Epidermal growth factor inhibits amiloride-sensitive sodium absorption in renal collecting duct cells
Departments of Pediatrics and Physiology and Biophysics, Rainbow Center for Childhood PKD, Case Western Reserve University, Cleveland, Ohio 44106-4948
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-"alpha " (TGF-"alpha " ; 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
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 "alpha " -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 "alpha " -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 "alpha " -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 "alpha " -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.
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-"gamma " . 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% "beta " -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 "alpha " -mENaC forward primer 5'-GCC AGT GCT CCT GTC A-3' and reverse primer 5'-GGG GTA CAG GGT ACC AA-3', "beta " -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 "alpha " -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.
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.
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-"alpha " , and PMA. mCT1 cells were treated (bilateral) for 24 h with vehicle, EGF (50 ng/ml), TGF-"alpha " (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).
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 (black-triangle; 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.
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.
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.
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.
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-"alpha " 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 "alpha " -, "beta " -, and "gamma " -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 "alpha " -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-"alpha " /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.
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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-"alpha " (TGF-"alpha " ; 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
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 "alpha " -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 "alpha " -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 "alpha " -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 "alpha " -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.
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-"gamma " . 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% "beta " -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 "alpha " -mENaC forward primer 5'-GCC AGT GCT CCT GTC A-3' and reverse primer 5'-GGG GTA CAG GGT ACC AA-3', "beta " -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 "alpha " -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.
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.
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.
fig-ommitted
Fig. 3. Effect on ISC of chronic exposure of mCT1 cells to EGF, TGF-"alpha " , and PMA. mCT1 cells were treated (bilateral) for 24 h with vehicle, EGF (50 ng/ml), TGF-"alpha " (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).
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 (black-triangle; 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.
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.
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.
fig-ommitted
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.
fig-ommitted
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.
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.
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-"alpha " 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 "alpha " -, "beta " -, and "gamma " -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 "alpha " -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-"alpha " /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.
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