Functional activity of epidermal growth factor receptors in autosomal recessive polycystic kidney disease

Department of Pediatrics, Rainbow Babies and Children's Hospital, and Case Western Reserve University, Cleveland, Ohio 44106-6003

	ABSTRACT

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Evidence from a number of laboratories suggests a potential role for the epidermal growth factor (EGF)-transforming growth factor--epidermal growth factor receptor (EGF-R) axis in promoting epithelial hyperplasia and cyst formation in autosomal recessive polycystic kidney disease (ARPKD). As previously reported, in the C57BL-6Jcpk/cpk (CPK), BALB/c-bpk/bpk (BPK), and C3H-orpk/orpk (ORPK) murine models of ARPKD, as well as in human ARPKD and human ADPKD, the EGF-R is mislocated to the apical surface of cystic collecting tubule (CT) epithelial cells. The present studies demonstrate that cells from cystic and control CTs can be isolated and that these cells maintain their in vivo EGF-R phenotype in vitro. Domain-specific high-affinity ligand binding was assessed by standard Scatchard analysis, and selective ligand stimulation of apical vs. basolateral EGF-R in these cells was followed by measurement of receptor autophosphorylation and determination of cell proliferation. These studies demonstrate that in vitro apically expressed EGF-Rs exhibit high-affinity binding for EGF, autophosphorylate in response to EGF, and transmit a mitogenic signal when stimulated by the appropriate ligand.

cyst formation; epithelial hyperplasia; receptor autophosphorylation

	INTRODUCTION

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AUTOSOMAL RECESSIVE polycystic kidney disease (ARPKD) is one of a number of human genetic and acquired diseases in which renal cysts are a central pathological feature. ARPKD has an incidence of 1 in 10,000 to 1 in 40,000, has a mortality of 40-65% in the newborn period, and accounts for ~5% of all end-stage renal disease in children (39). Although ARPKD has a wide spectrum of clinical and histopathological manifestations, two features of the disease are invariant: 1) progressive dilatation of the renal collecting duct, leading to eventual destruction of normal renal parenchyma, and 2) biliary ductal ectasia and dysgenesis associated with portal tract fibrosis (39). Utilizing positional cloning, Zerres et al. (45) have linked the gene responsible for ARPKD to chromosome 6p21-cen. Additional studies (13) suggest a lack of genetic heterogeneity in ARPKD. However, the gene associated with ARPKD has yet to be identified.

The significant morbidity and mortality produced by ARPKD and other types of renal cystic disease have provoked intensive investigation into the mechanisms of renal cyst formation and growth. These experimental studies have identified tubular hyperplasia as a necessary factor in cyst development and growth. The results of mathematical modeling suggest that tubular hyperplasia, in concert with any additional factors such as fluid secretion, obstruction, or abnormal tubular basement membrane compliance, is sufficient to explain the observed kinetics of cyst enlargement in polycystic kidney disease (PKD) (4). Evidence from a number of laboratories demonstrates a potential role for the epidermal growth factor (EGF)-transforming growth factor- (TGF-)-epidermal growth factor receptor (EGF-R) axis in promoting epithelial hyperplasia and cyst formation and enlargement. These studies demonstrate that 1) EGF and TGF- are cystogenic in a variety of in vitro systems (3, 29, 32), 2) cystic kidneys have increased TGF- mRNA expression (19), 3) renal cyst fluid from ADPKD, ARPKD, and murine and rat PKD models contain multiple EGF or EGF-like peptides in mitogenic concentrations (23, 25, 36, 41), 4) transgenic mice that overexpress TGF- develop renal cystic disease (21), and 5) EGF-R is overexpressed and mislocated to the apical surface of cystic tubular epithelium in human ADPKD, human ARPKD, and in the murine models of ARPKD [CPK, BPK, and ORPK (formerly TgN737Rpw)] (2, 30, 33, 42).

