Am J Physiol Renal Physiol
279: F195–F202, 2000.
Potassium depletion increases proton pump (H⫹-ATPase)
activity in intercalated cells of cortical collecting duct
RANDI B. SILVER,1 SYLVIE BRETON,2 AND DENNIS BROWN2
1
Department of Physiology and Biophysics, Joan and Sanford I. Weill Medical College of Cornell
University, New York, New York 10021; and 2Program in Membrane Biology, Massachusetts
General Hospital, Harvard Medical School, Charlestown, Massachusetts 02129
Received 4 October 1999; accepted in final form 9 March 2000
Address for reprint requests and other correspondence: R. B.
Silver, Dept. of Physiology and Biophysics, Joan and Sanford I. Weill
Medical College of Cornell Univ., 1300 York Ave., New York, NY
10021 (E-mail: [email protected]).
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.
proton-adenosinetriphosphatase; bafilomycin; intracellular
pH; 2⬘,7⬘-bis(2-carboxyethyl)-5(6)-carboxyfluorescein; immunocytochemistry
IT IS WELL ESTABLISHED THAT
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F195
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HCO3⫺ reabsorption increases in the cortical collecting duct (CCD) during
chronic hypokalemia (13). Two possible mechanisms
through which enhanced HCO3⫺ reabsorption can occur
in the CCD are via the vacuolar-type proton pump
(H⫹-ATPase) and the H⫹/K⫹ ATPase (9), both of which
are present in intercalated cells (ICs). Despite the
presence of both of these transporters in ICs, the signals responsible for their regulation and their relative
contributions to transepithelial proton transport and
intracellular pH homeostasis are not well defined.
Functional studies using microperfused late distal
tubules from control rats demonstrated active proton
secretion (JHCO3), one-third of which was blocked by
bafilomycin (27), a specific inhibitor of the H⫹-ATPase
(6). Under conditions of K⫹ depletion, as a consequence
of a low-K⫹ diet, JHCO3 almost doubles in this nephron
segment (27). Much of the enhanced proton (H⫹) secretion associated with the K⫹- restricted diet was inhibited by Sch-28080, a specific blocker of the gastric
H⫹-K⫹-ATPase (26). The contribution of bafilomycinsensitive H⫹-ATPase to net acid secretion in tubules
from K⫹-restricted rats was not studied. In microperfused terminal inner medullary collecting ducts from
rats maintained on a low-K⫹ diet, only one-half of the
measured total CO2 flux was sensitive to the H⫹-K⫹ATPase inhibitors Sch-28080 and ouabain (25), leaving
open the possibility that enhanced bafilomycin-sensitive H⫹-ATPase also contributes to the net acid flux
under K⫹-depleted conditions. Recently, it was shown
that electrogenic bafilomycin-sensitive H⫹ secretion is
increased with chronic hypokalemia in microperfused
distal rat nephron comprised of distal convoluted tubule, the connecting tubule, and the initial collecting
tubule (3, 4). Furthermore, immunocytochemical localization of the proton translocating H⫹-ATPase showed
that K⫹-deprivation resulted in an increase in the
percentage of intercalated cells with H⫹-ATPase localized in the apical membrane (4). However, in this study
the functional contribution of the ICs to net H⫹ secretion was not directly assessed.
The purpose of the present investigation was, therefore, to evaluate directly the bafilomycin sensitive H⫹pumping activity in ICs of the CCD. H⫹-ATPase activity was measured in individual cells from control and
Silver, Randi B., Sylvie Breton, and Dennis Brown.
