In rat
terminal inner medullary collecting duct (tIMCD), the Na,K-ATPase
mediates NH
uptake, which increases secretion of net
H+ equivalents. K+ and NH
compete for a common binding site on the Na,K-ATPase. Therefore,
NH
uptake should increase during hypokalemia
because interstitial K+ concentration is reduced. We
asked whether upregulation of the Na,K-ATPase during hypokalemia also
increases basolateral NH
uptake. To induce
hypokalemia, rats ate a diet with a low K+ content. In
tIMCD tubules from rats given 3 days of dietary K+
restriction, Na,K-ATPase
1-subunit (NK-
1)
protein expression increased although NK-
1 protein
expression and Na,K-ATPase activity were unchanged relative to
K+-replete controls. However, after 7 days of
K+ restriction, both NK-
1 and
NK-
1 subunit protein expression and Na,K-ATPase activity
increased. The magnitude of Na,K-ATPase-mediated NH
uptake across the basolateral membrane (J
) was
determined in tIMCD tubules perfused in vitro from rats after 3 days of
a normal or a K+-restricted diet.
J
was the
same in tubules from rats on either diet when measured at the same
extracellular K+ concentration. However, in either
treatment group, increasing K+ concentration from 10 to 30 mM reduced
J
>60%.
In conclusion, with 3 days of K+ restriction,
NH
uptake by Na,K-ATPase is increased in the tIMCD
primarily from the reduced interstitial K+ concentration.
sodium,hydrogen-adenosinetriphosphatase; terminal inner medullary
collecting duct; potassium; ammonium
 |
INTRODUCTION |
OUR LABORATORY
(36, 37, 40) has shown that along the basolateral membrane
of the rat terminal inner medullary collecting duct (tIMCD),
NH
uptake occurs by the Na,K-ATPase.
NH
uptake by the Na,K-ATPase provides a source of
H+ for secretion of net H+ equivalents and
titration of luminal buffers in this segment (36, 37).
Because NH
and K+ are competitive
substrates for a common extracellular binding site on this transporter (40), interstitial K+ concentration should
modulate NH
uptake by the Na,K-ATPase. Interstitial
concentrations of NH
and K+ have been
determined by sampling vasa recta plasma at the same level (9,
11, 14). These studies have demonstrated that K+
concentration in the interstitium of rat inner medulla is generally much higher than NH
concentration. For example, in
untreated rats, vasa recta NH
concentration is 8.4 mM
(14) whereas K+ concentration is 36 mM
(11). Moreover, the apparent affinity for the
extracellular binding site of the Na,K-ATPase is greater for
K+ than for NH
(40).
Therefore, uptake of K+ by the Na,K-ATPase should be much
greater than uptake of NH
under physiological
conditions. This raises the possibility that NH
uptake by Na,K-ATPase may be of little physiological significance in
the tIMCD in vivo. Our laboratory has explored the contribution of
NH
uptake by Na,K-ATPase to secretion of
H+ equivalents in tIMCD of normal rats eating a balanced
diet. tIMCD tubules from untreated rats were perfused at
NH
and K+ concentrations expected in the
interstitium of the inner medulla of these animals. Under physiological
conditions (K+ concentration = 30 mM;
NH
concentration = 6 mM), no change in
HCO
absorption (JtCO2) was detected with inhibition
of Na,K-ATPase by the addition of ouabain to the bath fluid
(38).
However, vasa recta K+ concentration varies widely with
changes in K+ homeostasis (9, 11). Dobyan and
collaborators (11) showed that after 3 days of a
K+-restricted diet, vasa recta K+ concentration
was reduced from 36 to 8.6 mM. These K+ concentrations
observed in vivo were used in experiments with tIMCD tubules perfused
in vitro. tIMCD tubules from rats eating a K+-restricted
diet were perfused in symmetrical
HCO
/CO2-buffered solutions that
contained K+ and NH
at concentrations
expected in the interstitium of this treatment group (K+
concentration = 10 mM; NH
concentration = 6 mM). Under these conditions, inhibition of NH
uptake
via Na,K-ATPase, by addition of ouabain to the bath, reduced net
secretion of H+ by 40-50% (38). Thus, in
tIMCD tubules from K+-depleted rats, when perfused in the
presence of physiological concentrations of NH
and
K+, an important role of NH
uptake by Na,K-ATPase in the process of secretion of net H+
equivalents was demonstrated.
When perfused and bathed under identical conditions (i.e., at a
K+ concentration of 10 mM and an NH
concentration of 6 mM), tubules from K+-depleted rats
secrete H+ equivalents at a higher rate than tubules from
K+-replete controls (41). These data indicate
that in the rat tIMCD a stable adaptation occurs during hypokalemia,
which increases net acid secretion. In rat outer medullary collecting
duct (OMCD), McDonough and colleagues (23) demonstrated
that dietary K+ restriction increases Na+ pump
expression threefold. We reasoned that if Na,K-ATPase
expression is upregulated at the level of the plasma membrane in the
tIMCD during hypokalemia, greater uptake of NH
across
the basolateral membrane should occur in tandem with increased secretion of H+ equivalents.
The purpose of the present study was therefore to determine whether
1) the Na,K-ATPase is upregulated during hypokalemia and 2)
if the increase in NH
uptake by Na,K-ATPase observed
during hypokalemia occurs because of upregulation of Na,K-ATPase at the
level of the plasma membrane or from the reduction in interstitial
K+ concentration observed in this treatment group.
 |
METHODS |
Animal conditioning.
Tubules from the tIMCD were dissected from pathogen-free male
Sprague-Dawley rats weighing 65-120 g (Harlan, Indianapolis, IN).
To isolate IMCD cells in suspension, 125- to 150-g rats were used.
Animal conditioning was similar to that reported previously by our
laboratory (38). Animals were housed in microisolator cages and fed a diet with 7.8 g K+/kg food and 0.664 g
Na+/kg food (Zeigler Brothers, Gardeners, PA) for 2-7
days. Rats were then divided into two treatment groups. Group
A rats ate a diet with a normal K+ content (diet no.
