Epidermal growth factor inhibits amiloride-sensitive sodiumabsorption in renal - 《American Journal of Physiology》美国生理学杂志干细胞之家



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发表于 2009-4-21 13:23 |只看该作者 |倒序浏览 |打印

作者：Jie-PanShen and Calvin U.Cotton作者单位：Departments of Pediatrics and Physiology and Biophysics,Rainbow Center for Childhood PKD, Case Western Reserve University,Cleveland, Ohio 44106-4948 . }' r6 S4 v: L! Y/ o. |9 p: G7 R5 w& _
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      【摘要】
   The effects of the ERK pathway onelectrogenic transepithelial Na absorption by renalcollecting duct cells were determined. Approximately 90% of theunstimulated short-circuit current (15 ± 1 µA/cm 2, n = 10) across conditionally immortalized murinecollecting duct epithelial cells (mCT1) is amiloride sensitive and islikely mediated by apical epithelial Na channels. Chronicexposure (24 h) of the epithelial monolayers to either EGF (50 ng/ml)or transforming growth factor- (TGF-; 20 ng/ml) reducedamiloride-sensitive short-circuit current by 60%. The inhibitoryeffect of EGF on Na absorption was not due to inhibitionof basolateral Na -K -ATPase, because the pumpcurrent elicited by permeabilization of apical membrane with nystatinwas not reduced by EGF. Chronic exposure of the mCT1 cells to EGF (20 ng/ml, 24 h) elicited a 70-85% decrease in epithelialNa channel subunit mRNA levels. Exposure of mCT1 cells toeither EGF (20 ng/ml) or PMA (150 nM) induced rapid phosphorylation of p42/p44 (ERK1/2) and pretreatment of the monolayers with PD-98059 (anERK kinase inhibitor; 30 µM) prevented phosphorylation of p42/p44. Similarly, pretreatment of mCT1 monolayers withPD-98059 prevented the EGF- and PMA-induced inhibition ofamiloride-sensitive Na absorption. The results of thesestudies demonstrate that amiloride-sensitive Na absorptionby renal collecting duct cells is regulated by the ERK pathway. Thispathway may play a role in alterations in ion transport that occur inpolycystic kidney disease.
      【关键词】 extracellular signalregulated protein kinase ERK/ polycystickidney disease mitogenactivated protein kinase
   INTRODUCTION

AMILORIDE-SENSITIVE ABSORPTION of Na is common to a variety ofepithelial tissues, including airway, renal collecting duct, urinarybladder, colon, and sweat and salivary glands ( 11 ). Expression of the epithelial Na channel (ENaC) isdocumented in each of these tissues, and the channel is thought to playa role in electrogenic Na absorption. ENaC belongs to thedegenerin/ENaC gene superfamily ( 5 ), and members of thedegenerin/ENaC superfamily share in common the predicted membranetopology: two hydrophobic membrane-spanning regions, intracellularNH 2 and COOH termini, and a highly conserved largeextracellular cysteine-rich loop ( 5, 15, 25, 30 ). ENaC isa heteromultimeric protein composed of some combination of threehomologous subunits: -, -, and -ENaC. Heterologous expressionof 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 potentatesamiloride-sensitive Na 100-fold ( 6, 19 ).