Clear delineation of the cellular pathophysiology involved in renal cystogenesis is a prerequisite for the development of rational treatment strategies. Given the hypothesized role for altered EGF-R expression in cyst formation, we sought to characterize the functional and signaling potential of abnormally expressed apical EGF-R in isolated collecting tubule (CT) cells from three murine models of ARPKD as well as human ARPKD kidneys. In these studies, immunolocalization of EGF-R in intact kidneys was followed by isolation of cystic and control CT cells. Domain-specific high-affinity ligand binding was assessed by standard Scatchard analysis, and selective ligand stimulation of apical vs. basolateral EGF-R in these cells was followed by measurement of receptor autophosphorylation and determination of cell proliferation. The results demonstrate that cystic ARPKD epithelia express functional apical EGF-Rs that bind ligands, autophosphorylate, and are mitogenic.

	MATERIALS AND METHODS

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Source of Cells

The C57BL/6Jcpk/cpk (CPK) model. The CPK murine model of ARPKD has been well characterized (1, 34). The CPK strain, as well as C57BL/6J control mice, was originally obtained from Jackson Laboratories and was maintained through controlled breeding in our laboratory.

The BALB/c-bpk/bpk (BPK) model. The BPK model, which arose as a spontaneous mutation in an inbred colony of BALB/c mice, has been recently characterized in our laboratory (26, 27). Inbred BALB/c wildtype mice were obtained from Simonsen (Gilroy, CA) and used as controls.

C3H-orpk/orpk (ORPK) model. The ORPK model, which arose by insertional mutagenesis, has been recently characterized in our laboratory (24, 43). ORPK mice were obtained from the Oak Ridge National Laboratories (Oak Ridge, TN). Noncystic littermates were used as controls for this study.

Human ARPKD kidneys. Fresh surgical samples were obtained within 30 min of kidney removal from five patients undergoing unilateral or bilateral nephrectomy. Patients were diagnosed by classic clinical and radiographic criteria and confirmed by histopathological tissue analysis of kidney and liver specimens. Four age-matched human control CT cell lines were kindly provided by Dr. Patricia Wilson (Mount Sinai School of Medicine, New York). Control CT cells were isolated as previously described (41).

Immunohistochemistry

Intact kidneys. The cellular localization of the EGF-R was determined by immunohistochemical staining of CPK and C57, BPK and BALB/c, ORPK and C3H, and human ARPKD and control sections with rabbit antiserum to the EGF-R (see Antibodies below). The methodology of fixation, embedding, and staining of kidney specimens has been described (37). Briefly, fresh tissue was fixed in 4% paraformaldehyde for 30 min, rinsed, and dehydrated through a series of graded acetone and embedded in Immunobed. Sections were cut, etched, trypsinized, and incubated overnight with primary antibody at 4°C (37). After primary and incubation, the sections were brought to room temperature, washed 2× for 10 min, incubated with a peroxidase-conjugated secondary antibody for 2 h at room temperature, and the chromagen was developed with 3,3'-diaminobenzidine (DAB)/H₂O₂.

Cell Isolations

The cell isolation procedure utilized is a modification of that devised by Van Adelsberg et al. (38). It takes advantage of the lectin specificity of cystic and noncystic CT cells [Dolichous biflorous agglutinin (DBA) reactivity for murine CT cells and peanut agglutinin (PNA) for human CT cells] as well as the predominant cell type [principal cells (PC)] present in CT cysts (1, 26, 42). With the use of a sterile technique, human and murine kidneys were removed, sliced into 150-µm slices, and incubated for 30 min in 1.5% (wt/vol) collagenase type IV in Hanks' balanced salt solution (HBSS) at 37°C. Small clumps were dispersed by aspiration through a 21-gauge needle. The dispersed cells were centrifuged at 800 g for 5 min, washed with ice-cold CT culture medium (see CT Cell Culture Medium) containing 10% fetal bovine serum (FBS) and recentrifuged as above. The resultant cell pellet was washed with 5 mM glucose in phosphate-buffered saline (PBS), centrifuged, and resuspended in 5 mM glucose-PBS. The cell suspension was dispersed onto lectin-coated plates (see below) and incubated for 30 min at 4°C. Unbound cells were removed by vigorous washing with ice-cold 5 mM glucose-PBS. Attached cells were eluted by adding 10 ml of 150 mM galactose in PBS and incubating for 5 min. The eluted cells were washed with 5 mM glucose-PBS and resuspended in CT culture medium. PCs were isolated from this cell population by panning the resuspended cells over F13/0121-coated dishes. F13/0121 is a monoclonal antibody to a PC surface antigen that cross-reacts with both human and murine tissue and was kindly provided by Dr. Geza Fejes-Toth (9). The cells were incubated for 30 min at 37°C, the medium removed, and the plate washed vigorously three times with room temperature CT cell culture medium. Cells that remained attached were then cultured for future experiments.

