Epidermal growth factor inhibits amiloride-sensitive sodium
absorption in renal collecting duct cells
Jie-Pan
Shen and
Calvin U.
Cotton
Departments of Pediatrics and Physiology and Biophysics,
Rainbow Center for Childhood PKD, Case Western Reserve University,
Cleveland, Ohio 44106-4948
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ABSTRACT |
The effects of the ERK pathway on
electrogenic transepithelial Na+ absorption by renal
collecting duct cells were determined. Approximately 90% of the
unstimulated short-circuit current (15 ± 1 µA/cm2,
n = 10) across conditionally immortalized murine
collecting duct epithelial cells (mCT1) is amiloride sensitive and is
likely mediated by apical epithelial Na+ channels. Chronic
exposure (24 h) of the epithelial monolayers to either EGF (50 ng/ml)
or transforming growth factor-
(TGF-
; 20 ng/ml) reduced
amiloride-sensitive short-circuit current by >60%. The inhibitory
effect of EGF on Na+ absorption was not due to inhibition
of basolateral Na+-K+-ATPase, because the pump
current elicited by permeabilization of apical membrane with nystatin
was not reduced by EGF. Chronic exposure of the mCT1 cells to EGF (20 ng/ml, 24 h) elicited a 70-85% decrease in epithelial
Na+ channel subunit mRNA levels. Exposure of mCT1 cells to
either EGF (20 ng/ml) or PMA (150 nM) induced rapid phosphorylation of p42/p44 (ERK1/2) and pretreatment of the monolayers with PD-98059 (an
ERK kinase inhibitor; 30 µM) prevented phosphorylation of p42/p44. Similarly, pretreatment of mCT1 monolayers with
PD-98059 prevented the EGF- and PMA-induced inhibition of
amiloride-sensitive Na+ absorption. The results of these
studies demonstrate that amiloride-sensitive Na+ absorption
by renal collecting duct cells is regulated by the ERK pathway. This
pathway may play a role in alterations in ion transport that occur in
polycystic kidney disease.
extracellular signal-regulated protein kinase; ERK1/2; polycystic
kidney disease; mitogen-activated protein kinase
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INTRODUCTION |
AMILORIDE-SENSITIVE
ABSORPTION of Na+ is common to a variety of
epithelial tissues, including airway, renal collecting duct, urinary
bladder, colon, and sweat and salivary glands (11). Expression of the epithelial Na+ channel (ENaC) is
documented in each of these tissues, and the channel is thought to play
a role in electrogenic Na+ absorption. ENaC belongs to the
degenerin/ENaC gene superfamily (5), and members of the
degenerin/ENaC superfamily share in common the predicted membrane
topology: two hydrophobic membrane-spanning regions, intracellular
NH2 and COOH termini, and a highly conserved large
extracellular cysteine-rich loop (5, 15, 25, 30). ENaC is
a heteromultimeric protein composed of some combination of three
homologous subunits:
-,
-, and
-ENaC. Heterologous expression
of the three subunits has shown that the
-subunit alone, but not
- or
-subunits, is sufficient to generate small amiloride-sensitive currents (4, 18, 20). However,
coexpression of
- and
-subunits with the
-subunit potentates
amiloride-sensitive Na+ currents >100-fold (6,
19).
Clinical disorders due to the malfunction of ENaC are associated with
gain- or loss-of-function mutations such as salt-sensitive hypertension
(Liddle's syndrome) or pseudohypoaldosteronism type I. Liddle's
mutations, characterized by hypertension (29), disrupt a
COOH-terminal proline-rich protein binding site in the
- or
-subunits, which cause an increase in ENaC channel number in the
cell membrane, leading to excess Na+ reabsorption
(26, 31). In pseudohypoaldosteronism type I, newborns
develop hyponatremia, hyperkalemia, salt wasting, and elevated
aldosterone concentrations (7, 32). The transition to air
breathing after birth requires rapid clearance of fetal lung fluid
(3) via an amiloride-sensitive Na+ absorption
pathway. Evidence for a functional role for ENaC in fetal lung fluid
clearance at birth is documented in
-ENaC subunit knockout mice that
fail to clear lung fluid and die within 48 h after birth
(12). Apart from functional disruption in ENaC, interactions with other ion channels, such as CFTR, may have important implications for cystic fibrosis (33). Moreover, aberrant
regulation of ENaC expression or activity may contribute to cyst
formation and enlargement in polycystic kidney disease (PKD). Cyst
formation in renal collecting tubules is thought to involve a
transition from an absorptive to a secretory epithelium and likely
involves downregulation of ENaC function via an as yet undetermined
pathway (22).
