Functional upregulation of
H+-ATPase by lethal acid
stress in cultured inner medullary collecting duct cells
Hassane
Amlal,
Zhaohui
Wang, and
Manoocher
Soleimani
Department of Medicine, University of Cincinnati School of
Medicine, and Veterans Affairs Medical Center, Cincinnati, Ohio
45267-0585
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ABSTRACT |
The response of
H+-ATPase to lethal acid stress is
unknown. A mutant strain (called NHE2d) was derived from cultured inner medullary collecting duct cells (mIMCD-3 cells) following three cycles
of lethal acid stress. Cells were grown to confluence on coverslips,
loaded with
2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein, and
monitored for intracellular pH
(pHi) recovery from an acid load. The rate of Na+-independent
pHi recovery from an acid load in
mutant cells was approximately fourfold higher than in parent cells
(P < 0.001). The
Na+-independent
H+ extrusion was ATP dependent
and K+ independent and was
completely inhibited in the presence of diethylstilbestrol, N, N'-dicyclohexylcarbodiimide,
or N-ethylmaleimide. These
results indicate that the
Na+-independent
H+ extrusion in cultured medullary
cells is mediated via H+-ATPase
and is upregulated in lethal acidosis. Northern hybridization experiments demonstrated that mRNA levels for the 16- and 31-kDa subunits of H+-ATPase remained
unchanged in mutant cells compared with parent cells. We propose that
lethal acid stress results in increased H+-ATPase activity in inner
medullary collecting duct cells. Upregulation of
H+-ATPase could play a protective
role against cell death in severe intracellular acidosis.
acid-base; intracellular pH regulation; proton-adenosinetriphosphatase; sodium-proton exchanger; acidosis
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INTRODUCTION |
MAMMALIAN CELLS RESPOND TO increased intracellular
acidosis by increasing H+
extrusion (31). Exposing renal cells to severe acid stress increases
the expression and activity of the ubiquitous
Na+/H+
exchanger (NHE) isoform NHE1 (30, 31). Cells overexpressing NHE1 show
increased survival in acidic media (19, 22, 23), consistent with a
vital role for this exchanger in regulating cell pH and growth. The
role of H+-ATPase in severe
(lethal) acid stress, however, remains unknown. Cultured inner
medullary collecting duct cells (mIMCD-3 cells) express isoforms NHE1
and NHE2 (32) and an ATP-dependent
H+ pump. The effect of lethal acid
stress on
Na+/H+
exchanger activity and isoform expression in mIMCD-3 cells was studied
(30). After three cycles of lethal acid stress, a mutant cell line
(called NHE2d) was isolated that demonstrated significant overexpression of NHE1 mRNA and activity (30).
The inner medullary collecting duct is a major site for renal acid
secretion (2, 3, 10, 13, 35) and shows appropriate adaptive regulation
in systemic acid-base perturbations (2, 3, 13). Whereas most studies
demonstrate that H+ secretion in
inner medullary collecting duct lumen is mediated via an electrogenic
H+-ATPase (2, 16, 27, 29, 35),
other studies suggest a role for the electroneutral
H+-K+-ATPase
(17, 21). The purpose of the current experiments was 1) to examine the mechanism of
Na+-independent
H+ secretion in mouse cultured
inner medullary collecting duct cells (mIMCD-3) and
2) to study the effect of lethal
acid stress on Na+-independent
H+ secretion in these cells. Our
results demonstrate that mIMCD-3 cells possess an ATP-dependent,
K+-independent
H+ secretion that is inhibited by
diethylstilbestrol (DES),
N, N'-dicyclohexylcarbodiimide (DCCD), N-ethylmaleimide (NEM), and
high concentrations of Schering 28080 (Sch-28080), consistent with
H+-ATPase. The results further
indicate that lethal acid stress increases
H+-ATPase activity via a
posttranscriptional process.
H+-ATPase may play an important
role in cell survival during severe acidosis.
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MATERIALS AND METHODS |
Cell Culture Procedures
Cultured mIMCD-3 cells, derived from simian virus transgenic mice, were
cultured in a 1:1 mixture of Ham's F-12 and Dulbecco's modified
Eagle's medium (DMEM-F12) containing 2.5 mM
L-glutamine and 2.438 g/l sodium
bicarbonate (GIBCO BRL) supplemented with 50 U/ml penicillin G, 50 µg/ml streptomycin, and 10% fetal bovine serum. Cultured mIMCD-3 and
NHE2d cells were incubated at 37°C in a humidified atmosphere of
5% CO2 in air. The medium was
replaced every other day.
Selection of Mutants by Lethal Acid Stress
Mutant NHE2d cells were obtained as described (30). Briefly, actively
proliferative, subconfluent mIMCD-3 cells were treated with
ethylmethylsulfonic acid and then subjected to a modified protocol of
lethal acid stress (22) as described below. Briefly, mIMCD-3 cells were
grown to confluence, centrifuged at room temperature, and resuspended
for 10 min at 37°C in an ammonium-containing solution that
consisted of (in mM) 20 NH4Cl, 120 tetramethylammonium chloride (TMA-Cl), 5 glucose, 1 CaCl2, 1 MgCl2, 2.5 K2HPO4,
and 5 N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES)-tris(hydroxymethyl)aminomethane (Tris), pH 7.40. This
procedure results in ammonium loading of the cells. The cells were then
pelleted and incubated for 30 min in a solution that consisted of (in
mM) 130 TMA-Cl, 5 KCl, 1 MgSO4, 2 CaCl2, 5 glucose, and 20 HEPES · Tris (pH 5.5). This step results in acid
loading secondary to passive diffusion of
NH3 from the cells. Thereafter, the cells were pelleted, washed, and incubated for 120 min at 37°C
in a solution that consisted of (in mM) 125 choline chloride, 5 NaCl, 5 KCl, 1 MgSO4, 1 CaCl2, and 20 2-(N-morpholino)ethanesulfonic acid at
pH 6.0. The cells were then centrifuged, recovered, and seeded to
culture-grade plastic dishes in DMEM-F12 medium (pH 7.40) for 10 days.
