Abnormal EGF-dependent regulation of sodium absorption in ARPKD collecting duct cells

【关键词】  cells

    Department of Pediatrics and Physiology and Biophysics, Rainbow Center for Childhood Polycystic Kidney Disease, Case Western Reserve University, Cleveland, Ohio

    ABSTRACT

    Amiloride-sensitive sodium entry, via the epithelial sodium channel (ENaC), is the rate-limiting step for Na+ absorption in kidney collecting ducts, and epidermal growth factor (EGF) inhibits Na+ transport and ENaC expression. A pathognomonic feature of polycystic kidney disease (PKD) is EGF receptor mislocalization to the apical plasma membrane and EGF/EGF receptor axis overactivity. Immunohistochemical and biochemical analysis revealed mislocalization of EGF receptor and excessive activation of the p42/44 extracellular signal-regulated protein kinase pathway (ERK1/2) in kidneys from cystic mice compared with noncystic littermates. Primary monolayer cultures of noncystic and cystic murine collecting duct principal cells were used to identify aberrant EGF-dependent ERK1/2 activation and regulation of Na+ transport associated with autosomal recessive PKD. Addition of EGF to the basolateral bathing solution of noncystic or cystic monolayers led to p42/44 phosphorylation and inhibition of Na+ transport (3035%), whereas apical EGF was effective only in monolayers derived from cystic mice. p42/44 Phosphorylation and inhibition of Na+ transport were prevented by prior treatment of the cells with an ERK kinase inhibitor. Chronic treatment (24 h) of noncystic and cystic monolayers with basolateral EGF elicited sustained inhibition of Na+ absorption (5055%) and a reduction in steady-state ENaC mRNA levels (5075%). In contrast, addition of EGF to the apical bathing solution (24 h) had no effect in noncystic monolayers but led to inhibition of Na+ transport (5060%) and decreased ENaC expression (4560%) in cystic cells. Pretreatment of the monolayers with an ERK kinase inhibitor abolished the chronic effects of EGF on Na+ transport. The results of these studies reveal that the mislocalized apical EGF receptors are functionally coupled to the ERK pathway and that abnormal EGF-dependent regulation of ENaC function and expression may contribute to PKD pathophysiology.

    mitogen-activated protein kinase; polycystic kidney disease; epidermal growth factor receptor; epithelial sodium channel; autosomal recessive polycystic kidney disease

    POLYCYSTIC KIDNEY DISEASES (PKDs) are genetic disorders characterized by formation and progressive enlargement of fluid-filled cysts in the kidney and liver (1, 8). Autosomal dominant (ADPKD) and recessive (ARPKD) forms of the disease ultimately lead to end-stage renal disease and kidney failure in both adults and children. Although the genetics of the PKDs have been defined, the pathogenesis of the disease remains unclear (12). ADPKD cysts originate from all segments of the nephron and frequently disconnect from the tubule (10), whereas ARPKD cysts are more correctly described as dilated collecting ducts (CDs) (13). The most important characteristics of disease development are neoplastic-like increase in cell proliferation (6, 18, 22) and abnormalities in fluid and electrolyte transport (36, 37, 45) that with time fill the cysts and accelerate disease progression. Cell proliferation and altered ion transport may be facilitated by a variety of factors abundantly present in luminal fluid such as growth factors [epidermal growth factor (EGF) and transforming growth factor-] (5, 20), nucleotides (31), and PGE (34) in an autocrine or paracrine fashion and by circulating hormones such as vasopressin (9).

    Cyst epithelial cells in human PKD also exhibit abnormalities in epithelial cell polarity. The EGF receptor (EGFR) localized to basolateral membrane of mature renal tubules is found in both apical and basolateral membranes of cyst epithelial cells (6, 24, 39). Similar mislocalization was observed in several murine models of PKD such as: cpk, orpk, bpk, Pkd1 mutant mice (11), and a recently developed Kif3A knockout model (15). The aberrant localization of EGFR does not reflect a generalized defect in cell polarity because aquaporins-2 and -3 (AQP2 and AQP3) and Na-K-ATPase are localized appropriately (2, 15) in the postnatal kidney. The localization of Na-K-ATPase still remains controversial as some investigators have found immunodetectable levels of the pump at the apical membrane of cystic cells, but in the mouse model of ARPKD used in these studies the Na-K-ATPase is found almost exclusively in the basolateral membrane of noncystic and cystic renal epithelial cells. Furthermore, overactivity of EGF/EGFR axis contributes to PKD pathophysiology, as EGFR inhibition slows disease progression in ARPKD models (27, 40, 42).

