Novel Schering and ouabain-insensitive potassium-dependent
proton secretion in the mouse cortical collecting duct
Snezana
Petrovic1,3,
Zachary
Spicer2,
Tracey
Greeley1,
Gary E
Shull2, and
Manoocher
Soleimani1,3
Departments of 1 Medicine and 2 Molecular Genetics,
Biochemistry, and Infectious Diseases, University of Cincinnati, and
3 Veterans Administration Medical Center, Cincinnati, Ohio 45267
 |
ABSTRACT |
The
intercalated (IC) cells of the cortical collecting duct (CCD) are
important to acid-base homeostasis by secreting acid and reabsorbing
bicarbonate. Acid secretion is mediated predominantly by apical
membrane Schering (SCH-28080)-sensitive
H+-K+- ATPase (HKA) and bafilomycin-sensitive
H+-ATPase. The SCH-28080-sensitive HKA is believed to be
the gastric HKA (HKAg). Here we examined apical membrane
potassium-dependent proton secretion in IC cells of wild-type HKAg
(+/+) and HKAg knockout (
/
) mice to determine relative
contribution of HKAg to luminal proton secretion. The results
demonstrated that HKAg (
/
) and wild-type mice had comparable rates
of potassium-dependent proton secretion, with HKAg (
/
) mice having
100% of K+-dependent H+ secretion vs.
wild-type mice. Potassium-dependent proton secretion was resistant to
ouabain and SCH-28080 in HKAg knockout mice but was sensitive to
SCH-28080 in wild-type animals. Northern hybridizations did not
demonstrate any upregulation of colonic HKA in HKAg knockout mice.
These data indicate the presence of a previously unrecognized K+-dependent SCH-28080 and ouabain-insensitive proton
secretory mechanism in the cortical collecting tubule that may play an
important role in acid-base homeostasis.
hydrogen-potassium-adenosinetriphosphatase; kidney; mouse
knockout; acid-base homeostasis
 |
INTRODUCTION |
ACTIVE PROTON
SECRETION by the collecting duct (CD) is coupled, in part, to
active K+ absorption via a membrane-bound
adenosinetriphosphatase (H+-K+-ATPase)
(24, 32). The H+-K+-ATPase (HKA)
that is expressed in renal CD under normal conditions shows striking
molecular, biochemical, and physiological similarities to the HKA found
in gastric parietal cells, which is responsible for the secretion of
acid into the gastric lumen (21, 24, 32). This exchanger
has been referred to as gastric HKA (HKAg) and is sensitive to
inhibition by SCH-28080 (24, 32). A distinct but
structurally related HKA is expressed in the distal colon, which
mediates active K+ reabsorption (6, 15). This
transporter is called colonic HKA (HKAc) or nongastric HKA and is
sensitive to inhibition by ouabain (24). Molecular studies
indicate expression of both HKAg and HKAc in CD (24, 32).
Functionally they are thought to be involved in H+
secretion and/or K+ reabsorption but appear to be regulated
differentially (24, 32). Furthermore, HKAc may also
mediate the exchange of extracellular K+ for
intracellular Na+ or NH
(5,
11), consistent with this transporter working as a
cation/K+ exchange mode.
To better understand the role of HKAg in acid-base regulation,
transgenic mice deficient in this gene were examined. HKAg null mice
have severe achlorhydria (25), consistent with the role of
this transporter as the major acid-secreting process in the parietal
cells of the stomach. The HKAg-deficient mice did not show any
significant abnormality in systemic acid-base balance or serum
potassium (25), despite the fact that this transporter plays an important role in net bicarbonate reabsorption in the collecting ducts of mouse kidney (12, 13). These results
suggest that another acid-secreting transporter(s) is likely
upregulated in the kidneys of HKAg null mice. To explore this issue
further, renal cortical collecting ducts (CCDs) of wild-type or HKAg
knockout mice were isolated and perfused, and their
- and
-intercalated cells (ICs) were examined. The results indicate that a
novel exchanger, which is distinct from HKAc, is upregulated in HKAg
null mice and maintains the K+-dependent H+
secretion at a comparable level to wild-type animals.
 |
METHODS |
Animals.
Wild-type and HKAg knockout mice were used for these
experiments. HKAg knockouts were described recently
(25). Animals were allowed free access to food and water
and were studied at 3-6 mo of age. For potassium depletion
studies, animals were placed on a potassium-free diet (11)
for 19 days.
Isolation of CCDs.
Animals were killed by intraperitoneal injection of 50 mg/kg
pentobarbital sodium. Kidneys were quickly removed and placed in
ice-cold dissection medium (solution 1, Table
1). Thin (~1 mm) slices were obtained
and transferred to the dissection chamber. The temperature of the
dissection medium in the chamber was kept at 4°C. Bovine serum
albumin, 0.1%, was added to the dissection medium to prevent sticking
of dissected tubules to the glass and forceps. CCDs were obtained by
free-hand dissection out of cortical medullary rays. Tubules were
measured using an eyepiece micrometer and generally were 0.3-0.5
mm in length. CCDs were distinguished from nearby proximal straight
tubules by their approximately one-third smaller diameter and more
turbid appearance compared with the "ground-glass" appearance of
the proximal straight tubules. Thick ascending limbs, on the other
hand, are thinner than CCDs and have a fragile, homogeneous, and
slightly shiny appearance in reflected light.
In vitro microperfusion.
Dissected tubules were quickly transferred to a 1.5-ml
temperature-controlled specimen chamber mounted on an inverted Zeiss Axiovert S-100 microscope (Carl Zeiss, Thornwood, NY). Tubules were
perfused using concentric glass pipettes according to the method of
Burg and Green (1) with modifications (19,
27) at 4.5 cmH2O pressure. Solutions that were used
to perfuse and bathe the tubules are listed in Table 1. All solutions
were delivered to the specimen chamber in CO2- and
O2-impermeable tubing (Cole Palmer, Chicago, IL) by a
peristaltic pump (Peristar, WPI, Sarasota, FL) at a rate of 1 ml/min.