The cells were trypsinized and subjected while in suspension to two
more rounds of lethal acid stress. The cells were subcultured and
passaged at very high dilutions (1:1,000) to isolate individual
colonies. A number of individual colonies were isolated with cloning
cylinder and then collected and subcultured. One strain (NHE2d)
was studied in detail for
Na+/H+
exchanger activity and isoform expression (30).
Intracellular pH Measurement
Changes in intracellular pH
(pHi) were monitored with the
use of the acetoxymethyl ester of the pH-sensitive fluorescent dye 2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein (BCECF) as
described (30, 32). mIMCD-3 and NHE2d cells were grown to confluence on
glass coverslips and incubated in the presence of 5 µM BCECF in a
solution consisting of (in mM) 140 NaCl, 0.8 K2HPO4,
0.2 KH2PO4,
1 CaCl2, 1 MgCl2, 10 HEPES, and 5 glucose
(solution B, Table
1). To measure
pHi, each coverslip was positioned
diagonally in a cuvette and the latter was then placed in a
thermostatically controlled holding chamber (37°C) in a Delta Scan
dual excitation spectrofluorometer (double-beam fluorometer, Photon
Technology International, South Brunswick, NJ). The monolayer was then
perfused with the appropriate solution (Table 1). The perfusion was
achieved with a Harvard constant infusion pump. Where indicated,
inhibitors were added to the experimental solution in a 1:1,000
dilution from a stock solution. The fluorescence ratio at excitation
wavelengths of 500 and 450 nm
(F500/F450)
was utilized to determine pHi
values in the experimental groups by comparison with the calibration curve. The emission wavelength was recorded at 525 nm. Calibration curves were established daily by incubating the BCECF-loaded cells with
3.3 µM nigericin in a medium containing (in mM) 120 KCl, 1 CaCl2, 1 MgCl2, 0.8 K2HPO4,
0.2 KH2PO4,
and 10 HEPES and adjusted at various pH values with Tris-buffered
solution.
F500/F450
was found to be linearly related to
pHi over the pH range of
7.40-6.30 (y = 2.1x
12.5;
r = 0.997). The initial rate of
pHi recovery (dpHi/dt,
pH/min) from an acid load following
NH3/NH+4 withdrawal was calculated by fitting to a linear equation. Correlation coefficients for these linear fits averaged 0.986 ± 0.003.
Isolation of Total RNA
Total cellular RNA was extracted from confluent cultured cells in
multiple 100-mm dishes by the method of Chomczynski and Sacchi (6). In
brief, cells were homogenized at room temperature in 3 ml of Tri
reagent (Molecular Research Center, Cincinnati, OH). RNA
was extracted by phenol-chloroform and precipitated by isopropanol (6).
RNA was quantitated by spectrophotometry and stored at
80°C.
Northern Hybridization
Total RNA samples (30 µg/lane) were fractionated on a
1.2% agarose-formaldehyde gel and transferred to Magna NT nylon
membranes (MSI), using 10× sodium chloride-sodium phosphate-EDTA
(SSPE) as a transfer buffer. Membranes were cross-linked by ultraviolet light and baked for 1 h (33). Hybridization was performed according to
the method of Church and Gilbert (7). Briefly, membranes were placed
for 1 h in 0.1× SSPE-1% sodium dodecyl sulfate (SDS) solution at
65°C. The membranes were then prehybridized for 1-3 h at
65°C with 0.5 M sodium phosphate buffer (pH 7.2), 7% SDS, 1%
bovine serum albumin (BSA), 1 mM EDTA, and 100 µg/ml sonicated carrier DNA. Thereafter, the membranes were hybridized overnight in the
above solution with 30-50 × 106 counts/min (cpm) of
32P-labeled DNA probe for the 16- or 31-kDa subunit of H+-ATPase.
The cDNA probes were labeled with
[32P]deoxynucleotides
using the RadPrime DNA labeling kit (GIBCO BRL). The membranes were
washed twice in 40 mM sodium phosphate buffer (pH 7.2), 5% SDS, 0.5%
BSA, and 1 mM EDTA for 10 min at 65°C, washed four times in 40 mM
sodium phosphate buffer (pH 7.2), 1% SDS, and 1 mM EDTA for 10 min at
65°C, exposed to PhosphorImager cassette at room temperature for
24-72 h, and read by PhosphorImager (Molecular Dynamics). For
the 16- or 31-kDa subunit cDNA, the EcoR
I-EcoR I fragment from a
pBluescript SK(
) plasmid containing the corresponding cDNA was
used as a specific probe.
Materials
DMEM-F12 medium was purchased from GIBCO BRL. BCECF and
nigericin were from Molecular Probes. DES, DCCD, NEM, bafilomycin A1, and other chemicals were
purchased from Sigma Chemical. The isotope
32P was purchased fom New England
Nuclear (Boston, MA). The RadPrime DNA labeling kit was purchased from
GIBCO BRL. Sch-28080 was a generous gift from Schering (via Dr.
Cuppoletti, Univ. Cincinnati). Mouse
H+-ATPase cDNAs were generous
gifts from Dr. Gary Dean, University of Cincinnati.
Statistics
Results are expressed as means ± SE. Statistical
significance between experimental groups was assessed by Student's
t-test or by one-way analysis of
variance.
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RESULTS |
Na+-Independent
pHi Recovery in Inner Medullary Collecting
Duct Cells
To study the
Na+-independent
pHi recovery, mIMCD-3 (parent) and
NHE2d (mutant) cells were incubated in an
Na+- and
-free,
HEPES · Tris-buffered medium (extracellular pH = 7.40) and gassed with 100% O2.
Na+ was replaced isosmotically
with TMA (solution A, Table 1). Under these conditions, resting pHi was
7.211 ± 0.018 (n = 6) and 7.244 ± 0.015 (n = 6) for mIMCD-3 and
NHE2d cells, respectively (P > 0.05, Fig.
1A).
When mIMCD-3 and NHE2d cells were acid loaded with
NH4Cl prepulse technique, the
pHi decreased to 6.204 ± 0.028 (n = 6) and 6.237 ± 0.017 (n = 6), respectively
(P > 0.05, Fig. 1A). The rate of recovery from an
acid load
(dpHi/dt)
was increased in mutant cells compared with parent cells (0.021 ± 0.002 and 0.078 ± 0.008 pH units/min in mIMCD-3 and NHE2d cells,
respectively, P < 0.001, Fig.