P1868, 2.3 g K+/kg food and 1.03 g
Na+/kg food; ICN Biochemicals, Aurora, OH). Group
B rats ate a diet identical to that of group A, but
with a low K+ content (diet no. 960189, 0.0041 g
K+/kg food and 1.03 g Na+/kg food; ICN
Biochemicals). Rats were maintained on diets with either low or normal
K+ content for 3 or 7 days before death and were pair fed.
To induce a rapid diuresis, animals were injected with furosemide (5 mg/100 g body wt ip) 30-45 min before death by decapitation. This
furosemide-induced diuresis reduces the inner medullary axial solute
concentration gradient (36, 43) and attenuates changes in
extracellular osmolality. Serum K+ was measured by the
Department of Veterinary Medicine, University of Texas at Houston,
using ion-sensitive electrodes (Vettest; Idex Laboratories, Westbrook, ME).
Dissection of tubules for immunoblots.
Rats were anesthetized with intraperitoneal pentobarbital (5 mg/100 g
body wt) before death. An abdominal incision was made, and the aorta
was cannulated with polyethylene tubing below the renal arteries. The
kidneys were perfused with ice-cold dissection media containing the
following (in mM): 144 NaCl, 5 KCl, 6 NH4Cl, 1 Na2HPO4, 2 CaCl2, 1.2 MgSO4, 5.5 glucose, and 10 HEPES, pH 7.4 bubbled with 100%
O2. This solution also contained 1 mg/ml collagenase and 1 mg/ml BSA. The kidneys were removed, and the inner medulla was excised.
The middle third of the inner medulla was incubated in the same
solution for 10 min at 37°C. The incubated tissue was washed once
with this solution, but in the absence of collagenase. tIMCD tubules
were dissected in the same solution at 11°C, but without albumin and
collagenase. On each experiment day, two 20-mm samples of tubules were
dissected from a single rat in each treatment group. Tubules were
transferred to an Eppendorf tube while being visualized under a
microscope. Tubules were centrifuged at 16,000 g for 3 min.
The supernatant was discarded, and the pellet was resuspended in the
same solution and recentrifuged. The supernatant was discarded and the
pellet was resuspended in 20 µl of buffer A, which
contained 10 mM triethanolamine, 250 mM sucrose, 1 mg/ml leupeptin
(Bachem, Torrance, CA) and 0.1 mg/ml phenylmethylsulfonyl fluoride
(PMSF; US Biochemical, Toledo, OH) pH 7.6. Membranes were solubilized
at 60°C for 15 min in Laemmli sample buffer before being loaded onto
SDS-PAGE gels.
Preparation of IMCD cell suspensions for immunoblots and for
ATPase assay.
IMCD cells were isolated as described previously (4, 30,
40). After the rats were killed by decapitation, the kidneys were removed and placed in chilled HCO
-buffered solution (suspension solution) containing (in mM) 118 NaCl, 25 NaHCO
, 5 KCl, 4 Na2HPO4, 2 CaCl2, 1.2 MgSO4, and 5.5 glucose bubbled with
95% air-5% CO2. On each experiment day, kidney
tissue from five rats eating a K+-replete diet and five
rats eating a K+-deficient diet was isolated. Kidney tissue
from rats in each treatment group was pooled. The inner medullas were
excised, minced, and then incubated in the suspension solution
containing 2 mg/ml collagenase B (Roche Molecular Biochemicals,
Indianapolis, IN) and 600 U/ml hyaluronidase V (Sigma, St. Louis, MO)
for 70 min at 37°C and bubbled continuously with 95% air-5%
CO2. DNase (0.001%, DNase I; Roche Molecular Biochemicals,
Indianapolis, IN) was then added to the suspension and incubated
another 20 min. The suspension was aspirated through a large-bore
pipette every 20 min. At the end of the incubation period, the
suspension was centrifuged at 65 g for 37 s at room
temperature. The supernatant was removed, and the pellet, which
contained the less buoyant structures such as the IMCD, was resuspended
in the suspension solution plus 0.1% BSA (without collagenase,
hyaluronidase, and DNase). This centrifugation step was repeated twice.
The final IMCD suspensions were placed on ice for 10 min to increase
the yield of IMCD cells. The supernatant was discarded, and the pellet
was resuspended in suspension solution (without albumin, collagenase,
hyaluronidase, and DNase) and centrifuged at 813 g for 10 min. When IMCD cells were prepared for immunoblots, the pellet was
suspended in 0.5 ml of homogenation buffer A and was
homogenized using an Omni tissue homogenizer (Omni/Tech Quest, Warrenton, VA) at 15,000 rpm on ice for 15 s. The homogenization step was repeated twice. The membranes were then centrifuged at 200,000 g for 1 h at 4°C. Expression of N,K-ATPase
1-subunit (NK-
1) or NK-
1
protein was not detected in the supernatant (data not shown). The
supernatant was therefore discarded. Membranes were resuspended in 100 µl of homogenization buffer A. Protein content was
measured using the method of Lowry et al. (20). When IMCD
cells were prepared for the ATPase assay, the protocol was varied.
After the 10-min centrifugation step at 813 g, the pellet
was suspended in 0.5 ml of homogenization buffer B, which contained 250 mM sucrose, 10 mM Tris · HCl, 1 mM EDTA-Tris, 1 mM PMSF, 3 mM benzamidine, and 1 µg/ml soybean trypsin inhibitor, pH
7.6. Membranes were homogenized and centrifuged at 200,000 g
for 1 h. The pellet was then resuspended in 100 µl of
homogenization buffer B.
Preparation of whole inner medulla and colon plasma membranes for
immunoblots.
