Human kidney proximal tubular cells (HK-2) were obtained from The Cell Bank of Type Culture Collection of Chinese Academy of Sciences (Shanghai, China). α-SMA (cat. no. BM0002), JAK2 (cat. no. BM1219), phosphorylated (p)-JAK2 (cat. no. BA3398), STAT3 (cat. no. BA0621), p-STAT3 (cat. no. BA1709) and β-actin (cat. no. BA2305) primary antibodies were obtained from Wuhan Boster Biological Technology, Ltd. (Wuhan, China). HGF and AngII were obtained from Santa Cruz Biotechnology, Inc. (Dallas, TX, USA).

HK-2 cells were cultured as described previously (22). The cells were maintained in RPMI-1640 (Sigma-Aldrich; Merck Millipore, Darmstadt, Germany), and supplemented with 100 IU penicillin, 100 µg/ml streptomycin (Invitrogen; Thermo Fisher Scientific, Inc., Waltham, MA, USA) and 10% fetal bovine serum (Gibco; Thermo Fisher Scientific, Inc.), in a humidified incubator at 37°C in 5% CO₂. Cells were divided into 4 groups and treated with AngII (1×10⁻⁶ M), HGF (8×10⁻³ M), AngII plus HGF or control conditions (RPMI-1640 with 10% FBS) for 24 h.

Total RNA was isolated from cells (1×10⁶) using Trizol reagent (Invitrogen; Thermo Fisher Scientific, Inc.) according to the manufacturer's instructions. First-strand cDNA was reverse transcribed using PrimeScript^™ RT reagent kit (Perfect Real Time; Takara Bio, Inc., Otsu, Japan). The protocol for conducting RT-PCR was identical to that described in a previous study (23). PCR products were separated by 1% agarose electrophoresis and DNA band intensities were quantified using Quantity One software (version no. 4.62; Bio-Rad Laboratories, Inc., Hercules, CA, USA). Target gene band densities were normalized to β-actin. The primers and parameters used for PCR are listed in Table I.

Cells (1×10⁶) were lysed in a sodium dodecyl sulfate (SDS) sample buffer containing 2% SDS, 10 mmol/l Tris-HCl (pH 6.8) and 10% (v/v) glycerol. The lysates were centrifuged at 12,000 × g for 15 min at 4°C, and the supernatant was stored at −70°C. Protein concentration was determined using a bicinchoninic acid assay kit (Bio-Rad Laboratories, Inc.). Total protein (50 µg) was loaded in each lane, before it was separated in a 10% SDS-PAGE gel and transferred to a nitrocellulose membrane. Following blocking in 4% non-fat dry milk in TBS, the membranes were incubated with primary antibodies (α-SMA, JAK2, p-JAK2, STAT3 or p-STAT3) at a 1:1,000 dilution in TBS overnight at 4°C. Following washing with TBS-0.5% Tween-20, the membranes were incubated for 1 h at 37°C with a horseradish peroxidase-conjugated anti-mouse IgG secondary antibody (cat. no. 7076; Cell Signaling Technology, Inc., Danvers, MA, USA) at 1:2,000 dilution, and immunoreactive proteins were detected using SuperSignal chemiluminescence reagent (Pierce; Thermo Fisher Scientific, Inc.). The blots were stripped and reprobed with β-actin antibody (dilution, 1:5,000). The immunoblots were analyzed by densitometry, and protein band densities were quantified using Quantity One software (version no. 4.62; Bio-Rad Laboratories, Inc.).

HK-2 cells (5×10⁴) were cultured in 24-well plates and divided into 4 groups that were treated with AngII (1×10⁻⁶ M), HGF (8×10⁻³ M), AngII plus HGF or control conditions. Following incubation for 24 h at 37°C in 5% CO₂, 5 µl (10 µg/ml) acridine orange and 5 µl (10 µg/ml) ethidium bromide were applied to each well, before the cells were incubated for 5 min at room temperature. The stained cells were analyzed using a fluorescence microscope (Olympus Corporation, Tokyo, Japan). The experiments were repeated three times.

In order to determine the level of apoptosis in HK-2 cells in each treatment group, Annexin V staining was performed using the Annexin V-FITC Apoptosis Detection kit (cat. no. ab14085; Abcam, Cambridge, UK) according to the manufacturer's instructions. Briefly, HK-2 cells (5×10⁵) were stained with Annexin V-fluorescein isothiocyanate (FITC) and propidium iodide (PI), and incubated in the dark at room temperature for 30 min following exposure to AngII, HGF, or AngII plus HGF for 24 h. Cells (1×10⁵) were subsequently analyzed using a flow cytometer (Beckman Coulter, Inc., Brea, CA, USA) following the addition of binding buffer and the results were analyzed with Navios tetra software (version no. 1.1; Beckman Coulter, Inc.). Annexin V⁺/PI⁻ cells were defined as cells in early apoptosis and Annexin V⁺/PI⁺ cells were defined as cells in late apoptosis or necrosis.