1B). As shown in Fig.
1A, NHE2d cells recovered faster and
reached baseline pHi in ~10 min
(
pHi = 0.685 ± 0.03 pH
units/10 min, n = 6), whereas mIMCD-3
cells recovered much slower
(
pHi = 0.198 ± 0.028 pH
units/10 min, n = 6).

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Fig. 1.
A:
Na+-independent
pHi recovery in mIMCD-3 and NHE2d
cells [representative intracellular pH
(pHi) tracings]. Cells
were loaded with BCECF, incubated in
Na+-free solution
[Na+ replaced with
tetramethylammonium
(TMA+)], and monitored for
baseline pH. Cells were then pulsed with 20 mM ammonium chloride for 10 min (20 mM TMA-Cl replaced with 20 mM
NH4Cl) and acid loaded by exposure
to an Na+- and ammonium-free
solution. pHi recovery was
recorded for 15 min. B: initial rates
of pHi recovery
(dpHi/dt,
pH units/min) in NHE2d cells (n = 6)
and mIMCD-3 cells (n = 6)
(P < 0.001).
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mIMCD-3 (Parent) Cells: Transport Properties and Inhibitory Profile
of the
Na+-Independent
pHi Recovery
Energy dependence of pHi recovery.
To determine whether the
Na+-independent
pHi recovery from an acid load is
dependent on ATP hydrolysis, pHi
recovery from an acid load was monitored in the presence of inhibitors
of cellular ATP production. Toward this end, cells were incubated for
45 min in either glucose-free medium or the presence of KCN before
being monitored for pHi. mIMCD-3
cells incubated in the absence of glucose for 45 min showed decreased
resting pHi (7.07 ± 0.007 in glucose-free medium vs. 7.234 ± 0.010 in the presence of 5 mM
glucose, P < 0.05, n = 4, Fig.
2A). In
the absence of glucose and after NH+4 withdrawal, pHi decreased to 6.240 ± 0.020 (n = 4) and did not recover. When 5 mM glucose was added to the perfusion solution, prompt
intracellular alkalinization was observed at a rate of 0.024 ± 0.006 pH units/min (n = 4, Fig.
2A), which was not different from
the control group (0.021 ± 0.002 pH units/min, Fig. 1). Incubation of mIMCD-3 cells in glucose-free medium had no effect on
Na+/H+
exchanger activity as shown by rapid cell alkalinization when perfused
with Na+-containing solution (data
not shown). These results suggest that Na-independent
pHi recovery from an acid load in
mIMCD-3 cells is dependent on glucose metabolism. To examine further
the mechanism of Na+-independent
pHi recovery, the rate of cell
alkalinization was studied in the presence of KCN (an inhibitor of
mitochondrial ATP production). A representative tracing (Fig.
2B) shows that preincubation of
mIMCD-3 cells with 4 mM KCN for 45 min decreased resting
pHi (7.234 ± 0.011, n = 4, in the absence and 7.07 ± 0.028, n = 5, in the presence of KCN,
P < 0.01). In the presence of KCN and following NH4Cl prepulse,
pHi decreased to 6.263 ± 0.017 with almost no recovery (0.004 ± 0.001 pH units/min, Fig.
2B). In the absence of KCN, the
pHi decreased to 6.28 ± 0.018 and then recovered at a rate of 0.021 ± 0.006 pH units/min
(P < 0.0003 vs. KCN group, Fig.
2B). In another group of experiments
designed so that each monolayer served as its own control, we observed
that removal of KCN in the perfusion solution was followed by a prompt
cellular alkalinization (0.019 ± 0.008 pH units/min,
n = 3). Together, these observations
are consistent with the presence of an ATP-dependent H+-extruding transporter in mouse
kidney inner medullary collecting duct cells.

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Fig. 2.
Energy dependence of
Na+-independent
pHi recovery in mIMCD-3 cells
(representative pHi tracings).
A: incubation of mIMCD-3 cells in
glucose-free medium for 45 min decreased the resting pHi
(n = 4, P < 0.05 vs. 5 mM glucose,
n = 6). Addition of 5 mM glucose
caused prompt cytosolic alkalinization
(n = 4).
B: representative pHi tracings depicting the effect
of KCN on Na+-independent
pHi recovery. Incubation of
mIMCD-3 cells with 4 mM KCN decreased the baseline
pHi
(n = 5, P < 0.01 vs. its own control).
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K+
independence of pHi recovery.
We next examined whether the ATP-dependent
H+ extrusion process was dependent
on extracellular K+. Accordingly,
mIMCD-3 cells were incubated in
Na+- and
K+-free medium
(solution C, Table 1) and assayed for
Na+-independent
pHi recovery in the absence or
presence of 5 mM K+
(solution D, Table 1). The resting
pHi was 7.200 ± 0.008 (n = 8). The
pHi following the acid load was
6.245 ± 0.017 and 6.282 ± 0.015 in the absence or presence of
K+, respectively
(P > 0.05, n = 4). The rate of
pHi recovery was 0.023 ± 0.004 and 0.020 ± 0.001 pH units/min in the absence or presence of
K+, respectively
(P > 0.05, n = 4). These results indicate that the ATP-dependent, Na+-independent
pHi recovery in mIMCD-3 cells was
independent of the extracellular
K+. The possibility that a
K+ leak from the cells into the
K+-free extracellular solution
could have increased extracellular K+ concentration was examined.
Although this possibility is unlikely due to constant perfusion of the
monolayer at 6 ml/min, collected solutions from several experiments
were analyzed for K+
concentration. Measured K+
concentration in K+-free solutions
was undetectable (<0.3 meq/l), whereas
K+ concentration in control
solution was 5.2 meq/l.
Inhibitory profile of
Na+-independent
pHi recovery in mIMCD-3 cells.