Acute and chronic regulation of ENaC is known to be influenced by
various hormones, including vasopressin and aldosterone (1, 13,
17). Furthermore, growth factors have been reported to play a
pivotal role in the regulation of epithelial transport independently of
cell proliferation. Keratinocyte growth factor inhibits the expression
of the
-subunit of ENaC mRNA in mouse fetal lung (43).
In adult rat alveolar type II cells, EGF significantly decreases the
expression of all three subunits of ENaC mRNA but increases the whole
cell conductance and the density of nonselective amiloride-sensitive
Na+ channels (14). Treatment of both human
primary cultures of cystic fibrosis airway cells and a cystic fibrosis
nasal epithelial cell line with hepatocyte growth factor reduces the
abnormally high amiloride-sensitive Na+ absorption observed
in cystic fibrosis airway cells (28). Expression of an
inducible Raf-1 kinase in a rat parotid gland cell line resulted in
downregulation of
-ENaC subunit expression (16, 42).
EGF stimulates Cl
secretion and inhibits Na+
absorption in primary cultures of endometrial cells (10).
In T84 cells, acute exposure to EGF inhibits Ca-activated
Cl
secretion (38). Several observations from
human and mouse PKD suggest that the overproduction and accumulation of
EGF in renal cysts and mislocalization of EGF receptors (EGFR) to the
apical plasma membrane contribute to PKD pathophysiology (24,
35). EGF is known to cause acute inhibition of Na+
absorption in isolated, perfused rabbit collecting tubules, presumably by means of an increase in intracellular Ca2+ (21,
23, 40, 41); however, the effects of long-term exposure of renal
collecting duct cells to EGF are not known. The present study was
undertaken to determine the role of the EGF/ERK pathway in modulating
amiloride-sensitive Na+ absorption in mouse renal
collecting duct. A conditionally immortalized mouse collecting duct
cell line, mCT1, was used. The results of these studies demonstrate
that amiloride-sensitive Na+ absorption is significantly
downregulated in renal collecting duct cells by ERK signaling.
Activation of this pathway may play an important role in ion transport
abnormalities associated with PKD.
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METHODS |
Cell culture.
Experiments were carried out with a conditionally immortalized murine
collecting tubule cell line (mCT1). The details of the generation and
characterization of the cell line were described previously
(37). Routine cell culture for expansion was carried out
on tissue culture dishes, and cells were seeded onto either Millicell-CM filters (12 mm; Millipore, Bedford, MA) or
Transwell-clear, tissue culture-treated polyester membrane filters (24 mm; Corning Costar, Cambridge, MA) for experiments. The cells were
grown in collecting tubule media consisting of a 1:1 mix of DMEM and
Ham's F-12 medium supplemented with 1.3 µg/l sodium selenite, 1.3 µg/l 3,3',5-triiodo-L-thyronine, 5 mg/l insulin, 5 µg/l
transferrin, 25 µg/l PGE1, 2.5 mM glutamine, 50 nM
dexamethasone, and 10 U/ml mouse interferon-
. The cultures were
maintained at 33°C in a humidified incubator with 5%
CO2. Media were changed every other day, and cells were
passed weekly. Cells used for experiments reported here were between
passages 10 and 25.
Transepithelial electrical measurements.
