Phenotypic analysis of conditionally immortalized cells isolated from the BPK model of ARPKD

William E. Sweeney Jr.1,*, Linda Kusner2,*, Cathleen R. Carlin2,3, Sharon Chang1, Lidia Futey1, Calvin U. Cotton1,2, Katherine MacRae Dell1, and Ellis D. Avner1

1 Rainbow Center for Childhood PKD, Department of Pediatrics, Rainbow Babies and Children's Hospital and Case Western Reserve University, Cleveland; and 2 Department of Physiology and Biophysics, 3 Case Western Reserve University Cancer Center, Cleveland, Ohio, 44106


    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

To study the pathophysiology of autosomal recessive polycystic kidney disease (ARPKD), we sought to develop conditionally immortalized control and cystic murine collecting tubule (CT) cell lines. CT cells were isolated from intercross breedings between BPK mice (bpk+/-), a murine model of ARPKD, and the Immorto mice (H-2Kb-ts-A58+/+). Second-generation outbred offspring (BPK × Immorto) homozygous for the BPK mutation (bpk-/-; Im+/±; cystic BPK/H-2Kb-ts-A58), were phenotypically indistinguishable from inbred cystic BPK animals (bpk-/-). Cystic BPK/H-2Kb-ts-A58 mice developed biliary ductal ectasia and massively enlarged kidneys, leading to renal failure and death by postnatal day 24. Principal cells (PC) were isolated from outbred cystic and noncystic BPK/H-2Kb-ts-A58 littermates at specific developmental stages. Epithelial monolayers were under nonpermissive conditions for markers of epithelial cell polarity and PC function. Cystic and noncystic cells displayed several properties characteristic of PCs in vivo, including amiloride-sensitive sodium transport and aquaporin 2 expression. Cystic cells exhibited apical epidermal growth factor receptor (EGFR) mislocalization but normal expression of ZO-1 and E-cadherin. Hence, these cell lines retain the requisite characteristics of PCs, and cystic BPK/H-2Kb-ts-A58 PCs retained the abnormal EGFR membrane expression characteristic of ARPKD. These cell lines represent important new reagents for studying the pathogenesis of ARPKD.

epidermal growth factor receptor, principal cells, cystic kidney


    INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

POLYCYSTIC KIDNEY DISEASES (PKD) cause significant morbidity and mortality and are characterized by the formation and enlargement of fluid-filled tubular and/or collecting duct cysts (18). Progressively enlarging cysts compromise normal renal parenchyma, eventually leading to renal failure. PKD is genetically transmitted as an autosomal dominant or autosomal recessive trait. Autosomal dominant polycystic kidney disease (ADPKD), which affects 1 in 500 to 1 in 1,000 individuals, is caused by a mutation in one of at least three distinct genetic loci (18). Two of the three genes responsible for ADPKD have been cloned. Mutations of the PKD1 locus on chromosome 16 account for ~85% of ADPKD cases (4, 5). Most of the remaining cases of ADPKD occur as a result of mutations of the PKD2 locus on chromosome 4 (19). A small number of cases have been reported that do not map to either the PKD1 or PKD2 loci (1, 7).

Autosomal recessive polycystic kidney disease (ARPKD), which affects 1 in 10,000 to 1 in 40,000 individuals, is invariably characterized by the formation and enlargement of renal collecting tubular (CT) cysts as well as hepatic biliary ectasia and fibrosis (18). Although the PKHD1 (polycystic kidney and hepatic disease 1) gene has been localized to a 1.0 CM interval on the short arm of chromosome 6 (6p21-p12), this gene has not yet been cloned (27). There is no evidence of genetic heterogeneity among ARPKD patients studied to date (45).

Despite the different loci responsible for the genetically distinct forms of PKD, a body of data has demonstrated that a number of similarities exist in the pathophysiology of progressive cyst formation and enlargement in all forms of PKD. These include abnormalities of epithelial cell growth, fluid secretion, and extracellular matrix biology (23). A number of laboratories have demonstrated a significant role for the epidermal growth factor (EGF)/transforming growth factor-alpha (TGF-alpha )/epidermal growth factor receptor (EGFR) axis in promoting epithelial hyperplasia, with resultant renal cyst growth and enlargement in murine and human ADPKD and ARPKD (8, 16, 17, 21, 22, 24-26, 29, 31-33, 41).

The study of disease pathophysiology in many disorders has been facilitated by the creation of immortalized cell lines via viral oncogene expression, provided that the cells retain essential differentiated and/or disease-specific phenotypic features. A number of strategies have been developed for immortalizing multiple cell types from different origins. These include transfection of isolated primary cells with SV40 early region DNA, the wild-type strain of SV40 (simian virus 40), or the temperature-sensitive mutant (SV40tsA58) of SV40 (3, 28). Many of these cell lines retain at least some of the characteristics of the tissue from which they were derived.

More recently, a transgenic mouse strain, H-2Kb-ts-A58 (13), also called the ImmortoMouse, that carries a temperature-sensitive mutant of SV40 under the control of the ubiquitous H-2Kb promoter [activated by gamma -interferon (gamma -IFN)] has been used to derive a variety of cell lines, including many of epithelial origin (13, 34-36). For example, a conditionally immortalized proximal tubule cell line, exhibiting proximal tubule characteristics at nonpermissive temperature, has been established from this transgenic mouse (36). We have previously reported the generation of a CT cell line derived from kidneys of the ImmortoMouse by immunoselection of CT cells. These cells form tight junctions and retain characteristics of CT cells, such as short-circuit currents stimulated by vasopressin, aquaporin 2 expression, and amiloride-sensitive sodium transport (34, 39).

In addition to derivation of cell lines directly from the ImmortoMouse, this transgenic mouse has been crossed with strains carrying disease-specific mutations such as the cystic fibrosis transmembrane regulator knockout (cftr-/-). Isolation and generation of epithelial cell lines from affected organs have provided valuable reagents for studying the cell- and organ-specific pathophysiology of cystic fibrosis (35).

We sought to employ a similar strategy to develop PKD-specific cell lines by genetic complementation of the BPK (Balb/C polycystic kidney) murine model of ARPKD with the ImmortoMouse. The creation of these double mutants permitted the generation and isolation of conditionally immortalized, segment-specific epithelial cells from noncystic and cystic animals at different developmental stages. These cell lines provide attractive alternatives to primary cultures because they can be expanded to provide large numbers of cells for systematic studies over an extended period of time. These cell lines will facilitate the systematic study of the complex cellular and molecular changes that occur as cystic disease progresses and may prove useful as a tool for screening potential pharmacological or gene-based therapies. This report describes the phenotypic characterization of cystic and noncystic conditionally immortalized principal cell (PC) cell lines isolated from BPK/H-2Kb-ts-A58 animals.


