The HK-2 human renal proximal tubular cell line was provided by Dr Honghong Chen (Institute of Radiation Medicine, Fudan University, Shanghai, China). Dulbecco's modified Eagle's medium (DMEM) F12 and foetal bovine serum (FBS) were purchased from Gibco; Thermo Fisher Scientific, Inc. (Waltham, MA, USA). CHBP was synthesized as previously described (9). The Cell Counting kit 8 (CCK-8), glutathione/glutathione disulphide (GSH/GSSG) assay kit, reactive oxygen species (ROS) assay kit, nuclear and cytoplasmic protein extraction kit, Annexin V apoptosis detection kit and one-step terminal deoxynucleotidyl transferase-mediated dUTP nick-end label (TUNEL) apoptosis assay kit were purchased from Beyotime Institute of Biotechnology (Haimen, China). DNA oligonucleotides were synthesized by Shanghai BoShang Biotechnology Co., Ltd. (Shanghai, China). Antibodies against cleaved caspase-3 (cat. no. 9661; 1:1,000), BiP (cat. no. 3177; 1:1,000), CHOP (cat. no. 5554; 1:1,000), HO-1 (cat. no. 5853; 1:1,000), beclin-1 (cat. no. 3495; 1:1,000), light chain 3 (LC3) A/B (cat. no. 12741; 1:1,000), phosphorylated (p)-mechanistic target of rapamycin (mTOR) Ser2481 (cat. no. 2974; 1:1,000), p-mTOR Ser2448 (cat. no. 5536; 1:1,000), p62 (cat. no. 5114; 1:1,000), mTOR (cat. no. 2972; 1:1,000), and β-actin (cat. no. 3700; 1:1,000) were purchased from Cell Signaling Technology, Inc. (Danvers, MA, USA). Antibodies against Nrf2 (rabbit anti-human monoclonal; cat. no. sc-722; 1:200) and lamin B (goat anti-human monoclonal; cat. no. sc-6216; 1:200) were purchased from Santa Cruz Biotechnology, Inc. (Dallas, TX, USA). The secondary antibody (cat. no. P0186; 1:1,000) used in the immunocytochemistry assay (Goat anti-Rabbit) were purchased from Beyotime Institute of Biotechnology (Haimen, China).

HK-2 cells were cultured in DMEM F12 medium supplemented with 10% FBS at 37°C and 5% CO₂ for 24 h. Confluent monolayers (80%) were cultures in 6-well plates and pretreated with or without 20 nmol/l CHBP for 1 h prior to treatment with 500 µmol/l H₂O₂ diluted in serum-free media, for 4 h.

Cell proliferation and viability were measured using a CCK-8 assay kit. Briefly, HK-2 cells were seeded in 96-well tissue culture plates for 24 h (8,000/well). Subsequently, cells were pretreated with or without 10, 20 and 40 nmol/l CHBP for 1 h prior treatment with 500 µmol/l H₂O₂ diluted in serum-free media for 4 h. Control cells were treated with media alone. Subsequently, cells were pretreated with or without 10, 20 and 40 nmol/l CHBP for 1 h prior to treatment with 500 µmol/l H₂O₂ diluted in serum-free media, for 4 h. Cells were washed twice with PBS and incubated with culture medium containing 10% CCK-8 solution at 37°C for 1 h. The absorbance of the wells was detected using a microplate reader at a wavelength of 450 nm, generating an optical density (OD) value. Culture medium containing 10% CCK-8 solution was utilized as a negative control.

ROS activity levels were determined using a dichloro-dihydro-fluorescein diacetate assay kit. The GSH/GSSG ratio was measured using a GSH/GSSG assay kit. HK-2 cells were washed twice with PBS and suspended in ice-cold 5% metaphosphoric acid. Cells were homogenized with a TissueLyser LT (Qiagen GmbH, Hilden, Germany), and suspensions were transferred to a microtube and centrifuged at 10,000 × g for 10 min at 4°C. The collected supernatant was utilized to analyse GSH and GSSG concentrations in addition to ROS levels, according to the manufacturer's protocol.