The chamber had a lid to minimize evaporation and heat loss and
maintain constant gas pressure and pH. Chamber pH was frequently
checked on a model B 213 Horiba pH meter that allows measurement of pH
in small samples.
To identify damaged cells, 0.07 mg/ml Fast Green dye (Sigma, St. Louis,
MO) was added to the perfusate. Damaged cells are stained green by Fast
Green dye. Tubules were carefully inspected and discarded if
green-staining cells or a perfusate leak were found.
Intracellular pH measurements.
After 20-min equilibration in solution 2, each tubule was
perfused with 6 µM of the fluorescent pH indicator
bis(carboxyethyl)-5(6)-carboxyfluorescein acetoxymethyl
ester (BCECF-AM) for 15 min. When BCECF-AM is introduced from the
luminal side of a CCD, intercalated, but not principal, cells take up
the dye (29). Charged and fluorescent molecules of the dye
are trapped in the cell after cleavage of the ester moiety by
cytoplasmic esterases. Another 10-15 min were allowed for the dye
washout. Fluorescent measurements were done on a Zeiss Axiovert S-100
inverted microscope equipped with the Attofluor RatioVision Digital
Imaging System (Attofluor, Rockville, MD). Achroplan ×40/0.8 water
objective with 3.6-mm working distance was used. Excitation wavelengths
were alternated between 488 and 440 nm, and emission was measured at
520 nm. Background fluorescence was measured before the dye was
introduced to the tubule and automatically subtracted from all
subsequent measurements. The Attofluor Digital Imaging System allows
for the control of light source intensity. Balance between the light
source intensity and the camera gain optimizes fluorescent imaging
while at the same time applies the smallest light intensity that is
sufficient to excite the dye. This helps to minimize
photobleaching and photodamage of the tubule cells, which can be
substantial (29).
To avoid tubule movements and out-of-focus fluorescence, the free end
of the tubule was allowed to loosely adhere to the
poly-L-lysine-covered part of the chamber coverslip
(28). Only cells in sharp focus in the tubule wall were
examined. Images were taken in duplicates at 2-s intervals. Attofluor
RatioVision software allowed for "regions of interest" to be
applied to individual cells so that multiple cells in a single tubule
were simultaneously examined. Generally, three to eight cells were
examined per tubule. Only one tubule per animal was used. Digitized
images were analyzed by using Attograph software. At the end of
each experiment, intracellular calibration was performed by using the
high-K+-nigericin method of Thomas et al.
(26). Calibration solutions were varied from pH 6.5 to
7.8, and calibration points were fitted to a linear regression curve,
which was then used for conversion of calculated ratios to cell pH.
Experimental procedures.
After a stable baseline cell pH reading in bicarbonate-buffered
solution (solution 2) was achieved, the bath solution was switched to a chloride-free solution (solution 3) to
distinguish between the IC types. As shown previously (14, 18,
20, 30), removal of bath Cl
causes alkalinization
of
-ICs due to blocking and/or reversing the basolateral
Cl
/HCO
exchanger. At the same time,
-ICs acidify, because this maneuver stimulates
HCO
exit via the apical
Cl
/HCO
exchanger. Basolateral
Cl
conductance (14, 18, 20, 30) of the
-ICs allows for intracellular Cl
to leave the cell on
basolateral Cl
removal. This adds to the apical membrane
Cl
gradient and stimulates apical
Cl
/HCO
exchange and
HCO
exit. Both the alkalinization of
-ICs as well
as the acidification of
-ICs is reversed on addition of
Cl
to the bath.
After identification of the IC type, HCO
was removed
from the bath and the lumen (solution 4). Also,
K+ was removed from the lumen to block
H+-K+-ATPase (solution 5). Luminal
H+-ATPase was blocked by adding 170 nM bafilomycin
A1 to the luminal solution. Each tubule was equilibrated in
those solutions for 20 min and then acidified by addition of 15 mM
NH4Cl (solution 6) to the bath. Basolateral
Na+/H+ exchange was blocked with 40 µM
ethylisopropylamiloride (EIPA) and basolateral
H+-ATPase of
-ICs with 170 nM bafilomycin
A1. The tubules were bathed in a solution containing
NH4Cl for 3 min. The bath solution was then switched back
to a HCO
-free HEPES-buffered solution
(solution 4) with 40 µM EIPA and 170 nM bafilomycin
A1. The lumen was kept K+ free, and luminal
H+-ATPase was blocked with 170 nM bafilomycin
A1. Under these conditions, no cell pH recovery was
observed. The luminal solution was then switched to solution
4 (containing 5 mM K+) and 170 nM bafilomycin
A1. K+-dependent cell pH recovery was followed
for the next 5-10 min. Inhibitors were then removed, and recovery
without inhibitors was followed for the next 10 min as an index of cell
viability. In a separate series of experiments designed to test the
inhibitor profile of H+-K+-ATPase, 10 µM
SCH-28080 or 1.5 mM ouabain was added to the appropriate luminal
solution (solution 4).
RNA isolation and Northern hybridization.
Total cellular RNA was extracted from whole kidney of wild-type or HKAg
null mice (on normal or K+-free diet for 19 days) by the
method of Chomczynski and Sacchi (2). In brief, 0.5-1
g of tissue was homogenized at room temperature in 10 ml Tri Reagent
(Molecular Research Center, Cincinnati, OH). RNA was quantitated by
spectrophotometry and stored at
80°C. RNA samples were fractionated
on a 1.2% agarose-formaldeyde gel. The samples were transferred to a
nylon membrane, cross-linked by ultraviolet light, and baked for 1 h. Hybridization was performed according to Church and Gilbert
(3). The cDNA probes were labeled with
32P-deoxynucleotide using the RadPrime DNA labeling kit
(Life Technologies). After hybridization, the membranes were washed,
blotted dry, exposed to a PhosphorImager cassette at room temperature
for 24-48 h, and read by PhosphorImager (Molecular Dynamics). The
K+-free diet protocol was similar to previous studies
reported from our laboratories (11). For HKAc, three PCR
products from the rat
-subunit cDNA (nucleotides 135-515,
2369-2998, and 3098-3678; Ref. 6) were pooled
and used as an isoform-specific probe.
