Immortalized epithelial cells from human autosomal dominant polycystic kidney cy - 《American Journal of Physiology》美国生理学杂志干细胞之家

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发表于 2009-4-21 13:43 |只看该作者 |倒序浏览 |打印

作者：Mahmoud Loghman-Adham, Surya M. Nauli, Carlos E. Soto, Barbara Kariuki,  Jing Zhou作者单位：Department of Pediatrics and Pediatric Research Institute, Saint LouisUniversity School of Medicine, St. Louis, Missouri 63104; and HarvardInstitutes of Medicine, Harvard Medical School, Boston, Massachusetts02115 6 r4 `4 e# Y0 u* D
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      【摘要】
   Autosomal dominant polycystic kidney disease (ADPKD) is the result ofmutations in one allele of the PKD1 or PKD2 genes, followed by "secondhit" somatic mutations of the other allele in renal tubule cells.Continued proliferation of clonal cells originating from different nephronsegments leads to cyst formation. In vitro studies of the mechanisms of cystformation have been hampered by the scarcity of nephrectomy specimens and thelimited life span of cyst-derived cells in primary culture. We describe thedevelopment of a series of immortalized epithelial cell lines from over 30individual renal cysts obtained from 11 patients with ADPKD. The cells wereimmortalized with either wild-type (WT) or temperature-sensitive (TS)recombinant adeno-simian virus (SV)40 viruses. SV40 DNA integration into thecell genome was verified by PCR analysis. The cells have been passaged over 50times with no apparent phenotypic change. By light microscopy, the cellsappear pleomorphic but mostly polygonal and resemble the primary cultures.Transmission electron microscopy shows polarized epithelia with tightjunctions. The SV40 large T antigen was detected by immunocytochemistry and byWestern blot analysis at 37°C in the WT cell lines and at 33°C in theTS cell lines. It disappeared in TS cells 72 h following transfer to 39°C.The majority ( 29 ) of the celllines show binding of Dolichos biflorus lectin, suggesting distal tubule origin. Three cell lines show binding of Lotus tetragonolobus lectin or express aminopeptidase N, suggesting proximal tubule origin. Threecell lines were derived from a mixture of cysts and express features of bothtubules. The PKD1 and PKD2 mRNA and protein were detected in all cells byRT-PCR and by immunocytochemistry. The majority of the cells tested alsoexpress the epidermal growth factor receptor, cystic fibrosis transmembraneconductance regulator, epithelial sodium channel, and renin. These new series of cyst-derived cell lines represent useful and readily available in vitromodels for studying the cellular and molecular biology of ADPKD.
      【关键词】 polycystic kidney disease SV( _9 V- ~0 Z; P
   AUTOSOMAL DOMINANT POLYCYSTIC kidney disease (ADPKD) is a common genetic disorder, which accounts for 8-10% of the end-stage renal diseasepopulation in the United States( 7 ). ADPKD is caused bymutations in at least two separate genes. The PKD1 and PKD2 genes have beenmapped to chromosomes 16p13.3 and 4q21  -23, respectively( 19, 35, 41 ). Mutations in PKD1 account for 85% of the cases( 41 ). Additional genes may beinvolved but have not been mapped. The gene products of PKD1 and PKD2, calledpolycystin-1 and polycystin-2, are large membrane-associated proteins whosefunction is not well known( 19, 41 ). On the basis of theirstructure, they are likely involved in cell-cell and cell-matrix interactions( 2, 41 ).
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Polycystin-1 is expressed in fetal and neonatal kidneys, with much lowerlevels in the adult kidney( 40 ). The highest expression is found in collecting ducts and distal nephrons( 13 ). Polycystin-1 is alsoexpressed in many cysts in adult ADPKD kidneys( 13, 40 ). Polycystin-2 expressionparallels that of polycystin-1, with primarily an intracellular localization( 31 ). The highlevel expressionof polycystin-1 and -2 in cysts of adult ADPKD patients is difficult toreconcile with a loss-of-function mechanism. Expression of immunoreactive butnonfunctional protein is a likely possibility( 31 ).
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Cysts are the result of overgrowth of clonal tubule epithelial cells,reaching several centimeters in diameter( 16 ). In ADPKD, cysts mayoriginate from any nephron segment, including both the proximal and distaltubules/collecting ducts ( 3 ).They are composed of an epithelial monolayer attached to an abnormal andthickened basement membrane( 43 ). Larger cysts develop their own capillary network, which provides nutrients to the cyst surface( 4 ). The mechanism of cystformation is not well understood but involves a combination of factorsincluding transepithelial fluid secretion, tubular epithelial cellproliferation, and increased sensitivity to cAMP and to growth factors such asepidermal growth factor (EGF)( 6 ). Recent studies showed thatpolycystin-2 can function as a nonspecific cation channel permeable to sodiumand calcium ( 8, 15, 18 ). Mutations in PKD2 (e.g.,R742X) that result in a truncated protein lacking the 181 COOH-terminal aminoacids result in partial translocation of polycystin-2 to the plasma membrane,altered Ca 2 permeability, and decreased sensitivity ofthe channel to intracellular Ca 2 concentrations( 15, 39 ). Binding betweenpolycystin-1 and polycystin-2 is also necessary for channel function( 18 ).

Studies of the mechanisms of cyst formation and of genotype-phenotype correlations have been hampered by difficulties in obtaining ADPKD kidneyspecimens and by the lack of available and useful cyst-derived cell lines.Several investigators have used cyst-derived cells in primary culture( 28, 29, 43 ). These cells maintain manyfeatures of the cysts of origin and have been used to describe the propertiesof cyst epithelium. Unfortunately, primary cultures of cyst-derived cellscannot be maintained beyond three passages, necessitating frequent harvest ofcells from nephrectomy specimens. The short life span of cyst-derived cells inprimary culture precludes repeated studies on the same cell isolate.Furthermore, most investigators have isolated cells from several cysts pooledand digested together ( 28, 43 ). In prior studies, thetubule origin of cyst-derived cells has not usually been identified, makingsome of the results difficult to interpret. For example, Wilson et al.( 43 ) reported reducedadenylate cyclase activity in response to parathyroid hormone (PTH) and AVP incultured cyst-derived cells compared with cultured proximal and distal tubulecells, regardless of the location of the cysts within the renal cortex. They correctly noted that superficial cysts are not necessarily derived fromproximal tubules and vice versa( 43 ). To ensure that cysts ofboth proximal and distal tubule origin are obtained, one must dissect multipleindividual cysts, isolate cells for culture, and then identify the desiredcells with specific tubule markers.& ~" T) g% }( N: ]

