1 Institute of Physiology, University of Innsbruck, A-6010 Innsbruck, Austria; and 2 Department of Surgery and Cellular and Molecular Physiology, Yale University, Medical School, New Haven, Connecticut 06510-8026
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ABSTRACT |
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The aim of the
present study was to obtain detailed information on MDCK
cell proton secretion characteristics under various growth conditions.
Confluent monolayers cultured on glass coverslips were adapted over 48 h to media with different osmolality and pH (200 mosmol/kgH2O, pH 7.4;
300 mosmol/kgH2O, pH 7.4; and 600 mosmol/kgH2O, pH 6.8)
corresponding to the luminal fluid composition of the collecting duct
segments found in the in renal cortex, the outer stripe of outer
medulla and inner medulla. Proton fluxes were determined from the
recovery of intracellular pH following an acid load induced by an
NH4Cl pulse times the
corresponding intrinsic buffering power
(i). The intracellular
buffering power was found to change only with culture
medium osmolality but not with culture medium pH. In addition to an
amiloride and Hoe-694-sensitive Na+/H+
exchange, Madin-Darby canine kidney (MDCK) cells possess a
Sch-28080-sensitive, K+-dependent
H+ extrusion mechanism that is
increased upon adaptation of monolayers to hyperosmotic-acidic culture
conditions. A significant contribution of the bafilomycin
A1-sensitive vacuolar
H+-ATPase could be found only in
cells adapted to hyposmotic culture conditions. Exposure of MDCK cells
to 10
5 or
10
7 M aldosterone for
either 1 or 18 h did not alter the
H+ extrusion characteristics
significantly. The results obtained show that different extracellular
osmolality and pH induce different MDCK phenotypes with respect to
their H+-secreting systems.
proton-adenosinetriphosphatase; potassium-proton-adenosinetriphosphatase; Sch-28080; bafilomycin A1; intracellular pH; buffering capacity
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INTRODUCTION |
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MADIN-DARBY CANINE KIDNEY (MDCK) cells, a widely used
renal epithelial cell line, were originally isolated from an unknown site along the dog nephron. They form epithelial monolayers and, when
grown on impermeable supports, develop domes resulting from unidirectional transport of solutes and water. These phenomena have led
to the postulation that the cells originate from distal nephron cells
(7). However, morphological as well as functional data exist [the
expression of a carbonic anhydrase activity (19) and the ability to
either secrete bicarbonate or protons (17)] which indicate that
MDCK cells could be of collecting duct origin (27, 19). This idea is
supported by the distribution of binding pattern (8) of peanut
agglutinin (PNA) and wheat germ agglutinin, described as specific
markers for intercalated (IC) and principal cells (PC) of the rabbit
collecting duct, respectively (11, 13). When cultivated using standard
culture conditions (300 mosmol/kgH2O and pH
7.4), PNA binding predominates in MDCK monolayers and is especially
pronounced in dome-forming cells, which have been demonstrated to
secrete bicarbonate (17). Morphologically these cells appeared as
"dark," mitochondria-enriched cells (20). In subsequent
investigations involving hyperosmotic-acidic growth conditions,
mimicking the environment of the outer medullary collecting duct, PNA
binding is abolished (8), but the number of electron microscopically
"dark," mitochondria-enriched cells increases (20). From these
observations, it was hypothesized that the dark, PNA-positive cells may
resemble -IC cells, and the dark, PNA-negative MDCK cells may be
related to H+-secreting
-IC-like cells. Surface pH measurements on these two respective dark
cell types using pH microelectrodes appear to support this hypothesis
(20). MDCK cells also exhibit an amiloride-sensitive Na+/H+
and a 4,4'-diisothiocyanostilbene-2,2'-disulfonic
acid-sensitive Cl
/
exchange, particularly in dome-forming cells (17). Although the
Na+/H+
exchange (NHE) plays an important role in MDCK cell
H+ secretion (17, 28), additional
evidence exists for
Na+-independent
H+ extrusion mechanisms (17, 18).
Therefore, an active, rheogenic V-type
H+-adenosinetriphosphatase
(H+-ATPase) and/or an
electrically silent,
K+-dependent P-type
ATPase previously found in the collecting duct (1, 10) may be
potentially involved in MDCK proton extrusion.
The present study was designed to discriminate between the various
mechanisms of acid secretion in MDCK monolayers and to functionally
prove the existence of a
K+-H+-ATPase
and/or a rheogenic
H+-ATPase and to further
dissect the contribution of these systems to MDCK proton extrusion. For
this purpose cells were adapted to hyposmotic and isosmotic growth
conditions at pH 7.4, indicative of the primarily
-secreting cortical collecting duct (CCD) regions, or to hyperosmotic and acidic growth conditions typical of the collecting duct transition segment from the outer to
inner medulla.