Clinical disorders due to the malfunction of ENaC are associated withgain- or loss-of-function mutations such as salt-sensitive hypertension(Liddle's syndrome) or pseudohypoaldosteronism type I. Liddle'smutations, characterized by hypertension ( 29 ), disrupt aCOOH-terminal proline-rich protein binding site in the - or -subunits, which cause an increase in ENaC channel number in thecell membrane, leading to excess Na reabsorption( 26, 31 ). In pseudohypoaldosteronism type I, newbornsdevelop hyponatremia, hyperkalemia, salt wasting, and elevatedaldosterone concentrations ( 7, 32 ). The transition to airbreathing after birth requires rapid clearance of fetal lung fluid( 3 ) via an amiloride-sensitive Na absorptionpathway. Evidence for a functional role for ENaC in fetal lung fluidclearance at birth is documented in -ENaC subunit knockout mice thatfail 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, aberrantregulation of ENaC expression or activity may contribute to cystformation and enlargement in polycystic kidney disease (PKD). Cystformation in renal collecting tubules is thought to involve atransition from an absorptive to a secretory epithelium and likelyinvolves downregulation of ENaC function via an as yet undeterminedpathway ( 22 ).
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Acute and chronic regulation of ENaC is known to be influenced byvarious hormones, including vasopressin and aldosterone ( 1, 13, 17 ). Furthermore, growth factors have been reported to play apivotal role in the regulation of epithelial transport independently ofcell proliferation. Keratinocyte growth factor inhibits the expressionof the -subunit of ENaC mRNA in mouse fetal lung ( 43 ).In adult rat alveolar type II cells, EGF significantly decreases theexpression of all three subunits of ENaC mRNA but increases the wholecell conductance and the density of nonselective amiloride-sensitiveNa channels ( 14 ). Treatment of both humanprimary cultures of cystic fibrosis airway cells and a cystic fibrosisnasal epithelial cell line with hepatocyte growth factor reduces theabnormally high amiloride-sensitive Na absorption observedin cystic fibrosis airway cells ( 28 ). Expression of aninducible Raf-1 kinase in a rat parotid gland cell line resulted indownregulation 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-activatedCl secretion ( 38 ). Several observations fromhuman and mouse PKD suggest that the overproduction and accumulation ofEGF in renal cysts and mislocalization of EGF receptors (EGFR) to theapical 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 Ca 2  ( 21, 23, 40, 41 ); however, the effects of long-term exposure of renalcollecting duct cells to EGF are not known. The present study wasundertaken to determine the role of the EGF/ERK pathway in modulatingamiloride-sensitive Na absorption in mouse renalcollecting duct. A conditionally immortalized mouse collecting ductcell line, mCT1, was used. The results of these studies demonstratethat amiloride-sensitive Na absorption is significantlydownregulated in renal collecting duct cells by ERK signaling.Activation of this pathway may play an important role in ion transportabnormalities associated with PKD.- D. n& o# C9 ]5 R5 G; B
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Cell culture. Experiments were carried out with a conditionally immortalized murinecollecting tubule cell line (mCT1). The details of the generation andcharacterization of the cell line were described previously( 37 ). Routine cell culture for expansion was carried outon tissue culture dishes, and cells were seeded onto either Millicell-CM filters (12 mm; Millipore, Bedford, MA) orTranswell-clear, tissue culture-treated polyester membrane filters (24 mm; Corning Costar, Cambridge, MA) for experiments. The cells weregrown in collecting tubule media consisting of a 1:1 mix of DMEM andHam'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/ltransferrin, 25 µg/l PGE 1, 2.5 mM glutamine, 50 nMdexamethasone, and 10 U/ml mouse interferon-. The cultures weremaintained at 33°C in a humidified incubator with 5%CO 2. Media were changed every other day, and cells werepassed weekly. Cells used for experiments reported here were between passages 10 and 25.4 g# Q' @' N1 `- A3 V$ N" `