Cells were seeded (1-3 × 105 cells/filter) on
collagen-coated, permeable supports (12-mm Millicell-CM filters). The
filter surface was coated with 125 µl/cm2 calfskin
collagen (Sigma) dissolved in acetic acid (7.5 mg/ml 0.2% glacial
acetic acid) and allowed to dry. The collagen coating was cross-linked
by exposure to ammonium hydroxide vapors (3.5% solution) for 10 min
followed by immersion in glutaraldehyde (2.5%) for 10 min. This
procedure was followed by a thorough rinsing in distilled water, 70%
ethanol, distilled water, and, finally, culture media. Cell monolayers
grown on modified supports were mounted vertically in a
thermostatically controlled Ussing chamber equipped with gas inlets and
separate reservoirs for the perfusion of apical and basolateral
compartments. Both sides of cell monolayers were bathed with equal
volumes (usually 6-10 ml) of Krebs-Ringer HCO
solution containing (in mM) 115 NaCl, 25 NaHCO3, 5 KCl, 2.5 Na2HPO4, 1.8 CaCl2, 1 MgSO4, and 10 glucose. The solutions were circulated
through the water-jacketed glass reservoir by gas lifts (95%
O2-5% CO2) to maintain solution temperature at
37°C and pH at 7.4. Transepithelial voltage difference (VT) was measured between two Ringer-agar
bridges, each positioned within 3 mm of the monolayer surface. Calomel
half-cells connected the bridges to a high impedance voltmeter. Current
from an external direct current source was passed by silver-silver
chloride electrodes and Ringer-agar bridges to clamp the spontaneous
VT to 0. The current required [short-circuit
current (ISC)] was corrected for solution and
filter series resistance. Monolayers were maintained under
short-circuit conditions except for brief 3- to 5-s intervals when the
current necessary to clamp the voltage to a nonzero value (usually +2
mV) was measured to calculate transepithelial resistance (RT).
Western blotting and RT-PCR.
Cell lysates were prepared from confluent culture filters (24 mm;
Corning Costar) in lysis buffer containing 50 mM
Tris · HCl (pH 7.5), 0.1% IGEPAL, 2 mM EDTA, and
1 mM EGTA for soluble cytosolic proteins and supplemented with 150 mM
NaCl, 0.1% SDS, and 0.5% sodium deoxycholate for membrane proteins.
Protein concentrations were measured by bicinchoninic acid protein
assay (Pierce, Rockford, IL). Whole cell lysates (20 µg protein) were
denatured in SDS-PAGE sample buffer containing 50 mM
Tris · HCl (pH 6.8), 2% SDS, 5%
-mercaptoethanol, 10% glycerol, and 0.1% bromphenol blue at
95°C. Proteins were separated on a 10% (for ERK1/2) SDS-PAGE gel and electrophoretically blotted onto a pure nitrocellulose transfer and
immobilization membrane (Schleicher & Schuell, Keene, NH). Membranes
were blocked 1 h at room temperature in Tris-buffered saline (TBS)
that contained 5% dried milk (wt/vol), 0.1% polyoxyethylenesorbitan monolaurate (Tween 20), and 0.01% sodium azide. After a brief wash to
remove Tween 20, the membranes were incubated at room temperature for
3 h with specific antibodies [anti-phospho-MAPK pAb (p-ERK1/2),
1:2,500 dilution, and anti-MAPK pAb (ERK1/2), 1:10,000 dilution,
Promega, Madison, WI] in TBS-1% BSA. The membranes were then
incubated with secondary antibody [horseradish peroxidase-conjugated donkey anti-mouse IgG (1:10,000 dilution for MAPK and phospho-MAPK)]. Membranes were rinsed with three changes of washing buffer (TBS), once
for 15 min and twice for 5 min after blocking and each antibody incubation. Peroxidase-labeled membranes were developed by enhanced chemiluminescence (Amersham, Arlington Heights, IL). Protein bands were
visualized on X-ray film (X-O-Mat, Kodak, Rochester, NY). Molecular
mass estimation of detected bands was determined by using prestained
high-molecular-mass protein standards (GIBCO-BRL, Life Technologies,
Rockville, MD). Quantification of the intensity of the bands on the
luminograms was determined with a Sci Scan 5000 densitometer. The
OS-Scan Image Analyses System density scan program (Oberlin Scientific)
was used to integrate the relevant peak areas in the protein bands.