Plasma membranes from rat distal colon and whole inner medulla were
isolated as described previously (7). Rats were fed the
K+-restricted diet for 7 days. Rat distal colon and rat
whole inner medulla were excised, minced, and suspended in 10 ml of
homogenization buffer C containing (in mM) 10 Tris · HCl, 1 EDTA-Tris, 1 PMSF, and 3 benzamide with 1 ug/ml
soybean trypsin inhibitor, pH 7.4. This solution also contained 27%
sucrose (wt/vol). Tissue was homogenized on ice using a Polytron (model
PT 10/35, Brinkman, Westbury, NY) for 15 s. The homogenization
step was repeated twice. Tissue was then homogenized with four or five
strokes in a Dounce homogenizer with pestle A (Kontes Glass,
Vineland, NJ) and centrifuged at 2,000 g for 4 min. The
pellet, which contained nuclei, was discarded. The supernatant was
applied to the top of 45% sucrose solution containing homogenization
buffer C. The suspension was centrifuged at 200,000 g
for 45 min at 4°C. Membranes in the 27/45% sucrose interphase
were diluted in buffer C and centrifuged at 25,000 g
for 30 min. The pellet was resuspended in buffer C and stored at
70°C. Protein was measured using the method of Lowry et
al. (20).
Preparation of immunoblots.
Membranes were solubilized at 60°C for 15 min in Laemmli sample
buffer. To confirm equal protein loading of IMCD homogenates in each
lane, electrophoresis was run on each pair of samples for a given
experiment in a single 12% polyacrylamide-SDS gel stained with
Coomassie blue. These gels were analyzed by densitometry to provide a
quantitative assessment of loading. SDS-PAGE was performed on minigels
of 10% polyacrylamide [NK-
1 and H,K-ATPase
2-subunit (HK-
2)] or 12% polyacrylamide
[NK-
1 and aquaporin (AQP)1 and 2]. The
proteins were transferred from the gels electrophoretically onto
nitrocellulose membranes. After being blocked with 5 g/dl nonfat dry
milk, membranes were probed with a mouse monoclonal antibody that
recognizes the NK-
1 subunit of rat Na,K-ATPase (Upstate
Biotechnology, Lake Placid, NY). This antibody was used at a 1:5,000
titer with individual IMCD tubules or at a 1:10,000 titer with IMCD
suspensions. To detect expression of NK-
1 protein, a
rabbit polyclonal antibody raised against a rat NK-
1
fusion protein (Upstate Biotechnology) was used at a 1:20,000 titer. Affinity-purified, peptide-derived polyclonal rabbit anti-rat AQP1 and
-2 (LL266 and LL752) antibodies were used at titers of 1:2,000 and
1:5,000, respectively (gift of M. A. Knepper, National Heart,
Lung, and Blood Institute; Refs. 10, 33).
Polyclonal antiserum raised in rabbits against a synthetic peptide that
corresponds to amino acids 686-698 of the rat HK-
2
sequence was also used (Ref. 5; a gift of J. Codina and
T. D. DuBose, Jr., University of Kansas, Kansas City, KS) at a
titer of 1:1,000. Antibodies were diluted in an antibody
dilution buffer solution containing 150 mM NaCl, 50 mM sodium
phosphate, 10 mg/dl sodium azide, 50 mg/dl Tween 20 and 0.1 g/dl BSA
(pH 7.5). For NK-
1 subunit immunoblots, sheep anti-mouse
IgG conjugated with horseradish peroxidase was used as a secondary
antibody (Amersham-Pharmacia Biotech, Piscataway, NJ) at a 1:5,000
dilution. For all other immunoblots, donkey anti-rabbit IgG conjugated
to horseradish peroxidase (no. 31458, Pierce, Rockford, IL) was used as
a secondary antibody at a 1:5,000 or a 1: 7,500 dilution. Sites of
antibody-antigen reaction were visualized using luminol-based enhanced
chemiluminescence (KPL; Kirkegaard and Perry, Gaithersburg, MD) before
exposure of X-ray film (X-OMAT AR; Eastman Kodak, Rochester, NY).
Relative quantification of the resulting band densities was performed
using densitometry software running on a SPARC station2 (Sun
Microsystems, Mountain View, CA) equipped with an image analysis system
(Bio Image, Ann Arbor, MI). Multiple exposures of autoradiograms were
made to ensure that densitometry gave a linear relationship between the amount of protein loaded onto the gel and band density, as described previously (22).
Deglycosylation of
1 subunit.
The
1 subunit of Na,K-ATPase was deglycosylated
using the protocol of Codina and collaborators (5).
Membranes (50 µg) in homogenization buffer A were added to
20 µl of buffer containing 10 mM Tris · HCl, pH 8, 1 mM EDTA,
1 mM PMSF, 1 mM benzamidine, 1 µg/ml soybean trypsin inhibitor, and
1% CHAPS, pH 7.6, and incubated at 4°C for 1 h. After this
incubation period, sodium phosphate (50 mM final concentration, pH 8)
and PNGase (1,000 U, no. 7045, New England Biolabs, Beverly, MA) were
added and the mixture was incubated for 1 h at 37°C. The
reaction was stopped by the addition of Laemmli sample buffer.
ATPase assay.
Ouabain-sensitive ATP hydrolysis was measured as reported
previously (7, 25). IMCD membranes were incubated for 30 min in 0.75 mg/ml sodium deoxycholate at room temperature. Total and ouabain-sensitive ATP hydrolysis is maximal when membranes are incubated for 30 min at this detergent concentration (data not shown).
The purpose of this incubation step is to expose unsealed vesicles
(25). The reaction was started by the addition of 2.5 µg
of sodium deoxycholate-treated rat IMCD membranes to the reaction mixture, such that the final mixture contained (in mM) 100 NaCl, 10 KCl, 50 Tris · HCl, 1 EDTA-Tris, 0.1 EGTA-Tris, 5 MgCl2, 3 ATP-Tris containing 1-10 × 106 cpm/200 µl of [
-32P]ATP (Amersham
Pharmacia Biotech), 1 PMSF, and 3 benzamidine with 1 µg/ml soybean
trypsin inhibitor, pH 7.32. The final volume of the reaction was 200 µl. The mixture was incubated for 30 min at 37°C. The reaction was
terminated by the addition of 1 ml of a 50% slurry of activated
charcoal in 10 mM Na2HPO4, pH 7.5. Samples were
vortexed twice, cooled on ice, and centrifuged at 18,544 g
for 5 min. The supernatant (450 µl) was used to quantify
32P released during the incubation. The ATP assay was
performed in the presence and absence of 5 mM ouabain in the reaction
mixture. Total and ouabain-sensitive [
-32P]ATP
hydrolysis was linear with time over the range of 0-60 min and was
linear with protein content over the range of 0-10 µg of protein
(not shown).
