TGF-β1 was obtained from R&D Systems, Inc. (Minneapolis, MN, USA). Lipofectamine^® 3000 Transfection Reagent (cat. no. L3000015) was purchased from Thermo Fisher Scientific, Inc. Anti-E-cadherin (cat. no. ab76055), anti-α-SMA (cat. no. ab5694), anti-FSP-1 (cat. no. ab41532), anti-collagen I (cat. no. ab34710), anti-vimentin (cat. no. ab92547), anti-Wnt3/3a (cat. no. ab172612) and anti-BMP-7 (cat. no. ab56023) antibodies were purchased from Abcam (Cambridge, UK). Anti-phospho-Smad2 (cat. no. 3108), anti-phospho-Smad3 (cat. no. 9520), anti-Smad2 (cat. no. 3122), anti-Smad3 (cat. no. 9513), anti-glycogen synthase kinase 3β (GSK-3β; cat. no. 12456), anti-phospho-GSK-3β (cat. no. 5558), anti-phospho-β-Catenin (Ser33/37/Thr41) (cat. no. 9561), anti-Non-phospho (Active) β-Catenin (Ser33/37/Thr41) (cat. no. 8814), anti-rabbit horseradish peroxidase (HRP)-linked secondary (cat. no. 7074) or anti-mouse HRP-linked secondary (cat. no. 7076) antibodies were obtained from Cell Signaling Technology, Inc. (Danvers, MA, USA). Anti-β-actin (cat. no. sc-8432) antibody was purchased from Santa Cruz Biotechnology, Inc. (Dallas, TX, USA).

The cell lines HK-2 (cat. no. CRL-2190™) and HEK 293T (cat. no. CRL-11268™) were purchased from the American Type Culture Collection (Manassas, VA, USA). HK-2 cells were maintained in Dulbecco's modified Eagle's medium/F12 (Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA) supplemented with 10% fetal bovine serum (FBS; Gibco; Thermo Fisher Scientific, Inc.) at 37°C with 5% CO₂ in a humidified incubator. HEK 293T cells were grown in DMEM media (Gibco; Thermo Fisher Scientific, Inc.) under the same conditions. For inducing EMT, cells were starved without serum for 12 h and subsequently treated with different concentrations of TGF-β1 (0, 2, 5 and 10 ng/ml) at 37°C for 0, 24 and 48 h as described previously (20).

pGCL-green fluorescent protein (GFP)-lentivirus carrying a full-length human BMP-7 cDNA sequence (LV-BMP-7) and a non-targeting sequence (LV-Control) were synthesized by Shanghai GeneChem Co., Ltd. Lentivirus packaging and infection were performed as described previously (21). One day before transfection, 1.2×10⁷ HEK 293T cells were plated at 90–95% confluence (in 15-cm dishes). At the time of transfection, the packaging HEK 293T cells were cotransfected with pGC-LV (20 µg), plasmid pHelper 1.0 (15 µg) and plasmid pHelper 2.0 (10 µg) using Lipofectamine^® 3000 (Thermo Fisher Scientific, Inc.). The titer of the recombinant lentiviral vector was 3×10⁸ TU/ml. The recombinant lentivirus was stored at −80°C. For lentiviral infection, add recombinant lentivirus (4 µl) at 3×10 ⁸ TU/ml to each well (2×10⁵ cells per well in 6-well plates). Polybrene (Shanghai GeneChem Co., Ltd.) was added to a final concentration of 5 µg/ml. Following incubation at 37°C for 24 h, the cell culture medium was then replaced with DMEM and 10% (vol/vol) FBS. Cells were incubated at 37°C for another 48 h and then the stable overexpression of BMP-7 in HK-2 cells was confirmed using reverse transcription-quantitative (RT-q)PCR and western blotting.

Total RNA from HK-2 cells was extracted using TRIzol^® reagent (Invitrogen; Thermo Fisher Scientific, Inc.), and cDNA was synthesized using a M-MLV kit (Takara Bio, Inc., Otsu, Japan) according to the manufacturer's protocol. Specific primers for α-SMA, collagen I, FSP-1, vimentin, E-cadherin, BMP-7 and GAPDH are listed in Table I. qPCR was performed using a SYBR-Green Real-Time PCR assay kit (Takara Bio, Inc.) on a CFX96 Touch Sequence Detection system (Bio-Rad Laboratories, Inc., Hercules, CA, USA). The thermocycling conditions were as follows: Initial denaturation at 95°C for 10 min, followed by 35 amplification cycles at 95°C for 15 sec and 60°C for 1 min. GAPDH was used as the endogenous control for normalization, and the expression was analyzed using the 2^−ΔΔCq method (22).