    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Immorto × BPK animals. Female heterozygotes from the BPK model (bpk+/-) were bred with H-2kb-ts-A58 transgenic males. Compound heterozygotes (bpk+/-;H-2Kb-ts-A58+/-) were identified (animals heterozygous for the Immorto gene were identified by PCR; bpk heterozygotes were identified by backcrossing to known bpk heterozygotes) and mated to generate cystic (bpk-/-;H-2Kb-ts-A58+/±) and noncystic (bpk+/-;H-2Kb-ts-A58+/±) offspring carrying at least one copy of the Immorto transgene (bpk-/-;H-2Kb-ts-A58+/±). Animals were genotyped for the H-2Kb-ts-A58 transgene by PCR analysis of DNA extracts from tail sections as previously described (34).

Renal function. Animals were deprived of liquid for 12 h before the collection of urine samples for urine osmolarity measurements. Blood samples for blood urea nitrogen (BUN) and creatinine analysis were obtained by orbital sinus collection. Serum BUN and creatinine were quantitatively determined using colorimetric assays from Sigma Diagnostics (St. Louis, MO).

Immunohistology and determination of segmental nephron cyst localization. Kidney and liver tissues were harvested for morphometric analysis as previously described (30, 33) at postnatal days 0, 7, 14, and 21. Briefly, kidney and liver were fixed in 4.0% paraformaldehyde in phosphate buffer (pH 7.4) for 30 min at 4°C. Tissues were then washed, dehydrated through a graded series of acetone, and embedded in Immunobed plastic embedding medium (Polysciences, Warrington, PA). Sections were cut at 4 µm on a Sorvall ultramicrotome, mounted on glass slides, and stained with hematoxylin (kidney or liver) or segment-specific lectins (kidney). Segmental nephron cystic localization and the cystic index were quantitated by combining morphometric analysis with light microscopy and immunohistological techniques as previously described (15, 24, 33).

Cell isolations. The cell isolation procedure utilized is our modification of that originally devised by Van Adelsberg et al. (38) and has been previously described in detail (32, 34). It takes advantage of the lectin specificity of cystic and noncystic CT cells for Dolichos biflorus (DBA). As previously described, the population of isolated CT cells is then further selected for PCs with F13/0121 antibody, an antibody to a PC surface antigen (9, 24, 32).

Cell culture of PCs. PCs were maintained in a serum-free defined medium consisting of a 1:1 mixture of Dulbecco's modified Eagle's medium and Ham's F-12 medium, supplemented with insulin (8.3 × 10-7 M), prostaglandin E1 (7.1 × 10-8 M), selenium (6.8 × 10-9 M), transferrin (6.2 × 10-8 M), triiodothyronine (2 × 10-9 M), dexamethasone (5.09 × 10-8 M), and recombinant gamma -IFN (10 U/ml; GIBCO-BRL, Gaithersburg, MD) at 33°C (permissive conditions). Cells were seeded on polycarbonate Transwell filter inserts (0.4-µm pore size; Costar, Cambridge, MA), which had been coated with a 20% Ethicon collagen dispersion in 60% ethanol (10, 43), at a density of 5 × 105 cells per 12-mm filters or 5 × 106 cells per 75-mm filter. Filter-grown cells were maintained until a tight monolayer was formed, as determined by measuring transepithelial electrical resistance (Rt) with a Millicell-ERS meter and electrode (Millipore, Bedford, MA). Cells were then cultured for an additional 6 days in medium lacking gamma -IFN at 37°C (nonpermissive conditions) before phenotype analysis.

Large T activity following shift to nonpermissive conditions. Cystic and noncystic cells were grown on plastic tissue culture dishes in permissive conditions (33°C with gamma -IFN) (day 0) and in nonpermissive conditions (37°C without gamma -IFN) for 2, 4, and 6 days. Cell lysates were obtained at the indicated days. Equal protein samples were resolved on a 12% SDS-PAGE gel and transferred to a nitrocellulose membrane, and Western analysis was performed with the use of a mouse monoclonal antibody to SV40 large T antigen (Oncogene, Boston, MA).

The activity of large T was assessed by cell proliferation of subconfluent monolayers by bromodeoxyuridine (BrdU) uptake after growth in permissive conditions (day 0) and in nonpermissive conditions for 2, 4, and 6 days. Cystic and noncystic CT cells were plated on plastic tissue culture dishes, and on the days indicated, BrdU was added at a concentration of 30 µM and cells were incubated for 3 h. Cells were methanol-fixed, and cellular uptake of BrdU was identified by immunohistology with biotinylated monoclonal anti-BrdU antibody (Zymed, South San Francisco, CA). Data are expressed as the percentage of BrdU-labeled cells per 500 cells counted.

Measurement of short-circuit current. PCs were grown on collagen-coated Millicell CM filter supports (Millipore) and mounted in Ussing chambers for measurement of electrogenic ion transport. The filters were clamped between Lucite flux chambers and bathed on both sides by equal volumes of Krebs-Ringer bicarbonate solution. The solutions were circulated through the water-jacketed glass reservoir by gas lifts (95%O2-5% CO2) to maintain solution temperature and pH. Transepithelial voltage difference (VT) was measured between two Ringer-agar bridges, each positioned within 3 mm of the monolayer surface. Calomel half-cells connected the bridges to a high-impedance voltmeter. Current from an external direct current source was passed by silver-silver chloride electrodes and Ringer-agar bridges to clamp the spontaneous VT to zero, and the resulting short-circuit current (Isc) was measured. At 1-min intervals, VT was clamped to +2 mV to calculate Rt. Amiloride (10-4 M) was added to the apical bathing solution, and 5 min later antidiuretic hormone (ADH, 10-8 M) was added to the basolateral bathing solution.

Confocal microscopy. Filter-grown cells were prepared for confocal laser scanning microscopy (CLSM) as previously described (10). Briefly, cells were rinsed three times with PBS, fixed with 3% paraformaldehyde for 10 min, and incubated with 50 mM NH4Cl in PBS for 10 min to quench excess paraformaldehyde. The fixed cells were permeabilized with 0.2% Triton X-100 in PBS for 10 min, rinsed three times with PBS, and then incubated with 3% BSA in PBS for 1 h at room temperature to block nonspecific binding. Cells were stained overnight at 4°C with one of the following primary antibodies: E-cadherin mouse monoclonal antibody (Transduction Laboratories, Lexington, KY), EGFR polyclonal antibody made in sheep (Upstate Biotechnology, Lake Placid, NY), and ZO-1 rat monoclonal antibody R26.4C (Developmental Studies Hybridoma Bank, University of Iowa, Iowa City, IA). Cells were rinsed three times with PBS supplemented with 10% fetal bovine serum and then preincubated with PBS supplemented with 5% normal serum from the host animal used to generate secondary antibodies to block nonspecific binding for 10 min. Cells were then stained with appropriate fluorophore-conjugated secondary antibody Fab fragments (Jackson ImmunoResearch Laboratories, Fort Washington, PA) for 1 h at 37°C. After being rinsed, filters were excised from plastic inserts and mounted cell side up on a glass slide with SlowFade reagent (Molecular Probes, Eugene, OR). Cells were examined with a confocal Zeiss LSM 410 microscope (Zeiss, Göttingen, Germany) by using the 488- to 568-nm wavelength lines of an argon-krypton laser. Image resolution obtained with the use of a Zeiss ×100 Plan-Neofluor oil objective and Zeiss LSM software was 512 × 512 pixels. Cell monolayers were optically sectioned every 0.5 µm, and vertical (x-z) optical sections perpendicular to the plane of the apical membrane were digitally compiled.