Measurement of protein-to-DNA ratio.
IMCD cells were isolated as described in Preparation of IMCD cell
suspensions for immunoblots and for ATPase assay.
The pellet was resuspended in 0.1 N NaOH plus 0.025% saponin and
incubated at 60°C for 1 h. Samples were then split into aliquots
for protein and DNA measurements. Protein was measured using the method
of Lowry et al. (20) with albumin as a standard. DNA was
measured using the Hoesch reagent (Hoesch 33258; Hoefer Scientific
Instruments, San Francisco, CA) with calf thymus DNA as a standard
(Hoefer Scientific Instruments) as described previously
(13). This assay detects DNA but not RNA
(13). IMCD suspensions (or DNA standards) were added to 2 ml of solution containing (in mM) 10 mM Tris · HCl, 1 EDTA, and
200 NaCl, pH 7.4 with 10
4 mg/ml Hoesch 33258, pH 7.4 at
22°C. Fluorescence was measured at excitation and emission
wavelengths of 365 and 460 nm, respectively, with a DNA fluorimeter
(TKO 100, Hoefer Scientific Instruments). Saponin at the concentrations
used did not affect the DNA standard curve (not shown). Nevertheless,
the standard curve was performed in the presence of NaOH and saponin at
final concentrations found in each of the unknown samples. With this
assay, DNA concentration is linear over the range of 0-150 ng of
DNA (13).
Dissection of tIMCD tubules for perfusion experiments.
The composition of the solutions used in the perfusion experiments is
given in Table 1. Coronal slices were cut
from the kidneys and placed into a dissection dish containing the
chilled experimental solution (11°C). Tubules were dissected in
solution 5 for buffering capacity measurements and in
solution 8 for experiments that measured changes in
pHi after ouabain addition. tIMCD tubules were dissected,
mounted on concentric glass pipettes, and perfused in vitro at 37°C
as reported previously by our laboratory (36, 43).
Osmolality was measured in all solutions (36). The bath
fluid was constantly bubbled with 100% O2. Bath pH was
measured continuously during all experiments as described previously
(36, 43).
Measurement of pHi in tIMCD tubules perfused in
vitro.
pHi was measured using
2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein
(BCECF)-AM. Tubules were perfused and bathed for 15 min at 37°C in
either solution 5 (buffering capacity experiments) or
solution 8 (ouabain-sensitive alkalinization experiments). The bath was then changed to the same solution but also containing 5 µM BCECF-AM. After 20 min of exposure to BCECF, the bath was changed
to the original solution without BCECF. pHi was determined by measuring the ratio of emitted light at >530 nm when BCECF was
excited alternatively at 440 and 495 nm. Readings were calibrated by
measuring the ratio of fluorescence at excitations of 495 relative to
440 nm when the tubule was perfused and bathed in either HEPES- or
PO
-buffered solutions containing 120 mM
K+ to approximate intracellular concentration of this ion
(solutions 1 or 3). All calibration solutions
contained 14 µM nigericin in the bath solution. The pH of this
solution was varied between 6.8 and 7.8. The other details of
pHi measurement in tubules perfused in vitro were described
previously by our laboratory (36, 37). A typical
calibration curve performed at K+ concentrations
of 90 and 120 mM is given in Fig.
1.1

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Fig. 1.
Effect of changes in extracellular K+
concentration on intracellular pH (pHi) calibration.
Tubules were perfused and bathed in solution 1, which
contained 120 mM K+ and 14 µM nigericin
(n = 7 tubules). pHi was measured when
extracellular pH was varied between 6.6 and 7.8. The bath and perfusate
were then switched to solution 2, which contained 90 mM
K+, and pHi was measured over the same range in
extracellular pH. In some tubules the order of these measurements was
reversed. The rate of change in the 495- to 440-nm fluorescence ratio
with changes in pHi was similar at K+
concentrations of 90 and 120 mM.
|
|
Calculation of H+ fluxes.
To quantitate NH
uptake through Na,K-ATPase,
proton efflux rates (JH,
pmol · mm
1 · min
1) were
calculated as JH = dpHi/dt ×
T × V/L, where dpHi/dt is the
initial rate of change in pHi (pH units/min) after ouabain addition,
T is the intrinsic intracellular buffering
power [mmol/(l · pH unit)], and V/L is
the cell volume/tubule length (nl/mm). Thus
T,
V/L, and dpHi/dt were each measured.
T was determined from the change in
pHi with the addition of a weak base (trimethylamine), as
described by Watts and Good (46). tIMCDs were perfused and
bathed at 37°C in symmetrical Cl
- and
Na+-free solutions (solution 6) that also
contained 10 µM Sch-28080, 2 mM BaCl2, 100 µM
amiloride, 100 µM bumetanide, and 5 mM ouabain. These transport
inhibitors were selected to inhibit pHi regulatory mechanisms and NH
transport mechanisms (36, 37,
42, 44, 45). pHi was clamped at values between 7.0 and 7.5 by setting the pH of the bath and perfusate between 7.2 and
7.9. Identical solution, but containing 2.5 mM trimethylamine (solution 7), was then added to the bath solution. Rapid
exchange from one bath composition to another was achieved by the
introduction of the new solution, which was preheated and pregassed
from a separate closed reservoir, at a rate of 30 ml/min with the
simultaneous introduction of the new solution to the continuous
exchange reservoir (39). Thus a bath exchange could be
made in <10 s. Addition of trimethylamine increased pHi
between 0.07 and 0.18 pH unit, followed by a stable pHi or
a plateau phase. pHi was also measured after trimethylamine
withdrawal.
T was calculated as
[HB+]i/
pHi, where
pHi is the increase (or decrease) in pHi
after addition (or withdrawal) of trimethylamine.