We describe the isolation and subsequent immortalization of over 30epithelial cells from individual cysts of 11 ADPKD kidneys. Twenty-one of the23 cell isolates (29 cell lines) obtained from individual cysts havecharacteristic features of distal tubules with two cell isolates (9-7 and9-12) showing features of proximal tubules. Four cell isolates [wild-type (WT)1-2, WT 3-2, temperature sensitive (TS) 7-0, and WT/TS 17-0] were derived froma mixture of several cysts and were not subcloned. They are presumably derivedfrom cysts of both proximal and distal tubule origin. However, based onlectin-binding properties and positive renin immunostaining (a feature of distal cysts), these cell mixtures are mainly of distal tubule origin. Some ofthe cells have been maintained in culture for over 50 passages without anyphenotypic changes. The major characteristics of the cell lines are summarizedin Table 1.3 P. B/ t6 K% e& O, v" g
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Table 1. Major characteristics of ADPKD cyst-derived cell lines
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METHODS1 I  P- W: |) H
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Culture of cyst-derived cells. We initially isolated cells from cysts obtained from three polycystic kidneys, using a collagenase digestionmethod, previously described by other investigators ( 29, 41, 43 ). The yield from thismethod was low, requiring several cysts to be pooled and digested together.Therefore, this method proved unsuitable for isolation of cells from single cysts. The cell isolates numbered 1-2, 3-2, and 7-0 were obtained by thismethod ( Table 1 ). Forsubsequent kidneys, we used a trypsin/EDTA digestion method similar to thatdescribed by McAteer et al.( 29 ) to release cells fromcyst walls ( Fig. 1 ). A total of11 polycystic kidneys was used for this study. Because there was no differencein the characteristics of the cells cultured by these two methods, the dataare combined. Briefly, cyst tops were excised, washed extensively in PBS, andincubated with 1 x trypsin/EDTA at 37°C for 15-20 min. Each cyst wasprocessed separately. The tubes containing the cyst fragments were vortexedvigorously every 5 min. Thereafter, ice-cold HBSS containing 10% FBS was addedto inactivate trypsin. The cells were further released from the fibrous cystwall by trituration, washed twice with HBSS, then centrifuged and resuspendedin fresh culture medium, and seeded on Pimaria culture plates. To allowattachment, the cells were grown for 24-48 h in DMEM containing 10% FBS.Thereafter, the medium was changed to a serum-free or to a low-serum (2% FBS)medium, consisting of a 1:1 mixture of DMEM:Ham's F-12, supplemented with 5µg/ml insulin, 5 µg/ml transferrin, 5 ng/ml selenium, 36 ng/ml(10 - 7 M) hydrocortisone, 10 - 8 Mtriiodothyronine, 10 ng/ml EGF, and 50 ng/ml PGE 1, as well as 100U/ml penicillin, and 100 µg/ml streptomycin, as described by Detrisac et al. ( 10 ). Except for higherhydrocortisone concentrations and the addition of EGF, the medium used issimilar in composition to the K1 medium described by Taub et al.( 34 ). The cells were grown at37°C in a humidified incubator in 5% CO 2 -95% air. The culturemedium was changed every 2-3 days until confluency was reached. The cells werepropagated by releasing them with 0.05% trypsin/0.53 mM EDTA and were seededon collagen I-coated culture dishes. Aliquots were frozen and stored in liquidnitrogen. The cells could be propagated for three passages, after which theybecame senescent and stopped dividing. They were, therefore, transformed andimmortalized as described below.

Fig. 1. Schematic diagram of isolation of epithelial cells from polycystic kidneys.Several cyst tops were individually dissected with a scalpel blade. Each cystcutout was rinsed extensively in ice-cold PBS and then placed in a separatetube containing 1 x trypsin/EDTA. The cysts were incubated for 20 min at37°C and vortexed vigorously every 5 min to allow separation of cells fromthe cyst wall. The cyst walls stripped of cells were removed and the cellswere washed twice in Hanks' buffered salt solution with 10% fetal bovineserum. The final pellet was resuspended in serum-deficienthormone-supplemented medium and seeded on Primaria culture plates. The cellswere transformed at passage 1 or 2 when 30-50% confluencewas reached.

Recombinant adeno-simian virus 40 viruses. Recombinantadeno-simian virus (SV)40 viruses were used to transform primary cultures ofcyst-derived and control kidney epithelial cells. These viruses contain anorigin-defective (ori - ) SV40 DNA cloned into the adenovirus vectorin place of early regions 1a and 1b( 38 ). Due to the removal ofearly region 1, the adenovirus vector has no transforming activity and thusdoes not interfere with SV40-induced transformation( 38 ). This ori - chimeric adeno-SV40 virus results in much higher efficiency of transformation compared with WT SV40 virus or to ori - SV40 virus. Furthermore, because the SV40 portion lacks the origin or replication, viral replicationdoes not occur within the cells( 38 ). Two chimeric adeno-ori - -SV40 viruses were used containing either the WT or TSA209 SV40 that carries a point mutation at position 209 in the early region( 25, 38 ). They were obtained fromDr. Janice Chou, National Institutes of Health. In this system, the chimericvirus attaches to the cell via the adenovirus receptor and allowsintracellular introduction of the SV40, followed by integration of the SV40DNA into the cell genome ( 1 ).The adenovirus acts as a vehicle and not as the transforming agent. Because itlacks the early region 1, the adenovirus does not replicate inside the cells( 32, 38 ). The recombinant adeno-SV40 viruses used in this study have been used previously to transformand immortalize human fibroblasts and human placental cells( 25, 38 ).
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Development of immortalized cells. The cells were seeded on 100-mmcollagen-coated culture dishes and grown in serum-free hormone-supplementedmedium until 50% confluent. They were transformed with recombinantori - adeno-SV40 viruses as detailed above. The viral supernatantswere obtained from infected HEK293 cells and kept at -80°C until use.Between 1.5 and 2.0 ml of viral supernatant ( 10 8 plaque-forming units/ml) were used per plate. Both WT and TS viruses wereused. The cells were incubated with the virus at 37°C for 60 min, and then the supernatant was aspirated and replaced with fresh culture medium.Thereafter, the medium was changed every 2-3 days. The cells infected with theWT virus were grown at 37°C, and those infected with the TS virus weregrown at 33°C. After viral infection, the majority of the cells becamedysmorphic, taking on the appearance of "ghost" cells. A fewdistinct colonies of normal-appearing cells remained, which becamesubconfluent over a period of 5-6 wk. These surviving cells were subcultured into additional collagen-coated plates and maintained in hormone-supplemented medium with 2% FBS until passage 6. Thereafter, DMEM with 10% FBS wasused. With the use of this method, we did not observe any fibroblastcontamination.
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In addition to cyst-derived cells, we immortalized normal human renalcortical tubule epithelial cells (RCTEC) to be used as the control for ourcyst-derived cell lines. The cells were purchased at passage 1 fromClonetics (Walkersville, MD). They were immortalized using the same adeno-SV40viruses and culture conditions used to immortalize the cyst-derived cells. The culture medium used for RCTEC was identical to that used for cyst-derivedcells, except that it did not contain EGF. Although the primary cultures ofRCTEC contained a mixture of 20% proximal and 80% distal tubule-derivedcells, continued culture with the above medium resulted in the preferentialgrowth of cells of distal tubule origin as evidenced by Dolichosbiflorus (DBA) lectin staining. These cells were further subcloned bylimited dilution to obtain pure populations of proximal and distal tubule celllines that were identified by their lectin-binding characteristics. Accordingly, the distal tubule cell line has been named RCTEC-DBA and theproximal tubule cell line named RCTEC- Lotus tetragonolobus agglutinin(LTA).8 t1 k3 s+ r% G2 a: \