The experiments described above showed that the
Na+-independent
pHi recovery in mIMCD-3 cells was
dependent on intracellular ATP and independent of extracellular
K+, consistent with the presence
of a plasma membrane H+ pump. The
results further showed that this
H+ pump is sharply upregulated in
inner medullary collecting duct cells subjected to lethal acid stress
(Fig. 1). To characterize this H+
pump further, the effect of several inhibitors on the
Na+-independent
H+ extrusion was examined.
The results of inhibitory profile experiments are summarized in Table
2. DES, a strong inhibitor of
vacuolar-type H+-ATPase (9, 21),
decreased the rate of pHi recovery
from 0.018 ± 0.03 in control to 0.002 ± 0.001 pH units/min
(P < 0.0002, n = 4, Fig.
3A and
Table 2). The effect of DCCD, another inhibitor of
H+-ATPase, was also examined.
Incubation of mIMCD-3 cells with 200 µM DCCD for 10 min (DCCD added
when cells were exposed to NH4Cl) completely inhibited the rate of
pHi recovery from an acid load (0.017 ± 0.006 in control vs. 0.001 ± 0.0001 pH units/min in
DCCD; P < 0.0001, n = 4, Fig.
3B and Table 2). The effect of
Sch-28080, an inhibitor of
H+-K+-ATPase
and vacuolar H+-ATPase (18, 26),
was next tested. These experiments were performed in the absence of
K+ in the perfusate
(solution C, Table 1) to avoid any
contribution to pHi recovery by
H+-K+-ATPase.
Sch-28080, at 300 µM, abolished the
Na+- and
K+-independent
pHi recovery (Fig.
3C). The rate of
pHi recovery decreased from 0.020 ± 0.002 in control to 0.003 ± 0.001 pH units/min in the
Sch-28080 group (P < 0.0002, n = 4, Fig.
3C and Table 2). NEM, an inhibitor of
nonmitochondrial H+-ATPase (1, 12,
14, 23), also decreased the rate of
pHi recovery from an acid load
(0.023 ± 0.006 in Fig. 3C,
n = 5, vs. 0.004 ± 0.001 pH units/min in NEM group, n = 5, P < 0.0001, Fig. 3D and Table 2).
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Table 2.
mIMCD-3 cells: pHi values and effects of DES, DCCD,
Sch-28080, and NEM on rate of Na+-independent
pHi recovery after an acid load
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Fig. 3.
Inhibition of Na+-independent
pHi recovery in mIMCD-3 cells by
diethylstilbestrol (DES),
N, N'-dicyclohexylcarbodiimide
(DCCD), and Schering 28080 (Sch-28080) (representative
pHi tracings). A: addition of 50 µM DES when
ammonium was withdrawn inhibited the
pHi recovery from an acid load.
B: incubation of mIMCD-3 cells with
200 µM DCCD for 10 min before the acid load prevented the pHi recovery
(n = 4). Changing the
perfusion solution from TMA-Cl to NaCl (with DES present) caused rapid
intracellular alkalinization via
Na+/H+
exchange. C: addition of 300 µM
Sch-28080 or its vehicle when ammonium was withdrawn inhibited the
pHi recovery from an acid load in
mIMCD-3 cells.
[K+]o,
extracellular K+ concentration.
D: initial rates of
pHi recovery
(dpHi/dt,
pH units/min) in mIMCD-3 cells were inhibited by 50 µM DES
(n = 4, P < 0.0002), 200 µM DCCD
(n = 4, P < 0.0001), 300 µM Sch-28080 (n = 4, P < 0.0002), and 200 µM
N-ethylmaleimide (NEM;
n = 5, P < 0.0001).
* Significant difference, compared with pooled controls (n = 16).
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We next examined the effect of bafilomycin
A1, a macrolide antibiotic that
specifically inhibits vacuolar
H+-ATPase (4), on
Na+- and
K+-independent
H+ extrusion. The experiments were
performed in Na+- and
K+-free solution
(solution C, Table 1). The rate of
pHi recovery from an acid load
remained unchanged in the presence of bafilomycin A1 (0.019 ± 0.003 in the
absence and 0.0178 ± 0.004 pH units/min in the presence of 10 nM
bafilomycin A1,
n = 4, P > 0.05). These results indicate
that the Na+- and
K+-independent
pHi recovery from an acid load in
mIMCD-3 cells is not inhibited by bafilomycin
A1.
NHE2d (Mutant) Cells: Transport Properties and Inhibitory Profile of
the
Na+-Independent
pHi Recovery
Energy dependence of pHi recovery.
To determine whether the
Na+-independent
pHi recovery from an acid load in
mutant NHE2d cells is dependent on ATP hydrolysis, cells were incubated
for 45 min either in glucose-free medium or in the presence of KCN
before being monitored for pHi.
Figure 4 shows that NHE2d cells incubated
in Na+-free media showed decreased
resting pHi [7.063 ± 0.010 (n = 4) in glucose-free vs.
7.153 ± 0.009 (n = 6) in the
presence of 5 mM glucose, P < 0.05]. After NH+4 withdrawal, the
pHi decreased to 6.24 ± 0.025 or 6.205 ± 0.032 in the presence or absence of glucose,
respectively (P < 0.05, Fig.
4A). The rate of
pHi recovery from an acid load was
sharply higher in the presence of glucose (0.074 ± 0.005 vs.
0.02 ± 0.004 pH units/min in the presence or absence of
glucose, respectively, P < 0.002, Fig. 4A). In separate experiments,
and during pHi recovery, addition of 5 mM glucose to glucose-free medium increased the rate of
pHi recovery in NHE2d cells (0.019 ± 0.008 in the absence and 0.058 ± 0.005 pH units/min in the
presence of glucose, n = 3, P < 0.001). Incubation of NHE2d
cells in glucose-free medium for 45 min had no effect on
Na+/H+
exchanger activity as shown in Fig. 4B
by rapid cell alkalinization when perfused with an
Na+-containing solution
(solution B, Table 1). These results
suggest that pHi recovery from an
acid load in the absence of Na+ is
dependent on glucose metabolism.

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Fig. 4.
Energy dependence of
Na+-independent
pHi recovery in NHE2d cells
(representative pHi tracings).
