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Growth, Immortalization, and Differentiation Potential of Normal Adult Human Proximal Tubule Cells

Posted on: Friday, 2 July 2004, 06:00 CDT

SUMMARY

Human proximal tubule epithelial cell lines are potentially useful models to elucidate the complex cellular and molecular details of water and electrolyte homeostasis in the kidney. Samples of normal adult human kidney tissue were obtained from surgical specimens, and S1 segments of proximal convoluted tubules were microdissected, placed on collagen-coated culture plate inserts, and cocultured with lethally irradiated 3T3 fibroblasts. Primary cultures of proximal tubule epithelial cells were infected with a replication-defective retroviral construct encoding either wild- type or temperature-sensitive simian virus 40 large T-antigen. Cells forming electrically resistive monolayers were selected and expanded in culture. Three cell lines (HPCT-03-ts, HPCT-05-wt, and HPCT-06- wt) were characterized for proximal tubule phenotype by electron microscopy, electrophysiology, immunofluorescence, Southern hybridization, and reverse transcriptase-polymerase chain reaction. Each of the three formed polarized, resistive epithelial monolayers with apical microvilli, tight junctional complexes, numerous mitochondria, well-developed Golgi complexes, extensive endoplasmic reticulum, convolutions of the basolateral plasma membrane, and a primary cilium. Each exhibited succinate, phosphate, and Na,K- adenosine triphosphatase (ATPase) transport activity, as well as acidic dipeptide- and adenosine triphosphate-regulated mechanisms of ion transport. Transcripts for Na+-bicarbonate cotransporter, Na+- H+ exchanger isoform 3, Na,K-ATPase, parathyroid hormone receptor, epidermal growth factor receptor, and vasopressin V2 receptor were identified. Furthermore, immunoreactive sodium phosphate cotransporter type II, vasopressin receptor V1a, and CLIC-1 (NCC27) were also identified. These well-differentiated, transport- competent cell lines demonstrated the growth, immortalisation, and differentiation potential of normal, adult, human proximal tubule cells and consequently have wide applicability in cell biology and renal physiology.

Key words: microdissection; SV40 large T-antigen; cilium; electrolyte transport.

INTRODUCTION

The renal tubule is a cytologically diverse, functionally complex system, which plays a critical role in the regulation of water and electrolyte homeostasis. Transgenic animals have provided new and decisive tools to study this system, particularly in relation to specific genes and their influence on renal physiology and pathophysiology (e.g., Ito et al., 1995; Oliverio et al., 1998). Segment-specific cell lines derived from these animal models (Obinata, 1997) extend the potential to acquire detailed physiological information about cell behavior in response to hormones, growth factors, nutrients, and other external stimuli. Furthermore, the techniques for generating cell lines have advanced significantly from the days when rare and unknown mutations in cellular genes were necessary to achieve continual growth in culture. The advent of immortalization genes, such as simian virus 40 (SV40) large T-antigen, not only increased the frequency with which cells acquired the potential to divide indefinitely, but, more importantly, cells were altered in a known and reproducible manner. For example, conditionally immortalized cell lines can be reproducibly generated from the Immortomouse, which carries the transgene, SV40 tsA58 TAg, a temperature-sensitive T-antigen (Jat et al., 1991). These cells can be expanded at the permissive temperature of 33 C, shifted to the nonpermissive temperature (39 C) where T-antigen levels are low, and studied reproducibly to any desired detail.

Cultures of normal human cells are characterized, in part, by a limited number of population doublings, followed by a permanent cessation of cell division, i.e., senescence (Hayflick and Moorhead, 1961; Yeager and Reddel, 1999). This process is tightly controlled, and spontaneous immortalization is rare. Yet, cell lines with a virtually unlimited proliferative potential can be developed from primary or early-passage cultures of normal human cells by the transduction of exogenous genes from deoxyribonucleic acid (DNA) tumor viruses (Katakura et al., 1998). Proximal tubule HKC (Racusen et al., 1997), cystinotic proximal tubule (Racusen et al., 1995), and proximal tubule (HK-2) (Ryan et al., 1994) epithelial cell lines, immortalized with adenovirus 12-SV40, temperature-sensitive SV40 large T-antigen, and HPV 16 E6/E7, respectively, have been generated. These cell lines retain many of the phenotypic properties of normal proximal tubular epithelium. In addition, American Type Culture Collection (ATCC) (see http://www.atcc.org/) lists a number of other human kidney cell lines; all have embryonic or tumor- derived origins and thus are poor models of normal adult tissue.

Yet, it has become clear that the establishment, growth, and maintenance of normal human epithelia are initial, albeit critical, steps in providing a cell culture model that appropriately mimics the in vivo situation. In addition, epithelial cells must also be highly and terminally differentiated, exhibiting the characteristic polarity, morphology, and function of the protoepithelial tissue. Al- Awqati et al. (1999) distinguished between protoepithelial and terminally differentiated states and the activation of a molecular switch that shifts cells toward terminal differentiation. In both states, cells were polarized with tight junctions but differed in the polarity of some membrane proteins. A distinguishing feature of the terminally differentiated renal epithelia is the formation of a solitary, primary sensory cilium arising from the centriole on the apical pole.

The primary cilium, long suspected of being a vestigial organelle, may be, in fact, critically important for the maintenance of a highly differentiated epithelial phenolype. In the mammalian nephron, all cells (except intercalated cells) have primary cilia that extend from the apical surface into the tubular lumen. Recent observations from the study of polycystic kidney disease (PKD) as well as normal kidney indicate that the cilium appears to function as a sensory organelle, chiefly as a mechanosensor (reviewed by Praetorius and Spring, 2001, 2002; Nauli et al., 2003; Pazour and Witman, 2003). A functional primary cilium, then, is characteristic of a terminally differentiated renal epithelium.

In the present investigation, human proximal tubule cell lines were generated from normal, discarded surgical specimens of adult human kidney. Proximal tubule segments were microdissected, and primary cell cultures were immortalized by a replication-defective retrovirus encoding either wild-type or temperature-sensitive SV40 T- antigen. Cells were grown on collagen-coated permeable supports with an optimized culture medium to promote a highly differentiated renal epithelial phenotype, including a primary cilium. The resulting cell lines were characterized by electron microscopy for morphology, Southern blotting for clonality, electrophysiology for electrolyte transport, as well as immunofluorescence and reverse transcriptase- polymerase chain reaction (RT-PCR) for differentiated phenotype. These cell lines retained the major, terminally differentiated phenotypic characteristics of normal human proximal tubular epithelium and are suitable for the study of renal cellular function, including the analysis of electrolyte transport by electrophysiologic and metabolic methods. Carnitine transport and its inhibition by sulfonylureas (Huang et al., 1999) and acidic dipeptide-induced Cl- secretion (Jin and Hopfer, 1997b) were previously described in some of these cell lines.

