The NRK-52E normal rat kidney epithelial cell line was purchased from the American Type Culture Collection (CRL-1571; Manassas, VA, USA). The NRK-52E cells were cultured in Dulbecco's modified Eagle's medium (low glucose; Gibco, Thermo Fisher Scientific, Inc., Waltham, MA, USA) containing 5% fetal bovine serum (FBS; Gibco), 1% streptomycin (100 µg/ml) and penicillin (100 U/ml), and incubated in a humidified atmosphere containing 5% CO₂ at 37°C. Upon reaching 90% confluency, the cells were passaged following detachment with 0.05% trypsin-ethylenediaminetetraacetate (Gibco), which was performed every 2 days.

One day prior to treatment, the NRK-52E cells were incubated with serum-free media for 24 h to synchronize the cell growth. The cells were randomly divided into the following five groups: control group (5.5 mmol/l glucose), high-glucose group (25 mmol/l glucose), high glucose with low dose of 1,25(OH)₂D₃ group [25 mmol/l glucose with 10⁻⁹ mol/l 1,25(OH)₂D₃], high glucose with a medium dose of 1,25(OH)₂D₃ group [25 mmol/l glucose with 10⁻⁸ mol/l 1,25(OH)₂D₃] and high glucose with a high dose of 1,25(OH)₂D₃ group [25 mmol/l glucose with 10⁻⁷ mol/l 1,25(OH)₂D₃]. Subsequent analyses were performed following for 24 h. Morphological observation was performed under a microscope (JS-500 binocular biological microscope; LIOO, Beijing, China) at ×40 magnification before the cells were lysed.

Following washing in cold phosphate-buffered saline (PBS), the cells were lysed with radioimmunoprecipitation assay lysis buffer (Beyotime Institute of Biotechnology, Haimen, China) containing protease and phosphatase inhibitor (both from Roche, Mannheim, Germany). A Bradford Protein Assay (Beyotime Institute of Biotechnology) was performed to determine the total protein content. Twenty milligrams of total protein from each sample were separated by 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred onto a polyvinylidene difluoride membrane (Wuhan Boster Biological Technology, Ltd., Wuhan, China). Following incubation in 5% bovine serum albumin for 2 h at room temperature to block non-specific binding, the membranes were incubated overnight at 4°C with primary antibodies against α-SMA (cat. no. ab32575; 1:2,000 dilution), E-cadherin (cat. no. ab76055; 1:1,000 dilution) or VDR (cat. no. ab109234; 1:1,000 dilution) (all from Abcam, Cambridge, MA, USA). Following three washes in Tris-buffered saline containing 0.1% Tween-20 for 10 min each, membranes were incubated with peroxidase-conjugated AffiniPure goat anti-rabbit immunoglobulin G (cat. no. 111035003; 1:10,000 dilution; Jackson ImmunoResearch Laboratories, Inc., West Grove, PA, USA) secondary antibody for at least 1 h at room temperature. Blots were visualized using an Enhanced Chemiluminescence Western Blotting Detection System (GE Healthcare Life Sciences, Chalfont, UK). The membrane was re-blotted with a rabbit anti-rat β-actin monoclonal antibody (cat. no. 4970; 1:1,000 dilution; Cell Signaling Technology, Inc.) to verify equal protein loading in each lane.

Total RNA from NRK-52E cells was extracted using TRIzol reagent (Invitrogen, Thermo Fisher Scientific, Inc.) according to the manufacturer's instructions. After determining the purity and quality of the total RNA, E-cadherin, α-SMA and VDR mRNA were quantified by RT-qPCR using the QuantiTect Reverse Transcription kit and the RNA SYBR-Green kit (both from Qiagen, Inc., Valencia, CA, USA) according to the manufacturer's instructions. PCR amplification was performed using the ABI 7500 Fast Real-Time PCR System (Applied Biosystems, Thermo Fisher Scientific, Inc.). The amplification program was 95°C for 15 min followed by 40 cycles consisting of 95°C for 10 sec and 60°C for 35 sec. The ABI PRISM 7900HT Sequence Detection System (Applied Biosystems) was used to analyze the data, and the ΔΔCq method was used to calculate the relative expression of the respective sample gene (19). The sequences of the primers, which were designed using Primer Premier (v5.0; Premier Biosoft International, Palo Alto, CA, USA) based on the relevant sequences deposited in GenBank (http://www.ncbi.nlm.nih.gov/genbank/), are listed in Table I.