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MATERIALS AND METHODS |
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Cell culture. Wild-type MDCK cells
(American Type Culture Collection, CCL-34) from passage 66-95 were
used for all experiments. Cultures were grown on glass coverslips in
Dulbecco's modified Eagle's medium (DMEM) supplemented with 10%
fetal calf serum (FCS), 100 U/ml penicillin and 100 µg/ml
streptomycin. After reaching confluence, cultures were adapted for a
further 48 h to either isosmotic (300 mosmol/kgH2O,
pH 7.4), hyperosmotic-acidic (600 mosmol/kgH2O, pH 6.8), or
hyposmotic (200 mosmol/kgH2O, pH
7.4) culture conditions. In an additional series, MDCK cells were
exposed to either hyperosmotic culture conditions (600 mosmol/kgH2O) at pH 7.4 or acidic
culture conditions (300 mosmol/kgH2O) at pH 6.8. Bicarbonate concentration and resulting pH of the culture media were
calculated for a constant, humidified incubator atmosphere at 5%
CO2
(PCO2 of 35 mmHg; total CO2 of 1.05 mM). Adjustment of medium pH was achieved by addition or
omission of NaCl and NaHCO3,
making up 44 mM in total, corresponding to the concentration of the
original formulation of DMEM (for pH 7.4, 21 mM
NaHCO3 and 23 mM NaCl; for pH 6.8, 5 mM NaHCO3 and 39 mM NaCl).
Osmolality was corrected by addition or omission of NaCl. In a second
series of experiments, cells were exposed to either FCS-free medium
supplemented with 105 or
10
7 M aldosterone for 1 h
or 18 h, respectively, or only to FCS-free medium as a control.
Fluorescence measurements of intracellular
pH.
All experiments were performed in the absence of bicarbonate to avoid
the participation of the
/Cl
exchanger or other potentially bicarbonate-dependent processes. All
intracellular pH (pHi)
measurements were carried out in isosmotic solutions. Glass
coverslip-grown cells were loaded with 3 µM of the pH-sensitive dye
2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein acetoxymethyl ester (BCECF-AM; Molecular Probes) for 15 min. Coverslips were washed twice in control buffer (solution
1) and mounted into a quartz cuvette at an angle of
60°. Ratiometric BCECF fluorescence (excitation 505/439 nm,
emission 535 nm) measurements were continuously taken with a
spectrofluorometer (F-4500, Hitachi) equipped with a thermostatically
controlled cuvette holder (37°C) over a period of 20 min.
H+ extrusion was obtained by
determining the pHi recovery rate
using the NH4Cl prepulse technique
(solution 2, for 2 min) (2). The pHi recovery was then monitored in
different solutions (Table 1) over a 12-min
period. To discriminate between
Na+- and/or
K+-dependent and
Na+- and
K+-independent proton
extrusion mechanisms, pHi changes
were measured under standard conditions (solution
1) or under
Na+-free
(solution 3) or
K+-free conditions
(solution 4), respectively. To
further delimit the contribution of a putative
K+-H+-ATPase
and/or H+-ATPase,
measurements were carried out in the presence of Sch-28080 (1-3 × 10
4 M, Schering), a
known specific, competitive inhibitor of the gastric
K+-H+-ATPase
(29), and bafilomycin A1 (1 × 10
6 M, Sigma), an
inhibitor of the V-type H+-ATPase
(3). Hoe-694 (1 × 10
4 M, Hoechst) (21) and
amiloride (1 × 10
4 to
2 × 10
3, Sigma), both
inhibitors of
Na+/H+
exchangers, were used to inhibit the NHEs. To block
Na+-K+-ATPase
and to exclude potentially involved, ouabain-sensitive isoforms of the
K+-H+-ATPase,
all solutions were prepared with 1 × 10
3 M ouabain (Sigma). At
the end of each measurement, BCECF calibration curves were obtained
using the nigericin and high-K+
method (26). The calibration solutions were composed of 140 mM KCl, 10 mM NaCl, and 20 mM
N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES), and pH was adjusted to 7.8 and 6.8, respectively. Nigericin (Sigma) was prepared as a stock solution and added just before use in a final concentration of
10
5 M. Following withdrawal
of NH4Cl, the
pHi recovery rates
(
pHi/
t, pH units/s) were measured once from the nadir
pHi and from the steepest slope,
if a further increase in
pHi/
t
could be observed during pHi
recovery.