[HB+]i is the change in intracellular
trimethylammonium concentration, which was calculated based on the
pKa of trimethylamine (9.8 at 37°C) and the
pHi measured after the addition or withdrawal of trimethylamine. This calculation assumes that the concentration of
trimethylamine base is equal in intracellular and extracellular fluids
at steady state. Only one trimethylamine pulse was obtained per tubule.
For a given tubule,
T was calculated based on the change
in pHi after both trimethylamine addition and withdrawal and the results were averaged. This mean value for
T was
reported for each tubule studied.
To determine NH
uptake through the Na,K-ATPase,
tubules were perfused and bathed in symmetrical physiological
HEPES-buffered solutions containing 6 mM NH4Cl and either
10 or 30 mM KCl (solutions 8 and 9). Baseline
pHi was measured. The bath was exchanged to an identical
solution but also containing 5 mM ouabain. When studied at a
K+ concentration of 30 mM, 5 mM ouabain fully inhibits rat
Na+,K+-ATPase (30, 32).
dpHi/dt was calculated from the slope of the
495-to-440 ratio measured over the first 45 s after the addition of ouabain to the bath (46).
V and L were determined on the basis of the
inner/outer diameter and length, which was measured in each tubule
using a calibrated optical micrometer. V and L
were measured in the same tubules used for measurement of
dpHi/dt after ouabain addition to the bath.
 |
RESULTS |
What gene product mediates NH
uptake by Na+ pump?
The relative abundance of the various Na,K-ATPase isoforms in kidney
has been studied previously (Ref. 23; see
DISCUSSION). We asked whether Na+ pumps other
than Na,K-ATPase, such as HK-
2, might mediate
NH
-dependent, ouabain-induced alkalinization reported
previously by our laboratory (36, 37). Thus
immunoreactivity of NK-
1 was compared with that of
HK-
2 in inner medulla from rats that ate the
K+-deficient diet for 7 days. As shown in Fig.
2, NK-
1 immunoreactivity is readily detectable in the inner medulla of the kidney and in the
colon. As shown, NK-
1 protein abundance is at least as
great in kidney as in colon. However, HK-
2
immunoreactivity is lower in inner medulla than in colon. Therefore, if
protein expression of HK-
2 and NK-
1 are
equivalent in colon, then expression of NK-
1 in kidney
is much greater than HK-
2. Low HK-
2
immunoreactivity may result from low affinity of the rabbit anti-rat
HK-
2 antibody. However, the most likely interpretation
of these data is that HK-
2 expression in the tIMCD is
low. Thus Na pump-mediated NH
uptake in tIMCD was
attributed to NK-
1 rather than HK-
2.
These results are consistent with previous studies (1, 6),
which detected relatively low levels of expression of
HK-
2 in the tIMCD. Further experiments therefore studied
the effect of hypokalemia on expression of the
1- and
1-subunits of the Na,K-ATPase.

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Fig. 2.
Expression of Na,K-ATPase 1-subunit
(NK- 1) and H,K-ATPase 2-subunit
(HK- 2) in K+-restricted rats. Rats received
a K+-replete or a K+-restricted diet for 7 days. HK- 2 protein expression was readily detected in
colon, but less expression was detected in the inner medulla (IM).
NK- 1 was readily detected in both colon and inner
medulla.
|
|
Effect of hypokalemia on expression of Na,K-ATPase.
The effect of 3 days of dietary K+ restriction on
NK-
1 subunit expression was examined. As shown in Fig.
3, the anti-rat NK-
1 antibody detected a protein that migrated at 100 kDa, the expected molecular mass of the
1-subunit of Na,K-ATPase
(27). No difference in NK-
1 subunit protein
abundance was detected in individual tIMCD tubules from rats that ate
either the normal or the low-K+ diet for 3 days.2

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Fig. 3.
Effect of 3 days of dietary K+ restriction on
NK- 1 protein expression in terminal inner medullary
collecting duct (tIMCD) tubules. A: rats ate either a normal
(N) or a K+-restricted diet (L) for 3 days. Two sets of
20-mm IMCD tubules were dissected from a single rat in each treatment
group on a single experimental day. Homogenates from each of these 2 samples taken from a rat in each treatment group were loaded in
separate lanes on a single gel. Thus each lane is loaded with equal
lengths of IMCD tubules. B: band density of immunoblots
given in A. Band density data were normalized to the means
observed in tubules from K+-replete control rats isolated
on the same day. No change in band density was detected with 3 days of
dietary K+ restriction. NS, not significant.
|
|
Immunoblots that use the anti-rat NK-
1 subunit antibody
require relatively high protein loading. Therefore, further experiments used IMCD suspensions. To characterize IMCD suspensions, AQP1 was used
as a marker of loops of Henle, whereas AQP2 was used as a collecting
duct marker. As shown in Fig. 4, in
homogenates of inner medulla, the rabbit anti-rat AQP1 and -2 antibodies recognized proteins at 35-40 and 29 kDa, the expected
mobility of the glycosylated and nonglycosylated AQP1 and -2 proteins,
respectively (10, 22, 33). Figure 4 shows that the IMCD
cells isolated are enriched in AQP2 relative to both non-IMCD cells and
whole inner medulla. In contrast, IMCD suspensions show little AQP1
immunoreactivity, consistent with low levels of contamination by
non-IMCD cells such as loops of Henle. Therefore, a relatively pure
suspension of IMCD cells was obtained with this protocol.

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Fig. 4.
Purity of IMCD suspensions. One-tenth of the inner
medulla suspension was collected and homogenized (whole IM). The
remaining nine-tenths of the inner medullary suspension was centrifuged
3 times (65 × g for 37 s) to separate the final pellet
(IMCD) from the lighter non-IMCD structures found in the pooled
supernatants (non-IMCD). Samples from each fraction were homogenized
and dissolved in Laemmli sample buffer and loaded on SDS-PAGE gels
(12%; Ref. 4). A: aquaporin-1 (AQP1)
immunoreactivity was low in IMCD suspensions relative to either whole
IM or non-IMCD structures, which indicated little contamination by
loops of Henle. B: AQP2 expression was high in IMCD
suspensions relative to either whole IM or non-IMCD structures, which
indicates enrichment of IMCD cells in these suspensions.
