Unless otherwise stated, all chemicals, including MTT and mouse monoclonal anti-β-actin, were obtained from the Sigma Chemical Company, St. Louis, MO, USA. The mouse anti-rat TfR1 antibody was purchased from Invitrogen, Carlsbad, CA, USA, and the rabbit polyclonal anti-mouse Fpn1 antibody from Chemicon International, Temecula, CA, USA. The rabbit anti-rat DMT1 + IRE and DMT1 - IRE polyclonal antibodies were bought from the Alpha Diagnostic International Company, San Antonio, TX, USA, and goat anti-rabbit or anti-mouse IRDye 800 CW secondary antibodies from LI-COR Biosciences, Lincoln, NE, USA. Dulbecco’s Modified Eagle’s Medium (DMEM)/Ham’s F-12 was obtained from GIBCO, Grand Island, NY, USA and BSA from the Calbiochem-Novabiochem Corporation, San Diego, CA, USA. The scintillation cocktail and tubes were obtained from the Beckman Coulter Company, Fullerton, CA, USA. All experimental protocols were performed according to the Animal Management Rules of the Ministry of Health of China, and approved by the Animal Ethics Committees of Fudan University and The Chinese University of Hong Kong.

Immortalized cells (WKPT-0293 Cl.2) from the S1 segment of the proximal tubule of normotensive Wistar-Kyoto rats (RPTC) were cultured as previously described Woost et al. (1996). Briefly, cells were maintained in renal tubular epithelium medium composed of DMEM and F-12 [nutrient mixture F-12 (Ham)] at a ratio of 1:1, supplemented with 15 mM HEPES, 1.2 mg/ml NaHCO₃, 5 μg/ml Tf, 10 ng/ml epidermal growth factor, 100 U/ml penicillin G, 100 mg/ml streptomycin sulfate, and 10% fetal calf serum. Cells were plated at a density of 5 × 10⁴/ml on collagen-coated flasks, passaged at 80% confluency, and split 1:10, twice a week.

Cell viability was assessed using an MTT assay as previously described (He et al., 2008; Du et al., 2009). Briefly, a total of 25 μl MTT (1 g/L in PBS) was added to each well before incubation was conducted at 37°C for 4 h. The assay was stopped by the addition of a 100 μl lysis buffer (20% SDS in 50% N’Ndimethylformamide, pH 4.7). Optical density (OD) was measured at the 570 nm wavelength by the use of an ELX-800 microplate assay reader (Bio-tek, Winooski, VT, USA) and the results were expressed as a percentage of the absorbance measured in the control cells.

Total iron in the WKPT-0293 Cl.2 cells was determined using a graphite furnace atomic absorption spectrophotometer (GFAAS, Perkin Elmer, Analyst 100) as previously described (Chang et al., 2005; Ke et al., 2005). The cells were diluted 1:20 (wt/v) with HEPES buffer and homogenized with a sonicator (MSE Soniprep 150 Ultrasonic Disintegrator, MSE Scientific Instruments, England). A 50-μl portion of the homogenate was added to an equal volume of ultra-pure nitric acid in a 0.5 ml polypropylene microfuge tube, digested for 48 h at 50°C, and diluted 1:40 with 3.12 mmol/L nitric acid for iron analysis. Standard curves (0–40 ppb) were prepared by diluting iron standard with blanks prepared from homogenization reagents in 0.2% HNO3. Standards and digested samples were read in triplicate by injecting 50 μl aliquots including 0.05 mg Mg(NO₃)₂ as matrix modification into graphite furnace. Absorbance readings at 248.3 nm, slit at 0.2 nm, pretreatment temperature at 1400°C, atomization temperature at 2400°C were recorded.

WKPT-0293 Cl.2 cells that received different treatments were washed with ice-cold PBS, homogenized with lysis buffer and then subjected to sonication using a Soniprep 150 (MSE Scientific Instruments, London, UK). After centrifugation at 10,000 g for 15 min at 4°C, the supernatant was collected and protein content was determined using the Bradford assay kit (Bio-Rad). Aliquots of the cell extract containing 50 μg of protein were diluted in 2 μl of sample buffer (50 mM Tris, pH 6.8, 2% SDS, 10% glycerol, 0.1% bromphenol blue, and 5% β-mercaptoethanol) and heated for 5 min at 95°C before SDS-PAGE on 10% gel and transfer to a pure nitrocellulose membrane. After the transfer, the membrane was blocked with 5% blocking reagent in Tris-buffered saline containing 0.1% Tween-20, overnight at 4°C. The membrane was then rinsed in three changes of Tris-buffered saline/Tween-20, incubated in fresh washing buffer once for 15 min and twice for 5 min, and then incubated overnight at 4°C with primary antibodies: rabbit anti-rat DMT1 + IRE, DMT1-IRE polyclonal antibodies, rabbit anti-mouse Fpn1 polyclonal antibody (1:5000), and mouse anti-rat TfR1 monoclonal antibody (1:1000). After three washes, the blots were incubated with goat anti-rabbit or anti-mouse IRDye 800 CW secondary antibody (1:5000, Li-Cor) for 1 h at room temperature. The intensity of the specific bands was detected and analyzed by the Odyssey infrared imaging system (Li-Cor) (Du et al., 2014; Qian et al., 2014). To ensure even loading of the samples, the same membrane was probed with mouse monoclonal anti-β-actin antibody at a 1:2000 dilution.

