The HK-2 cell line (CRL-2190; American Type Culture Collection, Manassas, VA, USA) consists of proximal tubular cells derived from a normal human kidney. The cells (5×10⁵) were seeded in 25T flasks (Corning Incorporated, Corning, NY, USA) and cultured with Dulbecco's modified Eagle's medium (DMEM) nutrient mixture (Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA) supplemented with 10% (v/v) fetal bovine serum (FBS; Gibco; Thermo Fisher Scientific, Inc.), 2% (v/v) penicillin/streptomycin (HyClone; GE Healthcare Life Sciences, Logan, UT, USA) and 1% (v/v) L-glutamine at 37°C in an atmosphere containing 95% environmental air and 5% CO₂. The cells were trypsinized using 0.05% trypsin-EDTA (Gibco; Thermo Fisher Scientific, Inc.). Prior to exposure to high glucose, cells were cultured in a minimum starvation media (0.1% FBS) for 24 h. And then the cells were cultured in DMEM containing 5.5 mM D-glucose (normal glucose, NG) as control group, 30 mM mannitol (Harbin Pharmaceutical Group, Harbin, China) or 30 mM D-glucose (Gibco; Thermo Fisher Scientific, Inc.) in culture flasks for 48 h to induce cellular fibrogenesis, and were subsequently treated with various concentrations of tanshinone IIA (98.0% pure; Xi'an Hongsheng Biotech Company, Xi'an, China) over 48 h. Three wells were allocated for each treatment, including a negative control (untreated cells). Tanshinone IIA was prepared as a 1-mM stock solution in dimethyl sulfoxide and stored in the dark at −20°C.

To detect apoptosis in vitro, TUNEL assays were performed using a one-step TUNEL fluorescent kit (Beyotime Institute of Biotechnology, Haimen, China). HK-2 cells were permeabilized with 0.1% Triton X-100, followed by fluorescein isothiocyanate-labeled TUNEL staining for 1 h at 37°C. TUNEL-positive cells were imaged under a fluorescent microscope and quantified as the number of green spots in each image (x100 magnification); three images were counted using Cellsens Imaging software version 1.4.1 (Olympus Corporation, Tokyo, Japan) in each group. In addition, treated cells were lysed using a lysis buffer [10 mM Tris, 1 mM EDTA, 1% Triton X-100, 1 mM Na₃VO₄, 20 µg/ml aprotinin, 20 µg/ml leupeptin, 1 mM dithiothreitol and 50 mM phenylmethane sulfonyl fluoride (PMSF)] and a bicinchoninic acid protein assay kit (cat. no. P0010S; Beyotime Institute of Biotechnology) was used to detect protein content.

RNAiso Plus reagent (Takara Biotechnology Co., Ltd., Dalian, China) was used to isolate total RNA from the cultured cells in accordance with the manufacturer's protocol. RNA (1 µg) from each sample was then reverse transcribed using an RNA PCR kit (Takara Biotechnology Co., Ltd.) according to the manufacturer's protocol. The resulting cDNAs were processed using RT-qPCR with SYBR-Green technology (Takara Biotechnology Co., Ltd.) according to manufacturer's protocol. Briefly, reactions were conducted in a StepOnePlus real-time PCR system (Applied Biosystems; Thermo Fisher Scientific, Inc.) for 40 cycles (95°C for 5 sec and 60°C for 40 sec) following an initial 10 min incubation at 95°C in a 20 µl volume. Subsequently, the quantification cycle (Cq) was determined and the relative expression levels of Snail, fibronectin and E-cadherin were calculated based on the Cq values that were normalized to GAPDH in each sample. The relative gene expression levels were calculated using a comparative Cq method formula 2^−∆∆Cq method (16). The primers for the RT-qPCR reaction were purchased from Sangon Biotech Co., Ltd. (Shanghai, China). The sequences were as follows: Snail, sense 5′-CATTCCACGCCCAGCTACCC-3′, antisense 5′-CGCCCAGGCTCACATATTCC-3′; fibronectin, sense 5-GTGATCTACGAGGGACAGC-3, antisense 5-GCTGGTGGTGAAGTCAAAG-3; E-cadherin, sense 5-GGGCTTGGATTTTGAGGC-3, antisense 5-AGATGGGGGCTTCATTCAC-3; GAPDH, sense 5-ATGCTGGTGCTGAGTATGTC-3, antisense 5-AGTTGTCATATTTCTCGTGG-3; and α-SMA sense 5′-AACTGTGAATGTCCTGTG-3′ and antisense 5′-CATAGGTAACGAGTCAGAG-3′.

