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 |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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 |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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- (TGF-
)/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 -interferon (
-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 |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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 × 107 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
-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
-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 -IFN) (day 0) and
in nonpermissive conditions (37°C without
-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).
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
(104 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 |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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).
|
|
|
|
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.
|
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.
|
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.
|
|
|
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.
|
|
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.
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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 -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
· cm2) and high (~400
· cm2) electrical resistance exhibit EGFR
mislocalization, whereas a noncystic cell line with an intermediate
(~200
· 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-/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 |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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
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
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-.
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
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- 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
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
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
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
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 2
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
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].