    Kidney CD is predominantly an absorptive epithelium and electrogenic Na+ entry into the principal cells (PC) is mediated by epithelial sodium channel (ENaC) (7, 28). ENaC is composed of three homologous subunits , , and  (3) and channel expression, trafficking, and gating are highly regulated. Although aldosterone is recognized as the major positive regulator of sodium transport in the colon and distal nephron (17, 19, 26), several signaling pathways appear to modulate ENaC-mediated Na+ transport in CDs including those activated by growth factors (EGF). In contrast to steroid hormones that increase Na+ transport, EGF inhibits CD Na+ transport by a poorly defined mechanism(s) (32, 43, 44). Interaction of EGF with its receptor elicits receptor dimerization and phosphorylation, recruitment of accessory proteins, and initiation of several downstream signaling pathways including sequential activation of Ras, Raf-1, Mek-1, and ERK1/2 kinases (30, 41). In parotid salivary epithelial cells, protein kinase C-dependent activation of ERK1/2 leads to transcriptional downregulation of the -ENaC expression (47). Furthermore, it was shown that ERK1/2 phosphorylation antagonized glucocorticoid-dependent activation of -ENaC gene transcription (16), which is evidence for a cross talk between nuclear receptor and ERK1/2 pathways. Rapid, nongenomic inhibitory effects of EGF on Na+ absorption have been reported (43); however, the precise mechanism of ENaC inhibition remains to be defined.

    The present study was undertaken to examine the effect of EGF on regulation of Na+ transport in CD PC isolated from noncystic and cystic kidneys. Short- and long-term exposure to EGF from either apical (AP) or basolateral (BL) surface was evaluated. The results of these studies demonstrate that acute addition of EGF to the BL bathing solution of noncystic or cystic CD PC stimulates phosphorylation of ERK1/2 and inhibition of amiloride-sensitive Na+ transport. In contrast, addition of EGF to the AP bathing solution had no effect on noncystic CD monolayers yet elicited robust activation of ERK1/2 and inhibition of Na+ transport in cystic cell monolayers. Long-term exposure to BL EGF was associated with a decrease in Na+ transport and steady-state ENaC mRNA expression in noncystic and cystic cells. A similar response was observed in cystic cells but not in noncystic cells when EGF was added to the AP bathing solution. These findings support the concept that mislocalization of EGFRs to the AP membrane of cystic cells allows access to activating ligand present in the luminal fluid and thereby contributes to elevated ERK1/2 phosphorylation in cystic tubules and may be responsible for enhanced cellular proliferation and ion transport abnormalities associated with PKD.

    METHODS

    CD cell isolation and primary culture. The mice used in this study were obtained by breeding the Hoxb7/EGFP transgenic line (35) with the BPK mouse model of ARPKD (25), and primary cultures of CD PC were prepared from kidneys taken from green fluorescent protein (GFP)-positive noncystic (Hoxb7/GFP-bpk/) transgenic mice and cystic (Hoxb7/GFP-bpk+/+) mice as described previously (45). Briefly, kidneys were dissected from 20- to 23-day-old cystic and noncystic GFP-positive littermates. Kidneys were washed in ice-cold sterile PBS, minced to small fragments, and incubated in 10 ml of collagenase solution (Type IV, 1 mg/ml in CT media, Sigma) for 2540 min. The digest was collected by centrifugation and small tubular fragments and cells were seeded in 10-cm tissue culture dishes and maintained in CT medium consisting of a 1:1 mixture of Dulbecco's-modified Eagle's medium and Ham's F-12 medium (Life Technologies, Rockville, MD), supplemented with 5 mg/l insulin, 2.5 mM glutamine, 25 μg/l prostaglandin E1, 1.3 μg/l sodium selenite, 5 mg/l transferrin, 1.3 μg/l triiodothyronine, 50 nM dexamethasone, 30 mg/l penicillin G, 50 mg/l streptomycin, and 5% FBS (GIBCO-BRL, Gaithersburg, MD) at 37°C. Five to 7 days later, cells were subjected to fluorescence-activated cell sorting (FACS) to collect GFP-positive CD cells. Cells were seeded on collagen-coated permeable supports (Millicell CM filter; 12-mm diameter) at a density of 2.5 x 105 cells/filter and grown in CT media containing 5% FBS, 2 ng/ml EGF, and 10 nM aldosterone for 46 days. At this point, serum and EGF were removed from CT media for at least 24 h before electrophysiological experiments or chronic treatment protocol.