Immunoprecipitations and immunoblotting. For immunoprecipitations, filter-grown cells were rinsed twice and then scraped in ice-cold PBS supplemented with 2 mM EDTA, 1 mM EGTA, and protease inhibitors (0.2 mM phenylmethylsulfonyl fluoride and 1 µM leupeptin). Cells were pelleted by centrifugation and lysed with 1% Triton X-100 in homogenization buffer (10 mM HEPES, pH 7.4, 0.25 M sucrose, and 1 mM EDTA) supplemented with 150 mM NaCl and protease inhibitors. Immunoprecipitations were carried out using antibodies adsorbed to protein A-Sepharose CL-4B beads (Sigma Chemical). For immunoblotting, cells were lysed with 1% Triton X-100 and 0.1% Nonidet P-40 (NP-40) in homogenization buffer supplemented with protease inhibitors, and protein concentrations were determined by Bradford assay (Bio-Rad Laboratories). Immunoprecipitates or aliquots of total cell protein were solubilized with Laemmli buffer, resolved by SDS-PAGE, and transferred to nitrocellulose using standard techniques (14, 37). Blots were incubated with appropriate horseradish peroxidase (HRP)-conjugated secondary antibodies for detection by enhanced chemiluminescence (ECL; Amersham Life Sciences or Jackson ImmunoResearch Laboratories). The following protein-specific antibody reagents were used: aquaporin (AQP) isoform-specific polyclonal antibodies to AQP2 and AQP3 made in goat (Santa Cruz Biotechnology, Santa Cruz, CA) or in rabbit (Calbiochem-Novabiochem, San Diego, CA) and the EGFR polyclonal antibody made in sheep.

Domain-specific EGFR detection. For domain-specific biotinylation, filter-grown cells were rinsed three times with PBS supplemented with 0.1 mM CaCl2 and 1 mM MgCl2 (PBS-CM) and then incubated on ice for 30 min with PBS-CM containing 1 mg/ml sulfosuccinimidyl-biotin (sulfo-NHS-bioten; Pierce Chemical, Rockford, IL) added to either the apical or basolateral side of the cells. The chemical cross-linker was quenched by rinsing cells three times with 50 mM NH4Cl, and cells were lysed with 1% Triton X-100 in 20 mM Tris, pH 8.0, supplemented with 150 mM NaCl, 5 mM EDTA, 0.2% BSA, and protease inhibitors. Cell lysates were immunoprecipitated using an EGFR-specific antibody made in rabbit, and bound proteins were solubilized with Laemmli buffer, resolved by SDS-PAGE, and transferred to nitrocellulose for immunoblotting with HRP-streptavidin (Pierce Chemical).

For domain-specific 125I-labeled EGF cross-linking, filter-grown cells were rinsed three times with HEPES-buffered MEM supplemented with 0.2% BSA and then incubated with ~10 nM 125I-EGF added to the apical or basolateral side of the monolayer for 3 h at 4°C. After extensive rinsing to remove unbound ligand, cells were incubated with 2 mM bis(sulfosuccinimidyl) suberate (BS3; Pierce Chemical) diluted in PBS, added to the same side of the epithelial monolayer as ligand, for 30 min at room temperature. The chemical cross-linker was quenched by 5-min incubation with 0.05 M Tris, pH 7.4, at room temperature. Cells were lysed with 1% NP-40 in 0.1 M Tris, pH 6.8, supplemented with 15% glycerol, 2 mM EDTA, 1 mM EGTA, and protease inhibitors. Aliquots of total cell protein were separated by SDS-PAGE for detection of 125I-EGF cross-linked receptors by autoradiography or for quantitation by phosphorstorage autoradiography (Molecular Dynamics, Sunnyvale, CA).

Domain-specific EGFR activation. Filter-grown cells were incubated with EGF (100 ng/ml) added to either the apical or basolateral side for 2 h on ice and then warmed to 37°C for 10 (basolateral stimulation) or 20 (apical stimulation) min. Cells were then placed on ice, rinsed twice with ice-cold PBS, and incubated with PBS supplemented with 0.1 mM pervanadate for 10 min. Cells were lysed with 1% Triton X-100, 0.5% deoxycholate, 0.1% SDS, and 0.5% NP-40 in 20 mM Tris, pH 7.4, supplemented with 150 mM NaCl, 1 mM EGTA, 1 mM orthovanadate, 0.1 mM ammonium molybdate, 0.2 mM phenylarsine oxide, and protease inhibitors. Clarified lysates were incubated for 2 h with 3 µg of biotin-conjugated anti-phosphotyrosine antibody (Transduction Laboratories) and then with streptavidin-agarose beads overnight at 4°C. Immunoprecipitates were washed three times with lysis buffer, solubilized with Laemmli buffer, separated by SDS-PAGE, and transferred to nitrocellulose for immunoblotting with an EGFR antibody and detection by ECL.


    RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

In vivo phenotypic characterization of cystic lesions in the cystic bpk/H-2Kb-ts-A58 animals. Offspring produced from double heterozygote breedings were analyzed for phenotypic changes due to alterations of the genetic background by analyzing renal function and for cyst localization. Cystic BPK mice with (BPK/H-2Kb-ts-A58) or without (BPK) the H-2Kb-ts-A58 transgene developed biliary ductal ectasia and massively enlarged kidneys, leading to renal failure and death by postnatal day 24. As shown in Table 1, renal cyst localization and renal cyst profiling of the cystic BPK/H-2Kb-ts-A58 animals were indistinguishable from those of cystic BPK animals. At postnatal day 0, <95% of the cystic lesions occurred in proximal tubules [Lotus tetragonobulus agglutinin (LTA+)]. The number and size of CT cysts increased with age in both the cystic BPK and cystic BPK/H-2Kb-ts-A58 animals. CT cysts (DBA+) in both the cystic BPK and cystic BPK/H-2Kb-ts-A58 animals were positive for F13/0121 and expressed AQP2 on the apical cell surface as well as AQP3 on the basolateral cell surface (Fig. 1), indicating that the CT cystic lesions were lined with PCs (9, 39). In addition, as shown in Fig. 2, CT cysts in BPK/H-2Kb-ts-A58 kidneys demonstrated apical and lateral expression of EGFR, a consistent and characteristic phenotypic marker for cystic CT epithelium (2, 8, 32).