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Determination of buffering capacity and
H+
fluxes.
The NH4Cl prepulse technique was
also used to determine the intrinsic buffering power
(i)
(mM/pHiU) for all adaptation
protocols listed above (2). The cells were exposed to a
Na+-free bath
(solution 3) containing Sch-28080,
bafilomycin A1 and including 20 mM
NH4Cl, which was then stepwise
reduced to 0 mM (10, 5, 2.5, 1, 0.5 mM). Calculation of
i was performed according to
the formula
i =
/
pHi, where intracellular NH+4 concentration
(
) was
calculated from the Henderson-Hasselbalch equation on the assumption that
[NH3]i = [NH3]o.
H+ fluxes
(JH+,
in
mmol · min
1 · l
1)
were determined as the product of recovery rate and the
respective buffer capacity as
JH+ =
pHi/
t ×
i.
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RESULTS |
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Buffer capacity dependence on osmolalities and
pH. As shown in Fig. 1 a
decrease of pHi induced by a
stepwise reduction of extracellular
NH4Cl increases
i for all culture conditions as expected. The slope of this
i
increment depends on the osmolality but not on adaptation to a lowered
pH in the culture media. Hyposmotic culture conditions induce an
increase of
i at low
pHi, whereas hyperosmotic culture
media induce a decrease of
i,
when pHi determinations were
performed in isosmotic measurement solutions. The
i values were utilized to
determine the proton fluxes
(JH+) as described above.
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The recovery rates proceeding from the nadir
pHi after removal of
NH4Cl in
Na+-free measuring
solution 3 as well as in solutions
containing the various blockers differed from maximal recovery rates.
The respective H+ fluxes, however,
are nearly identical due to the increment in pHi over the recovery period,
which is paralleled by a decrease of
i.
Adaptation to different osmolality and pH. Adapting confluent monolayers of MDCK cells to hyperosmotic-acidic (600 mosmol/kgH2O, pH 6.8), isosmotic (300 mosmol/kgH2O, pH 7.4), and hyposmotic (200 mosmol/kgH2O, pH 7.4) media for 48 h leads to different intracellular resting pHi values. After reexposure of the cells to the isosmotic extracellular solutions at a pH of 7.4 for fluorescence measurements (solution 1), MDCK cells grown under isosmotic conditions display a pHi of 7.48 ± 0.01 (±SE; n = 43 coverslips) (23, 28), whereas monolayers adapted to hyperosmotic-acidic conditions show a pHi of 7.37 ± 0.015 (n = 43) and cells adapted to hyposmotic conditions display a resting pHi of 7.54 ± 0.02 (n = 43). Furthermore, these differences in pHi values are maintained following removal of NH4Cl. It should be noted that the cells adapted to hyperosmotic-acidic culture conditions approach a minimal pHi of 6.56 ± 0.02 SE after NH4Cl removal. The response to acidification of cells kept under isosmotic conditions result in a slightly higher pHi (6.65 ± 0.022). In hyposmotically adapted cells, a pHi of only 6.83 ± 0.026 is achieved by NH4Cl prepulsing (Fig. 2).
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Complete recovery after NH4Cl prepulse to the initial resting pHi is achieved in solution 1 within 5 min in all types of adaptation. Return to initial pHi values is accelerated in hyperosmotically cultured cells compared with isosmotically and hyposmotically grown cells (Fig. 2).
JH+
in response to different extracellular
Na+ and
K+
concentrations.
Omission of K+ from the measuring
solution (solution 4) results in
consistent reduction of proton fluxes. Removal of extracellular Na+ (solution
3) results in a significant
(P 0.05) reduction of proton
extrusion rates to less than 2 mmol/min for all adaptation protocols
examined (Fig. 3).
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JH+
in the presence of inhibitors.
The addition of ouabain does not influence
H+ fluxes (data not shown). To
discriminate between the contribution of
Na+/H+
exchangers,
K+-H+-ATPase,
and a rheogenic
H+-ATPase, different
inhibitors were applied during the
pHi recovery period. In
experiments where the H+ extrusion
driven by the NHEs is eliminated (solution
3, Na+ free), a
clearly recognizable inhibition of
H+ extrusion by Sch-28080 (1 × 104 M) can be
detected for all adaptation protocols investigated (Fig.
4,
left). However, for hyperosmotically
grown cells, a significant reduction can be achieved only in the
presence of higher Sch-28080 concentrations (3 × 10
4 M) (Fig. 4,
left). Sch-28080 also reduces
H+ fluxes significantly when
applied under conditions where NHE-activity is normal
(solution 1, including
Na+) (Fig. 4,
right). The competitive mode of
action is clearly shown by the return of proton extrusion upon removal
of Sch-28080 from the measuring solution (Fig.
