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.
OCT2 activity
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.
Pgp activity
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.
Statistical analysis
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.