                              
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Table 1.   Cyst localization



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Fig. 1.   Immunohistological staining of serial sections from a BPK/H-2Kb-ts-A58 kidney for DBA (A), F13/0121 (B), AQP2 (C), and AQP3 (D). These data demonstrate that F13/0121 stains a subset of tubules that are positive for the lectin Dolichos biflorus (DBA) and that these tubules that are DBA+ and F13/0121+ have apical expression of AQP2 and basolateral expression of AQP3. AQP, aquaporin. Original magnification, ×100 Nomarski/differential interference contrast.



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Fig. 2.   Immunohistology of epidermal growth factor receptor (EGFR) expression in lectin (DBA)- and F10/0121-positive collecting tubule (CT) cysts in cystic BPK/H-2Kb-ts-A58 (A) and cystic BPK kidneys (B). Data demonstrate that CT cysts in both BPK and BPK/H-2Kb-ts-A58 kidneys have abnormal apical expression of EGFR.

The data in Table 2 demonstrate that at postnatal day 15, both renal function and maximal urinary concentration ability were equally impaired in both cystic BPK/H-2Kb-ts-A58 and cystic BPK animals and were significantly different from noncystic BPK and BPK/H-2Kb-ts-A58 controls. Serum BUN values for the cystic BPK/H-2Kb-ts-A58 and cystic BPK animals were five to six times higher than values for control mice (P < 0.024). Maximal urinary osmolarity in cystic BPK/H-2Kb-ts-A58 and cystic BPK animals was reduced to ~60% of values obtained from control mice (P < 0.05).

                              
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Table 2.   Renal function

These data demonstrate that outbred cystic BPK/H-2Kb-ts-A58 offspring were phenotypically indistinguishable from inbred cystic BPK animals. The progression of BPK cystic disease was unaltered by the ImmortoMouse genetic background.

Culture of immortalized CT cells. Cells isolated from cystic and noncystic kidneys were initially expanded on plastic, under permissive conditions, until they reached passage 5. Multiple aliquots were taken at each passage from passages 3 through 5 and stored in liquid nitrogen for future studies. Upon thawing, the early passage cells were again expanded on plastic, under permissive conditions, before they were seeded on collagen-coated permeable support filters for analysis of epithelial cell markers. Because the cells were then switched to nonpermissive conditions to turn off T antigen expression, cells were seeded on filters at high density to permit formation of a tight monolayer in the absence of cell proliferation. As shown in Fig. 3, Western analysis of large T expression in cells under nonpermissive conditions demonstrates decreasing expression of large T that correlates with a decreasing rate of proliferation as assessed by BrdU labeling. Although both the noncystic and cystic cells formed an electrically resistant monolayer within a few days, we found that the cystic cells maintained electrical resistance for at least 10 days, compared with the noncystic cells, which remained electrically resistant for up to 6 days (data not shown). Loss of electrical resistance in the noncystic cells occurred even sooner when they were seeded on filters that had not been precoated with a collagen-based extracellular matrix. We believe this occurred because the cystic cells continue to proliferate under nonpermissive conditions, consistent with the disease phenotype (18, 23), whereas the noncystic cells do not, consistent with conditional immortalization (13, 20, 40). Hence, cystic cells but not noncystic cells are capable of repairing epithelial breaches that may arise over time due to cell death.


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Fig. 3.   Analysis of large T antigen expression and cell proliferation under permissive and nonpermissive conditions. Cystic and noncystic cells were grown on plastic tissue culture dishes in permissive conditions [33°C with gamma -interferon (gamma -IFN)] and in nonpermissive conditions (37°C without gamma -IFN), and cell lysates obtained at the indicated days were probed for large T expression by Western analysis. Cystic and noncystic CT cells were plated on plastic tissue culture dishes, and cell proliferation of subconfluent monolayers was assessed by bromodeoxyuridine (BrdU) uptake after growth in permissive conditions (day 0) and in nonpermissive conditions for 2, 4, and 6 days. Data are expressed as the percentage of BrdU-labeled cells per 500 cells counted. These data demonstrate that the shift to nonpermissive conditions causes a loss of SV40 large T protein and a parallel decrease in cell proliferation.

In vitro characterization of immortalized CT cells. To characterize the phenotype of conditionally immortalized noncystic and cystic BPK/H-2Kb-ts-A58 CT cells, the cells were initially grown under permissive conditions on collagen-coated Transwell filters to form electrically resistant monolayers. Cells were then cultured for an additional 4-6 days under nonpermissive conditions. As shown in Fig. 4, both cystic and noncystic BPK/H-2Kb-ts-A58 cells exhibited normal expression of ZO-1 (green) at the apex of the lateral membrane as well as normal expression E-cadherin (red) along the entire length of the lateral cell membrane. These data demonstrate that both cystic and noncystic cell lines form polarized epithelial monolayers with normal expression and distribution of tight junction and epithelial cell adhesion molecules.


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Fig. 4.   Confocal laser scanning microscopy (CLSM) analysis of ZO-1 and E-cadherin localization in collecting duct principal cells derived from noncystic and cystic BPK/H-2Kb-ts-A58 animals. Filter-grown cells were fixed, permeabilized, and costained with a rat monoclonal antibody specific for the tight junction protein ZO-1 and a mouse monoclonal antibody specific for E-cadherin, followed by appropriate fluorochrome-conjugated secondary antibodies. ZO-1 staining is shown in green and E-cadherin staining in red. Vertical (x-z) optical sections perpendicular to the plane of the apical membrane were collected every 0.5 µm and digitally compiled for each monolayer (A: ZO-1; C: E-cadherin). Horizontal (x-y) optical sections shown for ZO-1 and E-cadherin were taken in the subapical plane at the level of the tight junction (B) and in the plane of the lateral membrane midway through the cell (D), respectively.

PC marker analysis. PCs are distinguished phenotypically from other epithelial cell types found in the kidney by the expression of AQP2 and amiloride-sensitive sodium absorption (39). Western analysis of cystic and noncystic BPK/H-2Kb-ts-A58 cells for aquaporin expression is shown in Fig. 5. These data show that cystic and noncystic BPK/H-2Kb-ts-A58 cells express both glycosylated and nonglycosylated forms of AQP2 as well as AQP3, and expression is maintained for at least 20 passages.