Transepithelial electrical measurements. Cells were seeded (1-3 × 10 5 cells/filter) oncollagen-coated, permeable supports (12-mm Millicell-CM filters). Thefilter surface was coated with 125 µl/cm 2 calfskincollagen (Sigma) dissolved in acetic acid (7.5 mg/ml 0.2% glacialacetic acid) and allowed to dry. The collagen coating was cross-linkedby exposure to ammonium hydroxide vapors (3.5% solution) for 10 minfollowed by immersion in glutaraldehyde (2.5%) for 10 min. Thisprocedure was followed by a thorough rinsing in distilled water, 70%ethanol, distilled water, and, finally, culture media. Cell monolayersgrown on modified supports were mounted vertically in athermostatically controlled Ussing chamber equipped with gas inlets andseparate reservoirs for the perfusion of apical and basolateralcompartments. Both sides of cell monolayers were bathed with equalvolumes (usually 6-10 ml) of Krebs-Ringer HCO 3 − solution containing (in mM) 115 NaCl, 25 NaHCO 3, 5 KCl, 2.5 Na 2 HPO 4, 1.8 CaCl 2, 1 MgSO 4, and 10 glucose. The solutions were circulatedthrough the water-jacketed glass reservoir by gas lifts (95%O 2 -5% CO 2 ) to maintain solution temperature at37°C and pH at 7.4. Transepithelial voltage difference ( V T ) was measured between two Ringer-agarbridges, each positioned within 3 mm of the monolayer surface. Calomelhalf-cells connected the bridges to a high impedance voltmeter. Currentfrom an external direct current source was passed by silver-silverchloride electrodes and Ringer-agar bridges to clamp the spontaneous V T to 0. The current required [short-circuitcurrent ( I SC )] was corrected for solution andfilter series resistance. Monolayers were maintained undershort-circuit conditions except for brief 3- to 5-s intervals when thecurrent necessary to clamp the voltage to a nonzero value (usually  2mV) was measured to calculate transepithelial resistance ( R T ).# N) e9 T) R& g; J: e' q
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Western blotting and RT-PCR. Cell lysates were prepared from confluent culture filters (24 mm;Corning Costar) in lysis buffer containing 50 mMTris · HCl (pH 7.5), 0.1% IGEPAL, 2 mM EDTA, and1 mM EGTA for soluble cytosolic proteins and supplemented with 150 mMNaCl, 0.1% SDS, and 0.5% sodium deoxycholate for membrane proteins.Protein concentrations were measured by bicinchoninic acid proteinassay (Pierce, Rockford, IL). Whole cell lysates (20 µg protein) weredenatured in SDS-PAGE sample buffer containing 50 mMTris · HCl (pH 6.8), 2% SDS, 5% -mercaptoethanol, 10% glycerol, and 0.1% bromphenol blue at95°C. Proteins were separated on a 10% (for ERK1/2) SDS-PAGE gel and electrophoretically blotted onto a pure nitrocellulose transfer andimmobilization membrane (Schleicher & Schuell, Keene, NH). Membraneswere 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 toremove Tween 20, the membranes were incubated at room temperature for3 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 thenincubated 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), oncefor 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 werevisualized on X-ray film (X-O-Mat, Kodak, Rochester, NY). Molecularmass estimation of detected bands was determined by using prestainedhigh-molecular-mass protein standards (GIBCO-BRL, Life Technologies,Rockville, MD). Quantification of the intensity of the bands on theluminograms was determined with a Sci Scan 5000 densitometer. TheOS-Scan Image Analyses System density scan program (Oberlin Scientific)was used to integrate the relevant peak areas in the protein bands.
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RT-PCR of total RNA obtained from mCT1 cells was performed withsubunit-specific primers. Total RNA was purified from mCT1 cultures byRNeasy Mini Kit (Qiagen, Valencia, CA). RT-PCR was performed by usingMoloney murine leukemia virus RT system (Life Technologies) accordingto 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 reverseprimer 5'-AAA GCG TCT GTT CCG TGA TGC-3', -mENaC forward primer5'-GCC AGT GAA GAA GTA CCT CC-3' and reverse primer 5'-CCT GGG TGG CACTGG 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 forwardprimer 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 cyclerwith 94°C/1 min of denaturing, 52°C/1 min of annealing, and72°C/1 min, 30 s of elongation in each cycle. The reaction samples were run on a 1.5% agarose gel and visualized withethidium bromide. The predicted product sizes are -ENaC, 546 bp; -ENaC, 632 bp; -ENaC, 671 bp; and GAPDH, 299 bp.
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Quantitative RT-PCR. The primers used for quantitative RT-PCR were -mENaC forward primer5'-GCC AGT GCT CCT GTC A-3' and reverse primer 5'-GGG GTA CAG GGT ACCAA-3', -mENaC forward primer 5'-CCC TTC CTT GCG TCC A-3' and reverseprimer 5'-CGC TCC TGA GAC AGG A-3', -mENaC forward primer 5-CGC TGTCAC TAT CTG CA-3' and reverse primer 5'-AAG CAG GTC ACC AGC A-3', andGAPDH forward primer 5'-CGT CTT CAC CAC CAT GGA GA-3' and reverseprimer 5'-CGG CCA TCA CGC CAC AGT TT-3'. The predicted product sizesare -ENaC, 500 bp; -ENaC, 500 bp; -ENaC, 499 bp; and GAPDH,299 bp. The appropriate product sizes were confirmed by running thesamples 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/16s of elongation in each cycle.