Cell culture plates were coated with the lectin DBA for murine CT cells or with Arachis hypogaea (PNA) for human CT cells by overnight incubation at 4°C with a sterile 10 µg/ml solution of lectin in PBS. F13/0121-coated plates were prepared by covering the bottom of the cell culture dish with concentrated cell culture supernatant for 2 h. Before use, the plates were rinsed two times with sterile PBS.

CT Cell Culture Medium

The basal medium consisted of equal volumes of Dulbecco's modified Eagle's medium and Ham's F-12 medium supplemented with insulin (8.3 × 10⁷ M), prostaglandin E₁ (7.1 × 10⁸ M), selenium (6.8 × 10⁹ M), transferrin (6.2 × 10⁸ M), triiodothyronine (2 × 10⁹ M), and dexamethasone (5.09 × 10⁷ M). All culture reagents, including EGF and bromodeoxyuridine (BrdU), were purchased from Sigma (St. Louis, MO).

Transwell Cultures

After the second passage of the isolated CT cells, the cells were cultured on Transwell tissue culture inserts to provide domain-specific access to monolayer cell surface domains (apical vs. basolateral). Monolayer tightness was assessed by [³H]inulin (Amersham Life Science, Arlington Heights, IL) incubation on the apical or basolateral compartment for 4 h at 4°C and by sampling the opposite compartment for [³H]inulin leakage. Leakage of <2% was considered a tight monolayer.

¹²⁵I Ligand Binding Assay

The binding assay was performed according to a modification of the procedure of Breyer et al. (6). Binding of EGF was performed on either the apical or basolateral cell surface for 4 h at 4°C. Incubation at 4°C prevents internalization of the receptor, and preliminary time course binding assays revealed equilibrium binding occurred at 4 h at 4°C (data not shown). ¹²⁵I-EGF labeled to a specific activity of 100 µCi/µg of EGF was purchased from Amersham Life Science. Total ligand binding was determined by incubation of either the apical or basal cell surface with ¹²⁵I-labeled EGF (5-100 ng). Nonspecific binding was determined by incubation of a paired cell monolayer with equal-labeled EGF and a 500-fold excess of unlabeled EGF. Cell monolayers were washed three times in 4°C PBS and dissolved in 1 N NaOH, and radioactivity was counted in a Beckman 5000 G gamma counter. Specific binding was calculated as total binding (¹²⁵I-EGF) minus nonspecific binding (¹²⁵I-EGF plus 500-fold excess unlabeled EGF). Triplicate values for each concentration were determined, and binding characteristics were determined by standard Scatchard analysis (18).