Data are expressed as mean ± standard error. One-way analysis of variance followed by the Tukey test for multiple comparisons was conducted to assess the differences among multiple groups. SPSS 17.0 (SPSS, Inc., Chicago, IL, USA) was used for data analysis. P<0.05 was considered to indicate a statistically significant difference.

To investigate the role of HGF and AngII in TEMT, the expression of α-SMA in HK-2 cells was examined at the RNA and protein levels. HGF significantly decreased the expression of α-SMA at the mRNA level when compared with the controls (P<0.05; Fig. 1). By contrast, AngII increased α-SMA expression when compared to control conditions (P<0.05; Fig. 1). In addition, HGF significantly attenuated AngII-induced expression of α-SMA mRNA (P<0.01 vs. AngII-only treated cells; Fig. 1). A similar α-SMA expression profile was observed at the protein level. Exposure to HGF significantly decreased α-SMA protein expression when compared with the controls (P<0.05; Fig. 2). However, AngII treatment significantly increased α-SMA expression relative to that of control cells (P<0.01; Fig. 2). In addition, exposure to HGF significantly attenuated AngII-induced increase α-SMA expression (P<0.01 vs. AngII-only treated cells; Fig. 2). As α-SMA expression is considered to provide a measure of TEMT, it is possible that AngII may promote the transdifferentiation process, whilst HGF may have the opposite effect. It is therefore possible that HGF may regulate TEMT by inhibiting AngII.

To investigate the association between HGF, AngII and the JAK2/STAT3 signaling pathway, the protein expression levels of JAK2 and STAT3, as well as the phosphorylated forms of these proteins, were examined. p-JAK2 protein expression was significantly decreased in the HGF treatment group when compared with the control group (P<0.05), whereas p-JAK2 protein expression was significantly increased in the AngII treatment group when compared with the controls (P<0.01; Fig. 3). p-JAK2 protein expression was significantly decreased in the AngII plus HGF treatment group when compared to the AngII-only treatment group (P<0.01; Fig. 3). These effects were comparable to those observed with α-SMA expression. However, a similar trend was not observed for JAK2 protein expression. JAK2 protein expression was higher in the HGF and control groups compared with the AngII and AngII plus HGF groups (Fig. 3).

HGF treatment significantly decreased p-STAT3 protein expression levels when compared with controls (P<0.05; Fig. 4). However, AngII treatment increased p-STAT3 expression when compared with controls (P<0.01; Fig. 4). In addition, expression of p-STAT3 protein following exposure to AngII and HGF was significantly decreased when compared to AngII-only treated cells (P<0.01; Fig. 4). This was similar to the trend in expression of α-SMA mRNA and protein among treatment groups. However, a similar trend to p-STAT3 was not observed for STAT3 protein expression. STAT3 protein expression was higher in the HGF and control groups compared with the AngII and AngII plus HGF groups. These results suggest that HGF may inhibit TEMT through the inhibition of AngII, and this effect may be mediated by inhibition of the p-JAK2/p-STAT3 signaling pathway.

Following exposure to AngII, HGF or AngII plus HGF, HK-2 cells were stained with acridine orange and ethidium bromide to determine the level of apoptosis in HK-2 cells exposed to different treatments. The results demonstrated that treatment of cells with AngII was associated with induction of apoptosis when compared with controls (Fig. 5). By contrast, treatment with HGF attenuated AngII-induced apoptosis (Fig. 5).

The results of the Annexin V-FITC/PI double-fluorescence staining assay revealed that AngII treatment significantly induced apoptosis in HK-2 cells when compared with controls (P<0.01; Fig. 6). This was demonstrated by an increase in the percentage of Annexin V⁺/PI⁻ and Annexin V⁺/PI⁺ subpopulations. In addition, the percentage of apoptotic cells significantly decreased following exposure to AngII plus HGF (P<0.01 vs. the AngII-only treated group; Fig. 6). The present study demonstrated that 15.2% of cells underwent apoptosis (Annexin V⁺/PI⁻) following treatment with AngII plus HGF (Fig. 6). By contrast, 28.4% of cells underwent apoptosis following treatment with AngII alone (Fig. 6). This indicated that HGF may prevent apoptosis induced by AngII.