Immunocytochemistry of cell monolayers. The WT cells were seeded on collagen-coated coverslips and grown at 37°C for 24 to 48 h beforestudy. The TS cells were initially grown at 33°C, then transferred to 37or 39°C for 72 h before study. For the majority of studies, the two-stepimmunofluorescence method was used. The cells were washed three times with PBSand then fixed for 15 min with 4% paraformaldehyde in PBS, followed by threewashes with PBS. The cells were permeabilized for 5 min at room temperaturewith 0.5% Triton X-100 in PBS, washed twice with PBS, and blocked with 0.5%BSA in PBS for 20 min. After incubation with primary antibodies for 1 h atroom temperature, the cells were washed three times with PBS followed byincubation with FITC-labeled secondary antibodies (anti-mouse or anti-rabbit IgG, used at 1:100 to 1:200 dilution) for 30 min in the dark. The dilutionsfor the primary antibodies are indicated in Reagents and supplies.For lectin-binding studies, FITC-labeled LTA, Arachis hypogaea (PNA),or DBA lectins were added directly to the cells at 1:50-1:100 dilution andincubated for 30 min at room temperature. LTA is a marker of proximal tubules,and PNA and DBA are markers of distal and collecting tubules. The cells werewashed with PBS and mounted on slides, using FluoroGuard antifade mountingsolution (Bio-Rad, Hercules, CA). The slides were viewed with a Zeiss Axioplanmicroscope equipped with epifluorescence and photographed using KodakElitechrome 400 film at either 8- or 15-s exposure.5 K: k6 s, r1 }, }- g( x

In some studies, immunocytochemistry was performed with the ABC method,using a commercial kit (Vectastain Elite kit, Vector Laboratories, Burlingame,CA). The cells were washed three times with PBS and then blocked with normalhorse serum for 20 min at room temperature, followed by the addition of the primary antibodies. The cells were incubated overnight at 4°C and thenwashed three times with PBS/0.1% Tween 20, followed by the addition of thesecond (biotinylated) antibody and incubated for 30 min at room temperature.The cells were washed three times with PBS followed by the addition of onedrop of the ABC reagent and incubated at room temperature for 30 min. They were washed three times with PBS, followed by the addition of peroxidasesubstrate for 6-10 min, then rinsed in distilled water, counterstained withhematoxylin, washed extensively in running water, and mounted. The slides wereviewed with a Zeiss Axioplan microscope and photographed with Kodak Ektachrome 64T film.

Electrical resistance measurements. The cells were grown at highdensity on 0.45-µm pore size collagen I-coated translucent permeable filterinserts (Biocoat, Collaborative Biomedical Products, Bedford, MA) and placedinside 12-well plates. When confluency was reached, electrical resistance wasmeasured across the filter, using a Voltohmeter (World Precision Instruments, Sarasota, FL). The resistance across empty filters was subtracted from theresistance across cell monolayers, and the value obtained was corrected forthe surface area of the filter and expressed as ohms per centimetersquared.

Electron microscopy. The cells were grown on filters as above andthen fixed with 2.5% glutaraldehyde/1% paraformaldehyde in cacodylate buffer(pH 7.4). They were postfixed with 1% osmium tetroxide in cacodylate buffer,dehydrated with a graded series of alcohols, and embedded in epoxy resin.Cross sections of the monolayers were cut with a diamond knife. The thinsections were counterstained with uranyl acetate and lead citrate. The specimens were observed and photographed with a JEOL 100S transmission electron microscope (JEOL, Tokyo, Japan).( A4 @0 u  c, k, x+ B0 G0 \
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RNA isolation and RT-PCR. The cells were grown to confluence in100-mm culture plates or 75-cm 2 flasks, then scraped, washed inPBS, and centrifuged. The cell pellet was snap-frozen in dry-ice ethanol, andstored at -70°C until used for RNA preparation. Total RNA was prepared,using commercial kits (either RNeasy, Qiagen, Valencia, CA or TRIzol Reagent,Invitrogen, Carlsbad, CA). mRNA for various genes was detected by reverse transcription of the RNA to obtain the complementary DNA (cDNA), followed bypolymerase chain reaction (RT-PCR), using a Commercial kit (Access RT-PCR,Promega, Madison, WI). Two different pairs of oligonucleotide primers weredesigned, corresponding to the 5'- and 3'-regions of the genes( Table 2 ). The primers weredesigned to span one or more exon-intron junctions to allow the distinction ofproducts amplified from genomic DNA. Parallel tubes were used in which thereverse transcriptase was omitted (designated -RT), which were then subjectedto the same steps as those containing this enzyme (designated  RT). PCRamplifications were performed for 30-35 cycles under fairly stringentconditions, using an MJ Research PTC-200 Thermocycler. Each cycle consisted ofdenaturation at 94°C for 30 s, annealing at 58°C for 1 min, andextension at 72°C for 1 min. Each experiment was carried out at leasttwice. In all experiments, one or more water blanks were used to verify thelack of illegitimate amplification. RTPCR reactions were repeated in parallelwith the same amount of RNA, using primers for the -actin gene, to checkfor the quality of the reaction and to allow for evaluation of the amount ofRNA used in each reaction. In some experiments, GAPDH or -tubulinprimers were used instead of -actin. The reaction products were sizefractionated on 1.5% agarose gels with ethidium bromide and photographed.

Table 2. Primers used in PCR amplification
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For detection of SV40 DNA integration into the cell genome, DNA wasisolated, using a commercial kit (QIAmp DNA mini kit, Qiagen). SV40 DNA wasamplified by PCR, using specific oligonucleotide primers( Table 2 ).
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Gel electrophoresis and Western blotting. Cells grown oncollagen-coated 60- or 100-mm culture dishes were washed twice with ice-cold PBS, scraped, and collected by centrifugation. The pellet was homogenized in asmall volume of lysis buffer (50 mM NaCl, 50 mM Tris·HCl, pH 8.0, 0.2%NP-40, 0.5 mM PMSF) and incubated on ice for 60 min, followed by twofreeze-thaw cycles. After centrifugation at 10,000 g for 10 min, thesupernatant was transferred to new tubes and frozen at -70°C./ I) N2 p. p  n) O! w$ n/ r0 S