Demonstration of Receptor Tyrosine Kinase Activity

Activated receptor was identified by measuring specific receptor autophosphorylation through modifications of the procedures of Honegger et al. (16) and Donaldson and Cohen (7). Monolayers with [³H]inulin-confirmed tight junctions were incubated with 20 ng/ml of EGF on either the apical or basolateral cell surface under equilibrium conditions (4 h at 4°C). These conditions were established by evaluation of a dose-response curve, utilizing varying concentrations of EGF as well as varying incubation times. Monolayers were then disrupted with ice-cold RIPA buffer, containing Nonidet P-40 (1%), sodium deoxycholate (0.5%), SDS (0.1%), aprotinin (100 µg/ml), leupeptin (5 µg/ml), pepstatin (50 µg/ml), EDTA (1 mM), phenylmethylsulfonyl fluoride (100 µg/ml), and sodium orthovanadate (1 mM). Cellular debris was pelleted by centrifugation, the supernatant transferred to clean conical microfuge tubes, protein content determined by the method of Bradford (5), and equal amounts of total protein immunoprecipitated with 4.0 µg of anti-EGF-R for 2 h at 4°C. After incubation with anti-EGF-R, 25 µl of protein A/G plus agarose (Santa Cruz Biotechnology, Santa Cruz, CA) was added to each supernatant sample to pull down resultant antibody-antigen complexes. The samples were incubated, with gentle rocking, overnight at 4°C. Immunoprecipitates were collected by centrifugation, washed, and processed for SDS-PAGE in equal volumes.

Immunoprecipitates were stored at 70°C until resolved under reducing conditions on 7.5% SDS-PAGE. Gels were loaded with equal volumes of immunoprecipitated sample per lane for SDS-PAGE, electrophoretically transferred to nitrocellulose, and probed with anti-phosphotyrosine (PY-20 or RC-20-HPRO, Transduction Laboratories, Lexington, KY) and detected by enhanced chemiluminescence (ECL, Amersham Life Science).

Domain-Specific Stimulation of Cell Division by EGF

Domain-specific EGF stimulation of cell division was evaluated by a modification of a previously described protocol for BrdU uptake (31). [³H]inulin-confirmed tight monolayers were stimulated by EGF (20 ng/ml) on either the apical or basolateral surface, and 10 µM BrdU (well in excess of media thymidine content) was added to the basolateral compartment. After 4-h incubations, the cells were rinsed and fixed for 15 min with ice-cold methanol, and cellular uptake of BrdU was identified by staining with alkaline phosphatase conjugated to a monoclonal anti-BrdU antibody (Boehringer Mannheim, Indianapolis, IN). Three monolayers stimulated from the apical surface and three monolayers stimulated from the basolateral surface were evaluated for each cell type. Data are expressed as percentage of labeled cells after EGF stimulation above basal, nonstimulated labeling rates.

Antibodies

Anti-EGF-R antibody RK-2, a rabbit polyclonal, was kindly provided by Dr. Ben Margolis (20) and used for immunohistochemistry and immunoprecipitation of both murine and human tissue. Additional antibodies were used to confirm the specificity of RK-2 in the tissues utilized. Monoclonal anti-EGF-R (clone F4, Sigma) was used for immunohistochemistry and immunoprecipitation of human tissue only. A rabbit polyclonal anti-EGF-R (Santa Cruz SC-03) was used for immunoprecipitation and immunohistochemistry of murine and human tissue. Anti-EGF-R 8506 was used for immunoprecipitation of human EGF-R. Anti-EGF-R 8506 antibody was generated by harvesting supernatant from a hybridoma cell line (cell line 8506; American Type Culture Collection, Rockville, MD) and by purifying the monoclonal antibody by elution from a column that specifically binds IgG (32). Supernatant from hybridoma cell line F13/0121 was generated in an identical manner as noted for HB 8506 described previously (32). Purified IgG was used for immunohistochemistry, and concentrated supernatant (at least 10-fold) was used to coat culture plates for cell selection.

Monoclonal anti-phosphotyrosine PY20 (1/800) and a recombinant conjugated anti-phosphotyrosine RC-20: HRPO (1/1,600) were used in Western analysis of activated EGF-R. Both were obtained from Transduction Laboratories.

Antibodies to cytokeratin and vimentin were purchased from Sigma.