HK-2 cells were washed twice in PBS and harvested. Extra-nuclear and intra-nuclear proteins were isolated using a protein extraction kit (Beyotime Institute of Biotechnology), according to the manufacturer's protocol. Western blotting was performed according to a previously published procedure (10). Expression levels of cleaved caspase-3, BiP, CHOP, Nrf2, HO-1, Beclin-1, LC3 A/B, p-mTOR Ser2448, p-mTOR Ser2481, p62 and mTOR were quantified using Image-Pro plus software version 6.0 (Media Cybernetics, Inc., Rockville, MD, USA). Extra-nuclear proteins were normalized to β-actin and intra-nuclear proteins were normalized to lamin B.

Apoptosis of HK-2 cells was determined using a one-step TUNEL assay kit according to the manufacturer's protocol. Briefly, cells were washed with PBS and fixed in 4% paraformaldehyde for 30 min. Cells were washed with PBS and incubated with cold PBS containing 0.1% Triton X-100 for 2 min in a light-proof container. Cells were washed with PBS and incubated with TUNEL reaction buffer for 1 h at 37°C in a humidified light-proof chamber. Following a final wash with PBS, cells were visualized using a laser scanning confocal microscope (Leica Microsystems, Inc., Wetzlar, Germany) at a wavelength of 530/485 nm.

Total RNA was extracted from the HK-2 cells using TRIzol reagent (Invitrogen; Thermo Fisher Scientific, Inc.) according to the manufacturer's protocol. cDNA was synthesized from 3–5 µg total RNA in a 20 µl reaction mixture using a RevertAid First Strand cDNA Synthesis kit (Thermo Fisher Scientific, Inc.). The specific primers for the genes that encode human BiP, CHOP and β-actin are listed in Table I. RT-qPCR was performed using ABsolute qPCR SYBR Green mix (Thermo Fisher Scientific, Inc.) on an Eppendorf Mastercycler^® ep realplex system as follows: Incubation for 2 min at 50°C and 10 min at 95°C. This was followed by a 2-step PCR program of 95°C for 15 sec and 60°C for 20 sec for 45 cycles. (Eppendorf, Hamburg, Germany). mRNA expression levels were normalized to those of β-actin in the same samples using the 2^−ΔΔCq method (11).

Experimental HK-2 cells were cultured in 12-well plate (10⁵/well) after H₂O₂ stimulation for 4 h were fixed in 4% paraformaldehyde for 10 min. Following a wash with PBS for 5 min at room temperature, cells were permeabilized with Triton X 100 for 5 min followed by PBS for 5 min and then incubated in blocking solution (5% BSA in PBS; Beyotime Institute of Biotechnology) for 1 h at room temperature. Cells were incubated with primary antibodies against Nrf2 (1:200) and LC3A/B (1:100) for 60 min. Following three rinses with PBS, cells were incubated with a secondary antibody conjugated to fluorescein isothiocyanate for 30 min at room temperature and washed with PBS. To visualize cell nuclei, slides were counterstained with DAPI (Beyotime Institute of Biotechnology). Samples were examined under a phase contrast microscope equipped with the appropriate fluorescence filters in a total of 10 fields per specimen.

Data was analysed using SPSS software version 13.0 (SPSS, Inc., Chicago, IL, USA). The results in two groups were compared using two-tailed independent t-tests, and the results among three or more groups were compared by one-way analysis of variance. Data are expressed as the mean ± standard deviation. P<0.05 was considered to indicate a statistically significant difference.