SDS-PAGE was performed according to Laemmli( 23 ). After separation, protein bands were electrophoretically transferred to polyvinylidene difluoride membranes (Immobilon-P) according to Towbin et al. ( 37 ). Nonspecific sites wereblocked by incubating the membranes for at least 2 h at room temperature in ablocking buffer consisting of Tris-buffered saline, pH 7.4, 0.2% Tween 20(TBS-T) and 3% BSA. After two washes in TBS-T, a 1:500 to 1:1,000 dilution ofthe primary antibody (in blocking buffer) was added to the membranes, followedby incubation at room temperature for 2 h. After three washes in TBS-T, thesecond antibody was added (1:20,000 to 1:40,000 dilution of a horseradishperoxidaseconjugated anti-mouse or anti-rabbit IgG), and the membranes wereincubated for 2 h at room temperature. After four washes in TBS-T, the proteinbands were visualized, using the SuperSignal West Pico chemiluminescencedetection method (BioRad Laboratories). Membranes were briefly exposed toradiographic film for a permanent record.* q4 B5 |+ Y# i# z% G
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Measurement of cAMP. Cells grown in 12-well collagen-coated plateswere transferred to serum-free DMEM for 15 h before the experiments. They werepreincubated for 1 h with serum-free DMEM containing 0.5 mM 3-isobutyl-1methylxanthine (IBMX), a phosphodiesterase inhibitor, followed by the additionof parathyroid hormone (10 - 6 M) or AVP(10 - 6 M). In some experiments, forskolin(10 - 5 M) was used as a positive control. After 10-min incubation at 37°C, the media were aspirated and the cells were washedthree times with ice-cold PBS. Thereafter, 1 ml of a 100:1 mixture ofethanol/1 N HCl was added to each well and the cells were incubated overnightat 4°C. After extraction, the ethanol was evaporated and the extract wasresuspended in 0.25 ml of ice-cold Tris-buffered saline (pH 7.4) containing 4mM EDTA and frozen at -20°C until assayed. cAMP was measured byradioimmunoassay, using a commercial kit (Biotrak, Amersham, ArlingtonHeights, IL).
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Each experiment was repeated at least two more times. Where appropriate,statistical analysis was performed by Student's t -test for groupcomparisons. A P value of 0.05 was considered significant.
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Reagents and supplies. Tissue culture media and FBS were purchased from GIBCO Invitrogen (Carlsbad, CA). Rat tail collagen I (no. 354236) waspurchased from Becton Dickinson (Bedford, MA). Monoclonal antibody againstpan-cytokeratin was purchased from Sigma (St. Louis, MO) and used at 1:100dilution for immunofluorescence studies and at 1:250 dilution with the ABCmethod. Monoclonal antibody against vimentin was purchased fromBoehringer/Roche (Indianapolis, IN) and used at 1:100 dilution forimmunofluorescence studies and at 1:250 dilution with the ABC method.Monoclonal antibodies against aminopeptidase N (CD13) and the SV40 large Tantigen were purchased from Santa Cruz Biotechnology (Santa Cruz, CA) and usedat 1:250 and 1:100 dilution, respectively. The rabbit polyclonal antibodyagainst the extracellular region of polycystin-1 (mr-3) and rabbit polyclonalantibody against polycystin-2 were prepared in Dr. Jing Zhou's laboratory and have been previously described( 14 ). Both were used at 1:250 dilution. FITC-labeled secondary antibodies were purchased from Pierce(Rockford, IL). FITC-labeled LTA, PNA, and DBA lectins were purchased fromSigma and used at 1:50 to 1:100 dilution. RNA and DNA isolation kits werepurchased from Qiagen. RTPCR kit (Access RT-PCR) was purchased from Promega.Oligonucleotide primers were synthesized by GIBCO Invitrogen. Other reagents of highest purity grades were purchased from Sigma, Fisher, or othercommercial suppliers.( }' y5 C3 q4 _6 X7 x

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Characteristics of ADPKD cells. The majority of the cells were transformed at the second passage after the initial seeding. After infectionwith the recombinant adeno-SV40 viruses, the cells divided rather slowly withcontinued presence of large ghost cells that were gradually replaced byhealthy appearing cells over 5-8 wk. The surviving cells were maintained in a hormone-supplemented medium containing 2% FBS to inhibit fibroblast growth.After passage 6, the medium was changed to DMEM with 10% FBS. Thecells have been maintained in this medium for 8-42 passages. From 30cyst-derived cell isolates, we obtained a total of 41 transformed cell lines,21 of which were transformed with WT SV40 viruses and 20 with TS SV40 viruses.Among these, 11 cell lines were successfully transformed with both WT and TSviruses. Additionally, SV40-transformed control proximal and distal renaltubule epithelial cells were obtained from a normal human renal corticalepithelial cell isolate ( Table1 ).
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The cells immortalized with the WT virus (WT series) are continuously grownat 37°C. Those immortalized with the TS virus (TS series) are maintainedat 33°C. To induce expression of a normal phenotype, the TS cells aretransferred to 39 or 37°C for 72 h before the experiments. The TS cellsremain viable for at least 7 days after transfer to the nonpermissivetemperature (not shown).