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Fig. 5.   Analysis of AQP isoform protein expression. Cell lysates were immunoprecipitated by using isoform-specific rabbit antibodies to AQP2 or AQP3. Principal cells were analyzed at 2 different passage numbers (p9 and p15 for noncystic cells; p7 and p20 for cystic cells). Immunoprecipitates were resolved on 12% polyacrylamide gels and transferred to nitrocellulose for immunoblot analysis with AQP2- or AQP3-specific antibodies made in goat. G, glycosylated; NG, nonglycosylated. Molecular weight standards: carbonic anhydrase, 31,000; soybean trypsin inhibitor, 21,500.

Electrogenic amiloride-sensitive sodium absorption and vasopressin receptor expression also are features of CT principle cells (39). Both cystic and noncystic BPK/H-2Kb-ts-A58 epithelial monolayers developed Isc, indicative of active cation absorption and/or anion secretion (Table 3 and Fig. 6). Addition of amiloride (100 µM) to the apical bathing solution reduced Isc by ~90% (Table 3 and Fig. 6). Subsequent addition of vasopressin (10 nM) to the basolateral bathing solution elicited an increase in Isc (Table 3 and Fig. 6) most likely due to activation of chloride secretion. Thus both cystic and noncystic BPK/H-2Kb-ts-A58 cells possess functional amiloride-sensitive sodium absorption and basolateral vasopressin receptors. Together, these data demonstrate that the cell lines maintain differentiated PC characteristics through at least 20 passages in culture.

                              
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Table 3.   Summary of ion transport properties of cystic and noncystic epithelial monolayers



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Fig. 6.   Effect of amiloride and antidiuretic hormone (ADH) on short-circuit current (Isc). Cystic (A) and noncystic cells (B) cultured on collagen-coated permeable supports were placed in Ussing chambers and bathed on both sides with Krebs-Ringer bicarbonate solution. The transepithelial voltage difference (VT) was clamped to 0, and the resulting Isc was measured. At 1-min intervals, VT was clamped to +2 mV to calculate transepithelial resistance. Amiloride was added to the apical bathing solution. Five minutes later, ADH was added to the basolateral bathing solution.

In vitro EGFR phenotype. In vivo, cystic CT lesions in both the cystic BPK and cystic BPK/H-2Kb-ts-A58 animals demonstrate apical as well as basolateral expression of EGFR (Fig. 2), a characteristic phenotypic feature of CT cysts in PKD (2, 8, 17, 32, 41). CLSM analysis of CT cells isolated from cystic BPK/H-2Kb-ts-A58 kidneys revealed that these cells maintain apical EGFR expression in vitro. Figure 7A shows digitally compiled vertical sections demonstrating a marked apical expression of EGFR in cystic BPK/H-2Kb-ts-A58 cells. Figure 7B, showing a section just above the level of tight junction ZO-1 staining, demonstrates apical EGFR expression in the cystic BPK/H-2Kb-ts-A58 cells but not in the noncystic BPK/H-2Kb-ts-A58 cells. Figure 7C, showing a horizontal section midway through the plane of the lateral cell membrane, shows lateral staining for EGFR in both cystic and noncystic BPK/H-2Kb-ts-A58 cells. The results of a more detailed CLSM analysis of EGFR and ZO-1 staining in the cystic BPK/H-2Kb-ts-A58 cells are shown in Fig. 8. Figures 7 and 8 demonstrate that in the presence of normal ZO-1 expression, the abnormal distribution of EGFR (i.e., apical as well as basolateral), a characteristic feature of CT cysts in PKD, is retained by the immortalized cystic CT cell line.


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Fig. 7.   CLSM analysis of EGFR membrane domain localization. Filter-grown, noncystic and cystic BPK/H-2Kb-ts-A58 cells were fixed, permeabilized, and costained with a rat monoclonal antibody specific for the tight junction protein ZO-1 (data not shown) and a sheep antibody specific for EGFR, followed by appropriate fluorochrome-conjugated secondary antibodies. Vertical (x-z) optical sections perpendicular to the plane of the apical membrane were collected every 0.5 µm and digitally compiled for each monolayer (A). Horizontal (x-y) optical sections are shown from a plane just above the level of the tight junction (B) and in the plane of the lateral membrane midway through the cell (C) to illustrate EGFR staining at the apical and basolateral surfaces, respectively.



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Fig. 8.   CLSM analysis of EGFR and ZO-1 in cystic BPK/H-2Kb-ts-A58 cells. Filter-grown cystic cells were costained with sheep anti-EGFR (red) and monoclonal antibody for ZO-1 (green). A: two independent vertical (x-z) optical sections perpendicular to the plane of the apical membrane were collected every 0.5 µm and digitally compiled. B: successive 1-µm horizontal (x-y) optical sections, starting from a section collected at the apical surface of the cell at the level of the tight junction (B1) and proceeding through a section collected at the basal surface at the level of attachment to the filter insert (B9).

Domain-specific EGFR expression. To further characterize domain-specific EGFR expression in the conditionally immortalized CT cells, we analyzed filter-grown cells by cell surface-specific biotinylation, 125I-EGF cross-linking, and domain-specific stimulation with EGF. Figure 9A shows the results obtained following domain-specific biotinylation. Biotinylated cell lysates were immunoprecipitated with an EGFR-specific antibody, and immunoprecipitates were analyzed for the presence of biotin by Western blotting with HRP-conjugated avidin. EGFRs were detected when either the apical or basolateral cell surface was biotinylated in cystic cells, but only when the basolateral surface was biotinylated in the noncystic cells. Similar results were obtained following domain-specific 125I-EGF chemical cross-linking. Figure 9B demonstrates that both apical and basolateral cell surfaces of cystic cells bind significant amounts of 125I-EGF detectable following cross-linking to surface receptors and autoradiography. When these data were quantitated by phosphorimaging, we found that total 125I-EGF cross-linking was essentially evenly distributed between the apical and basolateral receptors (45% apical/55% basolateral). In contrast, ~20% of the total radioactivity bound to the noncystic cells was bound to apical receptors, compared with 80% for basolateral receptors.


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Fig. 9.   Membrane domain-specific EGFR expression. A: filter-grown noncystic and cystic BPK/H-2Kb-ts-A58 cells were incubated with sulfosuccinimidyl-biotin (biotin) added to the apical (Ap) or basolateral (BL) surface, and cell lysates were immunoprecipitated with an EGFR-specific antibody. Immunoprecipitates (IP) were resolved by SDS-PAGE transferred to nitrocellulose, and immunoblotted (IB) with horseradish peroxidase (HRP)-avidin. B: filter-grown cells were incubated with 125I-EGF, followed by a chemical cross-linker [bis(sulfosuccinimidyl) suberate (BS3)] added to either the apical or basolateral surface. Equal aliquots of total cell protein were resolved on 7.5% polyacrylamide gels for detection of 125I-EGF cross-linked to EGFR by autoradiography. C: unstimulated cells (-) or cells that had been stimulated with EGF (100 ng/ml) added to the apical or basolateral surface were lysed and immunoprecipitated with a biotin-conjugated phosphotyrosine (pTyr)-recombinant monoclonal antibody. Cells were stimulated with apical EGF for 20 min or with basolateral EGF for 10 min, at 37°C. Antigen-antibody complexes recovered by streptavidin affinity chromatography were resolved by SDS-PAGE and transferred to nitrocellulose for immunoblotting with an EGFR-specific antibody.