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As shown in Fig. 5, in immunoblots of
IMCD suspensions, the anti-rat
1-fusion protein antibody
detected a broad band at 50-55 kDa in native IMCD cell homogenates
and a narrow band at 35 kDa in deglycosylated homogenates of IMCD
cells, consistent with the expected molecular mass of the native and
core
1-subunit of Na,K-ATPase (5).

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Fig. 5.
NK- 1 1 protein expression in
IMCD suspensions from rats eating a normal (N) or a
K+-restricted (L) diet for 3 days. Immunoblots of
NK- 1 1 subunits are shown. Each lane was
loaded with 6 ( 1), 20 (native 1), or 6 (core 1) µg protein.
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The effect of 3 days of a K+-restricted diet on
immunoreactivity of
1- and
1-subunit
expression as well as Na,K-ATPase activity is shown in Figs. 5 and
6. As shown, no increase in either
1-subunit protein abundance or Na,K-ATPase activity was
detected with 3 days of K+ restriction. However,
immunoblots of deglycosylated IMCD cell homogenates showed a threefold
increase in
1-subunit protein abundance relative to
K+-replete control rats (n = 5;
P < 0.05).

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Fig. 6.
NK- 1 1 protein expression in
IMCD suspensions from rats eating a normal or a
K+-restricted diet for 3 days. A: band density
of immunoblots given in Fig. 5. Band densities obtained from
homogenates of K+-restricted rats were normalized to the
band density of homogenates from K+-replete control animals
prepared the same day. B: Na,K-ATPase activity in tIMCD
suspensions from rats eating a normal or a K+-restricted
diet for 3 days. In this figure, ouabain-sensitive ATPase activity was
6.42 ± 0.70 µmol Pi · mg
protein 1 · h 1 (n = 5) in IMCD cell homogenates from K+-restricted rats and
5.03 ± 0.35 µmol Pi · mg
protein 1 · h 1 (n = 5) in K+-replete control rats. Ouabain-sensitive ATPase
activity obtained from homogenates of K+-restricted rats
was normalized to the ATPase activity observed in homogenates of
K+-replete control animals prepared the same day.
*P < 0.05. Values represent means ± SE.
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In rat OMCD, dietary K+ restriction upregulates expression
of Na,K-ATPase (23). However, in the present study
upregulation of Na,K-ATPase in the IMCD could not be demonstrated after
3 days of a K+-restricted diet. The reason for this
negative result in the IMCD was explored. We reasoned that our
inability to detect upregulation of Na,K-ATPase could result from
changes in cell volume. For example, if Na,K-ATPase activity per cell
is increased during hypokalemia to a similar extent as protein content
per cell, then no change in Na,K-ATPase activity would be detected if
expressed per milligram protein. To explore this possibility, relative
cell size was determined in the IMCD from rats in each treatment group
by measuring the ratio of protein to DNA content in IMCD cell
suspensions and cross-sectional area in tIMCD tubules perfused in
vitro. As shown in Fig. 7, with either of
these techniques, no change in relative IMCD cell size was detected
after 3 days of dietary K+ restriction. Therefore, our
inability to detect an increase in Na,K-ATPase activity or protein
expression after 3 days of K+ restriction is not due to
cellular hypertrophy.

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Fig. 7.
Relative cell volume of tIMCD tubules from rats eating a normal or
a K+-restricted diet for 3 days. Relative cell volume was
measured in IMCD suspensions (protein-to-DNA ratio, A) and
in tIMCD tubules perfused in vitro (cross-sectional area,
B). No effect of dietary restriction on relative cell volume
was noted. #P = NS.
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We asked whether Na+ pump expression is increased with
longer periods of dietary K+ restriction. Na+
pump activity and immunoreactivity of both the
1- and
1-subunits were compared in IMCD cells from rats eating
either a normal or a K+-restricted diet for 7 days (Figs. 8 and
9).3
After 7 days of dietary K+ restriction,
1-subunit protein abundance increased threefold relative
to expression in K+-replete control rats (n = 5; P < 0.05). Moreover, during K+
restriction protein expression of the native and core
1-subunit increased two- to fourfold (P < 0.05) and ouabain-sensitive ATPase activity increased twofold
(n = 5; P < 0.05). Thus, with 7 days of a K+-restricted diet, increased immunoreactivity of the
1- and
1-subunits of Na,K-ATPase and
increased Na,K-ATPase activity were observed in the IMCD.

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Fig. 8.
NK- 1 1 protein expression in
IMCD suspensions from rats eating a normal (N) or a
K+-restricted (L) diet for 7 days. Immunoblots of
NK- 1 1 subunit protein are shown. Each
lane was loaded with 6 ( 1), 20 (native
1), or 6 (core 1) µg protein.
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Fig. 9.
NK- 1 1 protein expression in
IMCD suspensions from rats eating a normal (N) or a
K+-restricted (L) diet for 7 days. A: band
density of immunoblots given in Fig. 8. Band densities obtained from
homogenates of K+-restricted rats were normalized to the
band density of homogenates from K+-replete control animals
prepared the same day. B: Na,K-ATPase activity in tIMCD
suspensions from rats eating a normal or a K+-restricted
diet for 7 days. In this figure, ouabain-sensitive ATPase activity was
11.55 ± 2.50 µmol Pi · mg
protein 1 · h 1 (n = 5) in K+-restricted rats and 5.47 ± 1.09 µmol
Pi · mg
protein 1 · h 1 (n = 5) in K+-replete control rats. Ouabain-sensitive ATPase
activity in homogenates of K+-restricted rats was
normalized to the ATPase activity observed in homogenates of
K+-replete control animals prepared the same day. Values
represent means ± SE. *P < 0.05.
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To confirm this finding,
1-subunit protein abundance was
determined in individual tIMCD tubules (Fig.