When seeded on either uncoated or on collagen I-coated culture dishes, thecells grow as monolayers with contact inhibition, as evidenced by theobservation of a plateau on growth curves. Their morphology varies from apolygonal or cobblestone appearance typical of epithelial cells to elongatedor fusiform shapes and swirls ( Fig.2 ). In one cell isolate (14-2), we observed dome formation at theinitial seeding but domes were not seen in cultures of immortalized cells( Fig. 2 B ). Todetermine the growth characteristics of the cells, representative samples fromboth WT and TS cyst-derived cells were seeded in 12-well multiwell plates at adensity of 20,000 cells/well. Beginning on the third day after seeding, thecells in duplicate wells were trypsinized every 24 h and counted with ahemocytometer. The WT cells were maintained at 37°C for the duration of the study. The TS cells were seeded on two different 12-well plates andinitially grown at 33°C. Seventy-two hours after seeding, one plate wasswitched to 37°C and daily cell counts were continued from duplicate wellsof each plate. The WT cells and TS cells grown at 37 and 33°C,respectively, showed typical "S"-shaped growth curves with fastergrowth rate for WT cells ( Fig. 3, A and B ). The doubling times estimated from thelinear portion of the exponential phase of growth curves were 33 h for WT9-7 cells grown at 37°C and 48 h for TS 9-7 cells grown at 33°C.We also calculated the "growth rate" of the same cells usinglog-linear transformation of the growth data( Fig. 3, C and D ). With the use of the values from log-lineartransformations, the doubling times were 38 h for WT 9-7 cells and 64 h for TS 9-7 cells. In TS cells, cell division and growth rate slowedand ceased 96 h after transfer to 39°C, coinciding with thedisappearance of the large T antigen on Western blots. In general, growth rates were more a function of the growth temperature necessitated by theimmortalizing SV40 virus used (faster for WT cells than for TS cells) than ofthe kidney or cyst from which the cells originated.
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Fig. 2. Phase-contrast microscopy of immortalized cyst-derived cells. A and B : confluent monolayers of cyst-derived cells (14-4 and 14-2) inprimary culture. B : note dome formation. C : confluentmonolayer of an immortalized temperature-sensitive (TS) cell line (TS 9-7)derived from a cyst of proximal origin; D : cell line (TS 11-7)derived from a cyst of distal origin, both grown at 33°C. E :confluent monolayer of an immortalized wild-type (WT) cell line (WT 9-7) froma cyst of proximal origin; F : WT cell line (WT 10-7) derived from acyst of distal origin. G : confluent monolayer of control human renaldistal tubule epithelial cell line (WT RCTEC). H : recentlytransformed cyst-derived cells with multiple "ghost" cells. Allcells were grown on collagen I-coated culture plates. Magnification x 100., a  i) q0 u+ `; F5 v
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Fig. 3. Growth curves of WT and TS immortalized cyst-derived cells. A :growth curve of a representative WT cell line (WT 9-7) at 37°C, showing atypical S-shaped curve with an initial lag time followed by exponentialgrowth, then slower growth, as the cells become confluent. B : growthcurve of a representative TS cell line (TS 9-7) under permissive andnonpermissive conditions. Cells were grown at 33°C, and then one-half weretransferred to 37°C (arrow) and the other one-half were kept at 33°C.The TS cells continue to divide when maintained at 33°C. In TS cellstransferred to 37°C, cell growth slows 48-72 h, following transfer to thenonpermissive temperature. The cells cease to grow and die when left 7 days ormore at this temperature. C and D : same data as in A and B plotted on a log scale. The slopes of the linescorrespond to growth rates of the cells.
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Previous studies showed increased life span and better differentiation whenrenal epithelial cells are grown on collagen or other extracellular matrixcomponents ( 10, 26 ). We therefore used collagen I-coated plates and dishes for all subsequent experiments. Regardlessof culture conditions (presence or absence of serum) and the type ofsubstratum, the nontransformed cyst-derived cells could not be maintained inculture beyond three passages.
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With the use of immunocytochemistry with a pancytokeratin antibody, wedemonstrated the expression of cytokeratin by all the immortalized cellsstudied, confirming their epithelial origin( Fig. 4, A and C ). The cells also express vimentin, a component of cells of mesenchymal origin ( Fig. 4, B and D ). The significance of this finding is not clear,but it may be related to the undifferentiated nature of these cells( 42 ). Other epithelial cellssuch as podocytes have also been shown to express vimentin( 44 ).. N5 ?1 T0 ^& W, }; E+ p5 W: W
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Fig. 4. Expression of cytokeratin and vimentin in cyst-derived cells. The cellswere seeded on collagen I-coated coverslips and used for immunocytochemistryusing either the ABC method ( A - C ) or FITC-labeled secondantibody ( D and E ). Strong cytokeratin staining ( A and D ) confirms epithelial origin of the cells. The cells alsoexpress vimentin ( B and E ), consistent with loss ofdifferentiation. C : cells incubated with preimmune IgG show nostaining. The results are representative of 3 experiments in differentcells.
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Characterization of tubule origin of the cysts and cells. To determine the tubule origin of the cyst-derived cells, we used lectins thatbind specifically to sugar moieties in proximal and distal tubules as well asantibodies to tubule-specific proteins. We used LTA and aminopeptidase N(CD13) as proximal tubule markers and DBA or PNA as distal tubule markers( 18 ). Twenty-one of 30transformed cell isolates corresponding to a total of 29 WT and TS cell linesbound DBA or PNA lectins, suggesting distal tubule origin of the cysts( Table 1 ). Two transformed cellisolates corresponding to three WT and TS cell lines (WT 9-7, TS 9-7, and WT9-12) bound LTA lectin or showed positive staining with CD13 antibody,suggesting proximal tubule origin of the cyst( Table 1 and Fig. 5 ). Five cell isolates(1-2, 3-2, 7-0, 13-3, 14-2, and 17-0) represent mixtures of DBA- andLTA-positive cells and two isolates (18-3 and 18-5) derived from single cystscould not be classified due to conflicting lectin-binding results. These twocell lines express angiotensinogen, a feature of proximal tubules( Table 1 ). Lectin-bindingcharacteristics did not change in those cells that were studied as primary cultures before immortalization and again after they were immortalized ( Fig. 5, G and H ). On the basis of these criteria, we have beensuccessful in isolating cyst-derived cells representing cysts of both proximaland distal tubule origin.

Fig. 5. Demonstration of tubule origin of cyst-derived cells. The cells were grownon collagen I-coated coverslips and either incubated with FITC-labeled D.biflorus (DBA), Arachis hypogaea (PNA), or L.tetragonolobus agglutinin (LTA) lectin or with an antibody toaminopeptidase N (CD13), followed by incubation with a FITC-labeled secondantibody. A : cyst-derived cells of proximal tubule origin (WT 9-7)showing positive staining for CD13. B : cyst-derived cells of distaltubule origin (WT 10-7), showing positive staining with DBA lectin. C : WT 9-7 cells incubated with preimmune IgG as primary antibody(control for A ). D - F : control human renal corticalepithelial cells (WT RCTEC) of distal tubule origin, showing negative stainingwith CD13 ( D ) and positive staining with DBA lectin ( E ).Same cells incubated with preimmune IgG as primary antibody (control for D ). G : cyst-derived cells in primary culture showingpositive staining with PNA lectin. H : cyst-derived cells in primaryculture showing positive staining with LTA lectin.
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We also measured the transepithelial electrical resistance across cellsgrown on collagen-coated permeable filter inserts. In four cell isolates (9-5,9-6, 10-7, 11-6) in primary culture, the mean transepithelial electricalresistance measured in duplicate filters was 128 ± 7 /cm 2 ( n = 4), which is comparable to the electricalresistance measured in leaky epithelia such as proximal tubules. Other cellisolates had significantly higher electrical resistances [2,204.3; 1,027.5;and 577.5 /cm 2 for 10-2, 11-6, and 14-2, respectively (mean1,270 ± 485, n = 3)]. After transformation, we were unable toshow a significant difference in electrical resistance between the two celltypes. For example, cell isolate 11-6 had electrical resistances of 1,645 and2,764 /cm 2 on 2 consecutive days when in primary culture,which decreased to 89 and 134 /cm 2 (duplicate measures),when the measurements were repeated in transformed WT 11-6 cells. However,many other characteristic features of proximal and distal tubules could beidentified in the immortalized ADPKD cell lines.* D1 t! W5 L; m

Electron microscopy. Electron microscopy was performed using cellsgrown on collagen I-coated filter inserts. The WT cells were grown at 37°Cuntil confluency was reached. The TS cells were grown at 33°C. Whenconfluent, they were transferred to 37°C for 72 to 96 h before beingprocessed for electron microscopy. The cells had large nuclei and appeared aspolarized epithelial cells with the apical side facing the medium and thebasolateral side facing the filter. We did not observe apical microvilli inthese limited studies. Both cyst-derived cells in primary culture and the TScells, studied after transfer to the nonpermissive temperature of 37°C,showed relatively well-developed junctional complexes( Fig. 6 ). As reported in otherimmortalized cells ( 24 ),multilayering could be observed when the cells were grown under suboptimalconditions., B8 Q3 _5 ~6 v# {# ~# [