MATERIALS AND METHODS

Tissue procurement and primary cell culture of human proximal tubules. Samples of normal adult human kidney were obtained from discarded tissue after surgery and placed on ice in a physiological saline solution, according to a protocol approved by the Institutional Review Board of University Hospitals, Cleveland, Ohio. Within 2 h of removal, the samples were washed several times with keratinoeyte serum-free medium supplemented with human recombinant epidermal growth factor (EGF) (5 g/L) and bovine pituitary extract (50 mg/L) (K-SFM, Life Technologies/GIBCO BRL, Grand Island, NY) and then incubated in Hanks' balanced salt solution (Life Technologies/ GIBCO BRL) containing 1 mg/ml collagenase type IV (Life Technologies/ GIBCO BRL) for 10-20 min al 37 C. Early (S1) segments of proximal tubule were microdissected from the surrounding tissue and placed on 12-mm Millicell-CM culture plate inserts (0.4 m; Millipore Corporation, Bedford, MA) that had been previously wetted with a 20% collagen dispersion (a generous gift of Ethicon, Inc., Princeton, NJ) in 60% ethanol, dried in a biological safety cabinet, and irradiated under an ultraviolet light to ensure sterility. Proximal tubule segments were maintained in a minimum volume of K-SFM without fetal bovine serum (FBS) or renal tubular epithelial (RTE) medium (Woost et al., 1996) supplemented with 5% FBS (Life Technologies/ GIBCO BRL) for the first 12-24 h to facilitate cell adhesion to the culture plate insert. Afterward, segments were cocultured with lethally irradiated (5000-6000 rads) NIH 3T3 \fibroblasts on both the collagen-coated insert and underlying plastic well, and the cultures were maintained in K-SFM without FBS or RTE plus 5% FBS at 37 C in a 5% CO2-humidified atmosphere. After approximately 5 d, cultures in K-SFM required 5% FBS for continued growth.

Immortalization of proximal tubule calls. Primary proximal tubule cell outgrowths were immortalized within 2 wk of initial plating by infection with a replication-defective retro viral construct encoding either U19 (Jat et al., 1986) or a temperature-sensitive U19tsA58 SV40 large T-anligen (Jat et al., 1989; a generous gift of Dr. P. Jat), as previously described (Woost et al., 1996).

Briefly, primary proximal tubule cells were dissociated with trypsin-ethylenediaminetetraacetic acid (EDTA) (0.05% trypsin, 1 mM EDTA-4Na^sup +^ in Hanks' balanced salt solution without calcium or magnesium; Life Technologies/GIBCO BRL) and then reseeded al a 1:1 split ratio onto new collagen-coated culture plate inserts with a fresh complement of lethally irradiated fibroblasts. Within 24 h, proximal tubule cells were infected with virus-containing culture medium (24-h conditioned Dulbecco modified Eagle medium [DMEM; Life Technologies/GIBCO BRL] containing 5% FBS) from one of the two producer cell lines. Cells were infected at 2-h intervals during the course of 6-8 h by successive addition of fresh virus-containing medium supplemented with 4 g/ml polybrene (hexadimethrmc bromide, Aldrich Chemical Co., Milwaukee, WI; Sladek and Jacobberger, 1990). After infection, cells were cultured in K-SFM with or without 5% FBS at either 33 C (for cells with temperature-sensitive T-antigen) or 37 C (for cells with wild-type T-antigen). Fetal bovine serum was added to the cultures if the proliferative potential of the cells diminished.

Nomenclature. Three cell lines are described: MPCT-03-ts, HPCT- 05-wt, and HPCT-06-wt, where H and PCT refer to human and proximal convoluted tubule, respectively. The two digits represent the specific isolate and are followed by the designation for the immortalizing gene, i.e., wt or ts, referring to wild-type or temperature-sensitive large T-antigen, respectively.

Culture of cell lines. Initially, each cell line was maintained in K-SFM or defined K-SFM (Life Technologies/GIBCO BRL), without FBS or antibiotics. Of these, HPCT-05-wt continued to grow well in the absence of serum. However, HPCT-03-ts and HPCT-06-wt required serum supplementation for optimal growth and are now routinely maintained in defined K-SFM containing 5% FBS. Alternatively, cell lines were maintained in RTE medium composed of DMEM (1 g/L glucose):Ham's F- 12 (1.8 g/L glucose), 1:1 (v/v), 5 g/ml insulin, 5 g/ml transferrin, 10 ng/ml EGF, 4 g/ml dexamethasone, 50 M L-ascorbic acid 2- phosphate, 15 mM N-2-hydroxyethylpiperazine-N'-2-ethane-sulfonic acid (HEPES), 1.2 mg/ml NaHCO^sub 3^, pH 7.4, and further supplemented with 5% FBS. Each cell line was grown on Ethicon collagen-coated 30-mm Millicell-CM culture plate inserts (0.4 m; Millipore) at cither 33 or 37 C in a 5% CO2-humidified atmosphere. Cells were subcultured with trypsin-EDTA at approximately 90% confluence, usually 5-7 d after the previous seeding, using a split ratio of 1:2 to 1:20.

To promote terminal differentiation, cells were grown al 33 C for temperature-sensitive "ts" cells or 37 C for wild-type "wt" cells until confluence, as judged by visual inspection and an increase in electrical resistance above background. Cells were either maintained at 37 C (wt cells) or shifted to 39 C (ts cells) in medium lacking EGF. In addition, serum was omitted from the apical side.