    Electrophysiological studies and experimental protocol. Transepithelial bioelectric properties of CD cell monolayers were evaluated as described previously (45). Briefly, confluent monolayers were mounted in thermostatically controlled Ussing chambers equipped with gas inlets and separate apical and basolateral bath solution reservoirs. Both sides were bathed with an equal volume (10 ml) of Krebs-Ringer bicarbonate solution 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 (VT) was measured and the current required to clamp VT to 0 mV was determined. The 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 was measured to calculate transepithelial resistance (RT).

    The experimental protocol for determining the effects of acute exposure to EGF on electrogenic sodium absorption consisted of the following sequence: transfer the monolayers to Ussing chamber, 5- to 10-min period of equilibration, exposure to vehicle or PD-98059 (30 μM AP and BL) for 1525 min, addition of EGF (20 ng/ml, AP or BL) for 1520 min, and finally amiloride was added to the AP bathing solution.

    The long-term effects of EGF on sodium transport and ENaC subunit mRNA levels were determined in cultures treated for 24 h with EGF. Confluent monolayers of CD cells isolated from noncystic and cystic mice were treated by addition of EGF (20 ng/ml) to the AP or BL media either with or without an ERK kinase inhibitor (30 μM PD-98059 was added to the apical and basolateral media 30 min before the addition of EGF). On the day of analysis, the cultures used for ion transport measurements were transferred to Ussing chambers, allowed to stabilize for 1520 min, and then amiloride was added to the AP side. The cultures used for RT-PCR analysis were washed and the cells were lysed for isolation of RNA.

    Immunohistochemistry. Kidneys were dissected from 18-day-old animals (cystic and noncystic GFP-negative littermates) and one kidney was fixed in ice-cold 3.7% paraformaldehyde in PBS and the contralateral kidney was quickly frozen for Western blot analysis. Kidneys were washed for 1 h under running water, dehydrated through serial ethanol and xylene solutions, and embedded in paraffin. Tissue sections (3 μm) were cut from paraffin-embedded kidneys. Immunohistochemistry was performed on deparaffinized, rehydrated sections as previously described (45). Sections were probed with antibodies to EGFR (RDI), E-cadherin (E-cadherin; Zymed), and anti-phospho-ERK1/2 (p-ERK1/2; Cell Signaling). Secondary antibodies (from Molecular Probes) were conjugated to either Alexa Fluor 488 or Texas Red. Photomicrographs were obtained with a confocal Zeiss LSM microscope.

    To examine EGFR localization in vitro, immunohistochemistry was performed as described (45) on confluent monolayers of cystic and noncystic CD cells grown on collagen-coated permeable supports. Image stacks were acquired with a Zeiss 200 M inverted microscope equipped with a DG4 light source (Sutter Instrument) and a 12-bit CoolSnapHQ camera (Roper Scientific) under control of Metamorph v4.5 (Universal Imaging). Images were deconvolved by Autoquant's Autodeblur software (AutoQuant Imaging).