RT-PCR of total RNA obtained from mCT1 cells was performed with
subunit-specific primers. Total RNA was purified from mCT1 cultures by
RNeasy Mini Kit (Qiagen, Valencia, CA). RT-PCR was performed by using
Moloney murine leukemia virus RT system (Life Technologies) according
to the manufacturer's directions. The primers used for the RT-PCR were
-mENaC forward primer 5'-CTA ATG ATG CTG GAC CAC ACC-3' and reverse
primer 5'-AAA GCG TCT GTT CCG TGA TGC-3',
-mENaC forward primer
5'-GCC AGT GAA GAA GTA CCT CC-3' and reverse primer 5'-CCT GGG TGG CAC
TGG TGA A-3',
-mENaC forward primer 5-AAG AAT CTG CCA GTT CGA GGC-3'
and reverse primer 5'-TAC CAC TCC TGG ATG GCA TTG-3', and GAPDH forward
primer 5'-CGT CTT CAC CAC CAT GGA GA-3' and reverse primer 5'-CGG CCA TCA CGC CAC AGT TT-3'. PCR reactions were performed on a thermal cycler
with 94°C/1 min of denaturing, 52°C/1 min of annealing, and
72°C/1 min, 30 s of elongation in each cycle. The reaction samples were run on a 1.5% agarose gel and visualized with
ethidium bromide. The predicted product sizes are
-ENaC, 546 bp;
-ENaC, 632 bp;
-ENaC, 671 bp; and GAPDH, 299 bp.
Quantitative RT-PCR.
The primers used for quantitative RT-PCR were
-mENaC forward primer
5'-GCC AGT GCT CCT GTC A-3' and reverse primer 5'-GGG GTA CAG GGT ACC
AA-3',
-mENaC forward primer 5'-CCC TTC CTT GCG TCC A-3' and reverse
primer 5'-CGC TCC TGA GAC AGG A-3',
-mENaC forward primer 5-CGC TGT
CAC TAT CTG CA-3' and reverse primer 5'-AAG CAG GTC ACC AGC A-3', and
GAPDH forward primer 5'-CGT CTT CAC CAC CAT GGA GA-3' and reverse
primer 5'-CGG CCA TCA CGC CAC AGT TT-3'. The predicted product sizes
are
-ENaC, 500 bp;
-ENaC, 500 bp;
-ENaC, 499 bp; and GAPDH,
299 bp. The appropriate product sizes were confirmed by running the
samples on agarose gels. PCR reactions were performed on LightCycler
(Roche Diagnostic, Indianapolis, IN) with 94°C/5 s of denaturing,
68~60°C/5 s of annealing (with step size of 0.5°C), and 72°C/16
s of elongation in each cycle.
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RESULTS |
mCT1 cells exhibit amiloride-sensitive ISC and express
-,
-, and
-ENaC mRNAs.
mCT1 cells, derived from murine collecting ducts, form polarized
epithelial monolayers when grown on collagen-coated permeable supports.
Mean values of RT and ISC
are 1,160 ± 119
· cm2 and
14.6 ± 1.1 µA/cm2, respectively (n = 10). After exposure to 100 µM amiloride, RT was increased to 1,510 ± 169
· cm2 and
ISC was decreased to 1.8 ± 0.1 µA/cm2 (n = 10). The basal
ISC in these cells (Fig.
1) is inhibited ~90% by submicromolar
concentrations of amiloride (K0.5 ~ 220 nM) and benzamil (K0.5 ~ 20 nM).
As illustrated in Fig. 2, mCT1 cells as
well as murine kidney express
-,
-, and
-subunit mRNAs for
ENaC. It is likely that the amiloride-sensitive
ISC represents ENaC-mediated Na+
absorption, a feature of collecting duct principle cells
(37).

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Fig. 1.
Dose-response relationships for inhibition of
short-circuit current (ISC) by amiloride and
benzamil. Cumulative addition of amiloride or benzamil to the apical
bathing solution of confluent monolayers of mCT1 cells caused
inhibition of ISC. Greater than 90% of
spontaneous ISC was inhibited. Calculated values
for IC50 were 20 and 219 nM for benzamil and amiloride,
respectively. Values are means ± SE for 4 monolayers exposed to
benzamil (basal ISC = 10.5 ± 0.7 µA/cm2) and 4 monolayers exposed to amiloride (basal
ISC = 13.3 ± 1.3 µA/cm2). Lines, fits of the data to a
Michaelis-Menten-type equation.
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Fig. 2.