A: incubation of NHE2d cells in the
absence of glucose for 45 min acidified baseline
pHi
(n = 4, P < 0.05). Furthermore,
pHi recovery from an acid load was inhibited in the absence of glucose (n = 4, P < 0.0002 vs. 5 mM glucose,
n = 6).
B: NHE2d cells were incubated in the
absence of glucose for 45 min and perfusion solution was switched from TMA-Cl to NaCl during pHi
recovery. Presence of Na+ caused a
rapid intracellular alkalinization via
Na+/H+
exchange (n = 3).
C: incubation of NHE2d cells with 4 mM
KCN decreased the baseline pHi
(n = 4, P < 0.004) and reduced the pHi recovery from an acid load
(P < 0.003 vs. control).
D: NHE2d cells were incubated in the
presence of 4 mM KCN for 45 min and perfusion solution was switched
from TMA-Cl to NaCl during pHi recovery. Presence of Na+ caused
rapid intracellular alkalinization via
Na+/H+
exchange (n = 3).
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To examine further the mechanism of
Na+-independent
pHi recovery, the rate of cell
alkalinization after an acid load was studied in the presence of KCN.
Baseline pHi in NHE2d cells
preincubated with KCN was 7.03 ± 0.0157 (n = 4) vs. 7.153 ± 0.021 pH
units/min in control (n = 6)
(P < 0.004, Fig.
4C). The nadir
pHi values following
NH+4 withdrawal were 6.196 ± 0.034 and 6.24 ± 0.025 in the presence and absence of KCN,
respectively (P > 0.05). The rate of
pHi recovery from an acid load
decreased by 69% in the presence of KCN (0.023 ± 0.005 vs. 0.074 ± 0.005 pH units/min in the presence or absence of KCN,
respectively, P < 0.0001, Fig.
4C). Incubation of NHE2d cells with
KCN had no effect on the
Na+-dependent
pHi recovery mediated by the
Na+/H+
exchanger (representative experiment in Fig.
4D). Together, these results
indicate that the pHi recovery of
NHE2d cells is mediated via an ATP-dependent
H+-extruding transporter.
K+
independence of pHi recovery.
To determine whether the ATP-dependent,
H+ extrusion mechanism was
dependent on extracellular K+,
NHE2d cells were incubated in an
Na+- and
K+-free medium
(solution C, Table 1) and assayed for
Na+-independent
pHi recovery in the absence or
presence of 5 mM K+
(solution D, Table 1). The
pHi following an acid load in
NHE2d cells was 6.194 ± 0.014 and 6.243 ± 0.018 in the absence
or presence of K+, respectively
(P > 0.05, Fig.
5). The rate of
pHi recovery was 0.095 ± 0.002 in the absence and 0.102 ± 0.008 pH units/min in the presence of
K+
(n = 5 for each group,
P > 0.05, Fig. 5). These results
indicate that the ATP-dependent,
Na+-independent
pHi recovery in NHE2d cells is
independent of extracellular K+.

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Fig. 5.
Effect of extracellular K+
([K+]o) on
Na+-independent pHi recovery in NHE2d
cells (representative pHi
tracings). Cells were pulsed with 20 mM
NH4Cl in
Na+- and
K+-free solution and then
acidified by ammonium withdrawal in the same solution
(n = 5) or in the presence of 5 mM
K+
(n = 5).
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Inhibitory profile of
Na+-independent
pHi recovery.
The experiments illustrated in Figs. 4 and 5 showed that the
Na+-independent
pHi recovery in NHE2d cells was
dependent on ATP and independent of extracellular
K+, consistent with the presence
of a plasma membrane H+ pump. This
H+ pump is significantly
upregulated in inner medullary collecting duct cells subjected to
lethal acid stress (Fig. 1). To characterize this
H+ pump further, the effects of
various inhibitors on the
Na+-independent
pHi recovery were examined.
The results of inhibitory profile experiments are shown in Fig.
6 and summarized in Table
3. DES inhibited the rate of
pHi recovery (Fig.
6A) in a dose-dependent manner, with
half-maximal inhibition and maximal inhibition achieved with 23 and 50 µM, respectively (Fig. 6A and Table
3). The effect of the H+-ATPase
inhibitor DCCD was also tested. When NHE2d cells were incubated with
200 µM DCCD for 10 min as described above, the rate of
pHi recovery from an acid load
decreased from 0.069 ± 0.004 in control to 0.027 ± 0.005 pH
units/min (P < 0.001, n = 4, Table 3 and Fig.
6B). Figure
6B and Table 3 also show that addition
of NEM, at 200 µM, significantly decreased the rate of pHi recovery from an acid load
(0.024 ± 0.004 in NEM group, n = 4, vs. 0.075 ± 0.006 pH units/min in control,
n = 5, P < 0.001). The effect of Sch-28080
was tested next. The experiments were performed in the absence of
K+ in the perfusate
(solution C, Table 1) to avoid any
possible contribution from
H+-K+-ATPase.
Sch-28080 inhibited the Na+- and
K+-independent
pHi recovery in a dose-dependent
manner (Fig. 6C), with a 50%
inhibitory concentration (IC50)
of 62 µM and maximal inhibition at 300 µM (Fig.
6C and Table 3).

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Fig. 6.
Dose-response inhibition of
Na+-independent
pHi recovery by DES and Sch-28080
in NHE2d cells (representative pHi
tracings). A: cells were incubated in
an Na+-free medium, loaded with
ammonium for 10 min, and then acidified in the presence of vehicle (0)
or in the presence of indicated concentrations of DES
(n = 4 for each). The 50% inhibitory
concentration (IC50) was ~23
µM DES. B: inhibition of
Na+-independent
pHi recovery
(dpHi/dt,
pH units/min) by 50 µM DES (n = 4, P < 0.0001), 200 µM DCCD
(n = 4, P < 0.001), 300 µM Sch-28080 (n = 4, P < 0.0001), and 200 µM NEM
(n = 5, P < 0.001). * Significant difference, compared with pooled controls
(n = 18).
C: NHE2d cells were incubated in an
Na+- and
K+-free solution and acidified in
the presence of vehicle (0) or in the presence of indicated
concentrations of Sch-28080 (n = 4-5 for each); IC50 for
Sch-28080 was ~62 µM.