    Immunoprecipitation and Western blot analysis. Kidneys were dissected from noncystic and cystic littermates, capsula was removed, and tissues were washed with PBS and quickly frozen on dry ice. Afterwards, 1 ml of ice-cold RIPA buffer (150 mM Tris, pH 8.0, 150 mM NaCl, 1% IGEPAL, 0.1% Triton X-100, 1 mM sodium orthovanadate, 2.4 mM EDTA, 0.5 mM PMSF) plus protease and phosphatase inhibitor cocktail (according to manufacturer's instructions, Sigma) was added to the noncystic kidney and 2 ml to cystic kidney. Tissues were homogenized on ice and the lysates were cleared by centrifugation (10 min to 10,000 g). The supernatant was collected and protein concentration was determined using BCA (Pierce, Rockford, IL). EGFR was precipitated from 500 μg of cell lysate protein by 2-h incubation at 4°C with 10 μg of anti-EGFR antibody. Complexes were bound to 25 μl of protein A-agarose (Santa Cruz Biotechnology) by coincubation overnight at 4°C. Immune complexes were recovered by centrifugation (14,000 g for 20 s), washed three times with 1 ml of ice-cold lysis buffer, and prepared for SDS-PAGE and probing with anti-EGFR antibody or anti-phosphotyrosine antibody. The remaining portion of the kidney lysate (not used for IPs) was retained for SDS-PAGE and Western blot analysis of MAP kinase signaling components. Cell lysates were also prepared from primary monolayer cultures of CD cells, treated the same as described under electrophysiological studies (acute EGF exposure), for SDS-PAGE analysis of total and phosphorylated ERK1/2. Samples of kidney lysates (20 μg), primary cell culture lysates (10 μg), and protein recovered by immunoprecipitation were boiled in SDS sample buffer containing 50 mM Tris?HCl (pH 6.8), 2% SDS, 5% -mercaptoethanol, 10% glycerol, and 0.1% bromphenol blue for 10 min. The denatured proteins were separated by either 7.5 or 10% SDS-PAGE. The protein was electrophoretically blotted onto a pure nitrocellulose transfer and immobilization membrane (Schleicher & Schuell, Keene, NH). Membranes were blocked 1 h at room temperature in TBS that contained 5% dry milk (wt/vol), 0.1% polyoxyethylenesorbitan monolaurate (Tween 20), and 0.01% sodium azide or TBS-3% BSA. After a brief wash to remove the Tween 20, the membranes were incubated overnight at 4°C with specific primary antibodies [anti-EGFR (Research Diagnostics); RC20 anti-p-Tyr (BD Biosciences); anti-pRaf, anti-p-Mek, anti-p-ERK1/2, anti-ERK1/2, anti-p-Elk1 (Cell Signaling)] in TBS-5% dry milk (or TBS-3% BSA for anti-p-Tyr). The membranes were then incubated with secondary antibody (horseradish peroxidase conjugated) at room temperature for 1 h. Membranes were rinsed three times and peroxidase-labeled membranes were developed by enhanced chemiluminescence (Amersham, Arlington Heights, IL) and protein bands were visualized on X-ray film (X-O-Mat, Kodak, Rochester, NY). Molecular mass estimation of detected bands was determined by using Precision Plus Protein Standards (Bio-Rad). Quantification of the intensity of the bands on the luminograms was determined with Versa Doc Imaging Systems (model 3000, Bio-Rad). The Quantity One (version 4.4.0) density scan program (Bio-Rad) was used to analyze the relevant densitities of protein bands.

    Quantitative RT-PCR analysis of ENaC mRNA. Cystic and noncystic CD cells were grown on collagen-coated filter inserts and divided in two groups: 1) nontreated (controls) and 2) EGF treatment (20 ng/ml) for 24 h (AP or BL). Total RNA was extracted using the RNeasy Mini Kit, which includes an on-column DNase digestion with RNase-free DNase (Qiagen, Valencia, CA). The concentration and quality of mRNA were determined photometrically (260/280 nm). RT-PCR was performed by using 0.51 μg of RNA, random hexamer primers, and Moloney Murine Leukemia Virus, an RT system (Life Technologies), in 25-μl reaction volume; 25 μl out of 25 μl of cDNA reaction were used for real-time PCR amplification (RT-PCR) using DNA amplification kit (SYBER Green I, Roche Diagnostics, Indianapolis, IN). Transcript levels of the housekeeping gene, GAPDH, and ENaC -, -, and -subunits were quantified by real-time PCR on a LightCycler (Roche Diagnostics). The following primer pairs were used: mouse GAPDH forward 5'-CGT CTT CAC CAC CAT GGA GA-3', reverse 5'-CGG CCA TCA CGC CAC AGT TT-3'; mouse -ENaC forward 5'-GCC AGT GCT CCT GTC A-3', reverse 5'-GGG GTA CAG GGT ACC AA-3'; mouse -ENaC forward 5'-CTC CGA TGT TGC CAT AAA G-3', reverse 5'-TCT CTC TCT GGG TCA CAC TC-3'; mouse -ENAC forward 5'-CTC GTC TTC TCT TTC TAC ACC G-3', reverse 5'-TTC CCA CTG ATT TTC CGC-3'. Hot start PCR was performed and reactions were continued for 3540 cycles with 94°C denaturing for 5 s, 6860°C annealing for 5 s, 72°C elongation for 16 s per cycle. Water instead of cDNA was used as a control for PCR contamination and primer-dimer formation. To further eliminate possible variation due to genomic DNA contamination, primers were designed to span intron/exon boundaries. Several ENaC primer pairs were investigated and the selected ones had good efficiency and no primer-dimer formation for up to 40 cycles. The amplified products were of the predicted size as demonstrated by electrophoresis (data not shown).