Expression of -, -, and -epithelial
Na+ channel (ENaC) subunit mRNA in mouse kidney and mCT1
cells. Total RNA from mCT1 cells and whole mouse kidney was isolated
and reverse transcribed. PCR amplification of the resultant cDNAs was
completed by using primers specific for GAPDH (G) and -, -, and
-ENaC ( , , and , respectively). Molecular weight marker
lanes (M; 100-bp DNA ladder) and a negative control lane ( ; without
cDNA addition) are also included. The predicted sizes of the amplified
fragments are GAPDH, 299 bp; -ENaC, 546 bp; -ENaC, 632 bp; and
-ENaC, 671 bp. The results are representative of 3 independent
experiments.
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Effects of EGF, TGF-
, and PMA on amiloride-sensitive
ISC.
Exposure of isolated, perfused rabbit collecting tubules to EGF is
known to cause rapid (5-10 min) inhibition (50%) of
amiloride-sensitive Na+ absorption (21, 40,
41). The effect is thought to be mediated by an EGF-induced
increase in intracellular Ca2+; however, the long-term
effects of EGF on collecting duct ion transport are not known. Effects
of chronic (24 h) bilateral exposure to EGF, TGF-
, and PMA on
ISC of mCT1 cell monolayers are summarized in
Fig. 3. Chronic exposure to EGF or
TGF-
reduced amiloride-sensitive ISC by
50-60% with no effect on RT. EGF and
TGF-
bind to receptor tyrosine kinases, which results in activation
of a MAPK cascade (ERK1/2). Phorbol esters also activate ERK1/2, and as
can be seen in Fig. 3, chronic exposure of mCT1 monolayers to PMA
reduces amiloride-sensitive ISC by ~25%, with
no effect on RT.

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Fig. 3.
Effect on ISC of chronic exposure
of mCT1 cells to EGF, TGF- , and PMA. mCT1 cells were treated
(bilateral) for 24 h with vehicle, EGF (50 ng/ml), TGF- (20 ng/ml), or PMA (150 nM), mounted in Ussing chambers, and the
amiloride-sensitive (100 µM, apical) ISC was
measured. Values are means ± SE; n = 4-8
monolayers. *P < 0.05, treated monolayers had
significantly lower amiloride-sensitive ISC
(paired t-test).
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Dose-response relationship and time course for EGF-induced
inhibition of Na+ absorption.
The dose-response relationship for EGF-induced inhibition of
amiloride-sensitive ISC is shown in Fig.
4. The EC50 for inhibition is
~20 ng/ml with ~65% inhibition at the highest dose tested (100 ng/ml). The time course for inhibition of amiloride-sensitive ISC by EGF (20 ng/ml) is illustrated in Fig.
5. The earliest time point at which
significant inhibition of ISC is seen is 6 h. EGF inhibition of ISC is fully developed by
12 h and persists for at least 48 h with continued exposure
to EGF. The EGF-induced inhibition of ISC is
fully reversed within 24 h of removal of EGF (Fig. 5).

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Fig. 4.
Dose-response relationship for EGF-inhibition of
amiloride-sensitive ISC. Monolayers of mCT1
cells were exposed to the indicated concentration of EGF (bilateral)
for 18-24 h. The monolayers were placed in an Ussing chamber, and
the amiloride-sensitive (100 µM, apical) ISC
was measured. The values are expressed as percentage of control
amiloride-sensitive ISC in paired monolayers not
treated with EGF. Significant inhibition of ISC
was observed at doses of EGF 3 ng/ml. The amiloride-sensitive
ISC of untreated monolayers was 11.1 ± 1.0 µA/cm2, n = 18. Values are means ± SE; n = 5-18 monolayers.
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Fig. 5.
Time-course for inhibition of amiloride-sensitive
ISC by EGF. mCT1 monolayers were exposed to EGF
(20 ng/ml, bilateral) for the indicated times (2, 6, 12, 24, or 48 h). Paired monolayers, not exposed to EGF, were measured in parallel
experiments for each time point, and the values for control monolayers
did not change significantly over the time interval of these
experiments. Significant inhibition of ISC was
observed at time 6 h. In 1 set of experiments ( ;
n = 3), EGF was removed after 24 h, and the
amiloride-sensitive ISC was measured at 48 h. Values are means ± SE; n = 3-10 for
treated and control monolayers at each time point.
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Basolateral membrane
Na+-K+-ATPase
pump current is not inhibited by EGF.