The Ang II secreted into the culture supernatant by NRK-52E cells was detected by ELISA. The culture supernatant was collected and centrifuged at 8,000–12,000 × g for 20 min. ELISA plates (Nalge Nunc International, Thermo Fisher Scientific, Inc.) were coated with anti-Ang II monoclonal antibody (MoAb; Ziker Biological Technology, Ltd., Shenzhen, China) according to the instructions of the Rat Ang II ELISA kit (100 µl of a 10 µg/ml solution in 0.07 M NaCl buffered with 0.1 M borate, pH 8.2) and left overnight at 4°C, followed by four washes with 200 µl of wash solution per well (0.9% w/v NaCl containing 0.05% v/v Tween-20). Subsequent to blocking with 200 µl PBS containing bovine serum albumin (1.0% w/v) and 0.05% v/v Tween-20 for 60 min at room temperature, wells were washed as described above. Centrifuged culture supernatant or serum samples (100 µl/well) were added, followed by incubation for 30 min at 37°C. Subsequently, wells were incubated with horseradish peroxidase-conjugated streptavidin (1:4,000 dilution) for 30 min at 37°C and tetramethylbenzidine (both from Zymed Laboratories, Inc., South San Francisco, CA, USA) as a substrate. The color reaction was allowed to develop for 30 min at 4°C in the dark and was stopped by addition of 100 µl 0.2 M H₂SO₄ per well (Baker, Mexico City, Mexico). The optical density of each well at a wavelength of 450 nm (OD 450) was determined using an ELISA processor (Benchmark Microplate Reader; Bio-Rad Laboratories, Inc., Hercules, CA, USA). The concentration of Ang II in the culture supernatant was determined from the OD 450 using a standard curve and the experiments were performed in triplicate.

Values are expressed as the mean ± standard deviation of results of experiments performed in triplicate. Statistical analysis was performed using SPSS 20.0 software (International Business Machines Corp., Armonk, NY, USA). The mean values were analyzed for their normality by using the Shapiro-Wilk normality test. All data passed the normality test and were tested for significant differences by one-way analysis of variance or Student's t-test. P<0.05 was considered to indicate a statistically significant difference between values. Each experiment was repeated three times.

Initially, the effects of high glucose on the morphology of NRK-52E cells were assessed, which were observed by light microscopy following incubation with high glucose (25 mmol/l) for 24 h (Fig. 1). Compared to the control cells, those in the high-glucose group showed obvious transdifferentiation with cell polarity disappearing and the shape changing from circular to elliptic, suggesting that high glucose can induce cell morphological changes associated with EMT.

As high-glucose treatment resulted in cell morphological changes indicative of EMT, changes in the expression of molecular markers associated with epithelial and mesenchymal phenotypes by NRK-52E cells following high-glucose treatment were assessed. As determined by RT-qPCR and western blot analyses, respectively, the mRNA and protein levels of E-cadherin were significantly increased, while those of α-SMA were significantly reduced following high-glucose treatment (P<0.01) (Fig. 2A-C). Furthermore, ELISA revealed that the Ang II content in the culture supernatant of the high-glucose group (105.9±6.6 pg/l) was significantly higher than that in the control group (4.3±1.8 pg/l; P<0.001) (Fig. 2D).

In order to assess the inhibitory effects of 1,25(OH)₂D₃ on the EMT of kidney cells, the mRNA and protein expression of α-SMA and E-cadherin by NRK-52E cells was detected after incubation with various concentrations of 1,25(OH)₂D₃ and high glucose. As shown in Fig. 3, the high glucose-mediated reduction of E-cadherin in NRK-52E cells was significantly and dose-dependently attenuated by treatment with 1,25(OH)₂D₃. Furthermore, 1,25(OH)₂D₃ suppressed the high glucose-induced expression of α-SMA in a dose-dependent manner.

To further investigate the mechanisms by which 1,25(OH)₂D₃ inhibits high glucose-induced EMT, the mRNA and protein expression of VDR was analyzed using RT-qPCR and western blot analysis, while Ang II was detected by ELISA. The results showed that VDR expression was significantly downregulated by high-glucose stimulation, while 1,25(OH)₂D₃ significantly inhibited this effect in a dose-dependent manner (Fig. 4). Furthermore, the Ang II content in the high-glucose group (105.9±6.6 pg/l) was significantly reduced by low, medium and high dose of 1,25(OH)₂D₃ in a dose-dependent manner (68.9±5.5, 44.6±4.3 and 17.4±5.23 pg/l, respectively) P<0.001 (Fig. 2D).