    The various transcript levels were determined by using a standard curve method for the gene of interest. Briefly, duplicates of five 10-fold dilutions (103-108 copies) of full-length cDNA for mouse GAPDH, or -, -, -ENaC were included in each RT-PCR run. GAPDH and -, -, and -ENaC mRNA (copy number) were measured for each EGF-treated sample and the respective control sample. Real-time PCR analysis of all samples (nontreated and EGF-treated cystic; nontreated and EGF-treated noncystic) was performed in a single run for each transcript. Variation in cDNA concentration in different samples was corrected by using the housekeeping gene concentration-GAPDH in each sample. The relative amount of ENaC mRNA subunit (calculated as a ratio of - or - or -ENaC/GAPDH) present in EGF-treated samples was normalized to the amount of ENaC mRNA subunit (calculated as a ratio of - or - or -ENaC/GAPDH) in the control samples.

    The studies with mice were performed in accordance with the Guide for the Care and Use of Laboratory Animals of National Institutes of Health and approved by the Institutional Animal Care and Use Committee of Case Western Reserve University School of Medicine.

    Statistical analysis. All results are expressed as means ± SE and statistical significance was evaluated by either unpaired or paired Student's t-test. P < 0.05 was considered significant.

    RESULTS

    EGFR and ERK1/2 activation in noncystic and cystic kidneys. EGF/EGFR axis overactivity is a feature of cystic tubules in dominant and recessive PKD and disruption of EGFR slows disease progression in animal models of ARPKD (39, 42), but the activity of potential downstream signaling pathways such as Ras/Raf/MEK/ERK has not been evaluated. As previously described, EGFR distribution in noncystic renal tubules is primarily at the basolateral membrane and overlaps that of E-cadherin (Fig. 1, AC). There is basolateral colocalization of EGFR and E-cadherin in cystic tubules, but there is also strong apical staining in these cells (Fig. 1, DF). The localization patterns of the EGFR in CDs from noncystic and cystic kidneys were retained in polarized primary cultures of CD PC as illustrated in Fig. 7, A and C. The expression levels of EGFR are similar in cystic and noncystic kidney, but the amount of the tyrosine-phosphorylated form of EGFR is significantly elevated in cystic kidneys compared with noncystic (Fig. 1G). Immunolocalization of p-ERK1/2 in sections of noncystic and cystic kidneys revealed a high level of p-ERK1/2 in epithelial cells lining the cystic tubules with almost no detectable p-ERK1/2 in sections from noncystic kidney (Fig. 2, A and B). Furthermore, Western blot analysis of lysates from noncystic and cystic kidney shows a dramatic increase (6- to 9-fold, n = 16) in the amount of p-ERK1/2 with no difference in tot- ERK1/2 (Fig. 2C).

    In vivo phosphorylation status of additional components of the EGFR-MAP kinase signaling pathway was evaluated by Western blot analysis of kidneys from three noncystic mice and three cystic littermates. As illustrated in Fig. 3, the phosphorylated forms of MAP kinase kinase kinase (cRaf), MAP kinase kinase (MEK), and MAP kinase (ERK1/2) were significantly increased in cystic compared with noncystic kidneys. Phosphorylation of a downstream transcription factor (Elk1), known to be a substrate of active ERK1/2, was also found to be increased in cystic kidneys. A sustained high level of MAP kinase signaling might be expected to impact cellular proliferation and function in cystic epithelial cells.

    Acute effects of EGF on noncystic and cystic CD monolayers. Primary cultures of cells isolated by FACS from Hoxb7/GFP-bpk/ transgenic mice (noncystic) and Hoxb7/GFP-bpk+/+ (cystic) form polarized epithelial monolayers and express marker proteins and ion transport functions attributable to distal nephron PC. Cultures derived from noncystic mice had mean Isc of 29 ± 4.5 μA/cm2 (n = 17), due primarily (90%) to amiloride-sensitive sodium absorption. Monolayer cultures derived from cystic mice exhibited 50% lower amiloride-sensitive sodium absorption (Isc = 14.2 ± 4.8 μA/cm2; n = 18). The bioelectric properties of normal and cystic cell cultures used in the studies reported herein are similar to those reported previously (45).