Potassium depletion increases proton pump (H⫹-ATPase) activity in intercalated cells of cortical collecting duct. Am J Physiol
Renal Physiol 279: F195–F202, 2000.—Intercalated cells (ICs)
from kidney collecting ducts contain proton-transporting ATPases (H⫹-ATPases) whose plasma membrane expression is
regulated under a variety of conditions. It has been shown that
net proton secretion occurs in the distal nephron from chronically K⫹-depleted rats and that upregulation of tubular H⫹ATPase is involved in this process. However, regulation of this
protein at the level of individual cells has not so far been
examined. In the present study, H⫹-ATPase activity was determined in individually identified ICs from control and chronically K⫹-depleted rats (9–14 days on a low-K⫹ diet) by monitoring K⫹- and Na⫹-independent H⫹ extrusion rates after an
acute acid load. Split-open rat cortical collecting tubules were
loaded with the intracellular pH (pHi) indicator 2⬘,7⬘-bis(2carboxyethyl)-5(6)-carboxyfluorescein, and pHi was determined
by using ratiometric fluorescence imaging. The rate of pHi
recovery in ICs in response to an acute acid load, a measure of
plasma membrane H⫹-ATPase activity, was increased after K⫹
depletion to almost three times that of controls. Furthermore,
the lag time before the start of pHi recovery after the cells were
maximally acidified fell from 93.5 ⫾ 13.7 s in controls to 24.5 ⫾
2.1 s in K⫹-depleted rats. In all ICs tested, Na⫹- and K⫹independent pHi recovery was abolished in the presence of
bafilomycin (100 nM), an inhibitor of the H⫹-ATPase. Analysis
of the cell-to-cell variability in the rate of pHi recovery reveals a
change in the distribution of membrane-bound proton pumps in
the IC population of cortical collecting duct from K⫹-depleted
rats. Immunocytochemical analysis of collecting ducts from
control and K⫹-depleted rats showed that K⫹-depletion increased the number of ICs with tight apical H⫹ATPase staining
and decreased the number of cells with diffuse or basolateral
H⫹-ATPase staining. Taken together, these data indicate that
chronic K⫹ depletion induces a marked increase in plasma
membrane H⫹ATPase activity in individual ICs.
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INCREASED H⫹-ATPase ACTIVITY WITH K⫹ DEPLETION
MATERIALS AND METHODS
Animals and Tubule Preparation
Pathogen-free Sprague-Dawley rats of both sexes (Charles
River Laboratories, Kingston, NY), weighing between 75 and
150 g, were used for these experiments. Rats were fed either
a normal diet (Purina Formulab 5008; Na⫹ content 0.28%,
K⫹ content 0.11%) or a K⫹-deficient diet that was otherwise
nutritionally balanced (Harlan Teklad 170550; Na⫹ content
0.4%) for 9–14 days.
CCTs were prepared and mounted in a perfusion chamber
as previously described (19). Rats were killed by cervical
dislocation, the kidneys were removed, and tubules were
dissected free and opened to form a flat epithelium. Sometimes, two tubules were used from each animal. Solutions
were gravity fed into a manually operated six-port Hamilton
valve. The solution leaving the valve passed directly into a
miniature water-jacketed glass coil (Radnoti Glass Technology, Monrovia, CA) for temperature regulation. The warmed
solution entered the experimental chamber, which was
mounted on the stage of an inverted epifluorescence microscope (Nikon Diaphot). The temperature of the superfusate
in the chamber was maintained at 37°C.
Solutions
Tubules were superfused with HEPES-buffered solutions
(Table 1) as described previously (19). A 1 mM solution of
bafilomycin A1 (LC Laboratories, New Bedford, MA; Alexis,
San Diego, CA) was prepared in dimethyl sulfoxide, and then
diluted 1:10,000 to give a final concentration of 100 nM in the
experimental superfusates. All chemicals were obtained from
Sigma Chemical unless otherwise stated. Nigericin (Molecular Probes, Eugene, OR) was added to K⫹-Ringer solutions
(solutions A and B) from a 10 mM stock (3 parts ethanol:1
part dimethyl formamide) to give a final concentration of 10
␮M. Individual vials (50 ␮g) of the acetoxymethyl derivative
of BCECF-acetyoxymethyl ester (BCECF-AM; Molecular
Probes, Eugene, OR) were stored dry at 0°C and reconstituted in dimethyl sulfoxide (at a concentration of 10 mM) for
each experiment. The final loading concentration of dye was
5 ␮M in Na⫹-Ringer solution.
Fluorescence Measurements of Intracellular pH (pHi )
With BCECF
Equipment. The basic components of the experimental
apparatus have been described previously (18, 19) and consist of the following: an inverted epifluorescence microscope
(Nikon Diaphot) equipped with a 75-W xenon lamp, Nikon
CF Fluor ⫻100/1.3-na oil-immersion objective, two Metal Tek
filter wheels back to back, a computer controllable excitation
light shutter, and a cooled charge-coupled device camera
(Princeton Instruments ) with a frame transfer chip (EEV37) and 12-bit readout. The emitted fluorescence signal is
relayed as real-time continuous output to an IBM PC/ATcompatible clone, and the image pairs were collected on a
Sierra Pinnacle Micro optical disk drive (1.3 Gbytes). The
imaging work station is controlled by using the Metafluor
software package (Universal Imaging). Quantitative image
pairs at 490- and 440-nm excitation with emission at 520 nm
were obtained every 15 s for the duration of the experiment.
