Novel conditionally immortalized human proximal tubule cell line expressing functional influx and efflux transporters
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Reabsorption of filtered solutes from the glomerular filtrate and excretion of waste products and xenobiotics are the main functions of the renal proximal tubular (PT) epithelium. A human PT cell line expressing a range of functional transporters would help to augment current knowledge in renal physiology and pharmacology. We have established and characterized a conditionally immortalized PT epithelial cell line (ciPTEC) obtained by transfecting and subcloning cells exfoliated in the urine of a healthy volunteer. The PT origin of this line has been confirmed morphologically and by the expression of aminopeptidase N, zona occludens 1, aquaporin 1, dipeptidyl peptidase IV and multidrug resistance protein 4 together with alkaline phosphatase activity. ciPTEC assembles in a tight monolayer with limited diffusion of inulin-fluorescein-isothiocyanate. Concentration and time-dependent reabsorption of albumin via endocytosis has been demonstrated, together with sodium-dependent phosphate uptake. The expression and activity of apical efflux transporter p-glycoprotein and of baso-lateral influx transporter organic cation transporter 2 have been shown in ciPTEC. This established human ciPTEC expressing multiple endogenous organic ion transporters mimicking renal reabsorption and excretion represents a powerful tool for future in vitro transport studies in pharmacology and physiology.
KeywordsProximal tubule cell Albumin endocytosis Phosphate transport Organic cation transport P-glycoprotein Human
In the kidney, the proximal tubular (PT) epithelium is responsible for reabsorption of filtered solutes and excretion of waste products and xenobiotics. Numerous solutes, such as phosphate, urate and amino acids, are filtered in the glomerulus and reabsorbed in the proximal tubules by active sodium-coupled transport, driven by an electrochemical gradient (Madsen et al. 2008). Other compounds of the glomerular filtrate, such as albumin and low-molecular-weight proteins, are reabsorbed by receptor-mediated endocytosis (Gekle 2005). The excretion of metabolic waste products or drugs is facilitated by multiple organic ion transporters mediating uptake from blood at the basolateral membrane and efflux across the apical membrane of PT epithelial cells (PTEC; Russel et al. 2002).
The function and regulation of these transport systems is the subject of physiological and pharmacological research and requires a suitable in vitro cell model. In the last two decades, a variety of human and animal renal tubular cell lines have been used for this purpose (recently reviewed by Bens and Vandewalle 2008). Currently available human PTEC have variable characteristics or express only a few transporters, such as the commercially available HK-2 cell line obtained from renal cortex and transfected with recombinant HPV16 E6/E7 genes (Ryan et al. 1994; Racusen et al. 1995). On the other hand, primary PTEC, isolated from either human or animal kidney material, can only yield a limited amount of material, as proliferation stops after a few passages and the cells dedifferentiate (Brown et al. 2008; Terryn et al. 2007; Weiland et al. 2007). The development of a human cell model with PT characteristics, including multiple influx and efflux transporters, would be useful to extend our current knowledge of renal solute reabsorption and drug excretion.
To overcome the limited availability of functional human PTEC, the combination of culturing exfoliated cells from human urine followed by immortalization steps can be applied (Racusen et al. 1997; Wilmer et al. 2005). Infection by using both temperature-sensitive mutant U19tsA58 of SV40 large T antigen (SV40T) and the essential catalytic subunit of human telomerase (hTERT) has recently proven to be effective for the development of conditionally immortalized podocytes and human glomerular endothelial cells and for the immortalization of human fibroblasts (O'Hare et al. 2001; Saleem et al. 2002; Satchell et al. 2006). Transfection with SV40T allows cells to proliferate at the permissive low temperature of 33°C, whereas the inactivation of the large T antigen at 37°C results in minor changes in gene expression (Stamps et al. 1994). The hTERT vector expresses telomerase activity to maintain telomere length, preventing the occurrence of replicative senescence (Bodnar et al. 1998).
Using a non-invasive technique of obtaining renal material from urine, we have developed conditionally immortalized human PTEC (ciPTEC) from a healthy volunteer. This cell line can be maintained for at least 45 passages and presents PT characteristics when cultured at the non-permissive temperature of 37°C for 10 days. The uptake of albumin and phosphate and the activities of the ATP-binding cassette (ABC) transporter P-glycoprotein (Pgp/MDR1/ABCB1) and organic cation transporter 2 (OCT2, SLC22A2) are demonstrated.
Materials and methods
Primary cell culture
Primary cells were cultured as described previously by collecting mid-stream urine. Informed consent was obtained from the parents of healthy volunteers with no clinical history of renal disease, nor with any other chronic disease. Urine sediment was transferred to supplemented DMEM-HAM’s F12 medium (Lonza, Basel, Switzerland) and cultured at 37°C, 5% CO2 (Wilmer et al. 2005).