    Addition of EGF (20 ng/ml) to the AP bathing solution of noncystic CD cell monolayers mounted on Ussing chambers had no effect on Isc (Fig. 4A). In contrast, addition of EGF to the BL bathing solution of noncystic CD cell monolayers elicited a monotonic decrease in Isc as illustrated in Fig. 4B. Pretreatment of the monolayer with an ERK kinase inhibitor (30 μM PD-98059, 15 min) completely prevented the EGF-induced inhibition of Isc (Fig. 4C). The data from multiple experiments are summarized in Fig. 6A.

    Apical mislocalization of the EGFR is a common feature in PKD, but the coupling to specific signal transduction pathways and the effects of apical EGF receptor signaling on cellular physiology in cystic cells have not been reported. To determine whether apical EGFRs are functional and can modulate ion transport, monolayers of cystic CD PC were exposed to EGF added to either the AP or BL bathing solution. Similar to what was observed with noncystic CD cell monolayers, addition of EGF to the BL bathing solution reduced amiloride-sensitive Isc by 35% (Fig. 5C). In contrast to the lack of response of noncystic cells to apical EGF, treatment of cystic monolayers caused inhibition of Isc (Fig. 5A) that was indistinguishable from the response to basolateral EGF (Fig. 6C). Furthermore, the inhibitory effect of apical or basolateral EGF on Isc is completely prevented by pretreatment with an ERK kinase inhibitor (30 μM PD-98059; Fig. 5, B and D). Treatment with PD-98059 has no significant effect on baseline amiloride-sensitive Isc in noncystic or cystic monolayers (Fig. 6, A and B) likely because ERK1/2 phosphorylation is very low under baseline conditions. The ion transport data from multiple experiments are summarized in Fig. 6B. In another set of experiments, EGF was added simultaneously to both apical and basolateral chambers and the inhibitory effects of EGF on Isc were not additive (data not shown). Most likely this is due to full stimulation of ERK1/2 signaling from either apical or basolateral EGF and/or to saturation of a downstream response element. The results presented in the preceding sections showed that the inhibitory effect of EGF on amiloride-sensitive Na+ current was completely abolished by pretreatment with PD-98059, presumably due to a reduction in EGF-induced ERK1/2 phosphorylation and activation. To test this, the effect EGF added to either the AP or BL bathing solution on p42/44 phosphorylation in noncystic and cystic CD cell monolayers was examined. Total and phosphorylated ERK1/2 in lysates from cystic and noncystic cell monolayers treated with vehicle, EGF (20 ng/ml; AP or BL), and EGF plus PD-98059 (30 μM; AP and BL) was measured. Whereas total ERK1/2 did not vary between the conditions, basolateral exposure to EGF dramatically increased the phosphorylation of ERK1/2 in both noncystic and cystic cells (Fig. 7, B and D). In contrast, addition of EGF to the AP side of noncystic CD cell monolayers did not increase ERK1/2 phosphorylation, but treatment of cystic CD cell monolayers elicited a robust phosphorylation of ERK1/2. Pretreatment with PD-98059 completely blocked EGF-induced ERK1/2 phosphorylation (Fig. 7, B and D).

    Modulation of electrogenic sodium transport by long-term exposure to EGF. Previous work from our laboratory demonstrated that prolonged exposure of mCT1 cells (mouse CD cell line) to EGF inhibits amiloride-sensitive Na+ absorption (32) due to a reduction in ENaC-mediated apical Na+ entry. The effects of chronic exposure to EGF in noncystic and cystic primary CD cell monolayers were determined. Confluent epithelial monolayers of noncystic or cystic cells were exposed to either apical or basolateral EGF (20 ng/ml) for 24 h and amiloride-sensitive Isc was measured. As summarized in Fig. 8A, addition of EGF to the apical side of noncystic monolayers had no effect on Isc, whereas basolateral EGF reduced Isc by 50% (28.7 ± 3.2 to 15 ± 2.6 μA/cm2; n = 4). Chronic exposure of cystic cell monolayers to EGF from either the apical or basolateral surface significantly inhibited Isc (Fig. 8B) by 6065% (control 16.7 ± 2.4, n = 4; apical EGF 8.2 ± 1.6, n = 5; basolateral EGF 6.7 ± 2.1 μA/cm2, n = 4). The inhibitory effects of EGF on amiloride-sensitive Isc were not observed in monolayers pretreated with the ERK1/2 kinase inhibitor PD-98059.