The fluorescence excitation was shuttered off except during
the brief periods required to record an image. To correct for
intrinsic autofluorescence and background, images were obtained by using the experimental acquisition configuration
on the split-opened portion of tubule before loading of the
dye. These background images were subtracted from the
corresponding images of cell fluorescence, and these corrected ratios were used for data analysis.
BCECF-loading and identification of intercalated cells.
Split-open tubules were loaded in the experimental chamber
with BCECF (5 ␮M) from both the basolateral and luminal
sides at room temperature for 15 min, after which they were
superfused with Na⫹-Ringer solution (solution 1) at 37°C for
at least 15 min before the start of the experiment. Calibration of the emitted signal from each cell in the tubule was
performed at the end of each experiment. Extracellular pH
Table 1. Composition of solutions
Component
NaCl
Na2HPO4
KCl
HEPES
MgCl2
Glucose
NH4Cl
NMDG/Cl
Phosphoric acid
CaCl2
pH
Na⫹-Ringer
(Solution 1)
135.0
2.5
5.0
10.0
1.0
2.0
2.0
7.4
NH4Cl
(Solution 2)
10.0
1.0
2.0
10.0
130.0
2.5
2.0
7.4
0 K, 0 Na
(Solution 3)
Calibration
(Solution A)
Calibration
(Solution B)
10.0
1.0
2.0
130.0
25.0
1.0
2.0
130.0
25.0
1.0
2.0
140.0
2.5
2.0
7.4
2.0
7.8
2.0
6.5
Solutions are expressed in mM. To titrate the solutions to the appropriate pH, NaOH was used in the Na⫹-Ringer solution, N-methyl
⫹
⫹
⫹
D-glucamine powder in the K - and Na -free solutions, and KOH in the Na -free calibration solutions.
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K⫹-depleted rats by following the K⫹- and Na⫹-independent H⫹ extrusion rate of ICs in response to an
acute acid load. Measurements were performed in ICs
from split-open rat cortical collecting tubules (CCTs) in
conjunction with ratiometric digital imaging techniques after the cells of the tubule were loaded with the
pH-sensitive fluorophore 2⬘,7⬘-bis(2-carboxyethyl)5(6)-carboxyfluorescein (BCECF). The results revealed
a marked increase in the rate of bafilomycin-sensitive
pHi recovery in response to an acute acid load in cells
from K⫹-depleted rats compared with ICs from control
rats. Also, immunocytochemistry showed a significant
increase in apical H⫹ATPase staining of ICs in the
CCD from the K⫹-restricted rats. Taken together,
these data indicate that chronic K⫹ depletion induces a
marked increase in plasma membrane H⫹ATPase activity at the single IC level.
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INCREASED H⫹-ATPase ACTIVITY WITH K⫹ DEPLETION
was varied from 6.8 to 7.8 (solutions A and B) in the presence
of the K⫹/H⫹ exchanger nigericin (10 ␮M) in 145 mM K⫹
according to the method of Thomas et al. (24) and as described previously in this preparation (19). Cells in the experimental field of view were analyzed singularly and independently from their neighbors, as previously described (18,
19). ICs were differentiated from principal cells by both their
visual appearance under transmitted light and by their much
greater loading of BCECF, as previously described (19, 20,
28). Because our measurements revealed a normal distribution of pHi recovery rates after an acid load in the ICpopulation under control conditions, no attempt was made to
distinguish ␣-type from ␤-type intercalated cells for the functional portion of this study. Values from all intercalated cells
measured were grouped together for statistical analysis.
Immunocytochemistry
Serum Analysis
Blood samples were obtained from some animals via the
dorsal aorta and analyzed for Na⫹, K⫹, and Cl⫺ by a commercial laboratory.
Statistics
Results are expressed as means ⫾ SE, where n refers to
the number of cells analyzed individually, unless otherwise
noted. Different IC subtypes have been described in CCDs,
but no apparent and consistent heterogeneity in the rate of
RESULTS
Effects of a Low-K⫹ Diet on Acid-Base and
Electrolyte Balance
Plasma K⫹, Na⫹, and Cl⫺ values were measured in
three control rats and six low-K⫹ rats and are presented in Table 2. Rats maintained on a low-K⫹ diet
were hypokalemic compared with control animals (P ⬍
0.0001).