5).
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DISCUSSION |
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In the collecting duct of the mammalian kidney, a potential source of the MDCK cell line, at least two morphologically discernible cell types, namely, PC cells and IC cells, which differ in structure and function (11, 15), have been identified. Three different proton extrusion mechanisms have been described (for review see Refs. 22 and 25) for the IC cells, whose primary function is urinary acidification; Na+/H+ exchanger(s) (14, 16), a bafilomycin A1-sensitive H+ pump, as well as a Sch-28080-sensitive K+-H+-ATPase (1, 10, 24, 30) have all been functionally identified in this cell type. The present study reports functional evidence for the presence of all of these proton extrusion mechanisms in MDCK monolayers. Furthermore, the data presented suggest that the activities of these H+ secretion mechanisms differ depending on the composition of the extracellular culture media to which the MDCK cells were exposed.
We chose hypo-, iso-, and hyperosmotic-acidic adaptations in an effort
to create culture conditions mimicking the environments of the
following tubules: the superficial renal distal convoluted and CCD
segment, the outer stripe of outer medulla collecting duct segment, as
well as that of the collecting duct segments contained in the
outer-inner medullary transition region. Previous experiments (20) have
shown that these maneuvers induce changes in MDCK phenotype and thus
should result in differences of H+
secretion. Hyposmotically adapted cells were expected to differentiate toward cells with functional characteristics similar to -IC cells in
the CCD, whereas hyperosmotic-acidic MDCK cultures should result in a
phenotype similar to the acid-secreting
-IC-like cells (15). In
addition, isosmotic-acidic and hyperosmotic-pH 7.4 adaptations were
investigated to differentiate between changes in
H+ secretion observed following
exposure of cells to hyperosmotic-acidic culture conditions. With these
modifications in the extracellular growth solution, we felt confident
that the question of whether acidification and/or changes of
medium osmolality are responsible for the alterations of
H+ extrusion could be answered.
Since net H+ extrusion can be
related to pHi recovery only if
intracellular buffering power stays constant, we have experimentally assessed changes in i under the
various culture conditions used in the present study. The results
obtained (Fig. 1) imply that the differences found must be attributed
primarily to changes in extracellular medium osmolality.
From the results depicted in Fig. 3, it is evident that NHEs represent the major mechanism for MDCK cell H+ secretion. The contribution of the other acid extruders, the vacuolar H+-ATPase and the K+-H+-ATPase depends predominantly on the form of adaptation imposed on the confluent MDCK monolayers.
Omission of Na+ from the measuring solution was chosen to maximally suppress all Na+-driven H+ extrusion mechanisms. From the data shown in Fig. 6 it could be suggested that, in addition to the amiloride and Hoe-964-sensitive "housekeeping" NHE-1 (17, 23, 27), MDCK cells may also contain other Na+/H+ exchanger isoforms. This was supported by the less-pronounced effect of Hoe-694 and/or amiloride on the pHi recovery compared with that under Na+-free measurement conditions. Further support for participation of other NHEs is the finding that these differences were only found following hyperosmotic-acidic and isosmotic pretreatment (Fig. 4, middle). In contrast to other adaptations, the hyposmotic pretreatment may reduce the activity of NHE isoforms to an extent, that the inhibitor concentrations used were sufficient to inhibit all NHEs. Alternatively, hyposmotic pretreatment may suppress the expression of NHE-3.
Participation of a bafilomycin
A1-sensitive vacuolar
H+-ATPase could unambiguously be
recovered only for MDCK monolayers adapted to hyposmotic growth
conditions comparable to the apical cellular environment given in the
outermost CCD portions (Fig. 4,
left). It could be assumed that an
active H+ secretion driven by a
V-type H+-ATPase in the model
presented is confined only to cells with functional characteristics
similar to -IC-like cells, which have an increased frequency in the
monolayer population following this type of adaptation (20). Indeed, a
V-type H+-ATPase localization in
-IC cells at the basolateral membrane domain of CCDs has been
previously described (6, 12). However, attempts to localize this enzyme
immunocytochemically in MDCK cells using antibodies against the 70- and
56-kDa subunits of bovine V-type
H+-ATPase have failed (20),
probably due to poor cross-reactivity between the bovine antibodies and
MDCK cell protein.
