicle). As in the cortex, band density for
each aquaporin protein in the outer medulla was not significantly
altered by furosemide infusion (AQP1, 80 ± 9 vs. 100 ± 9;
AQP2, 94 ± 11 vs. 100 ± 7; and AQP3, 72 ± 12 vs. 100 ± 22).
Chronic HCTZ infusion.
Chronic infusion of HCTZ revealed similar changes in urinary indices to
furosemide infusion. Table 2 shows
urinary data on the final day of HCTZ infusion. HCTZ produced a
significant increase in urine output and a decrease in urine
osmolality. As in the furosemide infusion study, urinary
Na+ and Cl
excretion were elevated but
K+ excretion was not significantly increased in
HCTZ-infused rats. Creatinine clearance was not affected.
In the HCTZ infusion study, the total amount of cortical protein
measured in the homogenates of cortex dissected from the kidneys of
HCTZ-infused rats was not different from the corresponding value of
vehicle-infused rats (68.6 ± 2.4 vs. 65.2 ± 1.5 mg/100 g
body wt). The two groups had no difference in total amount of protein
in the outer medullary homogenates.
Figure 7A shows an NCC
immunoblot in cortical homogenates from vehicle-infused and
HCTZ-infused rats. The abundance of NCC protein in HCTZ-infused rats
was significantly increased compared with vehicle-infused rats
(normalized band densities: 166 ± 14 for HCTZ vs. 100 ± 13 for vehicle, P < 0.05). Figure 7B shows a
loading control for the immunoblot, achieved by running an identically loaded polyacrylamide gel and staining it with Coomassie blue. There
was little or no variation in the densities of the major bands stained
with Coomassie blue, demonstrating that the increase in NCC band
density seen in Fig. 7A was not due to unequal loading.

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Fig. 7.
Effect of hydrochlorothiazide (HCTZ) infusion on
abundance of thiazide-sensitive NCC in cortex. Immunoblot of cortical
homogenates from Sprague-Dawley rats receiving a subcutaneous infusion
of HCTZ (3.75 mg/day) or vehicle via osmotic minipump is shown. Each
lane was loaded with protein sample from a different rat.
A: blot was loaded with 10 µg total protein/lane and was
probed with rabbit polyclonal anti-NCC antibody L573. Band density for
NCC protein was significantly increased by HCTZ infusion. B:
12% Coomassie blue-stained SDS-polyacrylamide gel from cortical
homogenates, confirming equal loading among lanes.
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Figure 8 shows NCC immunohistochemistry
from the cortex of HCTZ-infused and vehicle-infused rats. Compatible
with the immunoblot result, it reveals an increase in immunostaining in
an HCTZ-infused vs. a vehicle-infused rat. Similar observations were
made in three pairs of rats.

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Fig. 8.
Immunohistochemistry of thiazide-sensitive NCC in cortex from
Sprague-Dawley rats infused with vehicle or HCTZ. An increase in
immunostaining is noted in HCTZ-infused vs. vehicle-infused rat.
Top, ×100; bottom, ×400.
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Figure 9 shows the immunoblots from the
same cortical homogenates for the three ENaC subunits. In response to
HCTZ infusion, an increase in
-ENaC protein abundance was noted
(Fig. 9B). Normalized band density increased to 139 ± 7 of vehicle-infused controls (100 ± 4, P < 0.05). However, no significant increase was seen in the abundance of
the
-subunit (Fig. 9A; 111 ± 13 for HCTZ vs.
100 ± 11 for vehicle) or
-subunit (Fig. 9C;
122 ± 10 for HCTZ vs. 100 ± 12 for vehicle). Changes in the
abundance of ENaC in the outer medulla are depicted in Fig.
10. Band density of
-ENaC was not
significantly increased by HCTZ infusion (Fig. 10A; 122 ± 19 for HCTZ vs. 100 ± 9 for vehicle). In contrast, both
-ENaC (Fig. 10B; 201 ± 28 for HCTZ vs. 100 ± 16 for vehicle, P < 0.05) and
-ENaC (Fig.
10C; 212 ± 17 for HCTZ vs. 100 ± 8 for vehicle, P < 0.01) protein abundance were increased in the
outer medullary homogenates. A parallel Coomassie blue-stained
SDS-polyacrylamide gel demonstrated uniform loading among all samples
(Fig. 10D), ruling out the possibility that the increase in
band density in the outer medulla could be due to differences in
loading.

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Fig. 9.
Effects of HCTZ infusion on abundance of ENaC in cortex.
Each lane was loaded with cortical homogenate from a different rat.
A: blot was loaded with 30 µg total protein/lane and was
probed with rabbit polyclonal anti- -ENaC antibody L766. Band density
for -ENaC protein was not significantly altered by HCTZ infusion.
B: blot was loaded with 30 µg total protein/lane and was
probed with rabbit polyclonal anti- -ENaC antibody L558. Band density
for -ENaC protein was significantly increased by HCTZ infusion.
C: blot was loaded with 30 µg total protein/lane and was
probed with rabbit polyclonal anti- -ENaC antibody L550. Band density
for -ENaC protein was not significantly altered by HCTZ infusion.