Bafilomycin-Sensitive, Na⫹- and K⫹-Independent pHi
Recovery in Response to an Acute Acid Load
Control rats. H⫹-ATPase function was assayed as
the rate of Na⫹- and K⫹- independent intracellular
alkalinization in response to an acute pulse of 10 mM
NH4⫹. Figure 1A is a representative trace of this response as observed in an individual IC from a control
rat tubule. In this and all subsequent traces, the experimentally determined ratios have been converted to
pHi as described in MATERIALS AND METHODS. Ratio image
pairs were generated every 15 s and are represented by
the data points from the IC in this experimental trace.
After the steady-state pHi in Na HEPES-buffered solution (Table 1, solution 1) was monitored, an acute
acidosis was induced in the cells by superfusing the
tubule with a solution containing 10 mM NH4Cl, in the
absence of extracellular Na⫹ and K⫹, with these cations replaced in the solution with N-methyl-D-glucamine (Table 1, solution 2). The superfusate was then
changed to a Na⫹-, K⫹-, and NH4Cl-free solution (Table 1, solution 3), resulting in an abrupt intracellular
acidosis that, as shown in Fig. 1A, brought the pHi
down to 6.3. Within 90 s of reaching the nadir pHi, the
pHi began to increase steadily presumably due to H⫹
extrusion via the H⫹-ATPase. The initial H⫹ extrusion
rate measured in this cell (⌬pHi/⌬t), as calculated from
the slope indicated by the dashed line in Fig. 1A, is 0.07
pHi U/min, which eventually leveled off to a pHi of 6.7.
To be certain that this observed K⫹- and Na⫹-independent intracellular alkalinization was due to H⫹pump activity, the same acid pulse protocol described
Table 2. Plasma electrolyte values
Control rats
K⫹-depleted rats
n
K⫹ mEq/L
Na⫹ mEq/L
Cl⫺ mEq/L
3
6
3.7 ⫾ 0.1
1.8 ⫾ 0.1*
140 ⫾ 1
148 ⫾ 4
100 ⫾ 1
95 ⫾ 2
Values are means ⫾ SE. n, No. of animals. Plasma samples were
obtained from the dorsal aorta before removal of the kidneys. * P ⬍
0.0001.
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Tissue preparation. Control or K⫹-depleted Sprague-Dawley rats were anesthetized with pentobarbital sodium (Nembutal; 0.1 ml of a 50 mg 䡠 ml solution⫺1 䡠 100 g body wt⫺1), and
kidneys were fixed by perfusion through the left ventricle
with a fixative containing 4% paraformaldehyde, 10 mM
sodium periodate, 70 mM lysine [modified periodate-lysineparaformaldehyde (PLP)], and 5% sucrose, as previously described (7). After a 5-min perfusion, kidneys were removed,
sliced, and fixed by immersion for a further 6 h before rinsing
and storage in PBS (10 mM sodium phosphate buffer containing 0.9% NaCl, pH 7.4). For preparation of 5-␮m sections,
tissues were cryoprotected by immersion in 30% sucrose for
at least 1 h before sectioning with a Reichert Frigocut microtome using disposable knives.
Immunostaining procedure. Tissue sections (5 ␮m) picked
up on Fisher Superfrost Plus slides were rinsed for 10 min in
PBS and then treated with 1% SDS for 5 min. This step
increases antigenicity in frozen sections of PLP-fixed tissues,
as previously described (8). After three more rinses (5 min
each) in PBS to remove the SDS, sections were incubated for
20 min in PBS/1% BSA to block nonspecific background
staining. An affinity-purified primary anti-H⫹-ATPase antibody prepared in rabbit against the 11 COOH-terminal
amino acids of the 56-kDa B1-subunit of the bovine H⫹ATPase (“kidney” isoform) (14) was applied for 2 h at room
temperature at a dilution of 1:100. After washing 2 ⫻ 5 min
in high-salt PBS (PBS containing 2.7% NaCl) to reduce
nonspecific staining, and a further 1 ⫻ 5 min in normal PBS,
secondary goat anti-rabbit IgG (diluted 1:60) coupled to FITC
(Jackson Immunologicals) was applied for 1 h. After further
washing as above, sections were mounted in Vectashield
antifading solution (Vector Laboratories, Burlingame, CA)
diluted 1:1 in 0.1 M Tris 䡠 HCl, pH 8.0.
Sections were examined by using a Bio-Rad Radiance 2000
confocal microscope. Digital images were imported into
Adobe Photoshop (4.0) and printed on a Tektronix Phaser 440
dye sublimation color printer.
pHi recovery from an acid load that would be suggestive of a
differential response between ␣- and ␤-ICs, was found in the
present experiments. Therefore, data from all ICs examined
for each experimental group were pooled for final statistical
analysis. In addition, the numbers of animals and tubules
used in each protocol are indicated in the figure legends. The
NH4Cl pulse protocol was performed only once in each tubule, followed by the in situ nigericin calibration. Significant
differences were determined by ANOVA. Significance was
asserted if P ⬍ 0.05.