Immortalization and subcloning
Primary cells were infected with SV40T and hTERT vectors containing respectively geneticin (G418) or hygromycin resistance as described before (O'Hare et al. 2001; Satchell et al. 2006). Subconfluent cell layers were transferred to 33°C and selected by using G418 (400 µg/ml; Sigma-Aldrich) and hygromycin B (25 µg/ml; Sigma-Aldrich) for 10 days. To obtain a homogeneous cell culture, cells were subcloned by using irradiated NIH 3T3 fibroblast as non-dividing feeder cells (Saleem et al. 2002). After being cultured for 2 weeks at 33°C, single cell clones were visible and picked by using cloning discs drained in trypsin/EDTA. For the following experiments, cells were cultured at 33°C to 70% confluency, followed by maturation for 10 days at 37°C during which the cells formed a confluent monolayer. Propagation of cells was maintained by reseeding the cells at a dilution of 1:3 to 1:6 at 33°C. Experimental procedures were performed on the cloned cells between passages 15 and 40.
Morphology of ciPTEC was investigated by using phase-contrast microscopy. Additionally, cells cultured for 10 days at 37°C were scraped off flask by using a rubber policeman and embedded in paraffin for electron-microscopical analysis.
Characterization of ciPTEC
To investigate the epithelial origin of cells, confluent monolayers were fixed by using 2% paraformaldehyde, permeabilized in phosphate-buffered saline (PBS)-Tween (0.1%) and incubated with antibodies against the tight junction protein, zona occludens 1 (ZO-1; 1:25 dilution; Zymed Laboratories, South San Francisco, Calif., USA). Following treatment with secondary goat-anti-rabbit-Alexa488 conjugate (DAKO, Glostrup, Denmark) and 4,6-diamidino-2-phenylindole (DAPI, Molecular Probes, Invitrogen) to stain nuclei, cells were analysed by immuno-fluorescence microscopy. The presence of the brush-border membrane protein aminopeptidase N was detected by using mouse-anti-human CD13-fluorescein-isothiocyanate (FITC) antibody (DAKO) and endothelial marker CD31-FITC (DAKO) as described previously (Wilmer et al. 2005). Additionally, a sample of stained cells was transferred to a glass slide by cyto-spin (1000g, 10 min) and analysed by immuno-fluorescence microscopy. Alkaline phosphatase activity was determined in at least three independent experiments by using the BM Chemiluminescence ELISA substrate (AP) kit (Roche Diagnostics, Mannheim, Germany) as described previously (Wilmer et al. 2005). Values were compared with the HK-2 cell line as a positive control using shrimp alaline phosphate as standards and expressed as mean±SE.
To investigate whether the monolayers assembled sufficiently tightly for transport studies, ciPTEC was cultured on Transwell-Clear polyester membranes (Corning Costar, Cambridge, Mass., USA) for 10 days at 37°C. Both apical and basal compartments were washed in HEPES-TRIS buffer (10 mM HEPES-TRIS, 132 mM NaCl, 4.2 mM KCl, 1 mM CaCl2, 1 mM MgCl2, 5.5 mM D-glucose, pH 7.4), prior to the addition of 0.1 mg/ml inulin-FITC (Sigma-Aldrich) to the apical compartment. Inulin-FITC diffusion through the monolayer was monitored for 2 h by sampling 100 µl apical and basal compartments and by measuring fluorescence at 485 nm with emission at 535 nm. Data were expressed as mean±SE.
Polyacrylamide gel electrophoresis and Western blotting
Cellular homogenates of cells cultured for various days at 37°C were made by scraping cells off 75-cm2 tissue culture flask by using a rubber policeman and lysing them in 400 µl RIPA buffer containing 1% Igepal CA630, 0.5% Na-deoxycholate, 0.1% SDS, 0.01% phenylmethane sulphonylfluoride, 3% aprotinin and 1 mM Na-orthovanadate. Expression of SV40T antigen in cell homogenates was analysed by Western blotting using reduced 12% sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) and transference onto a polyvinylidene difluoride membrane (Immobilon; Millipore, Bedford, Mass., USA). Membranes were incubated with SV40T antibody (1:400 dilution; Santa Cruz Biotechnology, Santa Cruz, Calif., USA) and anti-D-glyceraldehyde-3-phosphate dehydrogenase (1:5000 dilution; Abcam, Cambridge, UK) as a house-keeping antigen, followed by incubation with goat-anti-mouse-horseradish peroxidase (HRP) conjugate (DAKO) and visualization by using Pierce ECL Western blotting substrate (Thermo Fisher Scientific, Waltham, Mass., USA).