The results of domain-specific EGFR activation with EGF are depicted in Fig. 9C. Cell lysates were immunoprecipitated with a phosphotyrosine-specific monoclonal antibody, and immunoprecipitates were resolved by SDS-PAGE and analyzed by Western blotting using an EGFR-specific antibody. These data show that cystic and noncystic cells both exhibit a low level of basal EGFR activity (Fig. 9C). EGF stimulation of cystic cells results in a marked increase in EGFR activation compared with unstimulated cells, when EGF is added to either the apical or basolateral cell surface. Although receptors were activated at both sides of the cell, we did observe that apical receptors were activated with slightly slower kinetics than basolateral receptors (20 min vs. 10 min to observe peak activity) and that there also were differences in the duration of apical vs. basolateral receptor activation (data not shown). In contrast, apical stimulation of noncystic cells produced only a minor increase in EGFR activation compared with basal levels, compared with dramatic stimulation of basolateral receptors. Together, these results indicate that, similar to other polarized kidney epithelial cell lines, EGFRs are expressed and activated predominantly at the basolateral surface in the noncystic PCs. This contrasts findings in PCs isolated from cystic animals, which express substantial EGFRs on both sides of the cell. Moreover, apical receptors in the cystic cells appear to be fully functional on the basis of their ability to bind ligand and to undergo ligand-induced tyrosine kinase activation. However, domain-specific differences in the kinetics and duration of receptor activation warrant further study.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Primary cultures of murine and human cells (32, 43) as well as immortalized epithelial cell lines (12-14, 42, 44) have been widely used for studies of renal epithelial cell biology. Many of these cell lines were generated by in vitro transfection of renal epithelial cells with SV40 large T antigen. Immortalized cell lines have also been developed from genetically modified mice that carry an SV40 large T antigen transgene. Recently, a transgenic mouse line (H-2Kb-ts-A58; ImmortoMouse) that carries a thermolabile mutant of SV40 large T antigen under the control of a ubiquitous gamma -IFN-inducible promoter has been developed and used to generate a number of conditionally immortalized cell lines (13, 20, 34-36). Generation of cell lines through the use of the ImmortoMouse has a number of advantages over in vitro transfection even with utilization of identical constructs. First, in vitro transfection produces unpredictable characteristics due to variable copy number and multiple sites of integration. Second, unlike in vitro transfection, heterogeneous cell populations can be isolated from an ImmortoMouse organ, and clones with specific characteristics may be expanded at a later date. Finally, the use of a temperature-sensitive immortalization construct provides the opportunity to control large T expression and promote differentiation by switching cells to nonpermissive culture conditions.

Previous work from this and other laboratories (13, 34-36, 40) has shown that organ-specific epithelial cell lines as well as specific cells within an organ can be isolated from conditionally immortalized animals and grown in culture for prolonged intervals. These isolated cells form electrically resistant monolayers in vitro and retain phenotypic and functional characteristics that define these cells in vivo. The current study describes the successful intercross of the ImmortoMouse with a murine model of PKD, thereby creating a model of PKD containing kidneys (and other organs) comprising cells that carry the immortalization transgene. Genetic complementation of the BPK mouse with the ImmortoMouse did not alter the time course of cystic disease progression, the site of cystic lesions, or the degree of renal failure due to growth and expansion of CT cysts. Cystic BPK/H-2Kb-ts-A58 animals demonstrated apical expression of EGFR in CT cystic lesions, a characteristic phenotypic manifestation of CT cysts in all forms of PKD. The cystic and noncystic cell lines derived from BPK/H-2Kb-ts-A58 animals retain properties characteristic of the CT principal cell. The junctional complex protein ZO-1, as well as the adhesion molecule E-cadherin, is abundantly expressed and appropriately localized. The cell lines express AQP2, a water channel that is unique to the apical membrane of the CT principal cells. As expected, the cell lines also express the basolateral water channel AQP3. The cell lines form polarized epithelial monolayers, as evidenced by the Rt and asymmetric electrogenic ion transport. The mislocalization of the EGFR in cystic cells is not due to defective junctions, because cystic cell lines of low (~60 Omega  · cm2) and high (~400 Omega  · cm2) electrical resistance exhibit EGFR mislocalization, whereas a noncystic cell line with an intermediate (~200 Omega  · cm2) electrical resistance shows normal basolateral EGFR localization. Cystic and noncystic epithelial monolayers also exhibit amiloride-sensitive sodium absorption, a major ion transport pathway in CT principal cells. There are quantitative differences in the electrical properties (Isc and Rt) of the various cell lines; however, it is likely that these represent variation inherent in the generation of cell lines, rather than PKD-specific properties. It is, however, intriguing that the cystic cell lines appear to have reduced sodium absorption and enhanced chloride secretion, a transport phenotype that would be consistent with cyst formation and that deserves further study.

Finally, confocal microscopy, cell surface biotinylation, and 125I-EGF cross-linking of cystic and noncystic BPK/H-2Kb-ts-A58 cells demonstrate that cystic cells retain abundant apical expression of EGFR in contrast to sparse apical expression in noncystic cells. Apical expression of EGFR in CT cystic lesions has been reported in human and multiple murine models of ARPKD and ADPKD and is a characteristic and consistent phenotypic feature of PKD renal epithelia (8, 32). Domain-specific stimulation of apical EGFR in these cystic BPK/H-2Kb-ts-A58 cells confirms earlier work in primary cells demonstrating that apical EGFRs are functional and that they autophosphorylate in response to EGF (32). Thus the "cystic phenotype" is maintained in cystic BPK/H-2Kb-ts-A58 PC even after long-term culture.

The current studies confirm that apical EGFR expression is a consistent and characteristic feature of the cystic CT phenotype. Such expression may be physiologically relevant in that apical receptors bind ligand, autophosphorylate, and mediate cell proliferation as well as chronic changes in ion transport (6). Apical expression of active EGFR in cystic CT epithelia suggests a mechanism by which a stimulatory TGF-alpha /EGF/EGFR autocrine-paracrine loop may be active in vivo. Preliminary investigations reveal that BPK cyst fluid can activate apically expressed EGFRs and transmit a mitogenic signal through these apical receptors at a magnitude comparable with EGF (31).