	RESULTS

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Immunohistochemical Localization of the EGF-R

Control (C57, BALB/c, C3H, and human) and cystic (CPK, BPK, ORPK, and human ARPKD) CTs were identified by their lectin specificity, and the cellular localization of the EGF-R was determined in serial sections by immunohistochemistry. At all ages examined, control kidneys (C57, BALB/c, and human control) demonstrated only basolateral localization of the EGF-R in lectin-identified CTs without apical EGF-R expression. A representative example of control tissue EGF-R localization is shown in Fig. 1A. In contrast, CPK, BPK, ORPK, and human ARPKD kidneys demonstrated apical as well as basolateral expression of EGF-R in all cystic CTs as shown in Fig. 1B-1E.

View larger version (170K): [in this window] [in a new window]   Fig. 1.   Immunohistochemical localization of EGF-R in BALB/c control day 14 kidney (A), CPK cystic kidney (B) , BPK cystic kidney (C), ORPK cystic kidney (D), and human autosomal recessive polycystic kidney disease (ARPKD) kidney (E). See MATERIALS AND METHODS section for complete description of models. A: representative epidermal growth factor receptor (EGF-R) localization in normal collecting tubules (CTs). B-E: lectin-identified cystic CT cysts demonstrate apical expression and basolateral expression of EGF-R. A is stained with RK-2 anti-EGF-R (1/100), B and D with RK-2 anti-EGF-R (1/300), C with Santa Cruz anti-EGF-R (1/100), and E with Sigma F4 anti-EGF-R (1/400). Noncystic CTs in A-E demonstrate only basolateral staining pattern of EGF-R. Magnification, ×450.

Cell Isolation

Day 14 control and cystic kidneys from CPK and BPK and day 45 control and cystic kidneys from ORPK murine models of ARPKD were used to isolate CT cells for culture and analysis. At this time point, there was still a considerable amount of normal renal parenchyma, and the majority of the cyst was of CT origin. All DBA-positive cysts were also F13/0121 positive, identifying them as predominantly composed of PCs.

Two day 14 cystic kidneys yielded approximately 6 × 10⁵ cells and approximately twelve day 14 control kidneys were utilized to achieve the same cellular yield. After the second passage, the isolated cystic PCs were 93% DBA positive and 88% F13/0121 positive, and the control cells were 89% DBA positive and 78% F13/0121 positive. Both control and cystic PCs were 100% positive for cytokeratin and negative for vimentin.

Human ARPKD cells were isolated from fetal and 2-mo-old kidneys. After the second passage, these cells were 87% PNA positive, 84% F13/0121 positive, 95% cytokeratin positive, and 100% negative for vimentin. Age-matched control CT cells were 92% PNA positive, 76% F13/0121 positive, 96% cytokeratin positive, and 100% vimentin negative.

¹²⁵I-EGF Binding Assay

¹²⁵I-EGF domain-specific binding in Transwell-cultured control and cystic cells detected specific binding, which could be displaced by excess cold EGF, in the basolateral domain of all control and cystic cells. Figure 2 shows the domain-specific binding data and Scatchard analysis for the CPK model (A), BPK model (B), ORPK model (C), and human ARPKD (D). Table 1 depicts the results of the Scatchard analysis of these data for each cell type. High-affinity binding was detected in the basolateral domain of control and cystic cells from CPK, BPK, ORPK, and human ARPKD. However, apical domain, high-affinity ligand binding was detected only in cystic cells from these four models. There was no specific binding detected in the apical domain of any control cells.

View larger version (33K): [in this window] [in a new window]   View larger version (33K): [in this window] [in a new window]   Fig. 2.   Domain-specific binding (BL, basolateral binding; A, apical binding) curve of 125I-epidermal growth factor (EGF)-stimulated isolated CT cells. Specific binding (nM) vs. label added is plotted for CPK model (A; CPK = cystic CT cells, C57 = control CT cells), BPK model (B; BPK = cystic CT cells, BALB/c = control CT cells), ORPK model (C; ORPK = cystic CT cells, C3H = control CT cells), and human ARPKD model (D). Curves generated from Scatchard analysis are also shown on right. Results of Scatchard analysis of these data are shown in Table 1.