    Effects of long-term EGF treatment on -, -, and -ENaC mRNAs. The acute effects of EGF on Isc probably reflect a change in channel gating or trafficking, whereas the sustained reduction of amiloride-sensitive Isc with prolonged exposure to EGF may involve genomic regulation. To investigate the mechanism of long-term (24 h) EGF-dependent, ERK1/2-mediated ENaC regulation of sodium transport, steady-state mRNA levels of all three ENaC subunits were measured in primary monolayer cultures of noncystic and cystic CD PC. Real-time RT-PCR was used to quantify the mRNAs for the three ENaC subunits and for GAPDH. For each treatment (EGF) and control sample, GAPDH mRNA and -, -, -ENaC mRNA were measured, and the data are normalized to GAPDH and expressed as a percentage of control (no treatment). The steady-state mRNA levels for GAPDH were not changed by EGF treatment of the monolayers [the mean values for GAPDH mRNA of treated noncystic cultures were 103 ± 5% (n = 4) and cystic 104 ± 4% (n = 6)]. The results are summarized in Fig. 9. Chronic exposure of primary CD cells to EGF from the basolateral side (20 ng/ml, 24 h) decreased the abundance of all three ENaC subunits in noncystic (, , and ; Fig. 9A) and cystic cells (, , and ; Fig. 9B). In contrast, exposure to EGF from the apical side caused downregulation of ENaC mRNA in cystic (, , and ; Fig. 9B) but not in noncystic CD cell cultures (Fig. 9A).

    DISCUSSION

    The primary objective of this study was to determine the impact of mislocalized EGFRs on MAP kinase signaling and regulation of sodium absorption in primary cultures of normal and ARPKD CD cells. Previous studies demonstrated that genetic or pharmacological modulation of EGFR activity results in attenuation or slowing of the development of renal disease (27, 40, 42). Mislocalized apical receptors can bind EGF, autophosphorylate, and are mitogenic (38) but the signaling pathway(s) downstream from the mislocalized apical receptors have not been delineated. Although our studies do not demonstrate directly that EGFR signaling is responsible for in vivo activation of MAP kinase cascade in cystic kidneys, it is a likely mechanism as ERK1/2 phosphorylation was prominent in cystic tubules known to express apical EGFRs (Figs. 1 and 2). Enhanced in vivo phosphorylation of ERK1/2 in cystic kidneys (Figs. 2 and 3) suggests a role for MAP kinase signaling in cell growth and function. Activation of basolateral EGFRs in perfused rabbit CDs (21) elicited rapid inhibition of sodium absorption and chronic treatment of a mouse CD cell line with EGF caused ERK1/2-dependent inhibition of sodium transport (32). It is clear that both noncystic and cystic cells respond to exogenous EGF added to the basolateral surface with robust phosphorylation of ERK1/2 (Fig. 7) and acute inhibition of amiloride-sensitive sodium absorption (Fig. 6). Perhaps more importantly, mislocalization of EGFRs to the apical membrane in cystic cells results in acquisition of sensitivity to EGF added to the AP bathing solution (Figs. 5 and 6). Phosphorylation of ERK1/2 and inhibition of Isc by EGF are prevented by pretreatment with an ERK kinase inhibitor; therefore, MAP kinase signaling is at least necessary for acute inhibition of sodium transport by EGF. Because primary cultures of cystic CD cells do not exhibit substantially enhanced ERK1/2 phosphorylation in the absence of exogenous ligand, the dramatically higher level of phosphorylation observed in vivo (Figs. 2 and 3) is probably due to enhanced ligand availability and/or aberrant expression of apical EGFRs that can be activated by ligand constituitively present in the luminal fluid. Alteration in ligand processing, increase in EGFR-independent signaling, or decrease in phosphatase activity could contribute to enhanced MAP kinase signaling in cystic kidneys. The finding that prolonged (24 h) exposure of cultured CD cells to EGF causes a significant reduction in amiloride-sensitive sodium transport and expression of ENaC subunits is consistent with ERK1/2-dependent changes in gene expression in CD PC. Similar effects would be predicted in vivo as: 1) EGFRs are mislocalized to that apical membrane in cystic CDs, 2) EGF and EGF-like molecules are present in urine, 3) there is overactivity of EGF/EGFR axis, 4) phospho-ERK1/2 is elevated in cystic CD cells, and 5) MAP kinase cascade signaling is substantially increased in cystic kidneys. Thus abnormal regulation of amiloride-sensitive sodium transport by apically localized EGFRs seems to be a feature of ARPKD.