10). As shown in Fig. 10, in tubules
from K+-restricted rats, protein abundance was increased
two- to threefold relative to that observed in tubules from
K+-replete control rats (n = 5;
P < 0.05).

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Fig. 10.
Effect of 7 days of dietary K+ restriction
on NK- 1 protein expression in tIMCD tubules.
A: rats ate either a normal (N) or a
K+-restricted (L) diet for 7 days. Two sets of 20-mm IMCD
tubules were dissected from a single rat in each treatment group on a
single experimental day. Homogenates from each of these 2 samples
obtained from a rat in each treatment group were loaded in separate
lanes on a single gel. Thus each lane is loaded with equal lengths of
IMCD tubules. B: densitometry of 1-subunit
protein expression obtained from normal and K+-restricted
rats. Band density data were normalized to the means observed in
tubules from K+-replete control rats isolated on the same
day and were expressed as means ± SE. Band density was increased
with 7 days of dietary K+ restriction. *P < 0.05.
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Effect of K+ restriction on
NH
uptake by Na,K-ATPase.
As an index of NH
uptake by Na,K-ATPase at the level
of the plasma membrane during steady-state conditions,
NH
-dependent, ouabain-induced alkalinization was
measured in tIMCD tubules. With this value, as well as
T
and V/L, ouabain-induced net H+
efflux (J
;
dpHi/dt ·
T · V/L)
was determined. tIMCD tubules from rats eating the
K+-restricted or the K+-replete diet for 3 days
were studied (36, 37). Values reported for
V/L (Fig. 7) and
T (Fig.
11) were similar in tIMCDs from rats in
both treatment groups.

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Fig. 11.
Buffering capacity in tIMCD tubules perfused in vitro
from rats eating a normal or a K+-deficient diet for 3 days.
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dpHi/dt after ouabain addition to the bath is
displayed in Fig. 12A. As
shown, in tIMCDs from rats in both treatment groups, pHi increased and then plateaued after the addition of
ouabain to the bath. To determine whether NH
uptake
by Na,K-ATPase is upregulated during hypokalemia through increased expression of Na,K-ATPase at the level of the plasma membrane,
was
compared between treatment groups (Fig. 12B).
J
was similar in
tIMCD tubules from both treatment groups when measured at the same extracellular K+ concentration. However, in both
treatment groups,
was reduced by >60% when extracellular K+ concentration
was increased from 10 to 30 mM (solutions 8 and 9; P < 0.05). Therefore, when perfused
under identical conditions, no change in NH
uptake by Na,K-ATPase was detected in tIMCD tubules from
K+-restricted rats. However,
J
varied markedly with changes in extracellular K+ concentration.

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Fig. 12.
Effect of K+ restriction on
NH uptake by the Na,K-ATPase in tIMCD
tubules perfused in vitro. A: the effect of ouabain on
pHi was studied in IMCD tubules from rats on a normal or
K+-restricted for 3 days. Tubules were studied at
K+ concentrations of 10 and 30 mM (solutions 8 and 9). Nine tubules were taken from previously
published data (38). B: NH
uptake by Na,K-ATPase
(J ) was taken to
be equal to
dpHi/dt · T · V/L,
where dpHi/dt is the initial rate of change in
pHi after the addition of ouabain to the bath
(A), T is buffering capacity (Fig. 11), and
V/L is the tubule volume per tubule length.
Initial (baseline) pHi measured in tIMCD tubules from
K+-restricted rats was 7.24 ± 0.03 (n = 6) at a K+ concentration of 10 mM and 7.18 ± 0.02 (n = 5) at a K+ concentration of 30 mM. In
tubules from K+-replete rats, initial pHi was
7.13 ± 0.03 (n = 8) at a K+
concentration of 10 mM and 7.10 ± 0.04 (n = 6) at
a K+ concentration of 30 mM.
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To explore the effect of extracellular K+ on
NH
uptake by Na,K-ATPase further, the effect of
changes in extracellular K+ concentration on resting
pHi was tested (Fig. 13).
As shown in Fig. 13, pHi rose when K+
concentration in the perfusate and bath was increased from 10 to 30 mM
(solutions 10 and 11). pHi fell to
baseline values when the K+ concentration was returned to
10 mM. Therefore, increased K+ concentration attenuates
uptake of net H+ equivalents. These results are consistent
with the hypothesis that changes in K+ concentration
regulate NH
uptake by Na,K-ATPase.

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Fig. 13.
Effect of extracellular K+ concentration on
resting pHi. Rats ate a K+-restricted diet for
3 days. Tubules were perfused and bathed in solution 10 (K+ = 10 mM, NH = 6 mM).
Resting pHi was 7.16 ± 0.04 (n = 6).
Bath and perfusate K+ concentration was increased to 30 mM
(solution 11), and pHi was measured 4 min later
(7.21 ± .04, P < 0.05). Bath and perfusate were
returned to the initial solution (solution 10), and
pHi was measured 4 min later (7.11 ± 0.03, P < 0.05). Thus pHi increased when
extracellular K+ concentration was increased, consistent
with inhibition of net H+ uptake at higher K+
concentrations (n = 6).
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DISCUSSION |
This study demonstrates that expression of both the
1- and
1-subunits of Na,K-ATPase is
upregulated during chronic hypokalemia. However, the dominant mechanism
for the increase in Na+ pump-mediated NH
uptake that occurs after 3 days of dietary K+ restriction
(38) is the change in interstitial K+
concentration, which occurs during hypokalemia in vivo, rather than
upregulation of Na+ pump expression at the level of the
plasma membrane.
Rates of NH
uptake by Na,K-ATPase after changes
in extracellular K+ concentration measured in the present
study were compared with rates predicted by the model of Kurtz and
Balaban (19). In rat IMCD, the Michaelis constant
(Km) and transport rate when all binding sites
of a carrier are saturated (Vmax) for the
Na,K-ATPase with NH
and K+ as
substrates were reported previously by our laboratory
(40).4
NH
concentration was taken to be 6 mM. Incorporating
these values, the model predicts that NH
uptake by
the Na+ pump is reduced by 61% when interstitial
K+ concentration is increased from 10 to 30 mM, a
prediction consistent with the experimental observations of this study.