Chemicals.
All chemicals were obtained from Sigma unless specified otherwise. EIPA
was dissolved in methanol as a 40 mM stock on the day of an experiment
and diluted 1:1,000 for the final concentration of 40 µM. A stock
solution of bafilomycin A1 was made fresh for each
experiment in methanol, and 1:1,000 dilution was used to make the final
concentration of 170 nM. Ouabain was directly added and dissolved in
appropriate solutions at a final concentration of 1.5 mM. This
concentration was used on the basis of studies indicating that HKAc
activity was inhibited partially by 1 mM ouabain (24).
Nigericin was dissolved in ethanol as a 10 mM stock and diluted 1:1,000
for the final concentration of 10 µM. BCECF-AM was obtained from
Molecular Probes (Eugene, OR) and kept frozen in small aliquots of
stock in DMSO. It was diluted 1:1,000 for the final concentration of 6 µM. SCH-28080 was kept frozen in small aliquots of 100 mM stock in
methanol and was used with a dilution of 1:10,000 in the final
concentration of 10 µM.
Statistics.
Results are give as means ± SE. Statistical comparisons between
the groups were performed according to Student's t-test for nonpaired data. The data were considered significant if
P < 0.05.
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RESULTS |
K+-dependent intracellular pH
recovery in
-ICs and
-ICs in wild-type mouse CCDs.
The first set of experiments was designed to examine
K+-dependent luminal proton secretion in each of the IC
types (
and
) in CCDs in wild-type animals. A representative
tracing is shown in Fig. 1. At the
beginning of each experiment, IC type was determined by basolateral
Cl
removal in the presence of HCO
. The same cells were then followed throughout the experiment. Nine of 19 cells in 5 CCDs of wild-type mice alkalinized on basolateral Cl
removal [change in intracellular pH
(
pHi) = 0.21 ± 0.01] and were therefore
considered
-ICs. Ten of 19 cells in 5 CCDs acidified on basolateral
Cl
removal (
pHi =
0.26 ± 0.02) and were considered
-ICs.

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Fig. 1.
Representative tracings demonstrate the effect of 5 mM
extracellular K+ on intracellular pH (pHi)
recovery from an acute acid load in intercalated cells (ICs) in
wild-type (WT) mouse. Upon identification of IC type (see
METHODS and first part of tracing), HCO
was removed from lumen and bath for the rest of the experiment to
render both luminal and basolateral HCO transporters
inactive. In the presence of bafilomycin A1 on both sides
(an inhibitor of H+- ATPase) and ethylisopropylamiloride
(EIPA) in the bath (an inhibitor of basolateral
Na+/H+ exchange), removal of K+
from lumen blocked pHi recovery from an acid load. The
return of 5 mM K+ to the lumen resulted in a significant
pHi recovery from acidosis in both cell types. The rate of
K+-dependent pHi recovery was similar in both
cells: 0.069 pH units/min in -ICs and 0.065 pH units/min in -ICs.
Removal of all inhibitors allowed for faster recovery at the rate of
0.142 pH units/min in -IC and 0.129 pH units/min in -ICs (to 7.27 and 7.28, respectively).
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After the IC cell types were examined, HCO
was
removed from the luminal and basolateral sides. The
Na+/H+ exchanger was blocked by addition of 40 µM EIPA to the bath solution. The H+-ATPase was blocked
by addition of 170 nM bafilomycin A1 to both luminal and
bath solutions, and K+ was removed from the lumen. Under
these experimental conditions, NH4Cl prepulse acidified
-ICs from a baseline pHi of 7.35 ± 0.02 to a nadir
pHi of 6.58 ± 0.03 (n = 9 cells in 5 CCDs) and
-ICs from a baseline pHi of 7.33 ± 0.02 to a nadir pHi of 6.53 ± 0.03 (n = 10 cells in 5 CCDs). No pHi recovery was observed in either cell type in the absence of luminal K+. As demonstrated in
Fig. 1 and summarized in Fig. 3, addition of 5 mM K+ to the
lumen resulted in pHi recovery at a rate of 0.109 ± 0.01 pH units/min in
-ICs and 0.093 ± 0.01 pH units/min in
-ICs (P > 0.05). These rates are similar to rates
reported in previous studies in normal rat and rabbit ICs (22,
23, 31). Cells recovered to 6.98 ± 0.04 and 7.01 ± 0.03 in
-ICs and
-ICs, respectively. Removal of the inhibitors
(EIPA and bafilomycin from bath and bafilomycin from the perfusate)
resulted in additional recovery to the baseline pH at a rate of
0.259 ± 0.01 and 0.268 ± 0.01 pH units/min in
-ICs and
-ICs, respectively. The lack of pHi recovery to baseline
levels after K+ addition is in agreement with observations
by other investigators (22, 23, 31). One explanation
that has been offered in this regard is that HKA activity might be
regulated by intracellular H+ concentration
(22).
Effect of SCH-28080 on K+-dependent
pHi recovery in
-ICs and
-ICs in wild-type mouse CCD.
Using the same protocol as described above, we also examined the effect
of HKAg inhibitor SCH-28080 on K+-dependent pHi
recovery in
-ICs and
-ICs of wild-type mouse CCD. A
representative tracing is shown in Fig.
2, and the results are summarized in Fig.