RESULTS; J5 E+ ~% T, i) E: c# i
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mCT1 cells exhibit amiloride-sensitive I SC and express -, -, and -ENaC mRNAs. mCT1 cells, derived from murine collecting ducts, form polarizedepithelial monolayers when grown on collagen-coated permeable supports.Mean values of R T and I SC are 1,160 ± 119 · cm 2 and14.6 ± 1.1 µA/cm 2, respectively ( n = 10). After exposure to 100 µM amiloride, R T was increased to 1,510 ± 169 · cm 2 and I SC was decreased to 1.8 ± 0.1 µA/cm 2 ( n = 10). The basal I SC in these cells (Fig. 1 ) is inhibited ~90% by submicromolarconcentrations of amiloride ( K 0.5 ~ 220 nM) and benzamil ( K 0.5 ~ 20 nM).As illustrated in Fig. 2, mCT1 cells aswell as murine kidney express -, -, and -subunit mRNAs forENaC. It is likely that the amiloride-sensitive I SC represents ENaC-mediated Na absorption, a feature of collecting duct principle cells( 37 ).4 V5 G' C9 F& t5 Y0 T- A8 c

Fig. 1. Dose-response relationships for inhibition ofshort-circuit current ( I SC ) by amiloride andbenzamil. Cumulative addition of amiloride or benzamil to the apicalbathing solution of confluent monolayers of mCT1 cells causedinhibition of I SC. Greater than 90% ofspontaneous I SC was inhibited. Calculated valuesfor IC 50 were 20 and 219 nM for benzamil and amiloride,respectively. Values are means ± SE for 4 monolayers exposed tobenzamil (basal I SC = 10.5 ± 0.7 µA/cm 2 ) and 4 monolayers exposed to amiloride (basal I SC = 13.3 ± 1.3 µA/cm 2 ). Lines, fits of the data to aMichaelis-Menten-type equation.) y; @6 _$ _( I# r" D
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Fig. 2. Expression of -, -, and -epithelialNa channel (ENaC) subunit mRNA in mouse kidney and mCT1cells. Total RNA from mCT1 cells and whole mouse kidney was isolatedand reverse transcribed. PCR amplification of the resultant cDNAs wascompleted by using primers specific for GAPDH (G) and -, -, and -ENaC (,, and, respectively). Molecular weight markerlanes (M; 100-bp DNA ladder) and a negative control lane (; withoutcDNA addition) are also included. The predicted sizes of the amplifiedfragments are GAPDH, 299 bp; -ENaC, 546 bp; -ENaC, 632 bp; and -ENaC, 671 bp. The results are representative of 3 independentexperiments.
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Effects of EGF, TGF-, and PMA on amiloride-sensitiveI SC. Exposure of isolated, perfused rabbit collecting tubules to EGF isknown to cause rapid (5-10 min) inhibition (50%) ofamiloride-sensitive Na absorption ( 21, 40, 41 ). The effect is thought to be mediated by an EGF-inducedincrease in intracellular Ca 2 ; however, the long-termeffects of EGF on collecting duct ion transport are not known. Effectsof chronic (24 h) bilateral exposure to EGF, TGF-, and PMA on I SC of mCT1 cell monolayers are summarized inFig. 3. Chronic exposure to EGF orTGF- reduced amiloride-sensitive I SC by50-60% with no effect on R T. EGF andTGF- bind to receptor tyrosine kinases, which results in activationof a MAPK cascade (ERK1/2). Phorbol esters also activate ERK1/2, and ascan be seen in Fig. 3, chronic exposure of mCT1 monolayers to PMAreduces amiloride-sensitive I SC by ~25%, withno effect on R T.

Fig. 3. Effect on I SC of chronic exposureof 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 theamiloride-sensitive (100 µM, apical) I SC wasmeasured. Values are means ± SE; n = 4-8monolayers. * P I SC (paired t -test).