Western blot analysis was used to evaluate the protein expression levels of fibronectin, α-SMA, vimentin, Snail and E-cadherin. The HK-2 cells were lysed using a lysis buffer (10 mM Tris, 1 mM EDTA, 1% Triton X-100, 1 mM Na₃VO₄, 20 µg/ml aprotinin, 20 µg/ml leupeptin, 1 mM dithiothreitol and 50 mM PMSF). Bicinchoninic acid protein assay kit (cat. no. P0010S; Beyotime Institute of Biotechnology) was used to detect protein content. Equal quantities of protein (30 µg) from each sample were separated by 8 or 10% SDS-PAGE, the protein was transferred onto a polyvinylidene difluoride (PVDF) membrane and the PVDF membrane was blocked with 10% (w/v) non-fat milk in Tris-buffered saline with 0.1% Tween-20 for 1 h at room temperature. The blots were probed overnight at 4°C with the following primary antibodies (all purchased from Abcam, Cambridge, UK) diluted to 1:2,000 (v/v): E-cadherin (ab133597), vimentin (ab184631), Snail (ab167609), fibronectin (ab194395) and α-SMA (ab124964). Following hybridization, the blots were washed and hybridized with 1:6,000 (v/v) dilutions of a goat anti-rabbit immunoglobulin G (IgG) or goat anti-mouse IgG horseradish peroxidase-conjugated secondary antibody (cat. no. A0208 and A0216; Beyotime Institute of Biotechnology) at room temperature for 1 h. Immunodetection was performed using enhanced chemiluminescence reagents (Beyotime Institute of Biotechnology). The blots were scanned and the intensity of each band was quantified using Image J software version 1.48u (National Institutes of Health, Bethesda, MD, USA). Signals were normalized against β-actin or GAPDH diluted to 1:1,500 (v/v) (cat. no. sc-130301 and sc-47724; Santa Cruz Biotechnology, Inc., Dallas, TX, USA) and the results were expressed as a relative expression of the control signal.

Following different treatments, HK-2 cells were plated onto 6-well plates with polylysine-coated coverslips at a density of 1.6×10⁵ cells/well. Cells were fixed in 4% paraformaldehyde for 15 min at 4°C and stained with anti-fibronectin antibodies diluted to 1:50 (v/v). Briefly, HK-2 cells were fixed, blocked with 10% skimmed milk, washed with phosphate-buffered saline (PBS), inactivate endogenous peroxidase with 0.3% H₂O₂ for 15 min and then incubated for 1 h with anti-fibronectin, washed with PBS, incubated for 1 h with secondary antibody 1:50 (v/v) (cat. no. A0216; Beyotime Institute of Biotechnology), Dako Real^™ Envision^™ detection system (cat. no. P0203; Beyotime Institute of Biotechnology) was used for detection. All procedures were performed at room temperature.

All experiments were repeated at least three times. Data are presented as the mean ± standard deviation. Data were analyzed using SPSS software version 13.0 (SPSS, Inc., Chicago, IL, USA). Statistical significance was assessed using analysis of variance followed by the least significant difference test for multiple comparisons and P<0.05 was considered to indicate a statistically significant difference.

The underlying effects of tanshinone IIA and HG on glucose-induced pharmacology were evaluated. Cell survival rates and protein content were analyzed in cells treated with glucose (30 mM) and tanshinone IIA (1, 10 and 50 µM). These observations demonstrated that treatment with tanshinone IIA or HG did not statistically affect cellular survival or protein content (Fig. 1).