Fig. 6. Transmission electron micrographs of immortalized cyst-derived cells. Thecells were grown on collagen I-coated permeable filters and processed forelectron microscopy. The TS cells were grown initially at 33°C and thenswitched to 37°C for 96 h before processing. A : section from acyst-derived cell in primary culture showing polarized appearance and awell-developed tight junction (arrows; magnification x 19,800 beforereduction). B and C : TS cells studied 96 h after transfer to37°C. B : two TS cells showing the presence of tight junctionsbetween them (arrows). C : another area from the same filter, showingjunctional complex between 2 cells (arrowhead). Magnification for B and C : x 15,000.
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Hormonal responses. The generation of cAMP in response to hormones such as PTH and AVP has been used to ascertain the tubule origin of culturedrenal cortical cells ( 26 ).Previous studies in cyst-derived cells in primary culture of unknown tubuleorigin showed reduced cAMP generation in response to PTH and AVP( 43 ). With the use ofimmortalized cyst-derived cells with defined tubule origin, we investigatedthe effect of PTH and AVP on intracellular cAMP generation. As expected, therewas a significant increase in intracellular cAMP, following exposure toforskolin (10 - 5 M) in all the cells tested( Fig. 7 ). In four experiments conducted in triplicate, we showed no change in intracellular cAMP generationin response to AVP in a cell line (WT 10-7) derived from a cyst of distaltubule origin. In contrast, we observed a significant increase inintracellular cAMP in response to PTH in a cell line (WT 9-7) derived from acyst of proximal tubule origin ( P n = 4).Interestingly, there was also a significant increase in cAMP in response toAVP in this cyst cell line of proximal tubule origin ( P n = 4). We observed increased intracellular cAMP (from 1.19 ± 0.03 to 2.47 ± 0.70 pmol/well, n = 3) in response to AVP inthe distal tubule control human kidney cell line (WT RCTEC). However, theincrease was significant only at P = 0.051, perhaps due tointra-assay variability ( Fig.7 ). On the basis of these experiments, we conclude that there is ablunted response to AVP in cyst-derived cells of distal tubule origin, whereas the response of cyst-derived cells of proximal tubule origin to PTH remainsintact.

Fig. 7. cAMP generation by immortalized cyst-derived cells. The cells wereincubated with forskolin (Forsk; 10 - 5 M), AVP(10 - 6 M), or parathyroid hormone (PTH; 1-34)(10 - 6 M) in the presence of 0.5 mM 3-isobutyl-1methylxanthine. Intracellular cAMP generated was measured by radioimmunoassay.Forskolin results in marked increase in cAMP in all the cell lines. A : cyst-derived cells of proximal tubule origin (WT 9-9) showincrease in intracellular cAMP in response to both AVP and PTH. B :cyst-derived cells of distal tubule origin (WT 10-7) fail to respond to AVP.WT RCTEC shows moderate increase in cAMP in response to AVP. The results aremeans ± SE of 3-4 experiments, each conducted in triplicate. A : significance at * P B : forWT 10-7, the difference between AVP or PTH and control was not significant. C : for WT RCTEC, the difference between AVP and control did not reachstatistical significance ( P = 0.051).

Expression of polycystin-1 and polycystin-2. We used both RT-PCR and immunocytochemistry to determine the distribution of PKD1 and PKD2 mRNAand polycystin proteins from several cyst-derived cell lines. Bysemiquantitative RT-PCR, we showed the presence of mRNA for PKD1 and PKD2 inall cells examined ( Fig. 8 ). Furthermore, both PKD1 and PKD2 band intensities appeared to be higher incells grown on collagen I, compared with cells grown on plastic. With the useof polyclonal antibodies against polycystin-1 and polycystin-2, we showed thepresence of both proteins in all the cells examined. Both polycystin-1 andpolycystin-2 showed a diffuse intracytoplasmic staining pattern, regardless ofthe degree of confluence of the cell monolayer( Fig. 9 ). We also detectedmoderate staining for both polycystin-1 and -2 in the control RCTEC cell lines( Fig. 9 and Table 1 ), indicating that theantibodies cannot distinguish between intact and mutated polycystins.
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Fig. 8. Demonstration of mRNA for PKD1 and PKD2 in immortalized cyst-derived cells.Cyst-derived cells (WT 1-2) were grown on either noncoated or on collagenI-coated culture dishes, followed by RNA isolation. RT-PCR was performed,using specific primers for human PKD1 and PKD2 genes. Strong PKD1 and PKD2mRNA bands are seen when the cells are grown on collagen. Weaker PKD1 and PKD2mRNA bands are seen when the cells are grown on uncoated plastic. Note thatthese cells also express renin mRNA, which is not normally expressed in renaltubules.# L6 g# g0 {+ q8 U1 M3 X
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Fig. 9. Polycystin expression by immortalized cyst-derived and control human kidneycells. The cells were seeded on collagen I-coated coverslips and used forimmunocytochemistry. Cells from a cyst of proximal tubule origin (WT 9-7)showing diffuse staining for polycystin-1 ( A ) and polycystin-2( B ). Cells from a cyst of distal tubule origin (WT 10-7) showingdiffuse staining for polycysin-1 ( C ) and polycystin-2 ( D ).WT RCTEC cells showing staining for polycystin-1 ( E ) and forpolycystin-2 ( F ). G : WT RCTEC cells stained with preimmuneIgG showing background staining. A - G : magnification x 200.0 K* B2 x) \( n3 q6 B# N
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Expression of tubule-specific channels and proteins. In addition to polycystins, the immortalized cells express a variety of other markerstypically associated with renal tubules. We studied the expression of mRNA forthe genes of interest in selected cyst-derived cells as well as in human RCTECusing RT-PCR. They included the mRNA for cystic fibrosis transmembraneconductance regulator (CFTR), epithelial Na channel (ENaC),Na -H exchanger (NHE3), aquaporin-1 (AQP1), andH -ATPase (Figs. 10 and 11 ). The epidermal growthfactor receptor (EGF-R) was demonstrated both by RT-PCR andimmunocytochemistry and was expressed by cyst-derived cells, of both proximaland distal tubule origin ( Table1 ). NHE3 was weakly expressed in cyst-derived cells, but strongNHE3 bands were seen in RCTEC lines. AQP1 was expressed in cyst-derived cellsof both proximal and distal tubule characteristics as well as in RCTEC lines.H -ATPase was weakly expressed by cyst-derived cells of distalorigin, but a strong band was seen in distal RCTEC. On the basis of theselimited experiments, we conclude that the immortalized cyst-derived cellsmaintain many features of their tubule of origin. Interestingly, we showed that cyst-derived cells of distal tubule origin express renin, whereas thecyst-derived cells of proximal tubule origin express angiotensinogen( 43 ). Both cell types expressangiotensin-converting enzyme and ANG I receptors (AT 1 subtype)(unpublished observations).