Dose-response relationship and time course for EGF-inducedinhibition of Na absorption. The dose-response relationship for EGF-induced inhibition ofamiloride-sensitive I SC is shown in Fig. 4. The EC 50 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 I SC by EGF (20 ng/ml) is illustrated in Fig. 5. The earliest time point at whichsignificant inhibition of I SC is seen is 6 h. EGF inhibition of I SC is fully developed by12 h and persists for at least 48 h with continued exposureto EGF. The EGF-induced inhibition of I SC isfully reversed within 24 h of removal of EGF (Fig. 5 ).) ~8 J, R7 T! R+ R) H7 F9 t% F
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Fig. 4. Dose-response relationship for EGF-inhibition ofamiloride-sensitive I SC. Monolayers of mCT1cells were exposed to the indicated concentration of EGF (bilateral)for 18-24 h. The monolayers were placed in an Ussing chamber, andthe amiloride-sensitive (100 µM, apical) I SC was measured. The values are expressed as percentage of controlamiloride-sensitive I SC in paired monolayers nottreated with EGF. Significant inhibition of I SC was observed at doses of EGF 3 ng/ml. The amiloride-sensitive I SC of untreated monolayers was 11.1 ± 1.0 µA/cm 2, n = 18. Values are means ± SE; n = 5-18 monolayers.; C8 m: I5 i$ i3 ?  p' [" i+ h8 k, r

Fig. 5. Time-course for inhibition of amiloride-sensitive I SC 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 parallelexperiments for each time point, and the values for control monolayersdid not change significantly over the time interval of theseexperiments. Significant inhibition of I SC wasobserved at time 6 h. In 1 set of experiments (; n = 3), EGF was removed after 24 h, and theamiloride-sensitive I SC was measured at 48 h. Values are means ± SE; n = 3-10 fortreated and control monolayers at each time point.# w: Y+ l+ W: s& m
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Basolateral membraneNa -K -ATPasepump current is not inhibited by EGF. Transepithelial Na absorption by renal collecting tubulesis mediated by amiloride-sensitive Na channels (mostlikely ENaC) in the apical plasma membrane and ouabain-sensitiveNa -K -ATPase and K channels inthe basolateral plasma membrane. In theory, the EGF-induced decrease in I SC could be due to an effect on apical and/orbasolateral transport proteins. Because EGF has been demonstrated toaffect Na -K -ATPase expression and activity inadult rat alveolar type II cells ( 9 ), experiments weredone to determine whether inhibition of basolateral Na pump activity contributes to EGF-mediated reduction in I SC. The apical plasma membrane of mCT1 cellmonolayers was rendered permeable to monovalent cations and anions byexposure to nystatin. The ouabain-sensitive I SC was unaffected by EGF treatment (Fig. 6 ). The data herein suggest that chronic exposure to EGF reducestransepithelial Na absorption by means of inhibition ofamiloride-sensitive Na entry rather than by inhibition ofbasolateral Na -K -ATPase activity.

Fig. 6. Effect of EGF onNa -K -ATPase-mediated current in permeablizedmCT1 monolayers. Monolayers were mounted in Ussing chambers, andamiloride (100 µM) was added to apical bathing solution to inhibitspontaneous Na absorption and reduce the I SC to similar levels in vehicle and EGF (20 ng/ml, bilateral, 24 h)-treated monolayers. The pore-formingantibiotic nystatin was added to the apical bathing solution at a finalconcentration of 30 µM to increase the permeability of the apicalplasma membrane. The resultant I SC (Na pump current) was measured. Values are means ± SE; n = 4 pairs of monolayers.. f- d" z& b6 B' I; H& J