3. In this group of experiments, 7 of 16 cells in 6 CCDs alkalinized on basolateral Cl
removal
(
pHi = 0.25 ± 0.02) and were therefore
considered
-ICs. Nine of 16 cells in 6 CCDs acidified on basolateral
Cl
removal (
pHi =
0.24 ± 0.02) and were considered
-ICs. In these experiments, the
NH4Cl prepulse acidified
-ICs from a baseline pHi of 7.26 ± 0.035 to a nadir pHi of
6.64 ± 0.047 (n = 7 cells in 6 CCDs) and
-ICs
from a baseline pHi of 7.33 ± 0.027 to a nadir
pHi of 6.49 ± 0.05 (n = 9 cells in 6 CCDs). In the absence of K+ in the lumen, no
pHi recovery was observed in either cell type. As
demonstrated in Figs. 2 and 3, the presence of 10 µM SCH-28080 in the
lumen blocked the K+-dependent pHi recovery by
>80% in both IC cell types. Rates of K+-dependent
pHi recovery were 0.019 ± 0.005 and 0.012 ± 0.004 pH units/min in
-ICs and
-ICs, respectively
(P < 0.001, compared with the rates of recovery in the
absence of SCH-28080). The HKAg is highly sensitive to SCH-28080, with
the ATPase activity inhibited by >90% in the presence of 1 µM
SCH-28080 (reviewed in Ref. 24). It has been shown that at
concentrations >10 µM, SCH-28080 can inhibit other transporters such
as Na+-independent H+-ATPase (16).
Furthermore, recent data demonstrated that prolonged exposure of kidney
CD cells to SCH-28080 as well as high concentrations of SCH-28080 cause
ATP depletion (4), which in turn can have nonspecific
inhibition of HKA activity. To avoid these complications, we did not
try higher concentrations of SCH-28080. On removal of SCH-28080 and
other inhibitors, all cells recovered to their baseline pHi
at the rate of 0.275 ± 0.02 pH units/min in
-ICs and
0.253 ± 0.022 pH units/min in
-ICs. In summary, the results in
Figs. 1-3 demonstrate comparable K+-dependent,
SCH-28080-sensitive H+-secreting activity in the apical
membrane of both
-ICs and
-ICs and are consistent with the
activity of luminal HKAg in both cell types.

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Fig. 2.
Representative tracings demonstrating the effect of Schering
(SCH)-28080 on K+-dependent pHi recovery in ICs
of WT mouse. The identification of IC type is demonstrated in the first
part of the tracings. HCO was removed from lumen and
bath for the rest of the experiment to render
HCO -dependent transporters inactive. In the presence
of bafilomycin A1 on both sides and EIPA in the bath,
removal of K+ from lumen blocked pHi recovery
from an acid load. SCH-28080 blocked the K+-dependent
pHi recovery in IC cells. Removal of the inhibitors allowed
for fast recovery at the rate of 0.211 pH units/min in -IC and 0.223 pH units/min in -IC.
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Fig. 3.
SCH-28080 inhibition of K+-dependent pHi
recovery from acid load of ICs in WT mice CCD (summary of results). The
effect of luminal K+ and SCH-28080 on the rate of
pHi recovery in - or -IC in wild-type mice is
shown.
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K+-dependent pHi recovery
in
-ICs and
-ICs in HKAg knockout mouse CCD.
In the next series of experiments, we examined K+-dependent
pHi recovery in both IC cell types in HKAg knockout
mice CCDs. On the basis of their pHi response to
basolateral Cl
removal, 12 of 25 cells (in 6 CCDs) were
identified as
-ICs, and the remaining 13 were identified as
-ICs.
In the presence of HCO
, baseline pHi was
comparable in
-ICs of HKAg knockout (7.353 ± 0.004, n = 12) and wild-type animals (7.346 ± 0.002, n = 9) (P > 0.05). Interestingly,
basal pHi in
-ICs was lower in HKAg knockout animals
(7.265 ± 0.005, n = 13) compared with wild-type
animals (7.326 ± 0.003, n = 10) (P < 0.05). Absence of HCO
did not significantly alter the baseline pHi levels (data not shown).
A NH4Cl prepulse acidified
-ICs from a baseline
pHi of 7.26 ± 0.035 to a nadir pHi of
6.64 ± 0.047 (n = 12 cells in 6 CCDs).
-ICs
acidified from a baseline pHi of 7.33 ± 0.027 to a
nadir pHi of 6.49 ± 0.05 (n = 13 cells in 6 CCDs). Similar to wild-type animals, in the absence of
luminal K+, no pHi recovery was observed in
either cell type. Surprisingly, as shown in representative tracings in
Fig. 4 and summarized and compared with
wild-type mice in Fig. 5, both IC cell
types in the CCDs of HKAg knockout mice demonstrate luminal
K+-dependent H+-secreting activity comparable
to that of wild-type mice: 0.118 ± 0.01 pH units/min
(n = 12) in
-ICs and 0.102 ± 0.01 pH units/min in
-ICs (n = 13) (P > 0.05 compared
with
-ICs and
-ICs without SCH-28080, respectively). On addition
of K+ to the lumen, cells recovered to pHi
7.05 ± 0.02 in
-ICs and 7.06 ± 0.03 in
-ICs. Removal
of the inhibitors resulted in further pHi recovery to the
baseline pH at the rate of 0.228 ± 0.01 pH units/min in
-ICs
and 0.249 ± 0.02 pH units/min
-ICs.

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Fig. 4.
Representative tracings demonstrating the effect of 5 mM
K+ on pHi recovery from acid load in ICs of
gastric H+-K+-ATPase (HKAg) null mice. After
the identification of - and -cells (as demonstrated in the first
part of the tracing), HCO was removed from the
experiments to render HCO transporters inactive. In
the presence of bafilomycin A1 on both sides and EIPA in
the bath, removal of K+ from lumen prevented
pHi recovery from acidosis. The return of 5 mM
K+ to the lumen resulted in significant pHi
recovery (to ~6.88) in both cells. Rate of K+-dependent
recovery was 0.083 pH units/min in -IC and 0.066 pH units/min in
-ICs. Removal of the inhibitors allowed for additional recovery at
the rate of 0.142 pH units/min in -IC and 0.143 pH units/min in
-IC.
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Fig. 5.