Gene transfer validation. High-molecular weight genomic DNA was extracted from each cell line (Strauss, 1998) and dialyzed, and 3 g; was digested with the restriction enzyme StuI, which cleaves once in the retroviral genome but not in the T-antigen complementary DNA (cDNA) (data not shown). After overnight electrophoresis (Voytas, 1998) on a 0.8% agarose gel with a buffer composed of 40 mM Tris- HCl, 18 mM NaCl, 20 mM Na-acetate, 20 mM EDTA, pH 8.0, the DNA was transferred by blotting (Southern blot; Brown, 1998) onto Zeta- Probe membrane (Bio-Had, Hercules, CA) and probed with a biotinylated cDNA for T-antigen (Random Octamer Labeling Kit, Tropix, Foster City, CA). Hybridization, stringency washes, and chemiluminescent probe detection using CDP-Star substrate, (Tropix, Bedford, MA) were performed according to the manufacturer's instructions. T-antigen expression and DNA levels were simultaneously measured by flow cytometry, as previously described (Jacobberger et al., 1986; Sladek and Jacobberger, 1990, 1992) Deoxyribonucleic acid content was compared with that of the diploid human fetal fibroblast cell line, IMR-90 (Nichols et al., 1977).

Transmission electron microscopy. The HPCT cells were grown to confluence on Ethicon collagen-coated 12-mm Millicell-CM culture plate inserts and on Ethicon collagen-coated plastic 12-well tissue culture plates (Becton Dickmson and Company, Franklin Lakes, NJ). Cells were fixed with glutaraldehyde (Polysciences, Warrington, PA), postfixed with OsO^sub 4^, dehydrated, embedded in LX-112 (Ladd Research, Williston, VT), and allowed to polymerize al 65 C, as previously described (Romero et al., 1992; Woost et al., 1996). The porous filler supports were removed, and cells were thin sectioned (80 m), counterstained with uranyl acetate and lead citrate, and examined with a JEOL 100CX transmission electron microscope.

Immunorescence-primary cilium, angiotensin (ATI) receptor, sodium phosphate cotransporter, vasopressin (V1a) receptor. HPCT-03-ts and HPCT-05-wt cells were grown on collagen-coated 12-mm Millicell-CM culture plate inserts or collagen-coated Lab-Tek chamber glass slides (Nalge Nunc International, Naperville, IL). At confluence, monolayers were incubated with serum free medium at the apical surface and medium supplemented with 5% FBS al the basal surface for 24 h to promote differentiation. After this incubation, cells were washed with warm (33 C) Dulbecco phosphate-buffered saline (DPBS; Life Technologies, Inc./GIBCO BRL, Rockville, MD) for 1 min to help preserve the microtubule architecture. Cell monolayers were fixed immediately with 4% paraformaldehyde (PFA; Electron Microscopy Sciences, Ft. Washington, PA) for 20 min al room temperature, washed briefly three limes with DPBS to remove the PFA, treated with quenching buffer (75 mM NH^sub 4^Cl and 20 mM glycine prepared in DPBS) for 15 min to stop fixation, and then washed three times with DPBS for 5 min. In some experiments, the glycoprotein-rich brush border of the apical surface was stained with wheat germ agglutinin labeled with Texas red (100 g; WGA; Molecular Probes, Inc., Eugene, OR) for 30 min al 4 C. The WGA was removed by three 5-min DPBS washes, and the monolayer was postfixed with 4% PFA for 10 min. Cells were permeabilized wilh 0.1% Triton X-100 and 0.05% saponin (Sigma Chemical Co., St. Louis, MO) in blocking buffer (5% donkey serum, 5% goal serum, 1% bovine serum albumin, and 5% KBS in DPBS) for 10 min.

All primary antibodies were applied to the apical side for 2 h at 33 C, whereas secondary antibodies were added to the apical surface for 1 h at 4 C. Primary antibodies included ID5 (neat), a mouse polyclonal antibody raised against detyrosinated tubulin (Rudiger et al., 1999, a generous gift of Dr. J. Wehland); AT1-N10 (1:50), a rabbit polyclonal antibody raised against a sequence in the amino terminus of the AT1 receptor (Santa Cruz Biotechnology, Santa Cruz, CA); NPT2 (1:50), a rabbit polyclonal antibody raised against a sequence in the first extracellular loop of the human sodium phosphate co-transporler type II (NPT2, Alpha Diagnostic International, Inc., San Antonio, TX); and V1a (1:50), a rabbit polyclonal antibody raised against a sequence in the cytoplasmic domain between TM5 and TM6 of the rat vasopressin receptor V1a (US Biological, Swampscott, MA). Secondary antibodies were either donkey anti-rabbit or donkey anti-mouse IgG conjugated with Alexa 488 (Molecular Probes) and were prepared in blocking buffer at 1:500. Excess antibodies were removed by washing three times with DPBS for 10 min. In addition, monolayers were incubated with Hoechst 33342 (Molecular Probes) diluted 1:500 in DPBS for 20 min at 4 C to stain nuclei. Finally, excess reagents were removed by washing with DPBS.

Monolayers on porous filters in culture plate inserts were carefully removed and mounted on a glass slide. All preparations were treated with ProLong Antifade (Molecular Probes), according to the manufacturer's directions. A coverslip, which was elevated by spacers to preserve cell height and morphology, was placed on top.

Image slacks covering the entire thickness of the monolayers were acquired with a Xeiss 200M inverted microscope (63 1.24 NA oil- immersion lens) with a Sutter DG4 fluorescent light source and a CoolSnapHQ camera (dynamic range of 12 bit; Roper Scientific, Trenton, NJ) under the control of Metamorph v4.5 (Universal Imaging Corp., Downingtown, PA). Images were deconvolved by Autoquant's Autodeblur (blind deeonvolution) software (AutoQuant Imaging, Inc., Troy, NY).

Immunofluorescence-CLIC-1. Polyclonal antiserum was raised against the entire CLIC-1 (NCC27) protein expressed in bacteria and purified (Tulk et al., 2000). The antiserum was affinity purified using a CLIC-1 affinity column (Tulk and Edwards, 1998). The affinity-purified antibody, designated AP1089, shows high specificity for CLIC-1 and does not react significantly with any other proteins on immunoblots of unfractionated human kidney protein. This antiserum is distinct from the previously reported anti-CLIC-1 antisera 823 (Tulk and Edwards, 1998) in that it will react with PFA-fixed material.