View this table: [in this window] [in a new window]   Table 1.   Scatchard analysis of domain-specific 125I-EGF binding in second passage control and cystic CT cells from each model

Demonstration of Activated Receptor (Receptor Autophosphorylation)

After detection of high-affinity EGF binding receptors in the apical domain of isolated cystic CT cells from all four experimental models, we sought to determine if this binding resulted in receptor activation (i.e., receptor autophosphorylation) (35). Control and cystic cells from [³H]inulin-confirmed tight monolayers were stimulated on the apical or basolateral cell surface with EGF (20 ng/ml). After 4 h incubations at 4°C to achieve equilibrium binding without receptor internalization, the EGF-R was immunoprecipated, resolved by SDS-PAGE, blotted to nitrocellulose, and probed with anti-phosphotyrosine and detected by ECL (Amersham Life Sciences). As seen in Fig. 3, A-D, stimulation of the basolateral domain of C57, CPK, BALB/c, BPK, C3H, ORPK, human control, and human ARPKD cells all resulted in a detectable level of phosphorylated EGF-R. Stimulation of the apical domain with EGF resulted in detectable phosphorylated receptor only in cystic cells (CPK, BPK, ORPK, and human ARPKD). Apical EGF stimulation of control cells failed to detect phosphorylated receptor even in human control cells where nonspecific ¹²⁵I-EGF binding was detected. These results demonstrate that the EGF-Rs present in the apical domain of isolated cystic CT epithelial cells autophosphorylate in response to EGF. This autophosphorylation of EGF-R is a prerequisite to signal transduction and suggests that these cells are capable of initiating a signaling cascade (35).

View larger version (70K): [in this window] [in a new window]   Fig. 3.   Western analysis of phosphorylated EGF-R after domain-specific stimulation with EGF. After stimulation, EGF-R was immunoprecipitated, resolved by SDS-PAGE, transferred to nitrocellulose, probed with anti-phosphotyrosine antibody, and visualized with enhanced chemiluminescence. Phosphorylated receptor was detected after BL stimulation in both cystic and control cells. There was no detectable phosphorylated receptor after apical (A) stimulation of control cells. Apical stimulation produced detectable phosphorylated receptor only in cystic cells derived from BPK (A), CPK (B), ORPK (C), and human ARPKD kidneys (D).

BrdU Uptake After Domain-Specific EGF Stimulation

To determine if apical, stimulated EGF-Rs are competent to initiate signal transduction events leading to mitogenesis, BrdU uptake after domain-specific EGF stimulation was assessed. As shown in Table 2, EGF stimulation of basolateral domains of all cells tested showed increased BrdU labeling. EGF stimulation of the apical domain of control cells demonstrated no significant difference in labeling index compared with the basal labeling rate. However, EGF stimulation of apical EGF-R in cystic cells led to a twofold increase in BrdU labeling index compared with the basal or nonstimulated labeling index.

View this table: [in this window] [in a new window]   Table 2.   BrdU labeling index following domain-specific EGF stimulation as percentage of basal labeling

These data establish that isolated cystic CT cells with apical EGF-Rs are able to mediate a signaling cascade that results in increased cell division.

	DISCUSSION

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Mathematical modeling of cyst formation and growth has indicated that epithelial hyperplasia is a necessary element in the formation and growth of cystic lesions in PKD. The mechanism behind this hyperplastic expansion of renal tubular epithelium is as yet unclear. The present studies suggest that an abnormal EGF-TGF--EGF-R autocrine or paracrine loop may mediate this proliferative expansion of CT epithelium in ARPKD.