Rate of K+-dependent pHi recovery from
acute acid load in ICs of WT and HKAg knockout (KO) mice (summary
of results). The effect of luminal K+ on the rate of
pHi recovery in - or -IC of WT and KO mice is
shown.
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Effect of SCH-28080 on K+-dependent
pHi recovery in
-ICs and
-ICs in HKAg knockout
mouse CCD.
To characterize the K+-dependent H+ secretion
in the CCDs of HKAg knockout mice, we sought to examine its
sensitivity to SCH-28080 or ouabain. Data from the experiments
examining the effect of SCH-28080 are shown as a representative tracing
in Fig. 6 and are summarized in Fig.
7. Based on their pHi
response to bath Cl
removal, 11 of 22 cells in 5 CCDs
were identified as
-ICs, and 11 cells were considered
-ICs. The
NH4Cl prepulse acidified
-ICs from a baseline
pHi of 7.29 ± 0.027 to a nadir pHi of
6.64 ± 0.019 (n = 11 cells).
-ICs acidified
from a baseline pHi of 7.27 ± 0.032 to a nadir
pHi of 6.67 ± 0.027 (n = 11 cells).
In the absence of K+ in luminal perfusate, no
pHi recovery was observed in either cell type. However, and
contrary to wild-type animals, SCH-28080 did not block the
pHi recovery in the presence of 5 mM potassium in the
lumen. This phenomenon was observed in both IC cell types. The rate of
K+-dependent H+ secretion in the lumen of HKAg
knockout mice CCDs was 0.097 ± 0.005 pH units/min in
-ICs
(n = 11) and 0.094 ± 0.007 pH units/min in
-ICs (11 cells in 5 CCDs). Compared with the cells in tubules of
knockout mice perfused in the absence of SCH-28080, no significant difference was noted (P > 0.05 with or without
SCH-28080), indicating that SCH-28080 did not affect the
K+-dependent pHi recovery in ICs of
knockout mice CCDs. pHi recovered to 6.99 ± 0.03 and 7.05 ± 0.03 in
-ICs and
-ICs, respectively. Removal of
the inhibitors resulted in additional recovery to the baseline pH at
the rate of 0.251 ± 0.012 pH units/min in
-ICs and 0.243 ± 0.006 pH units/min in
-ICs.

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Fig. 6.
Representative tracings demonstrating the effect of SCH-28080 on
K+-dependent pHi recovery from acid load in ICs
from a HKAg KO mice. H+-ATPase was blocked with bafilomycin
A1 on both sides, and basolateral
Na+/H+ exchange was blocked with EIPA.
Interestingly, the presence of SCH-28080 did not prevent the
K+-dependent pHi recovery. The cell recovered
to pHi ~6.8. Rate of K+-dependent recovery
was 0.078 pH units/min. Removal of the inhibitors allowed for
additional recovery at the rate of 0.301 pH units/min to
pHi 7.34.
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Fig. 7.
SCH-28080 does not inhibit K+-dependent pHi
recovery from acid load in ICs of HKAg KO mice (summary of results).
The effect of luminal K+ and SCH-28080 on the rate of
pHi recovery in - or -IC of HKAg KO mice is shown.
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Effect of ouabain on K+-dependent
pHi recovery in
-ICs and
-ICs in HKAg null mouse CCD.
The purpose of the next series of experiments was to examine the effect
of ouabain on K+-dependent pHi recovery in CCDs
of HKAg knockout mice. Figure 8 is a
representative tracing, and Fig. 9
summarizes the results. In this group of experiments, 4 of 11 cells
(from 2 CCDs) were identified as
-ICs, and 7 of 11 cells were
considered
-ICs. The NH4Cl prepulse acidified
-ICs
from a baseline pHi of 7.33 ± 0.031 to a nadir
pHi of 6.79 ± 0.005 (n = 4).
-ICs
acidified from a baseline pHi of 7.22 ± 0.03 to a
nadir pHi of 6.71 ± 0.03 (n = 7).
Similar to previous experiments, in the absence of K+ in
the lumen, no pHi recovery was observed in either cell
type. In the presence of 1.5 mM ouabain in the lumen, the rate of
K+-dependent pHi recovery in IC cells of HKAg
knockout mice remained unchanged vs. no ouabain. As indicated in
Fig. 9, the pHi recovery rate in the presence of ouabain
was 0.099 ± 0.007 pH units/min in
-ICs (n = 4)
and 0.097 ± 0.005 pH units/min in
-ICs (n = 7)
of the HKAg knockouts. These were not statistically different vs. no
ouabain in
-ICs and
-ICs, respectively (P > 0.05) (Fig. 9). Cells recovered to 7.06 ± 0.02 in
-ICs and
6.99 ± 0.03 in
-ICs. Removal of inhibitors resulted in
additional recovery to baseline pHi levels at the rate of
0.236 ± 0.013 pH units/min in
-ICs and 0.234 ± 0.01 pH
units/min in
-ICs.

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Fig. 8.
Representative tracings demonstrate the effect of ouabain on
K+-dependent pHi recovery in ICs from HKAg KO
mice. H+-ATPase was blocked with bafilomycin A1
on both sides, and basolateral Na+/H+ exchange
was blocked with EIPA. Under these conditions, K+ removal
from lumen prevented pHi recovery from cell acidosis.
Presence of ouabain at 1.5 mM did not inhibit the
K+-dependent pHi recovery from acidosis.
pHi recovered to ~7.2 in the presence of ouabain. Rate of
K+-dependent recovery was 0.13 ph units/min in -IC and
0.105 pH units/min in -ICs. Removal of the inhibitors allowed for
additional recovery at a rate of 0.178 pH units/min in -IC and 0.16 pH units/min in -ICs to pHi of 7.34 and 7.35, respectively.
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Fig. 9.
Ouabain does not inhibit K+-dependent pHi
recovery from acid load in ICs of HKAg KO mice (summary of results).
The effect of ouabain and luminal K+ on the rate of
pHi recovery in HKAg KO mouse is shown.
|
|
RT-PCR and Northern hybridization studies in wild-type and HKAg
null mouse.