Transepithelial Na+ absorption by renal collecting tubules
is mediated by amiloride-sensitive Na+ channels (most
likely ENaC) in the apical plasma membrane and ouabain-sensitive
Na+-K+-ATPase and K+ channels in
the basolateral plasma membrane. In theory, the EGF-induced decrease in
ISC could be due to an effect on apical and/or
basolateral transport proteins. Because EGF has been demonstrated to
affect Na+-K+-ATPase expression and activity in
adult rat alveolar type II cells (9), experiments were
done to determine whether inhibition of basolateral Na+
pump activity contributes to EGF-mediated reduction in
ISC. The apical plasma membrane of mCT1 cell
monolayers was rendered permeable to monovalent cations and anions by
exposure to nystatin. The ouabain-sensitive ISC
was unaffected by EGF treatment (Fig. 6). The data herein suggest that chronic exposure to EGF reduces
transepithelial Na+ absorption by means of inhibition of
amiloride-sensitive Na+ entry rather than by inhibition of
basolateral Na+-K+-ATPase activity.

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Fig. 6.
Effect of EGF on
Na+-K+-ATPase-mediated current in permeablized
mCT1 monolayers. Monolayers were mounted in Ussing chambers, and
amiloride (100 µM) was added to apical bathing solution to inhibit
spontaneous Na+ absorption and reduce the
ISC to similar levels in vehicle and EGF (20 ng/ml, bilateral, 24 h)-treated monolayers. The pore-forming
antibiotic nystatin was added to the apical bathing solution at a final
concentration of 30 µM to increase the permeability of the apical
plasma membrane. The resultant ISC
(Na+ pump current) was measured. Values are means ± SE; n = 4 pairs of monolayers.
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Phosphorylation of p42/p44 MAPK (ERK 1/2) is necessary for
EGF-mediated regulation of amiloride-sensitive ISC.
Activation of the ERK signaling cascade by EGF elicits rapid
phosphorylation of p42/p44 MAPK (ERK1/2). As illustrated in Fig. 7A, exposure of mCT1
monolayers to either EGF or PMA for 15 min resulted in a large increase
in phospho-p42/p44 MAPK compared with unstimulated cells. The total
amount of p42/p44 present in the cells was not altered. Pretreatment of
the mCT1 monolayers with PD-98059 (an inhibitor of ERK kinase)
prevented the phosphorylation of p42/p44 by subsequent exposure to
either EGF or PMA (Fig. 7, B and C, respectively).
Similarly, pretreatment of mCT1 monolayers with PD-98059 prevented both
EGF- and PMA-induced inhibition of amiloride-sensitive
ISC (Fig. 8, A and
B, respectively). Long-term exposure to EGF caused sustained phosphorylation of p432/p44 MAPK, albeit at lower levels compared with the level of phosphorylation at
shorter times. As illustrated in Fig. 9,
phosphorylated ERK1/2 was decreased at 6 h compared with 1 h
(the signal at 1 h was nearly as intense as that obtained at 15 min) but remained above the unstimulated level for up to 24 h.

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Fig. 7.
Expression and phosphorylation of ERK1/2 (MAPK) in mCT1
cells. Cells were treated as indicated, harvested and subjected to
Western blot analysis (20 µg protein/lane) for total and
phosphorylated p42/p44 (ERK1/2). A: confluent monolayers of
mCT1 cells were exposed on both sides to vehicle, EGF (20 ng/ml), or
PMA (150 nM) for 15 min. The blots were probed for phosphorylated
ERK1/2 (left) and total ERK1/2 (right).
B: confluent monolayers of mCT1 cells were exposed to
vehicle, EGF (20 ng/ml), PD-98059 (PD; 30 µM), or EGF (20 ng/ml) plus
PD-98059 (30 µM) for the indicated times. The blots were probed for
phosphorylated ERK1/2 (left) and total ERK1/2
(right). C: confluent monolayers of mCT1 cells
were exposed to vehicle, PMA (150 nM), PD-98059 (30 µM), or PMA (150 nM) plus PD-98059 (30 µM) for the indicated times. The blots were
probed for phosphorylated ERK1/2 (left) and total ERK1/2
(right). The results of these experiments are representative
of a total of 3 independent experiments.
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Fig. 8.