Western blotting was performed as described previously (23,24). Total protein was isolated from HK-2 cells using NE-PER™ Nuclear and Cytoplasmic Extraction reagents (Thermo Fisher Scientific, Inc.; cat. no. 78833), and the protein concentration was examined using a Pierce bicinchoninic acid assay (Pierce; Thermo Fisher Scientific, Inc.; cat. no. 23227). Equal quantities of protein (20 µg) were loaded on a 10–12% SDS-gel and resolved using SDS-PAGE. Resolved proteins were transferred to a 0.22 µm polyvinylidene difluoride membrane (EMD Millipore, Billerica, MA, USA). Subsequently, membranes were blocked using 5% non-fat milk for 1 h at room temperature and incubated overnight at 4°C with anti-E-cadherin (1:1,000), anti-α-SMA (1:1,000), anti-FSP-1 (1:1,000), anti-collagen I (1:1,000), anti-vimentin (1:1,000), anti-Wnt3/3a (1:500), anti-BMP-7 (1:1,000), anti-phospho-Smad2 (1:500), anti-phospho-Smad3 (1:500), anti-phospho-GSK-3β (1:1,000), anti-phospho-β-Catenin (1:1,000), anti-Smad2 (1:1,000), anti-Smad3 (1:1,000), anti-GSK-3β (1,000), anti-Non-phospho (Active) β-Catenin (1:1,000) and anti-β-actin (1:2,000). Subsequent to incubation with the primary antibodies, membranes were washed with 0.1% tris-buffered saline with 0.5% Tween-20 for 3 times, and the membranes were incubated at room temperature for 1 h with the anti-rabbit or anti-mouse HRP-linked secondary antibodies (1:2,000). Signals were visualized using an enhanced chemiluminescence detection system (Thermo Fisher Scientific, Inc.), according to the manufacturer's protocol. Densitometry analysis was performed using Quantity One version 4.62 (Bio-Rad Laboratories, Inc.).

A total of 3×10³ HK-2 cells were plated in 96-well plates and, after a 24 h incubation period, cells were treated with different concentrations of TGF-β1 (0, 2, 5 and 10 ng/ml) at 37°C for 72 h. Subsequently, 10 µl CCK8 solution (Dojindo Molecular Technologies, Inc., Kumamoto, Japan) was added to each well and incubated at 37°C for a further 2 h, and cell viability was determined by measuring the absorbance at 450 nm on a spectrophotometer. Cell morphological changes were observed by phase-contrast light microscopy (magnification ×100; Olympus Corporation).

HK-2 cells transfected with LV-BMP-7 and control untransfected cells were pre-treated with 10 ng/ml TGF-β1 at 37°C for 48 h. Cells were suspended in 200 µl serum-free medium. Cell suspensions containing 10 ng/ml TGF-β1 were added to the upper chamber of a Transwell insert (8 µm pores; Corning, Inc.) at a density of 1×10⁵ cells/ml (200 µl per chamber). The lower chamber contained culture medium (600 µl per chamber) supplemented with 10% FBS which was used as a chemoattractant. Cells were incubated at 37°C for 24 h. Cells on the upper surfaces of the chambers were gently scraped off with cotton swabs and the cells which had migrated to the lower surface of the chamber were fixed with 100% methanol at room temperature for 20 min, stained with 0.1% crystal violet at room temperature for 5 min, and the number of cells in 10 random fields of view were counted per well using a light microscope (magnification ×100; Olympus Corporation).

GraphPad Prism version 8 (GraphPad Software Inc., La Jolla, CA, USA) was used for all statistical analyses. Data are presented as the mean ± standard error of the mean. A one-way or two-way analysis of variance or an unpaired Student's t-test were used to compare differences between groups and the LSD test served as the post hoc test for multiple comparisons. P<0.05 was considered to indicate a statistically significant difference.

To assess the potential effect of BMP-7 on EMT in RIF, lentiviral vectors encoding the BMP-7 gene were used to infect HK-2 cells. Infection efficiencies were visualized by fluorescence microscopy (magnification, ×100). To evaluate the efficiency of transfection, RT-qPCR and western blotting were performed. Subsequent to viral infection, >90% of the cells were GFP-positive, suggesting a high infection efficiency (Fig. 1). Additionally, as presented in Fig. 2, BMP-7 mRNA (Fig. 2A) and protein (Fig. 2B) expression levels in the HK-2 infected cells were significantly increased compared with the LV-Control and normal control cells (all P<0.001).

The expression of EMT-associated biomarkers in infected and control HK-2 cells was determined. BMP-7 overexpression significantly increased the mRNA expression levels of E-cadherin, and significantly reduced the mRNA expression levels of α-SMA, collagen I, FSP-1 and vimentin compared with the LV-Control cells (P<0.01; Fig. 3A). Furthermore, western blotting exhibited similar changes in the protein expression of these proteins (Fig. 3B).