Statistical analyses were performed using Graphpad Prism. Data were presented as mean ± SEM. The differences between the means were all determined by two-way analysis of variance (ANOVA). A probability value of P < 0.05 was taken to be statistically significant.

We first investigated the effects of FAC and nifedipine on the viability of WKPT-0293 Cl.2 by treating the cells with FAC (100 μM) (Chen et al., 2005) or various concentrations (1, 10, 100 μM) of nifedipine alone for 16 h, and then evaluated cell viability using an MTT assay. The results showed that there were no significant differences in viability between the control cells and the cells treated with 100 μM of FAC or 1 and 10 μM of nifedipine. However, viability in the cells treated with 100 μM of nifedipine was found to be significantly lower than that of the control cells (Figure 1A).

To find out the effects of co-treatment with FAC and nifedipine on the viability of WKPT-0293 Cl.2 cells, the cells were treated with FAC (100 μM) plus 1, 10 or 100 μM of nifedipine for 16 h. The MTT assay demonstrated that viability in the cells treated with 1 μM of nifedipine plus FAC or FAC alone were not significantly different from that of the control cells, but treatment with 10 or 100 μM of nifedipine plus FAC did induce a significant reduction in cell viability (Figure 1B). The findings here demonstrated that nifedipine could promote the toxic effects of FAC on the viability of WKPT-0293 Cl.2 cells.

We then examined the effects of nifedipine on iron uptake in WKPT-0293 Cl.2 cells. The cells were treated with FAC (100 μM) with different concentrations (0, 1, 10, 100 μM) of nifedipine for 16 h, and iron content was then measured using a GFAAS. It was found that treatment with 100 μM of FAC alone induced a significant increase in iron content, 38.629 fold of that of the control cells (Figure 2A). The addition of nifedipine led to a further increase in cell iron levels. Iron contents in the cells treated with 1, 10, or 100 μM of nifedipine plus 100 μM of FAC were significantly higher than those in the cells treated with 100 μM of FAC alone. There were no significant differences between the cells treated with different concentrations of nifedipine plus FAC (Figure 2B). These results showed that nifedipine has a role in increasing the iron uptake of WKPT-0293 Cl.2 cells.

To find out the potential mechanisms involved in the positive role of nifedipine on iron uptake by WKPT-0293 Cl.2 cells, we investigated the effects of nifedipine on the expression of major iron uptake proteins TfR1, DMT1 + IRE and DMT1 - IRE, as well as iron release protein Fpn1 in the cells. We demonstrated that nifedipine induced a progressive increase in TfR1 (Figure 3A) and DMT1 + IRE (Figure 3B) expression but not in DMT1 - IRE and Fpn1 with the concentrations added. Although the expression of DMT1 - IRE in cells treated with 1, 10, or 100 μM of nifedipine was significantly higher than that of the control cells, there were no significant differences between cells treated with different concentrations of nifedipine (Figure 3C). Treatment with 1 or 10 μM of nifedipine did not induce any significant changes in Fpn1 expression, with no significant differences being found in Fpn1 content between the cells treated with 1 or 10 μM of nifedipine and the controls (Figure 3D). However, treatment with 100 μM of nifedipine was found to induce a significant reduction in Fpn1 expression in the WKPT-0293 Cl.2 cells. The data imply that nifedipine likely increases iron uptake in WKPT-0293 Cl.2 cells via its role in up-regulating expression of iron uptake proteins.

We also investigated the effects of iron-overload on the expression of TfR1, DMT1 + IRE, DMT1 - IRE and Fpn1 by treating WKPT-0293 Cl.2 cells with 100 μM of FAC for 16 h. Western blot analysis showed that FAC treatment induced a significant reduction in TfR1 (Figure 4A) and an increase in Fpn1 (Figure 4D) expression, and had no effect on DMT1 + IRE (Figure 4B) and DMT1 - IRE (Figure 4C) expression in WKPT-0293 Cl.2 cells.

Finally, we investigated the effects of co-treatment with Nifedipine and FAC on the expression of TfR1, DMT1 + IRE, DMT1 - IRE and Fpn1 by treating WKPT-0293 Cl.2 cells with 100 μM of nifedipine plus100 μM of FAC for 16 h. Western blot analysis demonstrated that co-treatment with nifedipine and FAC led to a significant increase in TfR1 (Figure 5A), DMT1 + IRE (Figure 5B) and DMT1-IRE (Figure 5C) expression and had no effect on Fpn1 expression (Figure 5D) in WKPT-0293 Cl.2 cells.