Cellular homogenates matured for 10 days at 37°C were analysed as described above by 6% or 12% SDS-PAGE as indicated, with the following antibodies: rabbit anti-aquaporin 1 (AQP1; 1:4000; 12%; Chemicon International, Millipore), rabbit anti-OCT2 (1:500; 12%, Alpha Diagnostics, San Antonio, Tex., USA), rabbit anti-CD26 (dipeptidyl peptidase IV [dpp-IV]; 1:200, 12%; Santa Cruz Biotechnology), rabbit anti-multidrug resistance protein 4 (van Aubel et al. 2002; MRP4, ABCC4; 1:5000; 6%), mouse anti-Pgp (1:200; 6%; Dako) goat-anti-mouse-HRP conjugate (DAKO) and goat-anti-rabbit-HRP conjugate (DAKO). Human kidney homogenate in RIPA buffer was used as a control.
Albumin uptake by endocytosis
The ability of ciPTEC to reabsorb albumin was investigated by the incubation of confluent monolayers in 24-well plates with 50 µg/ml bovine serum albumin (BSA)-FITC (Sigma-Aldrich) for 30 min at 37°C unless described otherwise. Uptake was arrested by using ice-cold PBS and cells were detached by using trypsin, fixed by paraformaldehyde (0.5%) in PBS and analysed by flow cytometry or immuno-fluorescence microscopy. Concentration- and temperature-dependent uptake was investigated over a concentration range of BSA-FITC (0, 3.7, 11, 33, 100, 300 µg/ml) at 37°C and on ice for 30 min. Uptake inhibition was studied in three independent experiments by incubating the cells with BSA-FITC (50 µg/ml) in addition to excess unlabelled BSA (10 mg/ml) or recombinant receptor-associated protein (RAP; 1 µM), which was a kind gift of Dr. M. Nielsen (University of Aarhus, Denmark). Uptake inhibition by RAP was further examined by using a dilution range of RAP. BSA uptake in saturation experiments were plotted as mean fluorescence intensity and, in inhibition experiments, as mean (±SE) percentage uptake compared with the control condition.
Sodium-dependent phosphate uptake
Phosphate uptake was performed in confluent monolayers with 32PO4 (Perkin Elmer, Waltham, Mass., USA) as described earlier (Malmstrom et al. 1988). Cells cultured for 10 days at 37°C were incubated with 0.2 mM KH2PO4 (10 µCi/ml) for 5 min in four independent experiments, in the presence of 137 mM sodium salt or 137 mM N-methyl-D-glucamine to study sodium-dependent transport. Additionally, time-dependent (0.5, 1, 2, 5, 10, 15, 30 or 60 min) and concentration-dependent (0.02, 0.07, 0.22, 0.66 or 2 mM PO4) uptake was studied. Data were expressed as mean±SE.
Transport of xenobiotics across the basolateral membrane was investigated in ciPTEC by measuring the activity and expression of OCT2 by using a method adapted from Brown et al. (2008). Cells were grown on Transwell-Clear polyester membranes as described above. The activity of OCT2 was measured by incubating 1 µM fluorescent OCT2 substrate 4-(4-(dimethylamino)styryl)-N-methylpyridinium iodide (ASP; Invitrogen) in HEPES-TRIS buffer for 1 min at 37°C at the basal compartment. To inhibit OCT2-mediated uptake, cells were exposed to 100 µM tetrapentylammonium (TPA) at both apical and basal compartments for 10 min prior to uptake of ASP. Additionally, one set of experiments was performed at 4°C. After incubation, transport was arrested by using 1 mM ice-cold TPA. Cells were homogenized in 250 µl HEPES-TRIS-Triton (0.1%) buffer for 30 min, followed by the analysis of fluorescence intensity (excitation 450 nm, emission 642 nm) by using a Victor3 Multiplate Reader (Perkin Elmer). Data were expressed as mean±SE.
The activity of the ABC efflux transporter Pgp was assessed by measuring the accumulation of calcein as described before (van de Water et al. 2007). Briefly, matured cells were incubated in two independent experiments for 1 h at 37°C with the lipophylic non-fluorescent Pgp substrate calcein-AM (Invitrogen) in the presence or absence of inhibitor PSC-833, which was a kind gift from Novartis Pharma (Basel, Switzerland). Intracellularly, calcein-AM is metabolized by esterase activity to fluorescent calcein. Fluorescence of cell lysates was measured at 488 nm with emission at 518 nm. Fluorescence was expressed as mean±SE.
Michaelis-Menten curve-fitting for the calculation of Km and Vmax values was performed by non-linear regression analysis with GraphPad Prism 4.03 software. Differences in substrate transport in the presence or absence of inhibitors or unlabelled analogues were assessed by a paired Student’s t-test.