TGF-β1 has been demonstrated to be a strong promoter of EMT in renal tubular epithelial cells (25). Additionally, TGF-β1-induced EMT results in reduced cell proliferation (26). The effect of TGF-β1 on cell viability in HK-2 cells overexpressing BMP-7 was assessed. A CCK-8 assay was performed to assess cell viability in the HK-2 cells. Cell viability of HK-2 cells was not significantly affected by 5 ng/ml TGF-β1 (Fig. 4A). After 72 h, 10 ng/ml TGF-β1 significantly inhibited the viability rate of HK-2 cells (P=0.0003 vs. cells treated with 2 or 5 ng/ml TGF-β1; Fig. 4A), however, in the BMP-7 overexpressing cells, the viability rate was partially restored (P=0.0022 vs. cells treated with LV-Control+10 ng/ml TGF-β1; Fig. 4A).

To further investigate the potential function of BMP-7 in regulating the migratory capacity of HK-2 cells, Transwell assays were performed. As presented in Fig. 4B, 10 ng/ml TGF-β1 significantly increased the migratory capacity of HK-2 cells (P=0.0008 vs. cells infected with the LV-Control or LV-BMP-7) and overexpression of BMP-7 significantly reversed this effect (P=0.0262 vs. cells treated with LV-Control+10 ng/ml TGF-β1). These results demonstrate that BMP-7 overexpression reversed the suppression of viability and the increase in the migration induced by TGF-β1 in HK-2 cells.

To investigate whether BMP-7 overexpression resulted in the suppression of TGF-β1-induced EMT, HK-2 cells were treated with 10 ng/ml TGF-β1 for 48 h and the morphological changes were observed in HK-2 cells. As presented in Fig. 5A, HK-2 cells, which traditionally exhibit a cobblestone-like morphology, exhibited a spindle-like morphology when treated with TGF-β1, and also exhibited reduced cell-cell adhesion. However, in cells overexpressing BMP-7, treatment with TGF-β1 did not result in morphological changes. Western blotting was performed to assess the expression levels of EMT-associated markers. As presented in Fig. 5B, 10 ng/ml TGF-β1 reduced BMP-7 expression in a time-dependent manner in the HK-2 cells. This reduction was accompanied by increased expression of the mesenchymal markers α-SMA, collagen I, FSP-1 and vimentin, and decreased the expression of E-cadherin, an epithelial marker. However, overexpressing BMP-7 in cells resulted in the upregulation of E-cadherin and downregulation of the mesenchymal markers α-SMA, collagen I, FSP-1 and vimentin compared with untransfected cells treated with TGF-β1.

To determine the effect of different concentrations of TGF-β1 on the expression of EMT markers, HK-2 cells were incubated with 0, 2, 5 and 10 ng/ml TGF-β1 for 48 h, and the expression of EMT markers were determined by western blotting. TGF-β1 substantially decreased the protein expression of the epithelial marker E-cadherin and increased the expression of the mesenchymal markers α-SMA, collagen I, FSP-1 and vimentin in a dose-dependent manner, with peak expression observed with 10 ng/ml TGF-β1 (Fig. 5C). Next, the effect of TGF-β1 on the BMP-7 overexpressing cells was determined with regards to the expression of EMT markers. Treatment with 10 ng/ml TGF-β1 did not result in a change in the expression of epithelial and mesenchymal markers in the overexpressing cells, with the cells possessing an epithelial expression profile (Fig. 5C). These data suggest that TGF-β1 induced EMT in a time- and dose-dependent manner in HK-2 cells, and that the overexpression of BMP-7 reversed the effects of TGF-β1.

To elucidate the mechanism by which BMP-7 treatment inhibited TGF-β1-induced EMT in HK-2 cells, the Wnt3/β-catenin and TGF-β1/Smad2/3 signaling pathways were assessed. As presented in Fig. 6A, TGF-β1 activated the Wnt3/β-catenin and TGF-β1/Smad2/3 signaling pathways in a time-dependent manner. However, in the BMP-7-overexpressing HK-2 cells, the activation of the two signaling pathways were notably reduced.

To determine the effect of different concentrations of TGF-β1 on the activation of the Wnt3/β-catenin and TGF-β1/Smad2/3 signaling pathways, HK-2 cells were incubated with various concentrations of TGF-β1 for 48 h, and the protein expression of members of the Wnt3/β-catenin and TGF-β1/Smad2/3 signaling pathways were determined. The expression of Wnt3/3a, active β-catenin, phospho-Smad2 and phospho-Smad3 were increased substantially, in addition to the expression of phospho-β-catenin and phospho-GSK-3β being decreased, whereas this effect was reversed in the BMP-7-overexpressing cells after 72 h of treatment (Fig. 6B). Altogether, these results demonstrate that BMP-7 overexpression notably attenuated the TGF-β1-induced activation of Wnt3/β-catenin and TGF-β1/Smad2/3 signaling pathways in HK-2 cells.