Fig. 10. Demonstration of mRNA for cystic fibrosis transmembrane conductanceregulator (CFTR) and epithelial Na channel (ENaC; -subunit)in immortalized cyst-derived cells. RT-PCR was performed using RNA isolatedfrom several cells derived from single cysts of distal tubule origin.Autosomal dominant polycystic kidney disease (ADPKD)-2 and -3 cells wereobtained from cystic tissue dissected from deep portions of ADPKD kidneys.Human kidney RNA was used as positive control. Strong mRNA bands are seen inall the cells tested (arrows).% U% n8 [% l% H' f4 R- y8 p
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Fig. 11. Demonstration of mRNA for renal tubule-associated channels and proteins.Agarose gel of RT-PCR-amplified products corresponding toNa -H exchanger (NHE3), aquaporin-1 (AQP1), epidermalgrowth factor receptor (EGF-R), and H -ATPase 1 -subunit. GAPDH and -tubulin are used as positivecontrols. WT 9-12 LTA and WT 9-12 DBA were subcloned from a cyst-derived cellline, based on lectin-binding characteristics. WT 9-2 LTA has proximal tubulecharacteristics and WT 9-12 DBA has distal tubule characteristics. Similarly,WT RCTEC cells were subcloned into proximal tubule (RCTEC LTA) and distaltubule (RCTEC DBA) cell lines. Weak NHE3 bands are seen in both cyst-derivedcells, whereas AQP1 is present in all cells. EGF-R band intensity is strongerin cells of proximal tubule origin. Conversely, H -ATPase mRNA bandis seen primarily in distal RCTEC and weakly in distal cyst-derived cells. MW,molecular weight.
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Immortalized cyst-derived cells express the SV40 large T antigen. Because all the cyst-derived cell lines and the control human renal corticalcell line were immortalized with adeno-SV40 recombinant viral constructs, weverified the expression of SV40 large T antigen in these cells. We initiallyused immunocytochemistry with a monoclonal antibody against the large Tantigen of SV40. Strong immunostaining was observed within the nucleus of all the cells studied (both ADPKD and RCTEC). In TS cells grown at the permissivetemperature of 33°C, the intensity of the large T antigen staining wassimilar to that found in WT cells grown at 37°C. When the TS cells wereswitched to the nonpermissive temperature of 39°C, the large T antigen staining was markedly reduced at 72 h( Fig. 12 ). In additional experiments, we tested for the presence of the SV40 large T antigen by Westernblot analysis of cell lysates obtained from representative cyst-derived cells.The large T antigen was seen as a 94-kDa band in WT cells grown at 37°Cand in TS cells grown at 33°C. There was a gradual decrease in theintensity of the large T antigen band in TS cells switched to 39°C, whichcompletely disappeared after 72 h ( Fig.13 ).

Fig. 12. Detection of simian virus (SV)40 large T antigen in immortalizedcyst-derived cells. The cells were seeded on collagen-coated coverslips andused for immunocytochemistry with an antibody to the large T antigen of SV40.The WT cells (WT 9-7) were studied at 37°C. The TS cells (TS 11-7) werestudied both at 33°C and after transfer to 39°C for 72 h. Strong largeT antigen immunostaining is localized to the nuclei of cells in both WT and TScells. The T antigen staining intensity is significantly reduced in TS cellskept at 39°C for 72 h. C : apparent persistence ofimmunofluorescence signal is due to computer enhancement. All pictures weretaken at 8-s exposure.  y4 {5 g: e+ |" I+ A

Fig. 13. Detection of SV40 large T antigen in immortalized cyst-derived cells. Thecells were grown either at 37°C (WT 9-9 cells) or at 33°C (TS 9-7cells) and then transferred to 39°C for different times before collection.Cell culture media were collected at times indicated and used for SDS-PAGE,followed by Western blotting. The large T antigen is seen as a band of 94-kDamolecular mass. Clone 5 is a murine collecting duct cell line derivedfrom a transgenic mouse (tsA58) harboring the SV40 large T antigen gene. The Tantigen band is seen in WT cells at 37°C and in TS cells at 33°C. Itcompletely disappears 72 h after transfer of the TS 9-7 cells to 39°C.
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Demonstration of SV40 DNA incorporation into the cellular genome. Previous studies showed that human cells are relatively resistant toimmortalization with SV40 oncogenes, whereas rodent cells can be easilytransformed and immortalized( 17 ). The (ori - ) chimeric adeno-SV40 viruses such as the ones used in this study result in muchhigher efficiency of transformation compared with WT SV40 virus or with(ori - ) SV40 virus( 25, 38 ). This was the reason forusing the chimeric viruses instead of simple (ori - ) SV40.
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To ascertain that the cell lines have been successfully transformed, westudied the incorporation of SV40 DNA into the cyst-derived cellular DNA usingPCR amplification. Because the TS SV40 virus used (TS A209) differs from theWT by a point mutation, the same PCR primers were used to amplify the genomicDNA obtained from both WT and TS cells. The results show the presence of astrong band for SV40 DNA in all cells tested, including the control RCTEC andcyst-derived cell lines ( Fig.14 ).. D* J* v4 P* q8 ^

Fig. 14. Demonstration of integration of SV40 DNA into cell genome. Agarose gel ofPCR products obtained from genomic DNA isolated from 2 immortalizedcyst-derived cell lines of proximal (WT 9-12 LTA) and distal (WT 9-12 DBA)tubule origin as well as from 2 immortalized control human renal proximal anddistal tubule cell lines (RCTEC LTA and RCTEC DBA) is shown. SV40 DNA is seenas a 421-bp band in all the cells studied, confirming the integration of viralDNA into the cell genome.

DISCUSSION

In ADPKD, cysts develop as a result of "second hit" somatic mutations in the normal or WT allele of the PKD1 or PKD2 gene, resulting ininactivation of the gene and production of a nonfunctional protein( 22, 36 ). A variety of mutationshas been described in PKD1 and PKD2 families( 30, 41 ). Because the cellsdescribed here were isolated from individual cysts, each cell line is likelyto have a different somatic mutation resulting in a unique genotype.Therefore, these cell lines will be very useful for studies of thegenotype-phenotype relationship, including studies aimed at rescuing thedefective gene mutation by overexpressing the WT PKD1 or PKD2 genes. Studiesare planned to obtain genotype analysis of all the cell lines described." Y" P. U& c5 e1 ^
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Primary cultures of cyst-derived cells have been used by a number ofinvestigators ( 28, 29, 43 ). The cells grow wellinitially and can be passaged up to three times. However, most experiments must be conducted by the second passage, as the growth slows significantlyafterward. Accordingly, the cells must be obtained from cysts of freshlyharvested polycystic kidneys, which considerably limits the availability ofsuch cells. Using cyst-derived cells obtained from different kidneys withpresumably different PKD mutations introduces significant experimentalvariability. The isolation of cells obtained from single cysts results in ahigh likelihood that each cell isolate has a defined tubule of origin(proximal or distal).