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Fig. 10.
Effects of HCTZ infusion on abundance of ENaC in outer
medulla. Immunoblots of outer medullary homogenates from Sprague-Dawley
rats receiving a subcutaneous infusion of HCTZ (3.75 mg/day) or vehicle
via osmotic minipump are shown. Each lane was loaded with protein
sample from a different rat. A: blot was loaded with 30 µg
total protein/lane and was probed with rabbit polyclonal anti- -ENaC
antibody L766. Band density for -ENaC protein was not significantly
altered by HCTZ infusion. B: blot was loaded with 30 µg
total protein/lane and was probed with rabbit polyclonal anti- -ENaC
antibody L558. Band density for -ENaC protein was significantly
increased by HCTZ infusion. C: blot was loaded with 30 µg
total protein/lane and was probed with rabbit polyclonal anti- -ENaC
antibody L550. Band density for -ENaC protein was significantly
increased by HCTZ infusion. D: 12% Coomassie blue-stained
SDS-polyacrylamide gel from outer medulla, confirming equal loading
among lanes.
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Figure 11 demonstrates ENaC
immunohistochemistry from the cortex of HCTZ-infused and
vehicle-infused rats by using the
-ENaC antibody. Compatible with
the immunoblot result, it shows an increase in immunostaining in an
HCTZ-infused vs. a vehicle-infused rat. Similar observations were made
in three pairs of rats.

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Fig. 11.
Immunohistochemistry of -ENaC in cortex from
Sprague- Dawley rats infused with vehicle or HCTZ. Each panel
shows an increase in immunostaining in HCTZ-infused vs. vehicle-infused
rat. Top, ×100; bottom, ×400.
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DISCUSSION |
In this study, we analyzed changes in the abundance of the major
apical Na+ transporters in the distal sites of the renal
tubule in response to chronic infusion of a loop diuretic (furosemide)
or a thiazide diuretic (HCTZ) in rats. Table
3 summarizes the percent changes in
NKCC2, NCC, and three subunits of ENaC protein abundances with either
diuretic treatment. The data provide important new information relevant
to clinical use of these diuretics and address an important physiological issue: What is the effect of increased distal fluid delivery on transporter abundances? The main results showed that the
Na+ channel expressed in connecting tubule and collecting
duct, ENaC, was increased in its abundance in response to chronic
infusion of either diuretic, consistent with previous reports showing
increased Na+ absorption in these segments (14,
38). In addition, furosemide induced an increased abundance of
NKCC2, whereas HCTZ induced an increased abundance of NCC. The
important clinical implication from our study is that the diuretic
tolerance associated with long-term diuretic use may be caused by
increases in the abundance of distal Na+ transporters.
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Table 3.
Percent changes in Na+ transporter abundances in response
to chronic furosemide or hydrochlorothiazide infusion
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Chronic diuretic infusion induces compensatory increase in ENaC
abundance.
Both furosemide and HCTZ caused a substantial diuresis and natriuresis,
although the response was greater with furosemide than with HCTZ. With
concomitant salt and water substitution, the animals gained body weight
steadily without apparent volume depletion during the experiment. On
the basis of the known sites of action of the diuretics, it is likely
that Na+ delivery to collecting duct was increased by both diuretics.
ENaC constitutes the rate-limiting step for Na+
reabsorption in the connecting tubule and collecting duct in the rat
kidney (6, 16, 36). Because ENaC is a heteromultimeric
protein formed by the association of three homologous subunits,
,
, and
(2), we tested its expression by using the
specific polyclonal antibodies to each subunit (26). In
our study, chronic furosemide infusion increased the protein abundance
of all three subunits in the cortex and more prominently in the outer
medulla. Chronic HCTZ infusion also increased the abundance of
-subunit in the cortex and
- and
-subunits in the outer
medulla. Although regulation of ENaC activity in the collecting duct
and connecting tubule involves several control processes, such as
regulated trafficking (16, 25), and posttranslational
modifications, such as methylation (34), it is likely that
an important element of ENaC regulation is achieved through control of
subunit abundance as seen in response to aldosterone or vasopressin
(7, 26). We did not investigate the mechanism of altered
ENaC subunit protein abundance in the present study, although
regulation of mRNA levels (31, 39) and regulation of ENaC
protein half-life (5, 27, 41) have been described in
previous studies.
Our animal protocol of salt and water substitution was apparently
effective in preventing extracellular fluid volume depletion, as
evidenced by the absence of an increase in serum aldosterone level.
Furthermore, NCC abundance, which is regulated by aldosterone (20), was not altered by chronic furosemide
administration. Thus it appears unlikely that the changes in ENaC
subunit abundance are mediated by either aldosterone or vasopressin.
Another factor previously shown to alter ENaC subunit abundance is
acid-base state. Alkali loading was demonstrated to increase both
-
and
-ENaC abundance in rat kidney (19), raising the
possibility that the increases in the abundance of these two subunits
could be related to the metabolic alkalosis seen with chronic diuretic adminstration.