In the present study, NH
-dependent, ouabain-sensitive J
was used as an
index of the rate of NH
uptake by Na,K-ATPase at
steady state. We observed that
J
was reduced by
84% when extracellular K+ concentration was increased from
10 to 30 mM in either normal or K+-restricted rats. Thus
changes in extracellular K+ concentration over the
physiological range observed in the interstitium of the inner medulla
in vivo produce a dramatic change in Na pump-mediated NH
uptake. However, we cannot exclude the possibility
that trafficking of the Na+ pump between the plasma
membrane and the cytosol occurs after changes in K+ concentration.
Our laboratory reported previously that in IMCD tubules from
K+-restricted rats,
JtCO2 is upregulated relative to
K+-replete controls (41). Because
tubules were perfused and bathed in identical solutions
(K+ = 10 mM, NH
= 6 mM), a stable
adaptation must occur during hypokalemia that upregulates net acid
secretion. Most likely, uptake of H+ equivalents across the
basolateral membrane is increased in series with increased secretion of
H+ equivalents into the luminal fluid. However, after 3 days of dietary K+ restriction, increased net
H+ uptake across the basolateral membrane does not occur
through increased expression of Na,K-ATPase. Expression of another
transporter that localizes to the basolateral membrane, such as anion
exchange, may be upregulated during hypokalemia, which augments
secretion of H+ equivalents.
A variety of techniques have been used to study transporter expression
at the cell surface. At the level of the plasma membrane, protein
expression has been studied extensively with immunocytochemical techniques. However, this technique is technically challenging when
studying changes in Na,K-ATPase expression along the collecting duct because of the extensive infolding of the basolateral membrane (31). Cellular fractionation has also been used
(47), but it requires considerable tissue, limiting its
use in IMCD suspensions. Ouabain binding and ouabain-sensitive
Rb+ uptake have been used in other cell types for study of
changes in Na+ pump expression along the basolateral
membrane. However, because the tIMCD suspensions represent tubule
fragments, functional measurements such as Rb+ uptake are
not useful because of the large variability in measurements (Wall SM and Trinh HN, unpublished observations). Moreover,
ouabain binding studies in rats are difficult because of the low
affinity of Na,K-ATPase for ouabain in rat (16). Our
laboratory therefore used tubules perfused in vitro as a means by which
to study expression of functional Na+ pumps along the
basolateral membrane. We showed previously (36) that in
the presence of NH
, pHi is increased
after ouabain addition to the bath. Through a series of ion
substitution experiments we showed (36, 37) that this effect of ouabain on pHi occurs through inhibition of
NH
uptake by Na,K-ATPase. Using this approach, we
observed that cell surface expression of functional Na,K-ATPase is
not changed with 3 days of dietary K+ restriction.
After a dietary K+ load, both Na,K-ATPase (12,
35) and ROMK channels (35) are upregulated in the
cortical collecting duct (CCD). Upregulation of these transporters
occurs in series and results in increased secretion of K+
into the luminal fluid of the CCD (21, 26). Conversely,
with dietary K+ depletion these transporters are
downregulated, which promotes K+ conservation in this
segment (18, 35). K+ absorption is observed in
the OMCD and IMCD after dietary K+ restriction
(17), which also promotes K+ conservation.
However, in the medullary collecting duct the mechanism of this
adaptive response probably differs from that of the CCD. The
Na+ pump is upregulated in the CCD during hyperkalemia
(12, 35), whereas in the medullary collecting duct it is
upregulated during hypokalemia
(23).5 Why
Na,K-ATPase protein expression is upregulated in the medullary collecting duct after 7 days of K+ restriction is unknown.
This increased expression observed during hypokalemia may generate a
latent pool of Na+ pumps, which are available for
translocation into the plasma membrane. Such a mechanism may serve to
maintain intracellular volume or intracellular K+
concentration after rapid changes in extracellular
K+ concentration. Whether Na,K-ATPase-mediated
transport across the basolateral membrane is increased in the medullary
collecting duct after >1 wk of K+ restriction has not been
explored. Therefore, it remains to be determined whether increased
Na+ pump expression augments
NH
-stimulated H+ secretion after
prolonged intake of a low-K+ diet.
The Na,K-ATPase is composed of
- and
-subunits. In some renal
segments, such as in rat inner medulla,
NK-
1
1 also assembles with a
-subunit
(2, 24). In kidney, although the
1-and
1-isoforms of Na,K-ATPase are very abundant, protein
expression of the other isoforms of the
- and
-subunits
(
2-4 and
2-3) has not been
demonstrated (15, 23, 28, 29, 34). In the IMCD
Na,K-ATPase activity correlates more closely with
1-
than with
1-subunit expression, similar to previous observations in rat OMCD (23).
Although changes in expression of NK-
1
1
during hypokalemia were studied in detail, changes in expression of the
-subunit in this treatment group remain to be determined. The
physiological role of the
-subunit is not understood. However, it is
known to alter the affinity of NK-
1
1 for
K+ and Na+ (2). The present study
did not discern the effect of hypokalemia on
-subunit expression,
nor did it determine the effect of changes in
-subunit expression on
Na+ pump-mediated NH
uptake. However, the
measurements of NH
uptake by the Na+ pump
in IMCD tubules perfused in vitro reported in the present study reflect
changes in expression of each subunit, including
, when assembled as
a heterotrimer in native tissue and studied under physiological conditions.
In conclusion, Na+ pump expression is upregulated in the
rat IMCD during hypokalemia. However, the primary mechanism for the increase in NH
uptake mediated by Na,K-ATPase that follows 3 days of dietary K+ restriction results from
changes in interstitial K+ concentration observed in vivo
rather than changes in protein expression.
We thank Drs. Juan Codina, Bruce Kone, and Thomas Pressley for
helpful suggestions.
This study was supported by National Institute of Diabetes and
Digestive and Kidney Diseases Grant DK-52935 (to S. M. Wall).