The HPCT cells were grown to confluence on culture plate inserts and fixed with 2% PFA, 75 mM lysine, and 75 mM sodium phosphat\e, pH 7.4, for 30 min at room temperature. The cells were rinsed with phosphate-buffered saline (PBS; 135 mM NaCl, 10 mM sodium phosphate, pH 7.4) and then blocked and permeabilized with 0.05% saponin in PBS- fg (PBS containing 0.02% fish gelatin and 5% goal serum) for 1 h at room temperature. Cell culture inserts were then incubated with 1:20 dilution of AP1089 in PBS-fg for 2 h, followed by four 10-min washes with PBS-fg. Inserts were then incubated with fluorescein isothiocyanale-conjugaled goat anti-rabbit IgG (Jackson Laboratories, West Grove, PA) for 45 min at room temperature, followed by four 10-min washes with PBS. The filters with cell monolayers attached were cut from the tissue culture inserts and mounted in Vectashield (Vector Labs, Burlingame, CA) face up on a glass slide. Images were collected with a Biorad Confocal Microscope using a 63 lens and manipulaled with NIH Image software.

Reverse transcriptase-polymerase chain reaction and Southern, hybridization. Poly A^sup +^ messenger ribonucleic acid (mRNA) was isolated using Oligotex Direct mRNA Micro Kit (Qiagen, Valencia, CA) and reversed transcribed into single-stranded cDNA using 1st Strand cDNA Synthesis for RT-PCR (avian myeloblastosis virus [AMV]) (Roche Molecular Biochemicals, Indianapolis, IN), both according to the manufacturer's instructions. Polymerase chain reactions (PCR) were conducted with 1-2 l reverse-transcribed product, as well as with the manufacturer's recommended concentrations of Taq DNA polymerase, 10 PCR buffer, and deoxynucleoside triphosphale mixture (1st Strand cDNA Synthesis for RT-PCR [AMV], Roche Molecular Biochemicals, or TaKaRa Taq, TaKaRa Biomedicals, Shiga, Japan). Primers (Table 1) were added to a final concenlration of 0.2-0.4 M. Annealing temperatures and magnesium concentrations (Table 1) were empirically determined for each reaction. The PCR reaction began with an initial denaturation step at 94 C for 3 min and continued for 20-28 cycles, i.e., denaturation at 94 C for 1 min, annealing al 42-66 C for 30 s, extension at 72 C for 1 min, and after the last cycle, a final extension at 72 C for 7 min. The PCR products were fractionated in 1- 2% agarose gels and transferred onto nylon membranes for Southern hybridization. Membranes were probed with oligonucleolides (Table 1) that had been labeled with ^sup 32^P- or digoxigenin-deoxyuridine triphosphate (DIG Oligonucleolide Tailing Kit, Roche Molecular Biochemicals). After stringency washes, hybridization products were visualized by X-ray autoradiography or antidigoxigenin-alkaline phosphatase conjugate and chemiluminescent detection (Roche Molecular Biochemicals).

Electrical conductance and electrophysiology. Electrical conductance (G^sub m^ in mS/cm^sup 2^) of visually confluent epithelial monolayers on culture plate inserts was measured under sterile conditions using the Millicell-Eleclrical Resistance System (Millipore), as described previously (Romero et al., 1992). Detailed electrophysiological measuremenls were conducted as described (Romero el al., 1992; Woost et al., 1996). Briefly, HPCT cells were grown to confluence on 12-mm Millicell-CM culture plate inserts and mounted in a modified Ussing chamber equipped with a conventional four-electro de system for measuring transepithelial conduclance, potential, and short-circuit current (Isc). Experiments were conducted in the voltage clamp mode unless noted otherwise. Apical and basal compartments were continuously perfused with a bicarbonate- free Ringer solution composed of (in mM) 114 NaCl, 5 KCl, 1 MgCl^sub 2^, 1 CaCl^sub 2^, 20 D-glucose, 5 L-glutamine, and 28 NaOH (titrated with HEPES acid such that the pH was 7.4 al 37 C). In phosphate transport experiment, the apical compartment was perfused with a bicarbonate-free Ringer-based solution composed of (in mM) 114 NaCl, 5 KCl, 1 MgCl^sub 2^, 1 CaCl^sub 2^, 20 D-glucose, 5 L- glulamine, and 28 NaOH (titrated with 2-[N- morpholino]ethanesulfonic acid such that the pH was 6.3 at 37 C), variations in voltage were recorded in current clamp mode, and Isc was calculaled using Ohm's law. All experiments were conducted al 37 C.

TABLE 1

PCR PRIMERS, HYBRIDIZATION PROBES, AND REACTION PARAMETERS(a,b)

Na,K-adenosine triphusphatase (ATPase) activity in permeabilized cell monolayers was also measured by electrophysiology. Monolayers were continuously perfused both apically and basolalerally with a bicarbonate-free solution composed of (in mM) 20 Na-gluconate, 120 N- methyl-D-glucamine), 5 K-gluconate, 1.2 CaCl^sub 2^, 1.1 MgCl^sub 2^, 25 n-glucose, 25 HEPES (titrated with acetic acid to pH 7.4). The apical membrane was permeabilized with 10 M amphotericin B (Sigma) and 0.1% dimethyl sulioxide, as previously described (Kirk and Dawson, 1983; Woosl et al., 1096: Jin and Hopler. 1997a).

RESULTS

Generation of human proximal tubule epithelial cell lines. Using the strategy that our laboratory previously applied to the immortalization of proximal tubule epithelial cells from rabbit (Romero et al., 1992) and rat (Woost et al., 1996), we have successfully generated and characterized three human kidney epithelial cell lines from the S1 segment of the proximal convoluted tubule (Table 2). This fivefold strategy includes mierodisseetion of the S1 segment of the proximal convoluted tubule, as opposed to chemical or enzymatic digestion of renal tissue (Todd et al., 1993; Hazen-Martin et al., 1994; White et al., 1996); coeullure of primary and early-passage cells with lelhally irradiated 3T3 fibroblasls to facilitale expansion and continued growth of cell cultures; infection with a replication-defective retrovirus, rather than transfection, osmotic shock, or electrophoration, to maximize the probability of incorporating the immortalization gene; maintenance of proximal tubule segments, primary cultures, and cell lines on collagen-coaled porous supports to promote a differentiated, proximal tubular phenotype; and early selection of confluent, electrically resistive nionolayers, which is a major epithelial cell characteristic.