Effect of ERK kinase inhibitor (PD-98059) on EGF- and
PMA-induced downregulation of amiloride-sensitive Na+
absorption. Confluent monolayers of mCT1 cells were treated on both
sides with vehicle, EGF (20 ng/ml), or PMA (150 nM) with or without
pretreatment with PD-98059 (30 µM). Approximately 24 h later,
the filters were placed in Ussing chambers and the amiloride-sensitive
ISC was measured. A: results from
experiments with EGF exposure (n = 6 for each group).
B: results from experiments with PMA treatment
(n = 7 for each group). Values are means ± SE.
*P < 0.05, significantly different from
vehicle-treated monolayers.
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Fig. 9.
Long-term exposure to EGF causes sustained
phosphorylation of ERK1/2. Confluent monolayers of mCT1 cells were
treated with EGF (20 ng/ml, bilateral) for 1, 6, or 24 h. Cells
were harvested and subjected to Western blot analysis (20 µg
protein/lane) for phosphorylated p42/p44 (ERK1/2). The results of this
experiment are representative of a total of 3 independent
experiments.
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Chronic exposure to EGF reduces the steady-state level of ENaC
subunit mRNA.
We used real-time RT-PCR to quantify mRNAs for the three ENaC subunits
and for GAPDH. As illustrated in Fig.
10, chronic exposure of mCT1 cells to
EGF (20 ng/ml) for 24 h decreased the abundance (normalized to
GAPDH expression) of all three ENaC subunit mRNAs by 70-85%.

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Fig. 10.
Quantitative real-time RT-PCR of ENaC subunit mRNAs.
Confluent monolayers were treated with EGF (20 ng/ml, bilateral) for
24 h, and total RNA was isolated and reverse transcribed. PCR
amplification of the resultant cDNAs was completed by using primers
specific for GAPDH and -, -, and -ENaC. The steady-state
expression levels were normalized to GAPDH mRNA levels and expressed as
percentage of control. Values are means ± SE from 3 independent
experiments. *P < 0.05, significantly different from
untreated control monolayers.
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DISCUSSION |
Acute exposure to EGF stimulates Cl
secretion and
inhibits Na+ absorption in primary cultures of endometrial
cells (10). In T84 colon carcinoma cells, EGF inhibits
Ca2+-activated Cl
secretion
(38). EGF is known to cause acute inhibition of
Na+ absorption in isolated, perfused rabbit collecting
tubules (21, 23, 40, 41). The response is elicited when
EGF is added to the basolateral side of the tubule but not when it is
added to the luminal perfusate. The precise mechanism of inhibition is
not known; however, an increase in intracellular Ca2+ is
required. The long-term effects of EGF and EGF-like molecules on renal
ion transport are not known. Our studies with conditionally immortalized mouse collecting duct cells demonstrate that chronic, bilateral exposure to EGF or TGF-
leads to sustained inhibition of
transepithelial Na+ absorption. On removal of the EGF,
Na+ absorption is fully restored within 24 h.
Electrogenic Na+ absorption is generally regulated by
changes in apical membrane Na+ entry pathways
(11). The apical entry step for Na+ absorption
in the collecting duct principle cells is mediated in part by the ENaC.
Several lines of evidence suggest that Na+ absorption in
the immortalized collecting duct cells used for this study (mCT1) is
mediated by ENaC. First, the IC50 values for amiloride and
benzamil are similar to those reported for heterologously expressed
ENaC (33). Second, the amiloride-sensitive
ISC across mCT1 cell monolayers is insensitive
to db-cGMP, unlike the amiloride-sensitive nonselective cation channel
detected in some renal cells (8, 39). Third, mRNA for
-,
-, and
-subunits of ENaC are expressed in mCT1 cells (Fig.
2).
A previous report from studies of endometrial cells suggested that
ENaC-mediated Na+ entry is reduced by chronic exposure to
EGF (10). In contrast, Danto et al. (9) and
Kemp et al. (14) found that EGF stimulated Na+
absorption in rat alveolar type II cells. They reported that EGF either
has no effect (9) or reduced (14) ENaC mRNA
levels. They concluded that EGF increased Na+ absorption
across alveolar type II cells by means of a direct effect on
basolateral Na+ pump expression and activity. Our results
with apical permeabilization revealed that basolateral Na+
pump activity was unaffected by EGF pretreatment, therefore suggesting that apical ENaC-mediated Na+ entry is the step that is
downregulated in collecting duct cells. Furthermore, the results of
quantitative RT-PCR analysis of steady-state ENaC subunit mRNAs (Fig.