Phosphorylation of p42/p44 MAPK (ERK 1/2) is necessary forEGF-mediated regulation of amiloride-sensitive I SC. Activation of the ERK signaling cascade by EGF elicits rapidphosphorylation of p42/p44 MAPK (ERK1/2). As illustrated in Fig. 7 A, exposure of mCT1monolayers to either EGF or PMA for 15 min resulted in a large increasein phospho-p42/p44 MAPK compared with unstimulated cells. The totalamount of p42/p44 present in the cells was not altered. Pretreatment ofthe mCT1 monolayers with PD-98059 (an inhibitor of ERK kinase)prevented the phosphorylation of p42/p44 by subsequent exposure toeither EGF or PMA (Fig. 7, B and C, respectively).Similarly, pretreatment of mCT1 monolayers with PD-98059 prevented bothEGF- and PMA-induced inhibition of amiloride-sensitive I SC (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 atshorter 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. 7. Expression and phosphorylation of ERK1/2 (MAPK) in mCT1cells. Cells were treated as indicated, harvested and subjected toWestern blot analysis (20 µg protein/lane) for total andphosphorylated p42/p44 (ERK1/2). A : confluent monolayers ofmCT1 cells were exposed on both sides to vehicle, EGF (20 ng/ml), orPMA (150 nM) for 15 min. The blots were probed for phosphorylatedERK1/2 ( left ) and total ERK1/2 ( right ). B : confluent monolayers of mCT1 cells were exposed tovehicle, EGF (20 ng/ml), PD-98059 (PD; 30 µM), or EGF (20 ng/ml) plusPD-98059 (30 µM) for the indicated times. The blots were probed forphosphorylated ERK1/2 ( left ) and total ERK1/2( right ). C : confluent monolayers of mCT1 cellswere 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 wereprobed for phosphorylated ERK1/2 ( left ) and total ERK1/2( right ). The results of these experiments are representativeof a total of 3 independent experiments.

Fig. 8. Effect of ERK kinase inhibitor (PD-98059) on EGF- andPMA-induced downregulation of amiloride-sensitive Na absorption. Confluent monolayers of mCT1 cells were treated on bothsides with vehicle, EGF (20 ng/ml), or PMA (150 nM) with or withoutpretreatment with PD-98059 (30 µM). Approximately 24 h later,the filters were placed in Ussing chambers and the amiloride-sensitive I SC was measured. A : results fromexperiments 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

Fig. 9. Long-term exposure to EGF causes sustainedphosphorylation of ERK1/2. Confluent monolayers of mCT1 cells weretreated with EGF (20 ng/ml, bilateral) for 1, 6, or 24 h. Cellswere harvested and subjected to Western blot analysis (20 µgprotein/lane) for phosphorylated p42/p44 (ERK1/2). The results of thisexperiment are representative of a total of 3 independentexperiments.

Chronic exposure to EGF reduces the steady-state level of ENaCsubunit mRNA. We used real-time RT-PCR to quantify mRNAs for the three ENaC subunitsand for GAPDH. As illustrated in Fig. 10, chronic exposure of mCT1 cells toEGF (20 ng/ml) for 24 h decreased the abundance (normalized toGAPDH expression) of all three ENaC subunit mRNAs by 70-85%.

Fig. 10. Quantitative real-time RT-PCR of ENaC subunit mRNAs.Confluent monolayers were treated with EGF (20 ng/ml, bilateral) for24 h, and total RNA was isolated and reverse transcribed. PCRamplification of the resultant cDNAs was completed by using primersspecific for GAPDH and -, -, and -ENaC. The steady-stateexpression levels were normalized to GAPDH mRNA levels and expressed aspercentage of control. Values are means ± SE from 3 independentexperiments. * P' m. K$ |. P* g2 N& p& ^* n

DISCUSSION( R, v- I6 A5 g

Acute exposure to EGF stimulates Cl secretion andinhibits Na absorption in primary cultures of endometrialcells ( 10 ). In T84 colon carcinoma cells, EGF inhibitsCa 2  -activated Cl secretion( 38 ). EGF is known to cause acute inhibition ofNa absorption in isolated, perfused rabbit collectingtubules ( 21, 23, 40, 41 ). The response is elicited whenEGF is added to the basolateral side of the tubule but not when it isadded to the luminal perfusate. The precise mechanism of inhibition isnot known; however, an increase in intracellular Ca 2  isrequired. The long-term effects of EGF and EGF-like molecules on renalion 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 oftransepithelial Na absorption. On removal of the EGF,Na absorption is fully restored within 24 h.