Apical EGFR expression in cystic CT lesions provides the basis for the development of innovative therapeutic approaches to ARPKD, which target the abnormally expressed EGFR (33). In addition, future studies of the specific mechanisms of EGFR signaling from the apical cell surface may provide new insights into the cell biology of ARPKD. BPK/H-2Kb-ts-A58 cells, derived from specific tubular segments at distinct developmental stages of noncystic and cystic kidneys, provide valuable reagents to study the changing cellular and molecular pathophysiology of PKD at different disease stages in specific nephron segments and identify new cellular targets for future therapeutic intervention.

In summary, BPK/H-2Kb-ts-A58 cell lines are unique reagents that retain PC properties and exhibit an EGFR expression pattern that recapitulates the disease phenotype. In addition, these cells are conditionally immortalized, thereby providing control of growth and differentiation in culture. Finally, BPK /H-2Kb-ts-A58 cells are the first cell lines developed from the BPK model of ARPKD and are the first cystic cell lines reported that are both nephron segment and disease stage specific. BPK/H-2Kb-ts-A58 cells provide valuable reagents that may be used to study the molecular and cellular pathophysiology of cyst development and progressive enlargement in PKD.


    ACKNOWLEDGEMENTS

This study was supported by Polycystic Kidney Research Foundation Grants 99003 (to E. D. Avner) and 99013 (to C. U. Cotton), National Institute of Diabetes and Digestive and Kidney Diseases Grant P50-DK-57306 (to E. D. Avner), and a March of Dimes Birth Defects Foundation grant (to C. R. Carlin).


    FOOTNOTES

* W. E. Sweeney, Jr., and L. Kusner contributed equally to this work.

Address for reprint requests and other correspondence: E. D. Avner, Dept. of Pediatrics, Rainbow Babies and Children's Hospital, 11100 Euclid Ave LC 6003, Cleveland, OH 44106-6003 (E-mail: eda{at}po.cwru.edu).

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

Received 6 February 2001; accepted in final form 6 July 2001.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

1.   Almeida, SD, Almeida ED, Peters D, Pinto JR, Tavora I, Lavinha J, Breuning M, and Prata MM. Autosomal dominant polycystic kidney disease: evidence for the existence of a third locus in a Portuguese family. Hum Genet 96: 83-88, 1995[ISI][Medline].

2.   Avner, ED, and Sweeney WE. Apical epidermal growth factor receptor expression defines a distinct cystic tubular epithelial phenotype in autosomal recessive polycystic kidney disease (Abstract). Pediatr Res 37: 359A, 1995.

3.   Briand, P, Kahn A, and Vandewalle A. Targeted oncogenesis: a powerful method to derive cell lines. Kidney Int 47: 388-394, 1995[ISI][Medline].

4.   Consortium, T. The polycystic kidney disease 1 gene encodes a 14 kb transcript and lies within a duplicated region on chromosome 16. Cell 77: 881-894, 1994[ISI][Medline].

5.   Consortium, T. Polycystic kidney disease: the complete structure of the PKD1 gene and its protein. Cell 81: 289-298, 1995[ISI][Medline].

6.   Cotton, C. Inhibition of amiloride-sensitive sodium absorption by EGF (Abstract). FASEB J 14: 339A, 2000.

7.   Daoust, MC, Reynolds DM, and Bichet DG. Evidence for a third genetic locus for autosomal dominant polycystic kidney disease. Genomics 25: 733-737, 1995[ISI][Medline].

8.   Du, J, and Wilson PD. Abnormal polarization of EGF receptors and autocrine stimulation of cyst epithelial growth in human ADPKD. Am J Physiol Cell Physiol 269: C487-C495, 1995[Abstract/Free Full Text].

9.   Fejes-Toth, N, and Fejes-Toth G. Immunoselection and culture of cortical collecting duct cells. J Tiss Cult Meth 13: 179-184, 1991.

10.   Hobert, M, and Carlin CR. The cytoplasmic juxtamembrane domain of the human EGF receptor is required for basolateral localization in MDCK cells. J Cell Physiol 162: 434-446, 1995[ISI][Medline].

11.   Hopfer, U, Jacobberger JW, Gruenert DC, Eckert RL, Jat PS, and Whitsett JA. Immortalization of epithelial cells. Am J Physiol Cell Physiol 270: C1-C11, 1996[Abstract/Free Full Text].

12.   Hopfer, U, Woost PG, Jacobberger JW, and Douglas JG. New methods for maintaining human renal epithelial cells and analyzing their ion transport functions: potential analysis of genetic disease. Ethn Health 1: 129-136, 1996[Medline].

13.   Jat, PS, Noble MD, Ataliotis P, Tanaka Y, Yannoutsos N, Larson L, and Kioussis D. Direct derivation of conditionally immortal cell lines from H-2Kb-tsA58 transgenic mouse. Proc Natl Acad Sci USA 88: 5096-5100, 1991[Abstract].

14.   Laemmli, UK. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227: 680-685, 1970[ISI][Medline].

15.   Loud, AV, and Anversa P. Morphometric analysis of biological processes. Lab Invest 50: 250-261, 1984[ISI][Medline].

16.   Lowden, DA, Lindemann GW, Merlino G, Barash BD, Calvet JP, and Gattone VH, 2nd. Renal cysts in transgenic mice expressing transforming growth factor-alpha . J Lab Clin Med 124: 386-394, 1994[ISI][Medline].

17.   Lu, W, Fan X, Babakanlou H, Law T, Rifal N, Harris PC, Perez-Atayde AR, Renneke HG, and ZJ Late onset of renal and hepatic cysts in Pkd1-targeted heterozygotes. Nat Genet 21: 160-161, 1999[ISI][Medline].

18.   McDonald, R, Watkins SL, and Avner ED. Polycystic kidney disease. In: Pediatric Nephrology (4th ed.), edited by Barratt TM, Avner ED, and Harmon WE.. Baltimore, MD: Lippincott Williams & Wilkins, 1999, p. 459-474.

19.   Mochizuki, T, Wu G, Hayashi T, Xenophontos SL, VB, Saris JJ, Reynolds DM, Cai Y, Gabow PA, Pierides A, Kimberling WJ, Breuning MH, Deltas CC, Peters DJ, and Somlo S. PKD2, a gene for polycystic kidney disease that encodes an integral membrane protein. Science 272: 1339-1342, 1996[Abstract].

20.   Morgan, JE, Beauchamp JR, Pagel CN, Peckham M, Ataliotis P, Jat PS, Noble MD, Farmer K, and Partridge TA. Myogenic cell lines derived from transgenic mice carrying a thermolabile T antigen: a model system for the derivation of tissue-specific and mutation-specific cell lines. Dev Biol 162: 486-498, 1994[ISI][Medline].