If one assumes that MDCK cells resemble IC of the collecting duct
cells, then these should potentially contain a
K+-H+-ATPase
as another H+ extruder in addition
to NHEs and vacuolar H+-ATPase as
reported for the IC cells of the mammalian collecting duct (1, 10, 30).
The first evidence for the possible existence of such a
K+-H+
pump was previously reported, showing an omeprazole-induced inhibition of net H+ current in dome-forming
cells of MDCK monolayers (18). According to observations on the
mammalian outer/inner medullary collecting duct,
K+-dependent mechanisms
significantly contribute to H+
secretion (10, 30). The maneuver of hyperosmotic-acidic adaptation, outlined above, should thus result in MDCK phenotypes with a clearly discernible K+-dependent
H+ extrusion. Figure 3 shows that
removal of K+ induced a reduction
of H+ extrusion in MDCK cell acid
secretion. Since, after blockade of all NHEs using a
Na+-free extracellular measuring
solution, a considerable H+
secretion capacity remains, the data suggest that other extrusion mechanisms such as the
K+-H+-ATPase
and/or
H+-ATPase are
activated or unmasked. That hyperosmotic-acidic cells indeed possess a
higher
K+-H+-ATPase
activity can be concluded from the fact that Sch-28080 concenrations of 3 × 104 M were needed to
maximally reduce
JH+. Following iso- and hyposmotic exposure, maximum reduction was already achieved with 1 × 10
4 M Sch-28080 (Fig. 4,
left). Such a
Sch-28080-sensitive JH+ flux upon an acid load in the absence of extracellular
Na+ strongly corroborates the view
of MDCK cells being related to cells provided with IC cells cell
properties. Support for this interpretation is given by the recent
observation of Silver and Frindt (24), who reported a
Sch-28080-sensitive, Na+
independent recovery of pHi after
an acid load only for collecting duct IC cells and not for PCs.
The investigations performed show that MDCK monolayers adapted to hyperosmotic-acidic (pH 6.8) culture media display the steepest slope of pHi recovery followed by cells adapted to isosmotic and hyposmotic culture conditions (Fig. 2). This behavior is also given for H+ fluxes driven by the amiloride and/or Hoe-694-sensitive Na+/H+ exchange and the Sch-28080-sensitive H+ extrusion.
For the hyposmotically adapted cells, Na+-independent H+ secretion appears largely to be the result of a bafilomycin A1-sensitive V-type H+-ATPase, which obviously does not contribute to acid secretion to the same extent in the other two experimental protocols investigated (Fig. 3 and Fig. 4, left and middle).
The counteracting effects of increasing medium osmolality and decreasing pH on H+ extrusion (Fig. 7) cannot yet be explained conclusively. Potentially, part of the further decrease of culture medium pH may be caused by the cells themselves during the adaptation period to an acid media pH resulting in values below 6.8 (the initial value of the culture medium).
Previous investigations on MDCK cells have shown a stimulation of the
NHEs upon exposure to aldosterone (17). In the present investigation no
prominent effect subsequent to aldosterone administration could be
observed, regardless of whether or not aldosterone was applied over
different periods of time (1 h or 18 h) or at different concentrations
(105M or
10
7 M). This would agree
with a recent report claiming the absence of aldosterone receptors in
IC cells (4, 5, 9) and thus support the view of MDCK cells resembling
IC cell equivalents or alternatively, that MDCK cells do not possess
the required cellular machinery necessary to process aldosterone.
Furthermore, it cannot be excluded that in contrast to earlier
investigations (18) MDCK cells used in this study did not respond to
aldosterone due to passage number and/or altered state of
differentiation.
From the results obtained in this study, it can be concluded that the major component of H+ secretion in MDCK monolayer cultures is mediated by NHEs. A K+-dependent Sch-28080-sensitive H+ extrusion contributes to acid secretion in MDCK cells grown under all culture conditions with a clear enhancement after adaptation to hyperosmotic-acidic media. The contribution of a bafilomycin A1-sensitive V-type H+-ATPase, considered to be the major extrusion system in the mammalian collecting duct, seems to play a significant role for acid secretion in MDCK cultures only when they are adapted to hyposmotic culture conditions.
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ACKNOWLEDGEMENTS |
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We thank Drs. P. Dietl, F. Lang, and N. P. Curthoys for critical comments.
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FOOTNOTES |
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This work was supported by the Austrian Science Foundation Grants P-7968-MED and P-9259-MED.
Address for reprint requests: W. Pfaller, Fritz-Preglstr. 3, Institute of Physiology, Univ. of Innsbruck, A-6010 Innsbruck, Austria.
Received 16 January 1996; accepted in final form 18 June 1997.
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