On the basis of prior literature, it appears quite plausible that the
increase in ENaC subunit expression seen with chronic diuretic
administration is related to the effects on the delivery of NaCl and
water to the collecting duct. Specifically, in furosemide-infused, volume-replaced rats, Stanton and Kaissling (18, 38)
demonstrated an increase in distal Na+ absorption
accompanied by an increase in epithelial cell volume of the distal
convoluted tubule, connecting tubule, and collecting duct. Consistent
with a general hypertropic effect of chronic furosemide infusion, we
found that the total amount of protein in cortical homogenates was
larger in furosemide-infused rats compared with vehicle-infused
controls. These findings seem to be compatible with a previous study
(23) showing that furosemide infusion with high-salt
intake caused an increase in DNA synthesis in distal segments
downstream of the thick ascending limb. The mechanism by which
the renal tubule cells sense an increase in flow rate is unknown but
may involve the central cilium present in renal tubule epithelial cells
(33).
An increase in Na+-K+-ATPase activity in the
cortical collecting duct was shown after either furosemide
(37) or HCTZ (14) administration. Although
the mechanism is unknown, the increase in basolateral
Na+-K+-ATPase activity would reduce
intracellular Na+, promoting Na+ influx via
apical ENaC in the collecting duct. A regulatory role of altered
intracellular and extracellular ion concentrations on ENaC function has
been reviewed (15), and it appears likely that
intracellular Na+ activity has effects on ENaC that are
independent of effects on the electrochemical driving forces for
Na+ ion movement through the channel. These effects could
possibly include altered abundance of ENaC subunit proteins. Although
changes in transporter abundance are by no means the only way in which transport capacity of renal tubule cells is regulated, the increase in
ENaC abundance that we have observed is likely to represent adaptive
responses to allow for the conservation of Na+ in response
to diuretic administration.
Abundance of NKCC2 and NCC is increased by furosemide and HCTZ
infusion, respectively.
Chronic furosemide infusion significantly increased the abundance
of NKCC2 in cortex and outer medulla, and chronic HCTZ infusion markedly increased the abundance of NCC in cortex. This may represent a
compensatory response and may contribute to diuretic resistance. Consistent with the effect of HCTZ administration on NCC abundance, a
previous study demonstrated that the binding density of
[3H]metolazone, an indirect measure of NCC abundance, was
increased by chronic HCTZ infusion (3, 29) despite a
decrease in NaCl transport capacity of distal convoluted tubule
(11). The distal convoluted tubules from HCTZ-infused rats
preserved epithelial structure and unipolar apical staining when they
were examined by the NCC immunohistochemistry (Fig. 8). These findings
are in contrast to the report of Loffing et al. (24) that
thiazide (especially metolazone) treatment in Wistar rats was
associated with injury of distal convoluted tubule cells, a basal shift
of NCC localization, and a significant decrease in mRNA transcripts of
NCC (24). We do not know whether the discrepancies result from differences in animals (Sprague-Dawley vs. Wistar rats) and doses
(3.75 mg·rat
1·day
1
in the present study vs. 40 mg·kg body
wt
1·day
1) of HCTZ.
Another interesting finding is that NCC abundance was not increased by
furosemide infusion. Previous micropuncture experiments in rat kidneys
have demonstrated that chronic furosemide infusion increases
Na+ reabsorption in the distal tubule (12,
38), an effect that is perhaps related to increased transport
via ENaC. However, in rats, chronic furosemide infusion increased
binding of [3H]metolazone to renal cortical membranes,
suggesting an increase in NCC abundance (3). Recently,
Abdallah et al. (1) reported that furosemide infusion
increased NCC protein abundance, but this effect was blocked by
spironolactone, suggesting that the response was dependent on a rise in
plasma aldosterone concentration. Compared with our study, they used
furosemide in much larger doses (125 mg· kg
1·day
1),
leading to a 10-fold increase in daily urine volume. Our finding that
NCC abundance was not increased by furosemide infusion may be related
to the lack of an increase in serum aldosterone in our study. In
addition, other neurohormonal factors can be considered as contributing
to the changes in NCC protein abundance during furosemide infusion.
Prostaglandin E2 production can be stimulated by furosemide
administration (28), and nitric oxide generation can be
increased by high-salt intake (42). The possibility that these factors might act to affect NCC protein abundance needs to be
investigated in future studies.
 |
ACKNOWLEDGEMENTS |
The authors thank So-Young Kim, Han-Na Jung, and Prof. Seoung-Wan
Chae for technical assistance.
 |
FOOTNOTES |
This work was supported by Seoul National University Hospital Clinical
Research Institute of Korea Grant 03-2000-033 and the intramural budget of the National Heart, Lung and Blood Institute, project no. Z01-HL-01282-KE (M. A. Knepper).
Address for reprint requests and other correspondence: J. S. Han, Dept. of Internal Medicine, Seoul National Univ. College of
Medicine, 28, Yongun-dong, Chongno-gu, Seoul 110-744, South Korea.
(E-mail: jshan{at}snu.ac.kr).
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.
September 24, 2002;10.1152/ajprenal.00227.2002
Received 17 June 2002; accepted in final form 10 September 2002.
 |
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