MDCK kidney cells were cultured according to AATC (American Association Tissue Culture) guidelines. Briefly, cells were grown as confluent monolayers in 10 cm diameter tissue culture plates in DMEM media specific for each cell line with 10% fetal calf serum at 37°C in the presence of 5% CO2 in a humidified incubator. Cells were plated 18 h before transfection and fed with fresh medium 4 h before transfection.
Construction of the reporter gene
The promoter region of TauT was identified in previous studies , and a p53-binding consensus site was found in the TauT promoter sequence, located at -663 to -695. In this study, ~1.1 kb of the TauT promoter region DNA was used as the template for PCR (GenBankTM/EBI accession number AR151716) and the PCR fragment was cloned into the promoter-less luciferase vector pGL3-Basic (Promega, Madison, WI) to generate the plasmid p963 for use in transfections and luciferase assays. The conditions used were 30 cycles of 1 min of denaturation at 94°C, 1 min of annealing at 58°C, and 1 min of elongation at 72°C. The sense primer (5'-GGGGTACCTTACTGAAGGTCACACAGC-3') designed for PCR contained a unique site for KpnI, and the antisense primer (5'-AAGATCTTGGCACGGGAGTTCA-3') contained a unique site for BglII. PCR products were digested with KpnI and BglII and re-ligated into KpnI and BglII sites of pGL3-Basic to generate plasmids containing segments of the TauT promoter sequence extending from the +48 nucleotide corresponding to the transcriptional start site. The constructs were verified by DNA sequencing. The p53-binding site deletion (del pGL-563) and p53 mutation (mt pGL-963) constructs were generated from the p963 plasmid by using sense primers 5'-GGGGTACCGAGTTGGGGAGGGA-3', and 5'-GGGGTACCAGATGAGG-AAACCCCCACACAGAAGGTCTGGGGCTTGCCTGATGTCA-3', respectively. The antisense primer used for these constructs was the same as described above.
Plasmid DNA was introduced into cultured MDCK cells using cationic liposomes (LipofectAMINE). Transfection was carried out for 16-18 h, and then cells were washed twice with phosphate-buffered saline and incubated in fresh medium for 24-48 h before harvesting. pGL-control, which contains a luciferase gene driven by the SV40 early region promoter/enhancer, and empty pGL-Basic vectors were used as positive and negative controls, respectively. To standardize the transfection efficiency, 0.1 µg of pRL-CMV vector (pRL Renilla reniformis luciferase control reporter vector; Promega) was cotransfected in all experiments. Cells were harvested 48 h after transfection and lysed in 200 µl of reporter lysis buffer (Promega). A luciferase assay was performed using a dual luciferase assay kit (Promega), and activity was measured with an Optocomp 1 luminometer (MGM Instruments, Inc., Hamden, CT). Promoter activity (mean ± S.D. of four samples in relative light units) of each construct is represented by relative light output normalized to pRL-CMV control. Graphs represent typical results of four separate experiments. The concentration of protein in the cell extracts was determined using the Bradford method (Bio-Rad, Hercules, CA).
Measurement of taurine transport
Taurine transport studies were performed on confluent monolayers three days after seeding cells. Briefly, cells were washed with Earle’s Balanced Salt Solution (EBSS) at 37°C. Uptake was initiated by the addition of uptake buffer (2 mM KCl, 1 mM MgCl2, 96 mM NaCl, 1.8 mM CaCl2, 5 mM Hepes, pH 7.6) to which 50 µM unlabeled taurine and 0.5 µCi/ml 14C-taurine (Perkin Elmer, Boston, MA) were added. After incubation for 30 min at room temperature, uptake was terminated by the removal of uptake buffer followed by three rapid washes with cold EBSS. Cells were solubilized in 1% SDS in 0.2 N NaOH and radioactivity was counted in a Packard 2000-CA Liquid Scintillation Analyzer.
In vivo model of cisplatin-induced AKI
Male mice (wild-type and TauT transgenic), 10-12 weeks old and weighing 28 to 30 g, were assigned to treatment groups (n = 8/group). For the experiment, eight TauT transgenic mice and eight wild-type mice received a single dose of cisplatin (15 mg/kg body weight) by intraperitoneal injection. Eight saline-injected mice were used as controls. To determine cisplatin-induced nephrotoxicity, mice were sacrificed three days after cisplatin injection. Kidney samples were collected and treated in 10% buffered formalin until used.
Western blot analysis
Cells were lysed in 50 µl M-PER mammalian protein extraction reagent (Pierce, Inc., Rockford, IL) supplemented with a protease inhibitor cocktail for use with mammalian cell and tissue extracts (Sigma). The lysates were cleared by centrifugation at 14,000 x g for 2 min, and the supernatants were transferred to clean tubes. Equal amounts of protein (50 µg) were separated by electrophoresis on a 12% SDS-polyacrylamide gel and transferred to a nitrocellulose membrane (Millipore, Bedford, MA) using a semi-dry electrophoretic transfer system (Bio-Rad). Membranes were incubated in 5% nonfat dry milk in Tris base/sodium chloride (TBS) buffer with 0.2% Tween 20 (TBST) at 4°C overnight. The membranes were incubated with primary antibodies for 1 h at room temperature. Blots were washed with TBST and incubated with horseradish peroxidase-linked secondary antibody (Sigma) for another hour, and then proteins of interest were detected using a chemiluminescence detection kit (Pierce, Inc.).
Immunohistochemistry was performed by following the manufacturer’s instructions (Pierce). Briefly, samples were rehydrated in decreasing ethanol series (100%, 95%, and 70%) for 5 min each. Samples were immersed in 1x PBS for 5 min at room temperature, then quenching solution (3% H2O2 in methanol) for 5 min, and then washed twice in dH2O for 10 min. Slides were blocked for 20 min with the blocking buffer. Primary antibodies (antibody against taurine transporter protein) were applied to slides and incubated for 1 h. Slides were washed for 10 min with PBS, then the biotinylated secondary antibody was applied and incubated for 1 h. After washing for 10 min with PBS, ABC reagent was applied for 30 min. Finally, immunostaining was detected by using a Metal Enhanced DAB Substrate Kit (Pierce).
Assessment of apoptosis
Apoptotic cells in kidney sections were detected by the terminal deoxynucleotidyl transferase (TdT)-mediated digoxigenin-deoxyuridine nick-end labeling (TUNEL) method following the manufacturer’s instructions (R&D Systems, Minneapolis, MN).
All experiments were performed in triplicate. Luciferase assays are expressed in units of relative light output. The data represent the mean ± standard error of three or four experiments. Statistical comparisons were made using one-way analysis of variance and Student's t test to determine significant differences in the means.