Electrogenic Na absorption is generally regulated bychanges in apical membrane Na entry pathways( 11 ). The apical entry step for Na absorptionin the collecting duct principle cells is mediated in part by the ENaC.Several lines of evidence suggest that Na absorption inthe immortalized collecting duct cells used for this study (mCT1) ismediated by ENaC. First, the IC 50 values for amiloride andbenzamil are similar to those reported for heterologously expressedENaC ( 33 ). Second, the amiloride-sensitive I SC across mCT1 cell monolayers is insensitiveto db-cGMP, unlike the amiloride-sensitive nonselective cation channeldetected 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 thatENaC-mediated Na entry is reduced by chronic exposure toEGF ( 10 ). In contrast, Danto et al. ( 9 ) andKemp et al. ( 14 ) found that EGF stimulated Na absorption in rat alveolar type II cells. They reported that EGF eitherhas no effect ( 9 ) or reduced ( 14 ) ENaC mRNAlevels. They concluded that EGF increased Na absorptionacross alveolar type II cells by means of a direct effect onbasolateral Na pump expression and activity. Our resultswith 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 isdownregulated in collecting duct cells. Furthermore, the results ofquantitative RT-PCR analysis of steady-state ENaC subunit mRNAs (Fig. 10 ) support the contention that chronic exposure of renal collectingduct cells to EGF leads to transcriptional downregulation of ENaCexpression. Lin et al. ( 16 ) and Zentner et al.( 42 ) found that expression of an inducible effector of EGFsignaling (Raf-1 kinase) in a parotid cell line was shown to act at thelevel of transcription to reduce expression of -ENaC mRNA. The EGFRsignaling pathways that control Na absorption in renaltubule cells are not known. Activation of the ERK1/2 pathway is likelybecause 1 ) exposure to EGF leads to phosphorylation ofp42/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 MAPKsignaling pathway (PD-98059). We also observed that acute addition ofEGF (20 ng/ml) to the basolateral bathing solution of immortalizedcollecting duct cell monolayers mounted in Ussing chambers causes,after a brief delay (~5 min), a small decrease (~15%) inamiloride-sensitive I SC that is prevented bypretreatment with PD-98059 (data not shown). Because both the acute andthe chronic inhibitory effects of EGF on amiloride-sensitiveNa absorption were prevented by pretreatment with the ERKkinase inhibitor (PD-98059) and because EGF caused both rapid andsustained phosphorylation (albeit at a diminished level) of ERK1/2, itis 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 ofEGF cannot be due to downregulation of ENaC expression, as appears tobe the case with long-term exposure to EGF. Thus multiple downstreameffectors of ENaC function and expression are likely involved in acuteand chronic inhibition of amiloride-sensitive Na absorption in renal collecting duct cells.' s' F( M2 @; r2 |! ]/ B  a2 i

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 isthat cystic epithelial cells exhibit inappropriate localization of theEGFR to the apical plasma membrane, whereas in noncystic tubulesexpression is restricted to the basolateral plasma membrane( 34 ). The presence of high levels of EGF and EGF-likemolecules in urine and cystic fluid raises the possibility thatmislocalized 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 activitycan dramatically reduce cell proliferation and disease severity in PKDmice ( 35 ). By analogy, abnormal EGFR expression and apicalsignaling in cystic epithelia might be predicted to cause chronicdownregulation of collecting duct Na absorption. On thebasis of the data presented herein, we envision one potential pathwayby which amiloride-sensitive Na absorption is reduced bymeans of activation of an aberrant ERK1/2 signaling pathway in cysticepithelial cells. Studies with primary cultures and/or cell linesderived from normal and cystic collecting ducts should provide insightinto the problem ( 36 ).
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ACKNOWLEDGEMENTS7 d/ z' o: s! O" A$ w1 R8 I5 M, J

The authors acknowledge helpful discussions with Cathy Carlin, BillSweeney, Stephanie Orellana, and Ellis Avner and thank Mike Haley,Elizabeth Carroll, and Mike Wilson for technical assistance.
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