Three human proximal tubule cell lines were generated from three different tissue samples, with each sample originating from a different patient. The cell lines and their origins are as follows: HPCT-03-ts from a 75-yr-old female; HPCT-05-wt from a 50-yr-old male; and HPCT-06-wt from a 55-yr-old male. Each cell line has been subcultured more than 50 times, and one line has been subcultured more than 100 times, i.e., HPCT-05-wt. This length of time in culture suggests that each cell line is immortal. Regardless, sufficient numbers of cells have been generated and cryopreserved, such that a virtually unlimited number of experiments can be performed. The regrowlh and continued use of HPCT cells from cryopreserved stocks are routinely and easily accomplished.

T-antigen expression in HPCT cell lines. These relatively high passage numbers are consistent with T-antigen expression. Southern blot analysis confirmed that each cell line is a distinct clone with a unique insert of SV40 large T-antigen cDNA (Fig. 1).

Expression of T-antigen was demonstrated by fluorescence flow cylomelry of HPCT-05-wt and HPCT-03-ts (grown under permissive conditions, i.e., 33 C) cell lines, in this assay, greater than 99% of the cells contained T-antigen above background (data not shown). The level of expression was similar to that previously observed in rat SKPT and WKPT cell lines (Woost et al., 1996).

Morphologic characterization of HPCT-05-wt. Light microscopy revealed that all HPCT cell lines exhibit a "cobblestone" appearance that is typical of epithelial cells (data not shown). Further analysis of HPCT-05-wt cells was conducted using transmission electron microscopy. When HPCT-05-wt cells were grown to confluence on collagen-coaled culture plate inserts, their epithelial nature wtis readily apparent (Fig. 2A and B). The cells formed simple cuboidal epithelium, with well-developed junctional complexes composed of tight junctions and desmosomes. They contained numerous mitochondria, well-developed Golgi complexes, extensive endoplasmic reticulum, convolutions in the basolateral membrane, and numerous apical microvilli that appear to be connected to a terminal web- like structure. In contrast, HPCT cells lose their epithelial-like morphology when they were grown to confluence on collagen-coated plastic tissue culture dishes (Fig. 2C). Consequently, all HPCT cells were maintained continuously on culture plate inserts from the time of microdissection to preserve a more differentiated, epithelial-like morphology.

TABLE 2

PROPERTIES OF THE HUMAN PROXIMAL CONVOLUTED TUBULE CELL LINES(a)

Fig. 1. Southern analysis of T-antigen insert in human proximal convoluted tubule cells. Genomic deoxyribonueleic acid (DNA) was isolated and digested with StuI, a restriction enzyme that cleaves once in the retroviral genome but not in the T-antigen region. Digests were electrophoresed, transferred to Zeta-Probe membrane, and probed with a T-antigen complementary DNA. See Materials and Methods for details. Lane 1, molecular weight markers: lane 2, HPCT- 05-wt; lane 3, HPCT-03-ts: lane 4, HPCT-06-wt.

Primary cilium. When EGF was removed from the medium and serum from the apical side, conditions that were designed to promote a terminally differentiated phenotype, abundant primary cilia were observed when cells were stained with ID5, an antibody raised against detyrosinated tubulin, which is relatively stable and specifically found in cilia. Figure 3 shows three cilia in a field of five to six cells. The primary cilium is a solitary structure arising from the supranuclear centriole and is reasoned to be a flow sensor converting mechanical energy to chemical energy, increasing intracellular calcium (Praetorius and Spring, 2001), opening Ca^sup 2+^-sensitive intermediate-conductance K+ channels (Praetorius et al., 2003), and modulating probably other as yet unknown \functions.

Identification of membrane receptors and transporters. The mRNA of HPCT cells was probed for some of the major transporters and receptors that are found in the proximal tubule and required for cellular growth, differentiation, and metabolism. In addition, HK-2 cells, an immortalized proximal tubule cell line from normal adult human kidney (Ryan et al., 1994), were also screened for comparison. The RT-PCR and Southern hybridization demonstrated the presence of transcripts for the Na^sup +^-bicarbonate colraiisporler, Na^sup +^- H^sup +^ exchanger isoform 3, Na,K-ATPase, parathyroid hormone receptor, EGF receptor, vasopressin V2 receptor, as well as actin (Fig. 4).

Interestingly, a transcript for V2, which is found typically in the collecting duct as well as in thick and thin ascending limbs (ImbertTeboul and Champignuelle, 1995; Bankir, 2001), was observed in both HPCT and HK-2 cells. Although unusual, this observation is not unprecedented. In the established porcine cell line ELC-PK1, a cell culture model that retains many of the morphologic and functional properties of the proximal tubule (Toutain and Morin, 1992), the V2 receptor is also expressed, albeit at low but detectable levels (Ausiello et al., 1987; Bouley et al., 2003). It may be that immortalization and long-term cell culture stimulate V2 expression over apparently undetectable levels in the native tissue.

The thiazide-sensitive Na^sup +^-Cl^sup -^ cotransporter (TSC), which is expressed in the distal convoluted tubule, was not detected in any of the HPCT cells but was observed in HK-2 cells.

Expression of NPT2, V1a, and CLIC-1. The NPT2 exhibits the transport characteristics associated with renal phosphate reabsorption and is expressed predominantly in the proximal tubule (Biber et al., 1998). When HPCT-05-wt cells were probed with an antibody against NPT2 (Fig. 5a), immunoreactive NPT2 was observed at or near the apical plasma membrane and throughout the cytoplasm. Cytoplasmic NPT2 is localized presumably to membrane vesicles, such as recycling endosomes.

FIG. 2. Cellular ultraslruclure. HPCT-05-wt cells were seeded at approximately 150,000 cells/cm^sup 2^ on collagen-coaled Millicell- CM culture plate inserts (A, B) and on collagen-coated plastic tissue culture dishes (C). Cells were grown to confluence, fixed in glutaraldehyde, and processed for electron microscopy as described in the Materials and Methods. Calibration, bar, 5 m in panels A and B and 2 m in panel C.

Vasopressin and its receptor subtypes (V1a, V1b, and V2) are involved primarily in the regulation of body fluid volume and blood pressure (Birnbaumer, 2000; Bankir, 2001). In rats, V1a receptors, according to one report (Imbert-Teboul and Champignuelle, 1995), are present in the thin ascending limb and collecting duct but absent in the proximal tubule. Yet, others have demonstrated that V1a receptors are present in proximal tubule cells of diabetic rats (Marcinkowski et al., 1997) and in proximal tubule cell lines established from transgenic mice harboring a gene for temperature- sensitive SV40 large T-antigen (Takeda et al., 1995). The nature of these differences is unclear. In our studies, when HPCT-05-wt cells were probed with an antibody against V1a receptors, immunoreactive V1a was observed throughout the cytoplasm, with some located at or near the apical plasma membrane (Fig. 5b).