Cell culture and morphology
Characterization of ciPTEC
Albumin uptake in ciPTEC
Sodium-dependent phosphate uptake
Organic cation transport activity
We have developed a human conditionally immortalized PT cell line from urine of a healthy volunteer; this line expresses transporters involved in renal reabsorption and excretion. The immortalization of non-invasively collected cells by using SV40T and hTERT vectors has enabled us to produce human cells maintaining PT characteristics and proliferating for at least 45 passages. Expression of SV40T decreases gradually in ciPTEC cultured for 10 days at 37°C, minimizing the influence of the transfection on cellular metabolism. Subcloning improves the homogeneity of the cell line as is shown by the decrease in morphological variations, which possibly occur because of the exfoliation of various cell types originating from the renal-urinary tract into urine (Dorrenhaus et al. 2000).
For the culture of renal cells from urine, we have used the methodology described by Racusen et al. (1997) who have developed conditionally immortalized PTEC clones from the urine of two patients with nephropathic cystinosis, an inherited disorder of PT transport attributable to lysosomal cystine accumulation (Gahl et al. 2002). These cells represent a suitable model for studying the pathogenesis of cystinosis. Obviously, such studies require healthy control cells obtained by the same methodology as described in this study. The culturing of PTEC from control urine is hampered by the small amounts of viable exfoliated cells in the urine of healthy volunteers. In the present study, the success rate for culturing proliferating cells is approximately 10%. Interestingly, only some urine samples from a few subjects contain viable PTEC with variable morphology.
The cells exfoliated in urine could be argued to be a less than ideal source for the establishment of a PTEC cell line, since cell damage could have resulted in their detachment from the tubular basement membrane. However, the detailed characterization of the ciPTEC presented here, including their viability, proliferation capacity, formation of a tight monolayer and preserved PT characteristics, indicates the feasibility of using urinary cells as a source for obtaining human renal material for in vitro research. Furthermore, the study of the pathogenesis of inherited PT disorders, which is often hampered by the limited availability of renal tissue, can be facilitated by collecting affected cells from the urine of these patients (Detrisac et al. 1983, 1984; Racusen et al. 1997).
In pharmacology and toxicology, the availability of a cell model of human origin expressing a broad range of functional transporters is of paramount importance. More specifically, the ciPTEC presented here is the first human cell line showing expression of MRP4, in concert with the expression and activity of OCT2 and Pgp. Probably because of different glycosylation, MRP4 in ciPTEC on Western blot shows a band of a different size from that of human kidney. We have therefore confirmed MRP4 expression by quantitative polymerase chain reaction after RNA isolation from ciPTEC. Together with the formation of a tight monolayer, as observed during the inulin diffusion experiments, these features make ciPTEC a valuable tool for the identification of substrates and inhibitors of renal drug excretion and the prediction of potential drug-drug interactions in pharmacological research. In addition to functional organic cation excretion, the human ciPTEC maintain sodium-dependent phosphate uptake and albumin endocytosis sensitive to inhibition by RAP. The apparent Km calculated for sodium-dependent phosphate transport in ciPTEC (0.12 mM) is approximately one-third of the apical phosphate transport value demonstrated in opossum kidney (OK) cells (0.37 mM) (Reshkin et al. 1990). This suggests a higher affinity for phosphate in ciPTEC compared with currently available cell models.
The reabsorption of albumin by ciPTEC in this study is most likely receptor-mediated endocytosis transport, since it is sensitive to RAP inhibition and the intracellular vesicular pattern of BSA-FITC. Inhibition of BSA-FITC in ciPTEC is similar to the inhibition found earlier in OK cells (Zhai et al. 2000), whereas the apparent Km calculated for ciPTEC (126 µg/ml) is approximately six times higher than the value reported for OK cells (20 µg/ml; Gekle et al. 1996). Although this transport might be linked to the multi-ligand receptor megalin, we have been unable to identify this receptor by Western blot or by immunofluorescence techniques (data not shown; Birn and Christensen 2006). This suggests the involvement of alternative albumin reabsorption mechanisms (Gekle 2005).
In conclusion, the present study introduces the first human cell line featuring functional sodium-dependent and endocytosis-mediated reabsorption together with the functional secretion capacity by Pgp and OCT2 and combined expression of MRP4. The ability of ciPTEC to proliferate for extended passages with maintained functional transport should allow standardized and high-throughput investigation of renal drug handling in pharmacological research.
The authors are grateful for fruitful discussions of the present study with E.I. Christensen and to M. O’Hare for giving permission to use the SV40T construct. Technical assistance with the Pgp activity experiments by Janny G.P. Peters is gratefully acknowledged. We thank Raf Ponsaerts, Eva Sammels and Djalila Mekahli for their assistance in preparing the microscopic images.
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