The gene-targeting strategy eliminated sequences encoding the catalytic
phosphorylation site, which is essential for enzyme activity. In the
stomach, the wild-type HKAg mRNA was eliminated (25),
although there were trace levels of an ~1-kb mRNA encoding some of
the NH2-terminal sequences. To test for the presence of HKAg mRNAs in kidneys of wild-type and mutant mice, RT-PCR analysis was
performed using primers from exons 6 and 10. As shown in Fig. 10, a PCR product of the appropriate
size (653 bp) was identified in wild-type kidney, but not in
knockout kidney. These results confirm that these critical
sequences, which span the major catalytic domain and the region
required for apical sorting (10) of the pump, are absent
in RNA from knockout kidney.

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Fig. 10.
RT-PCR analysis of RNA for the presence of HKAg. RT-PCR
analysis was performed on total RNA isolated from kidneys of WT and
HKAg KO animals using primers from exons 6 and 10. PCR products were
size fractionated on 1.2% agarose gel and visualized by ethidium
bromide staining. The results demonstrate a PCR product of expected
size (653 bp) for mouse HKAg in the kidneys of WT but not HKAg KO
animals. The specificity of the reaction is demonstrated by the absence
of an RT product in the absence of reverse transcriptase (RT ).
Bottom panel shows constitutive control -actin.
|
|
It is possible that in mice deficient in HKAg, the HKAc becomes
upregulated and compensates for HKAg deficiency, as both transporters are expressed in the CD and mediate the exchange of luminal
K+ for intracellular H+. Although the lack of
effect of ouabain on K+-dependent H+ extrusion
argues against such a possibility, a definite answer should come from
expression studies. Accordingly, we examined the renal expression of
HKAc by Northern hybridization. As indicated in Fig.
11, mRNA levels for HKAc remained
undetectable in whole kidney of both HKAg knockout and wild-type
animals on a normal diet (control groups). To determine the sensitivity
of the HKAc assay, both HKAg knockout and wild-type animals were
fed a K+-free diet according to established protocols
(11) and examined after 19 days. As indicated in Fig. 11,
both wild-type and HKAg null mice were able to upregulate their renal
HKAc expression in response to K+ depletion. These results
indicate that although the HKAg null mice can upregulate their HKAc in
response to pathophysiological states, they do not upregulate it in
response to HKAg gene deletion. Taken together, these results support
the conclusion that a yet unrecognized, luminal,
K+-dependent, SCH-28080- and ouabain-insensitive
H+-secreting activity contributes to acid secretion in the
CCD of HKAg null mice.

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Fig. 11.
Northern hybridizations of colonic HKA (HKAc) in whole
kidney of WT and HKAg KO mice. As indicated, HKAc mRNA levels remained
undetectable in WT and HKAg KO animals on a normal diet (control
groups). Both WT and HKAg KO mice upregulated their HKAc mRNA in
potassium depletion. The 28S rRNA is shown in the bottom of
the blot as an index of RNA loading. RNA (30 µg/lane) was loaded in
each lane.
|
|
 |
DISCUSSION |
The present studies examine the K+-dependent luminal
acid extrusion in CCDs of wild-type and HKAg null mice. HKAg null mice demonstrate an H+/K+ exchange activity that is
comparable to wild-type animals (Figs. 1-5). Whereas SCH-28080
inhibits H+/K+ exchange activity in wild-type
animals, H+/K+ exchange activity in the CCDs of
null mice is resistant to inhibition by SCH-28080 (Figs. 6 and 7).
H+/K+ exchange activity in null mice was also
insensitive to inhibition by ouabain, a known inhibitor of HKAc (Figs.
8 and 9). Northern hybridizations did not demonstrate any upregulation
of HKAc in HKAg null mice (Fig. 11).
HKAg is expressed in the CDs of mammalian kidneys, as supported by
molecular, biochemical, and functional studies (24, 32). Studies on the inhibitor profile of HKAg demonstrate that this transporter is inhibited by SCH-28080 but not ouabain
(24). Microperfusion studies in rabbit and rat kidney
indicate that a significant portion of K+ and
HCO
reabsorption in CCD is mediated via the
SCH-28080-sensitive HKAg (24, 32). Studies in
microperfused mouse CCD demonstrate that ~50% of
HCO
reabsorption is mediated via a
K+-dependent process that is inhibited by SCH-28080
(12, 13). Taken together, these studies indicate that HKAg
is responsible for a significant portion of HCO
reabsorption in the mouse kidney CD.
The present studies demonstrate that
-IC and
-IC in mouse CCDs
express apically oriented K+-dependent, SCH-28080-sensitive
acid-secreting activity, strongly suggestive of functional HKAg. This
is in agreement with functional studies in split-open rat and rabbit
CCDs and microperfused rabbit CCDs (22, 23, 31).
Interestingly, baseline pHi in
-ICs of HKAg knockout
mice was significantly lower compared with wild-type animals, whereas
it was comparable in
-IC cells (see RESULTS). On the
basis of present data, we cannot be sure about what causes this
difference in
-ICs or why there is not a difference in the basal
pHi in
-ICs. We can only speculate that the lower basal pHi in
-ICs in HKAg knockout mice raises the
possibility that these cells are either pumping HCO
out at higher rates or a H+-translocating transporter gets
downregulated. Although both
- and
-ICs seem to have apically
oriented HKAg, it may serve different functions in these two cell
types, or the way two cell types compensate for the lack of HKAg may be different.
HKAg null mice did not display any significant abnormality in systemic
acid-base balance or serum K+ under baseline conditions
(25). This suggests that acid-base transporter(s) distinct
from HKAg is upregulated in the CDs of HKAg null mice. Comparable
levels of K+-dependent pHi recovery in the CCDs
of both animal groups are consistent with this hypothesis.
Interestingly, the K+-dependent H+ secretion in
the CCDs of HKAg null mice is not inhibited by classical HKA inhibitors
such as SCH-28080 or ouabain, indicating the existence of a novel, yet
unrecognized isoform of HKA in the kidney.