CLIC-1 (or NCC27) is a 27-kDa member of a family of chloride channels (Edwards, 1999). In the kidney, CLIC-1 is highly expressed in proximal tubule cells, where it is located to the apical domain of the cell. It is also expressed in glomeruli and in periarterial smooth muscle but is not significantly expressed in distal nephron segments. As another marker of the differentiated proximal tubule phenotype, we assessed expression and distribution of CLIC-1 in HPCT- 05-wt cells. The cells stained inlcnscly for CLIC-1 using AP1089 (Fig. 6), whereas cells incubated with control antibody did not (not shown). Fifty-eight confocal images were collected at 0.28-micron intervals along the vertical axis. Images from focal planes near the apical (Fig. 6A) and basal (Fig. 6B) poles of the cells are shown. The stack of images was used to create an image of a cross section of the cell monolayer shown in Fig. 6C. The cells stain in a punctate intracellular pattern with marked apical polarization of CLIC-1.

Collectively, then, immunodetection of NPT2, V1a, and CLIC-1 is consistent with the known distribution of these proteins in the itilact proximal tubule and further evidence of their proximal tubule phenotype.

Epithelial functionality and proximal tubule origin of HPCT cell lines. Among its many characteristic functions, epithelia form a barrier to ions and solutes between its apical (lumen) and basal (blood) surfaces. In a similar manner, HPCT monolayers are able to organize as a tissue over at least 4.2 cm^sup 2^ (i.e., the surface area of a 30-mm-diameter culture plate insert) and exhibit low mean basal transepithelial conductances (G^sub m^) ranging from 5 to 20 mS/cm^sup 2^ (Table 2). The ability to establish and organize electrically resistive barriers is consistent with the epithelial nature of these cell lines.

FIG. 3. Immunofluorescence of primary cilium and AT1 receptor. After removal of epidermal growth factor from the medium and serum from the apical side, HPCT-05-wt cells were treated with antibodies ID5 and AT1-N10 to visualize the primary cilium and AT1 receptor, respectively. (A) Arrows indicate primary cilium. (B) AT1 receptor. (C) ID5-green and AT1-N10-red pseudocolored color-combined image of primary cilium and AT1 receptor. Arrows show the location of primary cilium. (D) Nuclei stained with Hoechst 33342. Bar, 10 m.

All epithelial cells possess tight junctions that regulate the paracellular transport of ions across the cell monolayer. According to the manufacturer, K-SFM nominally contains 90 M Ca^sup 2+^ to promote growth and slow differentiation of cultured human keratinocytes. Interestingly, when the calcium concentration was increased to 1.2 mM Ca^sup 2+^, the concentration found in many other cell culture media, including DMEM-F12-based media, the maximal transepithelial conductances decreased by 2- to 10-fold compared with control cells maintained in unsupplemenled K-SFM (data not shown). The well-established regulation of tight junctional permeability by calcium (Cereijido et al., 1993) is further evidence of the epithelial functionality of these cells.

The vectorial, transepithelial transport of ions across the cell monolayer is an additional and often defining characteristic of epithelial cells (Berry and Rector, 1991; Palmer and Sackin, 1992). Using conventional electrophysiology, changes in short-circuit current and conductance were measured in a basal state and after acute apical addition of 2.5 mM sodium succinate and 5 mM sodium phosphate. The basal transepithelial short-circuit current is slightly negative and representative of net cation secretion (or anion absorption) (Table 2). Consequently, the open-circuit potential is slightly lumen positive in these cell lines. These properties are in reasonable agreement with the electrical properties of primary cultures of human proximal tubule cells measured by Todd el al. (1993). In that study, conductances and short-circuit currents ranged from 0.8 to 5 mS/cm^sup 2^ and from - 2 to +7 A/cm^sup 2^, respectively.

In the proximal tubule, di- and tricarboxylic acids, e.g., intermediates of the Krebs cycle, such as succinate and citrate, are transported across both apical and basolateral membranes, and their movement across each membrane is coupled with the cotransport of sodium (Wright et al., 1980, 1982; Murer et al., 1992; Pajor, 1996). For example, the human renal Na^sup +^-dicarboxylate cotransporter (hNaDC-1) is electrogenic, mediating the transport of sodium and dicarboxylates with a 3 to 1 stoichiometry (Pajor, 1996). As shown in Fig. 7A, acule apical addition of sodium succinale to a confluent HPCT-05-wt monolayer resulted in a transient increase in short- circuit current. The change in direction of short-circuit current indicates that positively charged ions moved from the apical to basal compartments. This same result was also observed in HPCT-03- ts and HPCT-06-wt cells (Table 2).

The NPT2 is electrogenic and mediates the transit of three Na+ ions to one HPO^sub 4^^sup -2^ ion (Biber et al., 1998). Interestingly, in young hypothyroid rats, treatment with 3,3',5- triiodo-L-thyronine (T^sub 3^) resulted in significant increases in Na^sup +^-phosphate cotransport activity and protein and mRNA levels (Alcalde et al., 1999). As shown in Fig. 7B, acute apical addition of sodium phosphate to a confluent HPCT-06-wt monolayer, which had been previously pretreated with 0.3 nM T^sub 3^ for 3 d, produced an increase in [Delta]Isc. These results are consistent with the net movement of positively charged ions from the apical to basal compartments. Although the magnitude of the phosphate-induced change in Isc was dramatically reduced, qualitatively similar results were observed in the absence of T^sub 3^ (Table 2). T^sub 3^ is thought to contribute to the differentiated phenotype of proximal tubule cells. Taken together, these observations indicate lhe functional presence of Na^sup +^-succinate and Na^sup +^-phosphate cotransport and are further evidence of the proximal tubular origin of these cell lines.