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Table 3.
NHE2d cells: pHi values and effect of DES, DCCD,
Sch-28080, and NEM on rate of Na+-independent
pHi recovery after an acid load
|
|
We next examined the effect of bafilomycin
A1 on the rate of
Na+-independent
H+ extrusion. The rate of
pHi recovery from an acid load in
NHE2d cells in Na+- and
K+-free solutions
(solution C, Table 1) was 0.078 ± 0.008 in the control group (n = 5),
0.076 ± 0.009 in the presence of 10 nM bafilomycin
(n = 4), and 0.071 ± 0.010 pH
units/min in the presence of 200 nM bafilomycin
(n = 5)
(P > 0.05 between groups). These results demonstrate that bafilomycin
A1 at low or high concentrations did not inhibit the Na+- and
K+-independent
pHi recovery in NHE2d cells.
Northern Hybridization of
H+-ATPase
Subunits
The results of the above studies (Figs. 1-6) indicate that mIMCD-3
cells express a vacuolar-type
H+-ATPase that is sharply
upregulated in response to lethal acid stress. To examine the molecular
basis of H+-ATPase induction in
lethal acid stress, total RNA was isolated from mIMCD-3 or NHE2d cells,
size fractionated, transferred to a nylon membrane, and probed with
radiolabeled DNA encoding the 16- or 31-kDa subunit of
H+-ATPase. A representative
experiment is shown in Fig.
7A. As
demonstrated, mRNA levels for the 31-kDa subunit remained unaltered in
NHE2d cells compared with mIMCD-3 cells (Fig.
7A,
top, parent vs. mutant cells).
Similarly, expression of the 16-kDa subunit mRNA levels remained the
same in NHE2d cells (Fig. 7A,
bottom, parent vs. mutant cells).
Equal RNA loading in both lanes shown in Fig.
7A (top and
bottom) was verified by ethidium
bromide staining of nitrocellulose membrane-transferred RNA (Fig.
7B, a
and b). Three separate Northern
blots were performed in NHE2d and mIMCD-3 cells, and the results
invariably showed that none of the vacuolar
H+-ATPase subunits was affected in
response to lethal acid stress. These results indicate that functional
upregulation of H+-ATPase by
lethal acid stress is likely at posttranscription level. It has to be
mentioned that comparable mRNA levels for the 16- and 31-kDa subunits
do not unequivocally prove that transcriptional regulation has not
occurred.

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Fig. 7.
Northern hybridization of
H+-ATPase subunits.
A: representative Northern blots
showing 31-kDa subunit (top) and
16-kDa subunit (bottom) transcript
levels in parent (mIMCD-3) and mutant (NHE2d) cells. Corresponding
nitrocellulose membrane-transferred RNAs are shown in
B (a
corresponds to 31-kDa and b
corresponds to 16-kDa H+-ATPase
subunit Northern blot). The 31-kDa subunit transcript size was ~1.5
kb; 16-kDa subunit transcript size was ~1.1 kb. Thirty micrograms of
RNA were loaded on each lane.
|
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Role of
H+-ATPase in the
Maintenance of Baseline pHi
Effect of DES and Sch-28080 on the steady-state
pHi.
The objective of the next series of experiments was to determine
whether H+-ATPase plays any role
in the maintenance of baseline pHi
in mIMCD-3 or NHE2d cells. Accordingly, NHE2d or mIMCD-3 cells were
incubated in an Na+-free medium
(solution A, Table 1) and monitored
for baseline pHi in the presence
of DES or Sch-28080. Representative tracings for some of the
experiments are shown in Fig. 8.

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Fig. 8.
Effect of Sch-28080 and bafilomycin
A1 on steady-state
pHi in mIMCD-3 cells
(representative pHi tracings).
A: and
B: mIMCD-3 cells were incubated in an
Na+-free medium and exposed
acutely to either Sch-28080 (300 µM, A) or bafilomycin
A1 (200 nM,
B) or their vehicles.
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|
In mIMCD-3 cells, addition of DES (50 µM) decreased baseline
pHi by 0.180 ± 0.004 pH units
(from 7.150 ± 0.010 to 6.970 ± 0.009, n = 4, P < 0.001). Exposure of mIMCD-3
cells to 300 µM Sch-28080 (Fig.
8A) reduced baseline
pHi by 0.169 ± 0.006 pH units (from 7.122 ± 0.003 to 6.953 ± 0.005,
pHi = 0.169 ± 0.006 pH units, n = 5, P < 0.001). Interestingly,
bafilomycin A1 (200 nM), which had
no effect on the rate of pHi
recovery from an acid load (see above), decreased baseline
pHi (Fig.
8B). Baseline
pHi was reduced from 7.18 ± 0.006 in control to 7.05 ± 0.006 in the bafilomycin group
(
pHi = 0.129 ± 0.012 pH
units, n = 4, P < 0.001).
Effect of inhibitors on baseline
pHi in NHE2d cells was next
examined. Resting pHi was 7.18 ± 0.012 in NHE2d cells and decreased by 0.260 ± 0.005 pH units
when 50 µM DES was added (the new steady-state pHi was 6.92 ± 0.008, n = 4, P < 0.001 vs. control). Addition of
Sch-28080 (300 µM) in a similar manner decreased baseline
pHi in NHE2d cells by 0.180 ± 0.006 pH units (the new steady-state pHi was 7.02 ± 0.007, down
from 7.20 ± 0.010 in control, n = 4, P < 0.01).
To determine whether increased
H+-ATPase in mutant cells is a
true adaptive upregulation or is due to selection by lethal acid stress
of clones that express higher
H+-ATPase, three additional clones
from the parent cell line that were not subjected to lethal acid stress
were obtained. Cells were trypsinized and passaged at very high
dilution (1/1,000) to isolate individual colonies. A number of
individual colonies were isolated with a cloning cylinder and then
collected and subcultured. Three strains were studied for
H+-ATPase activity according to
MATERIALS AND METHODS. The results are
shown in Fig. 9 and demonstrate that the
rate of H+-ATPase activity was
comparable between these clones and the uncloned parent cell line.
These results suggest that increased
H+-ATPase activity by lethal acid
stress represents true adaptive upregulation of the pump.