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INCREASED H⫹-ATPase ACTIVITY WITH K⫹ DEPLETION
above was performed but with the addition of the
H⫹-ATPase inhibitor bafilomycin at a concentration
(100 nM) known to inhibit H⫹ pump activity in rat
distal tubules (4, 27) and CCDs (19). A representative
trace of the pHi recovery response to an acute acidosis
in the presence of bafilomycin, from a single IC from a
control split-open CCT, is shown in Fig. 1B. In the
presence of bafilomycin, the K⫹- and Na⫹-independent
pHi recovery response was virtually abolished compared with the representative trace in Fig. 1A. Taken
together, these results indicate that the IC-specific
H⫹ATPase is responsible for the pHi recovery induced
by the NH4Cl acid pulse and observed under control
conditions in these cells.
Fig. 2. K⫹- and Na⫹-independent pHi recovery after acute exposure to an NH4Cl acid pulse in an IC from a split-open tubule of
K⫹-depleted rat. Y-axis represents the pHi as determined from the
intracellular calibration of the dye in this tubule. The tubule was
initially superfused with solution 1 (Table 1) and then changed to
solution 2 (Table 1). Acute exposure to NH4Cl resulted in acidification after its removal. On removal of the NH4, Na⫹, and K⫹, the
pHi fell to ⬃6.6 from the initial value of 7.5. Within 15 s, the pHi
started to increase in this cell at an initial rate of 0.20 pHi U/min
and returned the pHi back to the initial pHi of 7.5.
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Fig. 1. A: K⫹- and Na⫹-independent intracellular pH (pHi) recovery after acute exposure to an NH4Cl acid pulse in an intercalated
cell (IC) from a split-open tubule of a control rat in the absence of
extracellular K⫹ and Na⫹. Y-axis represents the pHi as determined from the intracellular calibration of the dye in this tubule.
The tubule was initially superfused with Na⫹-Ringer solution
(NaR; solution 1, Table 1) and then changed to 10 mM NH4Cl
(solution 2, Table 1). Acute exposure to NH4Cl resulted in acidification after its removal. On removal of the NH4, Na⫹, and K⫹,
the pHi fell from an initial value of 7.00 to 6.30. Within 90 s, the
pHi started to increase, with intracellular alkalinization occurring
at a rate of 0.07 pHi U/min. The slope or rate of the K⫹- and
Na⫹-independent pHi recovery was calculated at the beginning of
the recovery process as illustrated in the trace. B: effect of bafilomycin. Y-axis represents the pHi as determined from the intracellular calibration of the dye in this tubule. The tubule was initially
superfused with solution 1 (Table 1) and then changed to solution
2 (Table 1). Bafilomycin A1 (100 nM) was present in the superfusate from the NH4Cl until the end of the protocol as shown.
Addition of the blocker prevented the K⫹- and Na⫹-independent
H⫹ efflux observed in Fig. 1A, demonstrating H⫹-ATPase activity
is responsible for the pHi recovery.
K⫹ deficiency. To test the hypothesis that K⫹ depletion stimulates acid secretion via increased H⫹ATPase activity in ICs, the above acid pulse protocol
performed in control ICs was carried out in tubule
preparations from low-K⫹ rats. Figure 2 is a representative response of an individual IC from a tubule of a
K⫹-restricted rat. On removal of the NH4Cl pulse, the
pHi fell to ⬃6.6 from an initial value of ⬃7.5. Within
15 s of the NH4Cl pulse, the pHi started to increase in
this cell at an initial rate of 0.20 pHi U/min, bringing
the pHi back to the pre-acid load initial value. This rate
of intracellular alkalinization was much greater than
that observed in the control IC of Fig. 1A. The mean
rate of Na⫹- and K⫹-independent pHi recovery for all of
the ICs from low-K⫹ rats was 0.22 ⫾ 0.02 pH U/min,
(n ⫽ 44 ICs, 6 tubules, 5 rats), which is almost threefold greater (P ⬍ 0.0001) than the mean rate measured
in control ICs (0.08 ⫾ 0.01 pH U/min, n ⫽ 57 ICs, 8
tubules, 6 rats) (Fig. 3).