FIG. 4. Transcripts of membrane receptors and transporters. Poly A+ messenger ribonucleic acid (mRNA) (1 g) from HK-2 or HPCT-05-wt cells was reversed transcribed, and the complementary deoxyribonucleic acid product (1 l) was used as template in the PCR reaction. Primers, annealing temperatures, and magnesium concentrations are listed in Table 1. The PCR products were fractionated in a 2% agarose gel, transferred onto nylon membrane, probed with the appropriate ^sup 32^P-labeled oligonucleotid\es (Table 1), and visualized by autoradiography. PTHR, parathyroid hormone receptor; NBC, Na^sup +^-bicarbonate cotransporter; NHE3, Na^sup +^-H^sup +^ exchanger isoform 3; TSC, thiazide-sensitive Na^sup +^-Cl^sup -^ cotransporter; V2R, vasopressin V2 receptor; EGFR, epidermal growth factor receptor. Analysis of poly A+ mRNA from HPCT-03-ts and HPCT-06-wt cells yielded the same results using similar methods.

Although it was tested, phlorizin-sensitive or glucose- stimulated changes in short-circuit current were not detected by electrophysiology in these cell lines and suggest that the activity of the Na^sup +^-glucose cotransporter is less than 15 pmol/(s cm^sup 2^) (Deetjen et al., 1992). The low activity or loss of Na^sup +^-glucose cotransport activity is not uncommon in proximal tubule cells maintained in culture (Todd et al., 1993; Woost et al., 1996) and may be due to the relatively high levels of glucose in the culture medium.

Regulated ion transport in HPCT cell lines. The degree of differentiation of a cell line can be assessed, in part, by the functional expression and regulation of ion transport and intracellular signaling pathways. Many epithelial cells express purinergic, receptor-mediated signal transduction pathways (Dubyak et al., 1993). Apical stimulation of purinergic receptors has been demonstrated to cause an increase in apical Cl- and K+ conductance (Friedrich et al., 1909; Stutts et al., 1992). As shown in a representative experiment (Fig. 8A), acute apical stimulation of a confluent HPCT-05-wt cell monolayer with a maximal concentration of adenosine triphosphate resulted in a transient increase in short- circuit current (7.3 1.4 A/cm^sup 2^, n = 11).

In addition, Jin and Hopfer (1997b) reported that acidic dipeptides, e.g., Ala-Asp, stimulated Cl- secretion in both human and rat proximal tubule cell lines. Similarly, acute apical addition of Ala-Asp to a confluent monolayer of HPCT-05-wt cells increased the short-circuit current (Fig. 8B), reflecting a net increase in anion secretion. Thus, these cell lines retain functional and regulated mechanisms of ion secretion.

Na,K-ATPase activity and sodium transport in HPCT cell lines. Na,K-ATPase plays an important role in electrolyte homcostasis in the kidney. The activity of Na,K-ATPase is indicative of the overall rate of salt absorption that is mediated by renal epithelial cells under steady-state conditions (Orosz and Hopfer, 1996). Because the Na,K-ATPase transports more Na+ out than K+ into the cell (3: 2 stoichiometry), its activity was measured as short-circuit current in intact monolayers after permeabilization of the apical plasma membrane with amphotericin B, as described previously (Woost et al., 1996; Jin and Mopfer, 1997a). Amphotericin B selectively increases the permeability of the apical membrane to monovalent ions without destroying the permeability barrier to divalent and larger organic ions. By bathing the apical and basal compartments in symmetrical solutions, changes in short-circuit current represent primary active transport (ATPase), Ca^sup 2+^ currents, or secondary active Iransport dependent on the Ca^sup 2+^ gradient between the extracellular solution and the cytosol. As shown in a representative; experiment (Fig. 9), apical, amphotericin B permeabilization of HPCT-05-wt revealed an increase, in short- circuit current, which is inhibited by the basolateral addition of ouabain. This current is attributable to the Na,K-ATPase. The mean maximal Na,K-ATPase current in HPCT-05-wt cell was 10 1 A/cm^sup 2^ (n = 7) (Table 2) and corresponds to a current density of 300 pmol of Na+ transported per s per cm^sup 2^ (normalized to filter support area). In comparison with values determined for rabbit, rat, and mouse, these current densities represent approximately 12% of that seen in mammalian primary cultured proximal tubule cells (Gullans and Mandel, 1992; Jorgensen, 1996). HPCT-06-wt cell monolayers exhibited a similar level of activity (Table 2).

FIG. 5. identification of sodium phosphate cotransporter type II (NPT2) and vasopressin receptor (V1a). (5a A, B, and C) A subapical, supranuclear slice of HPCT-05-wt monolayer stained with antibody against NPT2 (5a A). Apical surface staining of the same epithelial monolayer as in Fig. 5a A with wheat germ agglutinin (WGA) (labeled with Texas red) (5a B). Merged apical image slices from Fig. 5a A and 5a B (5a C). Z slice from the same image stack as shown in Fig. 5a C (taken at the red line) (5a D). Note the NPT2 staining at the apical plasma membrane and throughout the cytoplasm and WGA staining located at and near the apical membrane. (5b A, B, and C) A subapical, supranuclear slice of HPCT-05-wt monolayer stained with antibody against V1a (5b A). Apical surface staining of the same epithelial monolayer as in Fig. 5b A with WGA (labeled with Texas red) (5b B). Merged apical image slices from Fig. 5b A and 5b B (5b C). Z slice from the same image stack as shown in Fig. 5b C (taken at the red line) (5b D). Note the V1a staining dispersed throughout the cytoplasm with some located at and near the apical plasma membrane and WGA staining located at and near the apical membrane. Bar, 10 M.

FIG. 6. Confocal microscopy of CLIC-1 expression in HPCT-05-wt cells. Filty-eight confocal images were collected at 0.28-micron intervals along the vertical (z) axis. (A) Image near the apical pole of the cell monolayer. (B) Image near the basal pole of the cell monolayer. (C) Image of the y-z plane generated from the slack of images at the position indicated by the vertical lines in panels A and B. The apical pole is to the right. Calibration bar, 20 m. Analysis of HPCT-03-ts and HPCT-06-wt produced similar results.