With respect to nongastric HKAs, HKAc is the well-known
SCH-28080-insensitive, ouabain-sensitive isoform (7, 32).
HKAc expression did not increase in HKAg null mice, indicating that this transporter is not responsible for the upregulation of HKA activity in this animal. Furthermore, the ouabain insensitivity of HKA
activity in HKAg null mice points to an isoform distinct from HKAc.
Recent functional studies in the colon have described the presence of
another HKA isoform, which is insensitive to ouabain (9).
No distinct HKA molecule that is insensitive to both SCH-28080 and
ouabain has been identified.
Studies on HKAs in the kidney point to the discrepancies in the
inhibitor profile of "nongastric" HKAs in heterologous expression systems and native tissues and have concluded that the presence of a
novel isoform may account for the discrepancies (7, 24). Studies in cultured kidney cells (4) have questioned some
of the inhibitor profile studies by demonstrating nonspecific effects of high concentrations of SCH-28080 on ATPase activity. To avoid any
nonspecific effect of SCH-28080 on HKA transporters, the exposure time
of perfused tubules to SCH-28080 in the present experiments was kept short.
Recently, Laroche-Joubert et al. (8) examined the
properties of three functional K+-ATPases isoforms in
microdissected rat nephron segments. They concluded that type II and
III K+-dependent ATPase activities exhibit different
sensitivity profiles to SCH-28080 and ouabain compared with the type I
renal K+-ATPase. Type I K+-dependent ATPase is
sensitive to SCH-28080 but is insensitive to ouabain and resembles
HKAg. Type III is sensitive to ouabain but is insensitive to SCH-28080.
They further found that pharmacological properties and tubular
localization of type III K+-ATPase are not compatible with
that of HKAc. They suggested that a new kidney HKA isoform might exist
that may fit the properties of type III K+-ATPase. It is
worth mentioning that an HKA that is insensitive to both SCH-28080 and
ouabain has not been described in kidney epithelial cells. As such, the
molecule mediating the H+/K+ exchange in apical
membrane of ICs in HKAg knockout mice is distinct from all HKA
isoforms described so far.
In conclusion, our results suggest that a novel acid-base transporter,
distinct from HKAc, is upregulated in HKAg null mice and maintains the
K+-dependent proton secretion at a comparable level to
wild-type animals. This may account for the lack of any acid-base
abnormality in HKAg null mice.
 |
ACKNOWLEDGEMENTS |
These studies were supported by National Institute of Diabetes and
Digestive and Kidney Diseases Grants DK-52821 (to M. Soleimani) and
DK-50594 (to G. E. Shull), a merit review grant, and grants from
Dialysis Clinic, Incorporated (to M. Soleimani).
 |
FOOTNOTES |
Address for reprint requests and other correspondence: M. Soleimani, Div. of Nephrology and Hypertension, Dept. of Internal Medicine, Univ. of Cincinnati, 231 Albert Sabin Way, MSB 5502, Cincinnati, OH 45267-0585 (E-mail:
manoocher.soleimani{at}uc.edu).
The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
First
published August 8, 2001; 10.1152/ajprenal.00124.2001
Received 18 April 2001; accepted in final form 2 August 2001.
 |
REFERENCES |
1.
Burg, M,
and
Green N.
Bicarbonate transport by isolated perfused rabbit proximal tubules.
Am J Physiol Renal Fluid Electrolyte Physiol
233:
F307-F314,
1977[Abstract/Free Full Text].
2.
Chomczynski, P,
and
Sacchi N.
Single-step method of RNA isolation by acid guanidinium thiocynate-phenol-chloroform extraction.
Anal Biochem
162:
156-159,
1987[ISI][Medline].
3.
Church, GM,
and
Gilbert W.
Genomic sequencing.
Proc Natl Acad Sci USA
81:
1991-1995,
1984[Abstract].
4.
Codina, J,
Cardwell J,
Gitomer JJ,
Cui Y,
Kone BC,
and
DuBose TD, Jr.
Sch-28080 depletes intracellular ATP selectively in mIMCD-3 cells.
Am J Physiol Cell Physiol
279:
C1319-C1326,
2000[Abstract/Free Full Text].
5.
Cougnon, M,
Bouyer P,
and
Jaisser F.
Does the colonic H,K-ATPase also act as an Na,K-ATPase?
Proc Natl Acad Sci USA
26:
6516-6520,
1998.
6.
Crowson, MS,
and
Shull GE.
Isolation and characterization of a cDNA encoding the putative distal colon H+,K+-ATPase: similarity of deduced amino acid sequence to gastric H+,K+-ATPase and Na+,K+-ATPase and messenger RNA expression in distal colon, kidney and uterus.
J Biol Chem
267:
13740-13748,
1992[Abstract/Free Full Text].
7.
Jaisser, F,
and
Beggah A.
The nongastric H+-K+-ATPases: molecular and functional properties.
Am J Physiol Renal Physiol
276:
F812-F824,
1999[Abstract/Free Full Text].
8.
Laroche-Joubert, N,
Marsy S,
and
Doucet A.
Cellular origin and hormonal regulation of K+-ATPase activities sensitive to Sch-28080 in rat collecting duct.
Am J Physiol Renal Physiol
279:
F1053-F1059,
2000[Abstract/Free Full Text].
9.
Lee, J,
Rajendran VM,
Mann AS,
Kashgarian M,
and
Binder HJ.
Functional expression and segmental localization of rat colonic K-ATPase.
J Clin Invest
96:
2002-2008,
1995[ISI][Medline].
10.
Muth, TR,
Dunbar LA,
Cortois-Coutry N,
Roush DL,
and
Caplan MJ.
Sorting and trafficking of ion transport proteins in polarized epithelial cells.
Curr Opin Nephrol Hypertens
6:
455-459,
1997[ISI][Medline].
11.
Nakamura, S,
Amlal H,
Galla JH,
and
Soleimani M.