Bafilomycin significantly inhibited the K⫹- and Na⫹independent intracellular alkalinization rates in control ICs (to 0.02 ⫾ 0.02 pH U/min, n ⫽ 12 ICs, 3
tubules, 3 rats; P ⬍ 0.05) and in ICs from low-K⫹ rats
(to 0.02 ⫾ 0.01 pHi U/min n ⫽ 16 ICs, 3 tubules, 3 rats).
These results demonstrate that chronic K⫹ restriction
stimulates the activity of bafilomycin-sensitive H⫹ATPase in ICs of the CCD.
Another difference between the responses of the
control ICs and the K⫹-depleted ICs was the magnitude of the pHi recovery response as evidenced by a
comparison of Figs. 1A and 2. As shown in Fig. 2, the
pHi in the IC from the K⫹-restricted tubule leveled
off to the initial pHi value of ⬃7.5 whereas in the
control IC the pHi recovery was partial, returning to
6.7. Generally, the pHi recovery in response to the
imposed acidosis was more complete in the ICs from
the K⫹-depleted rats compared with control ICs (Fig.
4), but this difference was not statistically significant.
INCREASED H⫹-ATPase ACTIVITY WITH K⫹ DEPLETION
F199
ICs, and the response ranged from 0.02 to 0.55 pHi
U/min. The variability in the pHi recovery response
for the 57 control ICs studied is shown in Fig. 5B. In
the control ICs, 10 of the 57 ICs studied showed no
H⫹-ATPase-dependent pHi recovery from the acute
acid load, and the response ranged from 0 to 0.38 pHi
U/min, with a mean value of 0.08 ⫾ 0.01 pHi U/min.
Immunocytochemistry
To determine whether H⫹-ATPase activity is influenced by pHi, and accounts for the difference in rates
seen in control and low-K⫹ ICs, we compared the pHi
values measured during three different parts of the
experimental protocol. The three points represented in
Fig. 4 are the following: initial pHi (in HEPES-buffered
Na⫹-Ringer solution; solution 1, Table 1); the nadir pHi
reached on removal of the NH4Cl solution; and the
final steady-state pHi reached. There was no difference
in the initial pHi measured in the control ICs (7.58 ⫾
0.07) and low-K⫹ ICs (7.50 ⫾ 0.07). The nadir pHi
reached on removal of the NH4Cl pulse was also similar between the two groups (6.40 ⫾ 0.04 for control vs.
6.47 ⫾ 0.04 for low-K⫹ ICs). In a comparison of the final
pHi reached in the absence of extracellular K⫹ and
Na⫹, the cells from low-K⫹ rats tended to be more
alkaline than the control ICs (7.11 ⫾ 0.11 vs. 6.92 ⫾
0.07, respectively), but, as indicated above, this difference was not statistically significant. It appears that
factors other than pHi are influencing the activity of
the H⫹ pump under conditions of chronic K⫹ restriction.
To determine whether the changes in H⫹-ATPase
function observed at the single IC level reflect
changes in the CCD IC population as a whole, the
cell-to-cell variability in the rate of the pHi recovery
response was analyzed. When individual pHi recovery values were plotted, there was a clear cell-to-cell
variability in the pHi recovery response in the group
of control ICs and the group of ICs from K⫹-depleted
rats. This variability in the pHi recovery response is
shown in Fig. 5, where the number of cells within
each group (K⫹-depleted and control) is plotted
against pHi recovery rate, with the rates binned in
increments of 0.10 pHi U/min. As shown in Fig. 5A,
all of the 44 ICs from K⫹-depleted rats showed an
H⫹-ATPase-dependent recovery from the acid load.
The mean pHi recovery response was higher in these
cells (0.22 ⫾ 0.02 pHi U/min) compared with control
Fig. 4. Graph of pHi values measured before and after the NH4Cl
pulse in ICs from K⫹-depleted rats (A) and control rats (B). The
baseline pHi refers to the pHi measured at the beginning of the
experimental protocol in HEPES-buffered NaR solution. The nadir
pHi corresponds to the lowest value reached on removal of the acid.
The final pHi value shown represents the final value reached in the
IC in the absence of extracellular Na⫹ and K⫹.
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Fig. 3. Comparison of H⫹-ATPase activity (⌬pHi/⌬t) in the absence
and presence of bafilomycin in control ICs and K⫹-depleted ICs.
Values are means ⫾ SE. The K⫹- and Na⫹-independent ⌬pHi/⌬t (pH
U/min) rate is compared in ICs from control rats and in ICs from
K⫹-depleted rats with and without bafilomycin (100 ␮M) added to
the superfusate (*** P ⬍ 0.0001).