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Fig. 9.
Na+-independent
pHi recovery in clones
(A-C) isolated from mIMCD-3 cells. Three
separate clones were isolated from mIMCD-3 cells not subjected to
lethal acid stress and studied for
H+-ATPase activity in a manner
similar to Fig. 1. D, uncloned parent cell
line.
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|
Last, we examined the possible contribution of
H+-K+-ATPase
to the maintenance of baseline pHi
in mIMCD-3 cells. Addition of Sch-28080 (10 µM), which completely
inhibits the P-type
H+-K+-ATPase
had no effect on baseline pHi
(n = 5, Fig.
10A).
Similarly, addition of K+ (5 mM)
(solution D, Table 1) to a
K+-free perfusion solution
(solution C, Table 1) had no effect on
the steady-state pHi in mIMCD-3
cells (n = 5, Fig.
10B). Similarly, replacement of
K+-containing solution with a
K+-free solution had no effect on
baseline pHi
(n = 4, data not shown).

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Fig. 10.
Effect of Sch-28080, 10 µM, or
[K+]o, 5 mM, on steady-state
pHi in mIMCD-3 cells. A: acute addition of
Sch-28080, 10 µM, to mIMCD-3 cells in Na+-free solution
in the presence of 5 mM [K+]o
(n = 5). B: cells were incubated in
Na+- and K+-free solution and then exposed
abruptly to a Na+-free solution that contained 5 mM
K+ (n = 5).
|
|
 |
DISCUSSION |
The mechanism of Na-independent H+
extrusion in inner medullary collecting duct cells was studied. The
results of current experiments provide strong evidence that the
Na+-independent
pHi recovery in both mIMCD-3 and
NHE2d cells (Fig. 1) is mediated via an active
H+ translocating pump. The
recovery of cell pH following an acid load was observed only in the
presence of glucose (Figs. 2A and 4A) and was reversibly inhibited in
the presence of the aerobic metabolic inhibitor KCN (Figs.
2B and
4C), consistent with ATP dependence
of this transporter. NHE2d cells displayed a residual but significant
pHi recovery in the presence of
KCN (Fig. 4, C and
D), probably due to the energy
provided by the glycolysis pathway. These observations are in agreement
with the metabolic studies that have shown that papillary cells possess
two pathways of glucose metabolism: glycolysis and aerobic oxidative
phosphorylation (8).
The experiments demonstrated that the
Na+-independent
pHi recovery in NHE2d and mIMCD-3
cells was not affected by extracellular K+ (Fig. 5 and
RESULTS), indicating lack of
functional activity of the
K+-dependent
H+ translocating pump
(H+-K+-ATPase).
Given the ATP dependence, inhibitory profile, and
K+ independence of
H+ extrusion, we propose that an
H+-ATPase transporter is
responsible for the
Na+-independent
H+ extrusion in mIMCD-3 and NHE2d
cells. A recent study showed the presence of gastric and colonic
H+-K+-ATPase
mRNAs in mIMCD-3 cells and suggested a functional role for gastric
H+-K+-ATPase
(21). The functional evidence for the presence of gastric H+-K+-ATPase
in mIMCD-3 cells was based on the inhibition by Sch-28080 (10 µM) and
the lack of effect of bafilomycin on
Na+-independent
pHi recovery (21). The authors of
this study (21) further demonstrated that acute replacement of
K+-free solution with a
K+-containing solution during
NH+4 withdrawal-stimulated pHi recovery. The reason for the
conflicting results between our studies and these previous experiments
(21) remains speculative. It is possible that acute replacement of
K+ during
NH+4 withdrawal could indirectly stimulate the electrogenic H+-ATPase by
depolarizing the cell membrane (via
K+ channels). Other possibilities
like differences in experimental conditions could not be excluded.
Specifically, one plausible explanation with respect to the difference
in the nature of ATP-dependent H+
extrusion in mIMCD-3 cells could be differences in the experimental nadir pH. pHi following
NH+4 withdrawal in mIMCD-3 or NHE2d cells was
<6.30 (see RESULTS); however, the
pHi following NH+4 withdrawal in inner medullary collecting duct cells was significantly higher (21) than our results. Whether H+-K+-ATPase
and H+-ATPase have different
optimal pHi activation set points
cannot be excluded. Experiments in primary cultured cells derived from the rat terminal inner medullary collecting duct indicated two Na+-independent,
H+ extrusion mechanisms. A
K+-dependent, Sch-28080 (10 µM)-sensitive process accounted for nearly 60% of
Na+-independent
pHi recovery from an acid load.
The remaining Na+- and
K+-independent
H+ extrusion (presumably via
H+-ATPase) caused significant
pHi recovery from an acid load (at a rate of 0.019 ± 0.007 pH units/min) (17). These values are in
full agreement with the Na+- and
K+-independent
pHi recovery of mIMCD-3 cells in
our experiments (see Table 2). Interestingly, the
Na+- and
K+-independent
pHi recovery in rat inner
medullary collecting duct cells was insensitive to bafilomycin
A1 (17) and was similar to our
experiments. Our results, however, demonstrated that bafilomycin A1 inhibits
H+-ATPase activity at baseline
pHi and causes significant
intracellular acidification. To determine whether the reduction in
baseline pHi by bafilomycin is
indeed mediated via inhibition of
H+-ATPase, cells were incubated
with DES at resting pHi before
being exposed to bafilomycin. Incubation of mIMCD-3 cells with DES (50 µM) decreased baseline pHi,
consistent with inhibition of
H+-ATPase (see
RESULTS). However, addition of
bafilomycin (200 nM) to cells incubated with DES had no additional
effect on pHi (data not shown).
These results indicate that the inhibitory effect of bafilomycin on
baseline pHi is mediated via
suppression of H+-ATPase. The
reason for the paradoxical effect of bafilomycin on
H+-ATPase inhibition at baseline
pHi and in the acid-loaded state remains unclear. It is possible that intracellular acidosis induces conformational changes in
H+-ATPase subunits that could then
affect their assembly, making the bafilomycin-binding site
inaccessible. In support of this hypothesis, reduction in cell pH was
found to affect assembly of
H+-ATPase subunits (12).