Colonic H+-K+-ATPase mediates NH
secretion in inner medullary collecting duct in potassium depletion.
Kidney Int
56:
2160-2167,
1999[ISI][Medline].
12.
Nakamura, S,
Amlal H,
Schultheis PJ,
Galla JH,
Shull GE,
and
Soleimani M.
HCO
reabsorption in renal collecting duct of NHE-3-deficient mouse: a compensatory response.
Am J Physiol Renal Physiol
276:
F914-F921,
1999[Abstract/Free Full Text].
13.
Nakamura, S,
Amlal H,
Soleimani M,
and
Galla JH.
Pathways for HCO
reabsorption in mouse medullary collecting duct segments.
J Lab Clin Med
136:
218-223,
2000[ISI][Medline].
14.
Petrovic, S,
and
Schwartz GJ.
In vitro acidosis reduces apical Cl/HCO3 exchanger in B-intercalated cells of rabbit cortical collecting duct (Abstract).
J Am Soc Nephrol
10:
11A,
1999.
15.
Rajendran, VM,
Singh SK,
Geibel J,
and
Binder HJ.
Differential localization of colonic H+-K+-ATPase isoforms in surface and crypt cells.
Am J Physiol Gastrointest Liver Physiol
274:
G424-G429,
1998[Abstract/Free Full Text].
16.
Sabolic, I,
Brown D,
Verbatatz JM,
and
Kleinman J.
H+-ATPase of renal cortical and medullary endosomes are differentially sensitive to Sch-28080 and omeprazole.
Am J Physiol Renal Fluid Electrolyte Physiol
266:
F868-F877,
1994[Abstract/Free Full Text].
17.
Sangan, P,
Rajendran VM,
Mann AS,
Kashgarian M,
and
Binder HJ.
Regulation of colonic H+-K+-ATPase in large intestine and kidney by dietary Na depletion and dietary K depletion.
Am J Physiol Cell Physiol
272:
C685-C696,
1997[Abstract/Free Full Text].
18.
Schuster, VL.
Function and regulation of collecting duct intercalated cells.
Annu Rev Physiol
55:
267-288,
1993[ISI][Medline].
19.
Schwartz, GJ,
and
Al-Awqati Q.
Carbon dioxide causes exocytosis of vesicles containing H+ pumps in isolated perfused proximal and collecting tubules.
J Clin Invest
75:
1638-1644,
1985[ISI][Medline].
20.
Schwartz, GJ,
Satlin LM,
and
Bergman JE.
Fluorescent characterization of collecting duct cells: a second H+-secreting type.
Am J Physiol Renal Fluid Electrolyte Physiol
255:
F1003-F1014,
1988[Abstract/Free Full Text].
21.
Shull, GE,
and
Lingrel J.
Molecular cloning of the rat stomach (H+-K+)-ATPase.
J Biol Chem
261:
16788-16791,
1986[Abstract/Free Full Text].
22.
Silver, RB,
and
Frindt G.
Functional identification of H-K-ATPase in intercalated cells of cortical collecting tubule.
Am J Physiol Renal Fluid Electrolyte Physiol
264:
F259-F266,
1993[Abstract/Free Full Text].
23.
Silver, RB,
Mennitt PA,
and
Satlin LM.
Stimulation of apical H-K-ATPase in intercalated cells of cortical collecting duct with chronic metabolic acidosis.
Am J Physiol Renal Fluid Electrolyte Physiol
270:
F539-F547,
1996[Abstract/Free Full Text].
24.
Silver, RB,
and
Soleimani M.
H+-K+-ATPases: regulation and role in pathophysiological states.
Am J Physiol Renal Physiol
276:
F799-F811,
1999[Abstract/Free Full Text].
25.
Spicer, Z,
Miller ML,
Andringa A,
Riddle TM,
Duffy JJ,
Doetschman T,
and
Shull GE.
Stomachs of mice lacking the gastric H,K-ATPase
-subunit have achlorhydria, abnormal parietal cells, and ciliated metaplasia.
J Biol Chem
275:
21555-21565,
2000[Abstract/Free Full Text].
26.
Thomas, JA,
Buchsbaum RN,
Zimniak A,
and
Racker E.
Intracellular pH measurements in Erlich ascites tumor cells utilizing spectroscopic probes generated in situ.
Biochemistry
18:
2210-2218,
1979[ISI][Medline].
27.
Tsuruoka, S,
and
Schwartz GJ.
Adaptation of rabbit cortical collecting duct HCO3 transport to metabolic acidosis in vitro.
J Clin Invest
97:
1076-1092,
1996[Abstract/Free Full Text].
28.
Tsuruoka, S,
Swenson ER,
Petrovic S,
Fujimura A,
and
Schwartz GJ.
Role of basolateral carbonic anhydrase in proximal tubular fluid and bicarbonate absorption.
Am J Physiol Renal Physiol
280:
F146-F154,
2001[Abstract/Free Full Text].
29.
Weiner, DI,
and
Hamm LL.
Use of fluorescent dye BCECF to measure intracellular pH in cortical collecting tubule.
Am J Physiol Renal Fluid Electrolyte Physiol
256:
F957-F964,
1989[Abstract/Free Full Text].
30.
Weiner, DI,
and
Hamm LL.
Regulation of Cl/HCO3 exchange in the rabbit cortical collecting tubule.
J Clin Invest
85:
1553-1558,
1991.
31.
Weiner, DI,
and
Milton AE.
H+-K+-ATPase in rabbit cortical collecting duct B-type intercalated cell.
Am J Physiol Renal Fluid Electrolyte Physiol
270:
F518-F530,
1996[Abstract/Free Full Text].
32.
Wingo, CS,
and
Smolka AJ.
Function and structure of H-K-ATPase in the kidney.
Am J Physiol Renal Fluid Electrolyte Physiol
269:
F1-F16,
1995[Abstract/Free Full Text].
Am J Physiol Renal Fluid Electrolyte Physiol 282(1):F133-F143
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