Work from a number of laboratories has suggested that there may be specific sorting abnormalities of a number of proteins in PKDs. (4, 42). It is unlikely that abnormal EGF-R expression represents a general sorting defect because, as previously reported, other polarized proteins such as E-cadherin and ZO-1 and GP-135 are present in their proper cellular location in cystic epithelium (2, 30). Studies in which EGF-R is overexpressed in MDCK cells suggest that apical protein expression may be a manifestation of increased protein production (15). Thus it is likely that the EGF-R is initially sorted to both apical and basolateral cell membranes and that steady-state expression may reflect different rates of turnover in the microenvironment of each respective cellular domain (15). This is consistent with data reported for sorting of the sodium pump (28). These data are of particular interest given the recent preliminary report that the product of the Tg 737 gene, whose mutation produces the ORPK model, may interact with sorting nexins, which are critical determinants of EGF-R turnover in cell membranes (44).

Apical EGF-R expression noted in the current studies may be a function of the partially differentiated phenotype of cystic epithelium (8, 11). Gattone et al. (10) suggested that this immature phenotype is due to defective EGF production reported in murine ARPKD. However, additional studies have shown that although EGF production is decreased in murine cystic animals, cystic fluid possesses mitogenic quantities of EGF in concentrations higher than urine from cystic animals (17, 42). This suggests that elevated EGF concentrations within cysts are due to sequestration or localized production of ligand within the cyst. Gattone et al. (12) also reported that administration of EGF to CPK animals improved renal function and prolonged survival. These data are difficult to assess in light of the lack of morphological improvement in treated cystic animals and published reports that EGF increases GFR in animals and humans with chronic renal failure regardless of the etiology (14). In addition, these same authors report that overexpression of TGF- in transgenic mice is associated with renal cystic disease (21).

The current studies demonstrate that a consistent CT phenotype is seen in murine and human ARPKD. The current studies also demonstrate that apical EGF-R expression may be physiologically relevant in that such receptors bind ligands, autophosphorylate, and mediate cell proliferation. Apical expression of an active EGF-R in cystic CT epithelia suggests a mechanism by which a stimulatory EGF-TGF--EGF-R autocrine-paracrine loop may be active in vivo. Preliminary investigations reveal that BPK cystic fluid can activate apically expressed EGF-Rs and transmit a mitogenic signal through these apical receptors at a magnitude comparable with EGF (14).

An essential role for EGF-R activity in cyst formation has also been established in vivo by crossing a hypomorphic EGF-R allele [waved-2 (wa-2)] into mice carrying the recessive orpk (Oak Ridge Polycystic Kidneys) mouse mutation (33). The wa-2 mutation is due to a single amino acid change in the EGF-R and results in a decreased tyrosine kinase activity of the EGF-R (22). Animals homozygous for both mutant genes (orpk and wa-2) demonstrated a marked reduction in cyst formation that was directly correlated with tyrosine kinase activity in the double mutants (33).

The results of this study do not address the specific mechanisms by which a mutated murine or human ARPKD gene leads to qualitative and quantitative abnormalities in EGF-R expression. The fact that the cpk gene, the bpk gene, and the orpk gene are on different chromosomes and that none of these genes is syntenic with the human ARPKD gene indicates that abnormal CT EGF-R expression is a common cellular phenotype downstream from a number of different primary gene mutations (2, 26, 30, 42).

Apical EGF-R expression in cystic CT lesions provides the basis for the development of innovative therapeutic approaches to ARPKD that target the abnormally expressed EGF-R (32). In addition, future studies of the specific mechanisms of EGF-R signaling from the apical cell surface may provide new insights into the cell biology of ARPKD.

	ACKNOWLEDGEMENTS

This study was supported by funding from the Polycystic Kidney Research Foundation and National Institute of Diabetes and Digestive and Kidney Diseases Grants DK-44875 and DK-51068.

	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. §1734 solely to indicate this fact.

Address for reprint requests: E. D. Avner, Director of Pediatrics, Rainbow Babies and Children's Hospital, 11100 Euclid Ave., Cleveland, OH 44106-6003.

Received 28 January 1998; accepted in final form 13 May 1998.

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