Alternatively, it is possible that the H+-ATPase transporter in the
terminal inner medullary collecting duct is distinct from the
H+-ATPase in other nephron
segments, i.e., cortical collecting duct. In support of this
hypothesis, we find that, whereas several studies have suggested the
presence of H+-ATPase activity in
inner medullary collecting duct cells, immunocytochemical studies with
the vacuolar H+-ATPase-specific
antibodies have failed to detect any labeling (5).
The effect of Sch-28080 on
Na+-independent
pHi recovery in inner medullary
collecting duct cells in our studies as well as others (17, 21) needs
further elaboration. Sch-28080 inhibits the
Na+- and
K+-independent
pHi recovery in NHE2d cells in a
dose-dependent manner (Fig. 6C). The
IC50 for Sch-28080 inhibition of
Na+-independent
H+-extrusion was 62 µM, which is
6- to 60-fold higher than that reported for
H+-K+-ATPase
activity in cultured inner medullary collecting duct cells (17) or
gastric microsomes (36), respectively. Our results are in full
agreement with recent studies showing inhibition of vacuolar-type
H+-ATPase by Sch-28080 in turtle
bladder (with an IC50 of 42 µM) (24) and in renal cortical and medullary endosomes (26).
Altogether, our results indicate that a vacuolar
H+-ATPase is present in the plasma
membrane of mIMCD-3 cells. This transporter is involved in the
maintenance of baseline pHi and is
responsible for Na+-independent
pHi recovery from an acid load.
The H+-ATPase is insensitive to
bafilomycin A1 at acidic
pHi but is sensitive to other
inhibitors of vacuolar H+-ATPase.
Specifically, our results demonstrate that the
Na+-independent
pHi recovery in mIMCD-3 cells was
completely inhibited by DES (Fig.
3A), a potent inhibitor of plasma
membrane H+-ATPase in
Neurospora crassa (4), rabbit renal
endosomes (12), lysosomes (9, 28), and chromaffin granules (14) as well as mitochondria (4). The results further demonstrated that the
ATP-dependent H+-pump in mIMCD-3
cells was inhibited by two known inhibitors of the vacuolar
H+-ATPase, NEM (1, 11, 16, 26)
(Fig. 3D) and DCCD (1, 26) (Fig. 3,
B and
D). Although NEM is a less specific
inhibitor of H+-ATPase than DES,
the existence of a modest but significant amount of NEM-sensitive but
Na+- and
K+-independent and
vanadate-resistant ATPase activity has been demonstrated in the inner
medulla (25).
The more intriguing aspect of the present studies is functional
overexpression of H+-ATPase in
mIMCD-3 cells that were subjected to lethal acid stress. The
H+-ATPase in NHE2d cells shows
greater than a fourfold increase in activity and demonstrates similar
inhibitory profile to mIMCD-3 cells (compare Figs.
3D and
6B). Given the brief time of
exposure of mIMCD-3 cells to the lethal acid medium (120 min),
synthesis of new transport proteins seems unlikely. Indeed, Northern
hybridization experiments (Fig. 7) demonstrate that mRNA levels for 16- and 31-kDa subunits of the vacuolar
H+-ATPase remained the same in
cells exposed to lethal acid stress compared with control. These
results indicate that functional upregulation of
H+-ATPase in response to lethal
acidosis is likely due to a posttranscriptional event. These
possibilities, including activation of currently inactive membrane
proteins (phosphorylation) or incorporation of intracellular proteins
into the membrane (exocytosis), are potential mechanisms for the
observed changes. Differentiating between these possibilities, however,
is very difficult at the present, as no antibodies are available that
could recognize the H+-ATPase
subunits in the renal medulla. It is also worth mentioning that
subunits other than the 16 and 31 kDa of
H+-ATPase pump (which were
examined in the present study) might have been affected by lethal acid
stress in inner medullary collecting duct cells. In addition, other
possibilities such as increased enzyme activity, increased mass, or an
alteration in the driving force against which the pump functions (such
as membrane potential) cannot be excluded as the cause of functional
upregulation of H+-ATPase in
response to lethal acid stress.
H+-ATPase plays an essential role
in the maintenance of baseline
pHi, as shown by intracellular
acidification in the presence of DES (50 µM) or Sch-28080 (300 µM)
in mIMCD-3 and NHE2d cells (see
RESULTS and Fig.
8A). Bafilomycin
A1 (200 nM) also decreased baseline pHi in mIMCD-3 cells
(Fig. 8B), whereas Sch-28080 (10 µM) had no effect on baseline
pHi (Fig.
10A). Resting
pHi was also decreased in
glucose-free medium or in the presence of KCN (Figs. 2 and 4). These
findings provide strong evidence that an
H+-ATPase is present in the plasma
membrane of both mIMCD-3 and NHE2d cells and is involved in the
maintenance of baseline pH. These results are in agreement with recent
studies showing that an Na+- and
K+-independent
reabsorption is present in the
papillary rat inner medullary collecting duct tubule perfused in vitro
(34).
In summary, terminal mIMCD-3 cells express an ATP-dependent
H+ extruding pump in their plasma
membrane, consistent with
H+-ATPase. This transporter plays
an important role in the maintenance of baseline pH in inner medullary
collecting duct cells. Exposing mIMCD-3 cells to lethal acid stress
resulted in overexpression of
H+-ATPase in surviving cells via a
posttranscriptional event. Overexpression of
H+-ATPase may play a protective
role against cell death in severe intracellular acidosis.
 |
ACKNOWLEDGEMENTS |
We acknowledge the excellent technical assistance of Holli Shumaker.
 |
FOOTNOTES |
These studies were supported by the National Institute of Diabetes and
Digestive Kidney Diseases Grant DK-46789, a Merit Review Grant from the
Department of Veterans Affairs, and a grant from Dialysis Clinic
Incorporated (to M. Soleimani).
Address for reprint requests: M. Soleimani, Univ. of Cincinnati Medical
Center, 231 Bethesda Ave., MSB 5502, Cincinnati, OH 45267-0585.
Received 18 February 1997; accepted in final form 17 June 1997.
 |
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