    The molecular mechanisms responsible for acute and chronic inhibition of sodium transport by EGF are unknown. Because both the acute and chronic inhibitory effects of EGF are prevented by pretreatment with an ERK kinase inhibitor, ERK1/2 phosphorylation is required for EGF-dependent regulation of ENaC activity. The characteristic rapid stimulatory effect of EGF on ERK1/2 phosphorylation and resultant inhibition of amiloride-sensitive Isc suggest a change in ENaC gating (i.e., decrease in channel open probability, perhaps due to phosphorylation of the channel or a regulatory protein) or an increase in endocytosis and decrease of the number of active channels present in apical membrane. Indeed, ERK1/2-dependent phosphorylation of ENaC near the PY motif present in the COOH terminus of the - and -subunits (33) has been suggested to increase its affinity for a ubiquitin ligase implicated in channel retrieval from the plasma membrane (4, 29).

    The inhibitory effect of long-term exposure to EGF is probably mediated by transcriptional downregulation or a decrease in mRNA stability as EGF leads to a decrease in steady-state mRNA levels for all three ENaC subunits. Sustained ERK1/2 activity may interfere with nuclear receptor function (glucocorticoid or mineralocorticoid receptor) by either direct phosphorylation of nuclear receptors (14) or by activation of a repressor of the ENaC transcription complex (46). Additional studies will be required to define the precise mechanisms of acute and chronic regulation of ENaC activity by EGF.

    We reported previously that primary cultures of CD PC isolated from normal and BPK mice differ primarily in the magnitude of amiloride-sensitive sodium absorption, with 50% reduction in cystic monolayers. The molecular basis for the lower rate of amiloride-sensitive sodium transport in the cystic cells is not known. However, synthesis of EGF or EGF-like ligands may be increased in primary cultures of cystic cells or cystic cells may be more sensitive to autocrine/paracrine regulation by released ligands, but there is not an obvious increase in ERK1/2 phosphorylation in cystic cell monolayers under basal in vitro conditions (Fig. 7).

    Many of the epithelial cysts present in advanced ADPKD lack upstream connections and fluid accumulation must be driven by net secretion of salt and water (36, 37). Because ADPKD is a slowly progressing disease and cysts are thought to form at multiple sites along the nephron, it is difficult to ascertain with certainty the segment of origin of a particular cyst and document changes in ion transport that accompany cyst development. In contrast, ARPKD progresses rapidly, and the cysts are derived primarily from collecting tubules and actually represent dilated nephrons with upstream and downstream connections rather than anatomically isolated cysts. Net fluid secretion is not required for cyst enlargement, as an increase in secretion or a decrease in absorption would favor retention of luminal fluid and enlargement of cystic tubules in face of destruction of kidney architecture and pseudoobstruction. We recently reported that primary cultures of noncystic and cystic CD PC maintained under basal conditions (i.e., without exogenous EGF) had similar Cl-secretory responses to elevation of cAMP or calcium (45). Elimination of a cAMP-regulated Cl channel, implicated in ADPKD fluid secretion, did not affect renal cystic disease in a murine model of ARPKD (23). Non-CFTR Cl channels could make a significant contribution to in vivo fluid secretion that is not apparent when cells are placed in primary culture (e.g., hyperactivity of the EGF/EGFR axis and enhanced ERK1/2 activation may promote fluid secretion). These observations suggest that abnormal regulation of sodium reabsorption, perhaps in combination with enhanced Cl secretion, would be expected to contribute to luminal fluid retention and tubule dilatation in ARPKD.

    GRANTS

    This work was supported by PKR Foundation Grant 99013 and National Institutes of Health Grants P30-DK-27651 and P50-DK-57306.

    ACKNOWLEDGMENTS

    The authors gratefully acknowledge helpful discussions with C. Carlin, B. Sweeney, and E. Avner and thank M. Haley, E. Carroll, and M. Wilson for expert technical assistance. We also thank F. Costantini (Columbia University) for providing the HoxB7/GFP mouse line.

    FOOTNOTES

    The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

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