10) support the contention that chronic exposure of renal collecting
duct cells to EGF leads to transcriptional downregulation of ENaC
expression. Lin et al. (16) and Zentner et al.
(42) found that expression of an inducible effector of EGF
signaling (Raf-1 kinase) in a parotid cell line was shown to act at the
level of transcription to reduce expression of
-ENaC mRNA. The EGFR
signaling pathways that control Na+ absorption in renal
tubule cells are not known. Activation of the ERK1/2 pathway is likely
because 1) exposure to EGF leads to phosphorylation of
p42/p44, 2) pretreatment with a MAPK inhibitor (PD-98059)
prevented p42/p44 phosphorylation and downregulation of Na+
absorption, and 3) exposure of mCT1 cells to PMA resulted in phosphorylation of p42/p44 and downregulation of Na+
absorption and both effects were prevented by interruption of the MAPK
signaling pathway (PD-98059). We also observed that acute addition of
EGF (20 ng/ml) to the basolateral bathing solution of immortalized
collecting duct cell monolayers mounted in Ussing chambers causes,
after a brief delay (~5 min), a small decrease (~15%) in
amiloride-sensitive ISC that is prevented by
pretreatment with PD-98059 (data not shown). Because both the acute and
the chronic inhibitory effects of EGF on amiloride-sensitive
Na+ absorption were prevented by pretreatment with the ERK
kinase inhibitor (PD-98059) and because EGF caused both rapid and
sustained phosphorylation (albeit at a diminished level) of ERK1/2, it
is likely that the proximal portion of this signaling pathway is important for both acute and chronic regulation of ENaC function. However, it is reasonable to assume that the acute inhibitory effect of
EGF cannot be due to downregulation of ENaC expression, as appears to
be the case with long-term exposure to EGF. Thus multiple downstream
effectors of ENaC function and expression are likely involved in acute
and chronic inhibition of amiloride-sensitive Na+
absorption in renal collecting duct cells.
A role for the TGF-
/EGF/EGFR axis has been suggested in PKDs
(24). Both autosomal dominant PKD and autosomal recessive PKD involve enhanced cell proliferation, remodeling of extracellular matrix, and alterations in fluid and electrolyte transport
(2). A common finding in human and animal models of PKD is
that cystic epithelial cells exhibit inappropriate localization of the
EGFR to the apical plasma membrane, whereas in noncystic tubules
expression is restricted to the basolateral plasma membrane
(34). The presence of high levels of EGF and EGF-like
molecules in urine and cystic fluid raises the possibility that
mislocalized apical EGFRs may contribute to PKD pathophysiology.
Indeed, recent studies have shown that apical EGFRs are functional
(34) and that inhibition of EGFR tyrosine kinase activity
can dramatically reduce cell proliferation and disease severity in PKD
mice (35). By analogy, abnormal EGFR expression and apical
signaling in cystic epithelia might be predicted to cause chronic
downregulation of collecting duct Na+ absorption. On the
basis of the data presented herein, we envision one potential pathway
by which amiloride-sensitive Na+ absorption is reduced by
means of activation of an aberrant ERK1/2 signaling pathway in cystic
epithelial cells. Studies with primary cultures and/or cell lines
derived from normal and cystic collecting ducts should provide insight
into the problem (36).
 |
ACKNOWLEDGEMENTS |
The authors acknowledge helpful discussions with Cathy Carlin, Bill
Sweeney, Stephanie Orellana, and Ellis Avner and thank Mike Haley,
Elizabeth Carroll, and Mike Wilson for technical assistance.
 |
FOOTNOTES |
This study was supported by Polycystic Kidney Research Foundation Grant
99013 and National Institute of Diabetes and Digestive and Kidney
Diseases Grants P50-DK-57306 and P30-DK-27651.
Address for reprint requests and other correspondence: C. U. Cotton, 825 BRB, Case Western Reserve Univ., 2109 Adelbert Rd., Cleveland, OH 44106-4948 (E-mail:
cuc{at}po.cwru.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.
August 27, 2002;10.1152/ajprenal.00028.2002
Received 22 January 2002; accepted in final form 20 August 2002.
 |
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