21.   Moskowitz, DW, Bonar SL, Liu W, Sirgi CF, Marcus MD, and Clayman RV. Epidermal growth factor precursor is present in a variety of human renal cyst fluids. J Urol 153: 578-583, 1995[ISI][Medline].

22.   Munemura, C, Uemaso J, and Kawasaki H. Epidermal growth factor and endothelin in cyst fluid from autosomal dominant polycystic disease cases. Am J Kidney Dis 24: 561-568, 1994[ISI][Medline].

23.   Murcia, NS, Sweeney WE, and Avner ED. New insights into the molecular pathophysiology of polycystic kidney disease. Kidney Int 55: 1187-1197, 1999[ISI][Medline].

24.   Nauta, J, Ozawa Y, Sweeney WE, Jr, Rutledge JC, and Avner ED. Renal and biliary abnormalities in a new murine model of autosomal recessive polycystic kidney disease. Pediatr Nephrol 7: 163-172, 1993[ISI][Medline].

25.   Nauta, J, Sweeney WE, Rutledge JC, and Avner ED. Biliary epithelial cells from mice with congenital polycystic kidney disease are hyper-responsive to epidermal growth factor. Pediatr Res 37: 755-763, 1995[Abstract].

26.   Orellana, SA, Sweeney WE, Neff CD, and Avner ED. Epidermal growth factor receptor expression is abnormal in murine polycystic kidney. Kidney Int 47: 490-499, 1995[ISI][Medline].

27.   Park, JH, Dixit MP, Onuchic LF, Wu G, Goncharuk AN, Kneitz S, Santarina LB, Hayashi T, Avner ED, Guay-Woodford L, Zerres K, Germino GG, and Somlo S. A 1-Mb BAC/PAC-based physical map of the autosomal recessive polycystic kidney disease gene (PKHD1) region on chromosome 6. Genomics 57: 249-255, 1999[ISI][Medline].

28.   Racusen, LC, Wilson PD, Hartz PA, Fivush BA, and Burrow CR. Renal proximal tubular epithelium from patients with nephropathic cystinosis: immortalized cell lines as in vitro model systems. Kidney Int 48: 536-543, 1995[ISI][Medline].

29.   Richards, WG, Sweeney WE, Yoder BK, Wilkinson JE, Woychik RP, and Avner ED. Epidermal growth factor receptor activity mediates renal cyst formation in polycystic kidney disease. J Clin Invest 101: 935-939, 1998[Abstract/Free Full Text].

30.   Sweeney, WE, and Avner ED. Intact organ culture of murine metanephros. J Tiss Cult Meth 13: 163-168, 1991.

31.   Sweeney, WE, and Avner ED. BPK cyst fluid contains EGF and TGF-alpha like peptides which are motogenic and phosphorylate apical EGFR (Abstract). J Am Soc Nephrol 7: 1606, 1996.

32.   Sweeney, WE, Jr, and Avner ED. Functional activity of epidermal growth factor receptors in autosomal recessive polycystic kidney disease. Am J Physiol Renal Physiol 275: F387-F394, 1998[Abstract/Free Full Text].

33.   Sweeney, WE, Chen Y, Nakanishi K, Frost P, and Avner ED. Treatment of polycystic kidney disease with a novel tyrosine kinase inhibitor. Kidney Int 57: 33-40, 2000[ISI][Medline].

34.   Takacs-Jarrett, M, Sweeney WE, Avner ED, and Cotton CU. Morphological and functional characterization of a conditionally immortalized collecting tubule cell line. Am J Physiol Renal Physiol 275: F802-F811, 1998[Abstract/Free Full Text].

35.   Takacs-Jarrett, M, Sweeney WE, Avner ED, and Cotton CU. Generation and phenotype of cell lines derived from CF and nonCF mice that carry the H-2Kb-tsA58 transgene. Am J Physiol Cell Physiol 280: C228-C236, 2001[Abstract/Free Full Text].

36.   Takeda, M, Hosoyamada M, Shirato I, Obinata M, Suzuki M, and Endou H. Establishment of vasopressin-responsive early proximal tubular cell lines derived from transgenic mice harboring temperature-sensitive simian virus 40 large T-antigen gene. Biochem Mol Biol Int 37: 507-515, 1995[ISI][Medline].

37.   Towbin, H, Staehelin T, and Gordon J. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci USA 76: 4350-4354, 1979[Abstract].

38.   Van Adelsberg, J, Edwards JC, Herzlinger D, Cannon C, Rater M, and Al-Awquati Q. Isolation and culture of HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>-secreting intercalated cells. Am J Physiol Cell Physiol 256: C1004-C1011, 1989[Abstract/Free Full Text].

39.   Verkman, AS. Physiological importance of aquaporins: lessons from knockout mice. Curr Opin Nephrol Hypertens 9: 517-522, 2000[ISI][Medline].

40.   Whitehead, RH, VanEeden PE, Noble MD, Ataliotis P, and Jat PS. Establishment of conditionally immortalized epithelial cell lines from both colon and small intestine of adult H-2Kb-tsA58 transgenic mice. Proc Natl Acad Sci USA 90: 587-591, 1993[Abstract].

41.   Wilson, PD, Du J, and Norman JT. Autocrine, endocrine and paracrine regulation of growth abnormalities in autosomal dominant polycystic kidney disease. Eur J Cell Biol 61: 31-42, 1993.

42.   Wilson, PD, Geng L, Li X, and Burrow CR. The PKD1 gene product, "polycystin-1," is a tyrosine-phosphorylated protein that colocalizes with alpha 2beta 1-integrin in focal clusters in adherent renal epithelia. Lab Invest 79: 1311-1323, 1999[ISI][Medline].

43.   Wilson, PD, Hovater JS, Casey CC, Fortenberry JA, and Schwiebert EM. ATP release mechanisms in primary cultures of epithelia derived from the cysts of polycystic kidneys. J Am Soc Nephrol 10: 218-229, 1999[Abstract/Free Full Text].

44.   Woost, PG, Orosz DE, Jin W, Frisa PS, Jacobberger JW, Douglas JG, and Hopfer U. Immortalization and characterization of proximal tubule cells derived from kidneys of spontaneously hypertensive and normotensive rats. Kidney Int 50: 125-134, 1996[ISI][Medline].

45.   Zerres, K, Mücher G, Becker J, Steinkamm C, Rudnik-Schöneborn S, Heikkilä P, Rapola J, Salonen R, Germino GG, Onuchic L, Somlo S, Avner ED, Harman LA, Stockwin JM, and Guay-Woodford LM. Prenatal diagnosis of autosomal recessive polycystic kidney disease (ARPKD): molecular genetics, clinical experience, and fetal morphology. Am J Med Genet 76: 137-144, 1998[ISI][Medline].


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