DISCUSSION

Cell lines present major advantages when it comes to probe cellular physiology and biochemistry because their environment is fully controllable. However, their usefulness in terms of understanding cell physiological processes depend on the degree to which differentiated in vivo features can he reproduced in vitro. The latter depends not only on the cell line but also on the culture conditions. We have shown in this study that adult HPCT epithelial cell lines can be derived from limited amounts of renal tissue acquired after surgery. Furthermore, under appropriate conditions that promote differentiation, each cell line has the morphologic and molecular characteristics as well as the functional properties of normal proximal tubular epithelium. Cells even develop primary cilia, which have been shown to be important in the cell biology of renal epithelial cells (Pazour and Whitman, 2003) but have been neglected in most cell culture studies of proximal tubule cells. These cell lines represent a potentially important, widely useful, and transport-competent cell model to probe the complex cellular and molecular details of the human proximal tubule.

The availability of normal adult human proximal tubule cell lines is very limited. To our knowledge, only one adult human proximal tubule cell line (i.e., HK-2; Ryan et al., 1994) is commercially available. For more information, see the ATCC (http://www.atcc.org/ ) and European Collection of Cell Cultures (http:// fuseiv.star.co.uk/ camr/) Web sites. This cell line, however, may not form electrically resistive monolayers and thus may not be suitable for transepithelial ion transport studies, such as those typically conducted in Ussing-type chambers. Furthermore, our results showed that HK-2 cells express the TSC, a marker for the distal tubule. Even though at least one other adult human proximal tubule cell line exists, one or a few cell lines from the same tissue can hardly express all the variation that is present in the human species. Thus, additional models are significant.

FIG. 7. Succinate- and phosphate-stimulated short-circuit current in HPCT-05-wt cells. Transepithelial short-circuit current (Isc) was measured in a confluent monolayer of HPCT-05-wt cells, as described in the Materials and Methods. Apical and basal compartments were perfused independently with a bicarbonate-free Ringer medium. In the phosphate experiment, the pH of the apical Ringer medium was 6.3 to increase the concentration of HPO^sub 4^^sup 2-^, the substrate for the sodium phosphate cotransporter. Measurements were made in the voltage clamp mode for the suecinate experiment or current clamp mode for the phosphate experiment. Acute apical addition (arrow) of 3 mM Na^sub 2^-succinate (A) or 5 mM Na^sub 2^HPO^sub 4^ (B) resulted in an increase in Isc or [Delta]Isc, respectively, which corresponds to the movement of net positive charge from the apical to the basal side of the monolayer and is consistent with increased Na+ absorption.

Early passages of human proximal tubule cells can be obtained commercially and are appropriate for some studies. See the Clonetics Web site (http://www.clonetics.com/). However, cell line generation is a more suitable approach to investigate the cellular physiology of human genetic diseases because specimens are invariably rare and in limited quantities. Our studies indicate that it is possible to produce sufficient material for a virtually unlimited number of experiments from small amounts of discarded surgical samples. Hence, this work opens the way for renal cell models from patients with essential hypertension, PKD, or other renal diseases with a genetic component. To this end, we have established and are currently characterizing a cell line isolated from a renal cyst of a patient with autosomal dominant PKD. The potential usefulness of such cell lines has been demonstrated by us and others with proximal tubule cell lines from spontaneously hypertensive and normotensive rats (Woost et al., 1996; Jin and Hopfer, 1997a, 1997b; Albrecht et al., 2000; Xu et al., 2000).

FIG. 8. Purmergic and peptidergic stimulation of ion conductances in HPCT-05-wt cells. Transepithelial short-circuit current(Isc, solid lines) and conductance (G, dotted lines) were measured on confluent HPCT-05-wt cells as described in the Materials and Methods. Apical and basal compartments were perfused independently with a bicarbonate-free Ringer medium. Acute apical additions (arrow) of 1 mM adenosine triphosphate (Tris^sup +^ salt) (A) and 10 mM Ala-Asp (B), both stimulated an increase in G^sub m^ and a change in Isc. A positive Isc value is representative of a net positive charge movement from the apical to the basal side of the monolayer.

FIG. 9. Na,K-adenosine triphosphatase (Na,K-ATPase)-mediated shortcircuit current in HPCT-05-wt cells. Na,K-ATPase activity in HPCT-05-wt cells was measured as mm bain-sensitive short-circuit current (Isc) in intact monolayers after selective permeabilization of the apical plasma membrane to monovalent ions with 10 M amphotericin B and 0.1% dimethyl sulfoxide. Apical and basal compartments were perfused symmetrically with a bicarbonate-free solution composed of 20 mM Na+, 5 mM K+, and low Cl- as described in the Materials and Methods. Ouabain (100 M) was added by basolateral perfusion.

Immortalization of cells with oncogenes, such as SV40 large T- antigen, is thought to proceed via inactivation of the proteins encoded by the p53 tumor suppressor and relinoblastoma genes (Jha et al., 1998; DeCaprio, 1999; Sheppard et al., 1999; Yeager and Reddel, 1999). Decreased activity of these gene products allows cells to proliferate beyond normal senescence, but, in most cases, proliferation eventually slows as cells enter a period of crisis. Continued proliferation through crisis is thought to arise as a result of additional, although rare, genetic changes, activating a telomerase maintenance mechanism, from which a truly permanent or immortalized cell line can emerge. However, we have not yet observed crisis in these human proximal tubule cell lines after more than 50 passages. Regardless, the differentiated nature of the cells is clearly advantageous in terms of studies on the cellular functions of the proximal tubule phenotype. Recent advances have shown that induction of telomerase activity increases the proliferative potential of some types of human cells and may result in less transformation and a more differentiated phenotype (Yeager and Reddel, 1999).

A disadvantage of immortalized cell lines, regardless of immortalizing vector, is some degree of biohazard. Although the cell lines in this report were infected with a replication-defective retrovirus, helper virus production remains a possibility, albeit a low one. Consequently, a prudent level of care needs to be exercised when working with these as with any immortalized cell line, and for these particular cells, that care should include routine screening for helper viruses.

ACKNOWLEDGMENTS

This research was supported by a grant from NIH (HL-41618). R. J. K. was supported by NIH training grant DK07678. Image acquisition and analysis was supported by DK27651.

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DAVID E. OROSZ, PHILIF G. WOOST,1 ROBERT J. KOLB, MARGARET B. FINESILVER, WENWU JIN, PHYLLIS S. FRISA, CHEE-KEONG CHOO, CHUNG-FAI YAU, KWOK-WAH CHAN, MARTIN I. RESNICK, JANICE G. DOUGLAS, JOHN C. EDWARDS, JAMES W. JACOBBERGER, AND ULRICH HOPFER

Departments of Physiology and Biophysics (D. E. O

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