To investigate whether HG could induce EMT in HK-2 cells, cells were treated with DMEM containing 5.5 mM D-glucose, 30 mM mannitol or HG (30 mM glucose). Cells treated with 5.5 mM glucose exhibited a typical epithelial cuboidal shape with a characteristic confluent monolayer and cobblestone morphology; however, cells that were exposed to HG exhibited distinct morphological alterations and possessed an elongated, spindle shape (Fig. 2C). The expression of the epithelial phenotypic marker E-cadherin was significantly decreased in the HG group (P<0.05; Fig. 3A) and mesenchymal phenotypic markers, α-SMA and vimentin, were markedly increased (P<0.05; Fig. 3B and C). In addition, the expression of fibronectin was enhanced following treatment with HG, according to immunocytochemistry and western blot analysis (Fig. 3B and D). No significant differences were identified between the mannitol and NG groups. The downregulation of E-cadherin and the concomitant upregulation of α-SMA, vimentin and fibronectin in the tubular epithelial cells strongly supported the case for HG as a potent stimulus of EMT in HK-2 cells.

To examine whether tanshinone IIA inhibits glucose-induced changes in HK-2 cells, cells were pretreated with glucose or mannitol for 48 h, followed by tanshinone IIA at the indicated concentrations (1, 10 and 50 µM) for 48 h. To demonstrate the osmotic effects of glucose, mannitol was used as a control in the present study (Fig. 3). It is evident that 30 mM glucose, but not mannitol, statistically increased the expression of protein markers. Therefore, it may be proposed that glucose (not osmosis) serves a pivotal role on the regulation of HK-2 cell fibrosis. HK-2 cells produced a confluent monolayer with cobblestone morphology in the control group (Fig. 2A and B). Following treatment with 30 mM glucose, a marked number of cells exhibited an elongated, spindle shape (Fig. 2C, as indicated by arrows). As presented in Fig. 2D-F, cells were incubated with 30 mM glucose for 48 h and treated with tanshinone IIA to investigate cellular morphology. It is evident that tanshinone IIA significantly reversed HG-induced changes. In addition, tanshinone IIA appeared to be able to restore HK-2 cell epithelial morphology.

The expression of EMT marker proteins (α-SMA, vimentin and fibronectin) was examined. HG (30 mM) induced a notable increase in α-SMA, vimentin and Snail expression, and a decrease in E-cadherin (Fig. 3). The aforementioned effects were significantly reversed by 50 µM tanshinone IIA. Subsequently, α-SMA, vimentin and Snail were decreased and E-cadherin was increased following treatment with tanshinone IIA in proximal tubule cells.

In the HK-2 cells treated with HG (30 mM), the expression of Snail was significantly greater than that of cells treated with 5.5 mM glucose or 30 mM mannitol; however, the expression of Snail following pretreatment with tanshinone IIA was significantly decreased, as compared with HG treatment (Fig. 3C). It is evident that 30 mM glucose, but not mannitol, significantly increased the expression of Snail. In addition, the mRNA expression levels of Snail were increased in HK-2 cells following treatment with HG; however, the expression of Snail mRNA was reduced following treatment with 50 µM tanshinone IIA (Fig. 4), which concomitantly increased E-cadherin protein expression, as the protein expression of E-cadherin is increasing in HG+Tan (50 µM) group (Fig. 3A).

EMT marker proteins (including α-SMA, vimentin and fibronectin), epithelial phenotypic markers (E-cadherin) and transcriptional factors for EMT (Snail) were assessed using immunocytochemistry, western blotting and RT-qPCR. HG (30 mM) significantly increased extracellular fibronectin, α-SMA and vimentin in HK-2 cells, compared with in the control group (Fig. 3; P<0.05). Notably, tanshinone IIA dose dependently (1, 10 and 50 µM) and significantly suppressed HG-induced expression of fibronectin, α-SMA, vimentin and Snail. Detection of mRNA expression was performed using RT-qPCR. E-cadherin, fibronectin, α-SMA and Snail mRNA expression was assessed in the control, HG and HG+tanshinone IIA groups (Fig. 4). E-cadherin mRNA expression was markedly reduced in the HG group, whereas Snail, fibronectin and α-SMA mRNA expression was significantly increased. However, following treatment with 50 µM tanshinone IIA, the mRNA expression levels were reversed, resulting in levels similar to those in the control group. HG increased the expression of EMT marker proteins, whereas tanshinone IIA significantly reversed the increase of EMT marker proteins in HK-2 cells. The aforementioned observation indicated that tanshinone IIA has the potential to downregulate HG-induced EMT in HK-2 cells.