Because the functional results presented above do
not take into account in which membrane of the IC
the H⫹-ATPase resides, we performed immunocytochemistry in the two groups of rats using H⫹-ATPase
antibodies. As previously described (1) heavily
stained ICs alternated with unstained cells in connecting segments and collecting ducts. In control rat
kidneys, the apical, basolateral, and diffuse patterns
of H⫹-ATPase staining that characterize different IC
subtypes were readily distinguished (Fig. 6A). As
shown in Fig. 6A, many of the stained cells have
basolateral, bipolar, or diffuse staining, and some
have unique apical staining. A very different staining pattern was observed in tubules from K⫹-deprived rats, as shown in Fig. 6B. In collecting ducts
from low-K⫹ rats, the basolateral staining pattern
was seen much less frequently, and apical staining
predominated.
F200
INCREASED H⫹-ATPase ACTIVITY WITH K⫹ DEPLETION
DISCUSSION
The BCECF-loaded, split-tubule preparation was
used in combination with dual excitation digital imaging to study H⫹-ATPase activity in individual ICs
in response to an imposed acidosis. ICs were visually
identified under epi-illumination by their much
brighter appearance after BCECF loading compared
with the neighboring principal cells, as previously
described (19). Our results indicate that maintaining
rats on a low-K⫹ diet stimulates bafilomycin-sensitive H⫹-pump activity in the plasma membrane of
ICs to a level that is almost three times greater than
that observed in ICs from control rats. In addition,
the lag time before the start of the intracellular
alkalinization process, once the nadir pHi had been
reached, was much shorter in the ICs from the K⫹depleted CCDs (24.5 ⫾ 2.1 s, n ⫽ 30 ICs) compared
with control ICs (93.5 ⫾ 13.7 s, n ⫽ 35 ICs). This
more rapid onset of alkalinization may indicate that
the ICs from CCDs of low-K⫹ rats are poised to deal
with an intracellular acid load more efficiently than
control cells. This is consistent with the hypothesis
that the H⫹-ATPase content of the plasma membrane may be initially greater in ICs from K⫹-depleted rats. In contrast, ICs from control rats may
have to mobilize more pumps from an intracellular
vesicular pool to the cell surface before significant
proton extrusion can occur, a process that has been
shown to occur after both acute (16) and chronic
systemic acidosis (5), as well as after exposure of
isolated collecting ducts and turtle bladder to basolateral CO2 (11, 17, 23).
Downloaded from http://ajprenal.physiology.org/ by 10.220.33.5 on June 17, 2017
Fig. 5. Histogram showing variability of pHi recovery rates in K⫹depleted (A) and control ICs (B). The ordinate represents the no. of
cells, and the abscissa the pHi recovery rates that have been binned
in increments of 0.10 pH U/min. A: variability of the response of 44
ICs from K⫹-depleted rats. B: the response of 57 ICs from control
rats.
It is generally accepted that the electroneutral,
gastric-like H⫹-K⫹-ATPase is also functional in the
CCD. At the molecular level, mRNA expression of
the ␣-subunit of the gastric pump is seen in the cells
of the CCD under K⫹-replete conditions and is enhanced with K⫹-depletion (22). Functionally, a Sch28080-sensitive gastric-like H⫹-K⫹-ATPase has been
identified at the individual IC level in rabbit and rat
CCD (19, 20) and in microperfused rabbit CCD
(29, 30) and rat distal tubule (15, 27) with
K⫹depletion. These results suggest that the ICs of
the CCD possess functional H⫹-ATPase and H⫹-K⫹ATPase under conditions of chronic K⫹depletion.
The relative contributions of both ATPases to net acid
secretion under hypokalemic conditions remain to be
elucidated.
Our study demonstrates an increased H⫹ secretion
via membrane-associated H⫹-ATPase in ICs from
K⫹-depleted rats. These results are in agreement
with previous work by Bailey et al. (3, 4) showing
that K⫹ depletion induced an increase in electrogenic
H⫹-ATPase activity in the rat distal tubule perfused
in vivo. These findings at first appear to differ from
the enzymatic results of others measuring H⫹ATPase activity in individually microdissected CCDs
from control and K⫹-depleted rats (9, 10). The enzymatic
studies showed there was no effect of chronic
⫹
K depletion on H⫹-ATPase activity in rat collecting
ducts (9, 10). However, the apparent discrepancy
may just reflect differences in what is actually being
measured. The biochemical assay used for assessing
H⫹-ATPase activity is performed on permeabilized
microdissected nephron segments and represents the
total pool of enzyme, consisting of the relative
amounts of intracellular (vesicular) plus the plasma
membrane H⫹-ATPase activity. Our measurements
as well as those of Bailey (3, 4) represent functional
plasma membrane-bound H⫹-ATPase only. Our immunocytochemical results support the concept that
the increase in H⫹-ATPase activity measured at the
individual cell level represents movement of H⫹ATPase from an intracellular vesicular pool to the
plasma membrane. This suggests that K⫹ depletion
results in a redistribution of preexisting pumps and
not synthesis of new pumps, which is consistent with
the enzymatic findings.