We describe the development and characterization of a series ofimmortalized cell lines derived from individual cysts of ADPKD kidneys andcontrol cell lines from normal human kidney cortex. About half of the celllines are conditionally immortalized with a TS SV40 virus and the restimmortalized with WT SV40 virus ( Table1 ). These immortalized cyst-derived cells offer severaladvantages. 1 ) They can be maintained in culture and used for longperiods of time, thus eliminating the experimental variability associated withthe use of cells derived from different ADPKD kidneys. 2 ) Theyobviate the need for scarce fresh human ADPKD kidneys for cell isolation. 3 ) They allow experiments to be planned ahead of time and to beperformed in a controlled manner at the investigator's convenience. 4 ) Identical cell lines can be distributed among many investigators,allowing comparison of the data between different laboratories andestablishment of collaborative studies. 5 ) They facilitate detailedstudies of the genotype-phenotype relationship by allowing comparisons betweencells derived from different cysts of the same kidney or cells derived fromcysts of different kidneys. 6 ) Immortalized cells can be stablytransfected with expression vectors, using antibiotic selection, to obtaincontinued expression of a variety of genes and proteins. Such studies have,until now, been possible only in non-ADPKD cell lines such as Madin-Darbycanine kidney cells. The availability of control human kidney cell lines immortalized with the same SV40 viruses further increases the value of thesecyst-derived cell lines as experimental models of ADPKD.
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The cell lines were derived from individual cysts of 11 ADPKD kidneys byinfecting the primary cyst-derived cell cultures with both WT and TSadeno-SV40 recombinant viruses. Thirty cyst-derived cells in primary culturewere used, of which 10 were immortalized with WT SV40 virus, 9 wereimmortalized with TS SV40 virus, and 11 were immortalized with both WT and TS viruses ( Table 1 )." o3 b7 W6 D: G+ I7 z1 J* L5 y6 z% O) _
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SV40 has been used successfully to transform a variety of cells and prolongtheir life span ( 9 ). Small etal. ( 33 ) showed that thefrequency of transformation can be enhanced with the use of SV40ori - mutants. Human cells are semipermissive for SV40 viruses, andviral replication occurs in 1-2% of the cells ( 38 ). A portion of the cellpopulation can become transformed and integrate the viral DNA into the cellgenome ( 38 ). However, successful transformation and subsequent immortalization occur rarely evenwhen ori - SV40 viruses are used( 25 ). To overcome theselimitations, chimeric adenovirus-ori - -SV40 recombinants have beendeveloped ( 38 ). They containan ori - SV40 DNA cloned into the adenovirus vector in place ofearly regions 1a and 1b. In previous studies, no transcripts of adenovirusearly regions were detected in cells transformed by hybrid adeno-SV40 viruses( 32 ). Therefore, theadenovirus acts as a vehicle and not as the transforming agent. Because itlacks the early region 1, the adenovirus does not replicate inside the cells ( 32, 38 ).

Successful transformation of cyst-derived cell lines was confirmed byobserving continued cell division beyond the third passage and, morespecifically, by demonstrating the presence of the SV40 large T antigenprotein within the nucleus of the cells and the integration of SV40 DNA withinthe host cell DNA. Because of the unique structure of the recombinantadeno-SV40 viral construct used, the amplified SV40 DNA most likely represents chromosomal DNA and not free DNA( 38 ). We further showed that the TS form of the large T antigen undergoes degradation and disappears incells grown for at least 72 h at the nonpermissive temperature of 39°C.Large T antigen degradation was also observed at the more convenienttemperature of 37°C. The loss of the T antigen has been generallyassociated with a more differentiated phenotype( 9, 26 ). Differentiation may alsobe improved by growing the cells on collagen-coated plates, instead ofplastic. For example, we showed that renin mRNA expression is higher incyst-derived cells grown on collagen compared with those grown on plastic(unpublished observations). The expression of PKD1 and PKD2 mRNA is alsoincreased in cyst cells grown on collagen-coated plates( Fig. 8 ).& z+ I1 u: m# W

In some experiments, the expression of SV40 large T antigen may be used toan advantage. Because the T/t common exon of SV40 can functionally replace theJ domain of molecular chaperones, continued expression of the SV40 large T mayfacilitate membrane translocation of channel proteins such as polycystins( 21, 39 ). Pairs of cyst-derivedcells, transformed with both WT and TS SV40 viruses, offer the ability to testthe effect of large T antigen on intracellular localization of polycystin-1 and polycystin-2.
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Although we observed high transepithelial electrical resistance across somecyst-derived cells in primary culture, lower electrical resistances wererecorded after the cells had been immortalized. The reason transepithelialelectrical resistance declines is not clear but may be related to reduceddifferentiation after immortalization or the possibility that the cell couldbe of proximal tubule origin. Among the cell lines, only a few TS cells weretested. Therefore, additional studies are needed to test all the cells underconditions that would induce maximum differentiation (nonpermissivetemperature, appropriate substratum). It is possible that under optimalconditions, the immortalized cyst-derived cells of distal origin might develophigher transepithelial electrical resistance close to values seen in cells inprimary culture.7 O& w- E7 `1 y6 L8 {3 z" N
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The immortalized cyst-derived cells maintain the expression of somechannels and proteins present on renal tubular epithelial cells. These includeproximal tubule markers such as angiotensinogen ( 37 ), AQP1( 11 ) or NHE3( 5 ), and distal tubule andcollecting duct markers such as ENaC( 24 ) and CFTR( 20 ). The cyst-derived cellsalso maintain the expression of the EGF-R. Although the expression of channelsin cyst-derived cells generally followed the pattern expected based on tubuleorigin, AQP1 and NHE3 were expressed in cyst-derived cells of both proximaland distal tubule origin. Additional studies are required to clarify this apparent discrepancy with previous reports( 11 ). Of particular interestis the expression of renin and all the other components of therenin-angiotensin system by many cyst-derived cell lines (unpublishedobservations). Currently, there are no human kidney cell lines that expressrenin, making these cell lines useful for studies of renin regulation.
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In summary, we developed two series of immortalized cell lines from cystepithelium of individual cysts of 11 human ADPKD as well as immortalizedcontrol cell lines from normal human renal cortex. One line of cells wasimmortalized with a WT adeno-SV40 virus, resulting in continued expression ofthe SV40 large T antigen. Another line of cells was immortalized with a TSadeno-SV40 virus, in which the large T antigen can be turned off when thecells are transferred to a nonpermissive temperature, resulting in a moredifferentiated phenotype. These new cell lines should facilitate studies ofthe mechanism of cyst formation including cell proliferation, cell-cell or cell-matrix interaction, signal transduction, and genotype-phenotype correlation.

DISCLOSURES

This study was supported in part by grants from the National Institutes ofHealth (to J. Zhou) and by grants from Polycystic Kidney Research Foundationand the Fleur-de-Lis Foundation (to M. Loghman-Adham). S. M. Nauli wassupported by a postdoctoral fellowship from the Polycystic Kidney ResearchFoundation. The shipping of nephrectomy specimens from various surgical programs was facilitated by the PKD Foundation and by patients and surgicalspecialists who offered to participate in the study.

ACKNOWLEDGMENTS6 d( _/ ~) y) c. q" G$ ~% _

The authors thank Dr. J. Ryerse, Dept. of Pathology, St. Louis University,for performing the electron microscopy.
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