The signal responsible for the enhanced H⫹ATPase activity in individual ICs is not yet known.
Although it has been suggested that plasma aldosterone levels modulate H⫹-ATPase activity (9), chronic
hypokalemia is characterized by low plasma aldosterone levels (4). It has also been speculated that
hypokalemic intracellular acidosis may contribute to
the increased insertion of H⫹-ATPase into apical
membrane (4); however, under conditions of our
study the initial pHi measured in HEPES-buffered
solutions was not different between the control and
the hypokalemic ICs (Fig. 4). In addition, it appears
that the buffering capacity of the ICs is similar
between the control and K⫹-depleted groups in that
the nadir pHi reached on removal of the NH4Cl pulse
INCREASED H⫹-ATPase ACTIVITY WITH K⫹ DEPLETION
F201
is also comparable between the two groups. We cannot exclude the possibility, however, that an in vivo
cell acidosis plays a role in increased insertion of
H⫹-ATPase into the plasma membrane.
Under conditions of low K⫹, the distribution of
⫹
H -ATPase in ICs was considerably modified compared with cells from control rats. In particular, cells
with basolateral, bipolar, or diffuse staining were
not as abundant, and more cells with a tight apical
band of staining were found in K⫹-depleted rats.
Although it is generally accepted that all ␣-type ICs
have apical H⫹-ATPase (either as a tight band or a
diffuse subapical staining), some ␤-type ICs (identified by the absence of basolateral AE1 staining) can
also have tight apical H⫹-ATPase staining (2, 12).
Therefore, the increased abundance of apically
stained IC found in the present study could result
from an increased apical polarization of H⫹-ATPase
in ␣-type ICs and/or a redistribution of H⫹-ATPase
to the apical pole of ␤-type ICs. Further studies will
be required to resolve this issue.
The analysis of the cell-to-cell variability in the pHi
response rates (Fig. 5) provides insight into the functional heterogeneity of the IC population under control
and K⫹-depleted conditions. These data support the
immunocytochemical results that K⫹-depletion stimulates insertion of H⫹-ATPase into the plasma membrane, because no IC failed to recover from an acid
load. Presumably, the control ICs that showed little or
no pHi recovery were those in which proton pumps
were mainly intracellular.
In conclusion, this investigation demonstrates enhanced H⫹-ATPase activity at the single IC level,
as evidenced by the functional data and confirmed
by the immunocytochemical results. The implication
of our finding is that H⫹-ATPase residing in the
apical membrane of ICs is actively contributing to
the increased proton secretion associated with
chronic hypokalemia, often leading to systemic metabolic alkalosis. This upregulation may contribute to
the increased secretory H⫹ transport that occurs
under low-K⫹ conditions, and it could also reflect a
cellular response involved in maintaining intracellular ion and pH homeostasis in this pathophysiological state.
This work was supported by National Institute of Diabetes and
Digestive and Kidney Diseases Grants DK-45828 and the Underhill
Downloaded from http://ajprenal.physiology.org/ by 10.220.33.5 on June 17, 2017
Fig. 6. Immunocytochemical staining using antibodies
against the 56-kDa H⫹-ATPase B1-subunit in control
(A) and K⫹-depleted (B) rat kidney cortex. In collecting
ducts from control rats, cells with basolateral and diffuse/bipolar staining are frequently found (arrows), and
some cells with tight apical staining can also be seen
(arrowhead). In collecting ducts from K⫹-depleted rats,
cells with basolateral and diffuse/bipolar staining (arrow) were much less frequent whereas cells with tight
apical staining were common (arrowheads). Bars ⫽
10 ␮m.
F202
INCREASED H⫹-ATPase ACTIVITY WITH K⫹ DEPLETION
and Wild Wings Foundations (R. B. Silver) and DK-42956 (D. Brown
and S. Breton).
15.
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