The effect of CHBP on viability in HK-2 cells following oxidative stress induced by H₂O₂ was determined. Cells were treated with H₂O₂ for 1, 4, 8 and 16 h prior to measurement of cell viability via the CCK-8 assay. Results revealed that pretreatment with CHBP significantly reduced the decrease in cell viability caused by H₂O₂ alone, and the greatest protection observed at 4 h (Fig. 1A). Treatment with H₂O₂ alone reduced the viability of HK-2 cells by ~3-fold; however, viability of cells treated with 20 nmol/l CHBP alone was not significantly altered compared with the control (Fig. 1B). Pretreatment with CHBP resulted in a significant and dose-dependent increase in the viability of HK-2 cells in the presence of H₂O₂ compared with cells treated with H₂O₂ alone (P<0.001; Fig. 1B). The protective effect of CHBP peaked at 20 nmol/l (Fig. 1B). In addition, cell viability was greatest following pretreatment with CHBP for 1 h prior to H₂O₂ exposure for 4 h (Fig. 1C). To determine whether CHBP reduces oxidative stress induced by H₂O₂, ROS activity levels and the GSH/GSSG ratio in HK-2 cells were measured. ROS activity levels in HK-2 cells were enhanced following exposure to H₂O₂ compared with the control, and this effect was significantly reduced by CHBP pretreatment (P<0.001; Fig. 1D). In addition, the GSH/GSSG ratio in HK-2 cells was reduced following exposure to H₂O₂ compared with the control, and this effect was reversed by pretreatment with CHBP (P<0.001; Fig. 1E). These results suggested that CHBP pretreatment enhances HK-2 cell viability and reduces oxidative stress in HK-2 cells.

To determine whether CHBP inhibits apoptosis induced by H₂O₂, apoptosis in HK-2 cells was measured following treatment. In addition, as activation of caspase-3 is a marker for apoptosis, caspase-3 activity was measured in HK-2 cells following treatment. The percentage of apoptotic cells was enhanced following H₂O₂ treatment alone, whereas pretreatment with 20 nmol/l CHBP in the presence of H₂O₂ significantly reduced the activity levels of caspase-3 (P<0.01; Fig. 2A). Caspase-3 and cleaved caspase-3 expression levels were enhanced in HK-2 cells following H₂O₂ treatment alone, and were significantly reduced by pretreatment with CHBP (Fig. 2B). Additionally, Pretreatment with CHBP significantly reduced the number of TUNEL positive cells in the presence of H₂O₂ (P<0.001; Fig. 2C). These results suggested that CHBP inhibits apoptosis induced by H₂O₂.

Elevated expression levels of BiP and CHOP are features of ER stress (2). To determine whether CHBP decreased ER stress in HK-2 cells, mRNA and protein expression levels of BiP and CHOP were measured in HK-2 cells following H₂O₂ treatment. The mRNA expression levels of the genes encoding BiP and CHOP were enhanced in cells treated with H₂O₂ alone, and these levels were significantly reduced by pretreatment with CHBP (P<0.01; Fig. 3A). This was supported by the protein expression levels of BiP and CHOP (P<0.01; Fig. 3B). These results suggested that CHBP pretreatment reduces ER stress in HK-2 cells exposed to H₂O_2.

Nrf2 is a transcriptional regulator of antioxidant proteins, including HO-1 (12). To determine whether CHBP activates the Nrf2 signalling pathway in HK-2 cells treated with H₂O₂, expression levels of intranuclear and extranuclear Nrf2 and HO-1 were measured following pretreatment with CHBP. Nrf2 expression levels were enhanced in the nuclei of HK-2 cells after H₂O₂ treatment alone, (P<0.001; Fig. 4A), and were further enhanced by pretreatment with CHBP (P<0.001; Fig. 4A). Similar results were observed in the extranuclear portion of HK-2 cells (P<0.001; Fig. 4B) via western blotting. In addition, these results were supported by the immunocytochemistry data for the Nrf2 protein (Fig. 4C). These results suggested that CHBP pretreatment enhances activation of the Nrf2 signalling pathway in HK-2 cells exposed to H₂O₂.

Our previous in vivo study demonstrated that the renoprotective effect of CHBP against IRI is mediated by induction of autophagy via inhibition of mTOR complex (C) 1 and activation of mTORC2 (9). As oxidative stress may crosstalk with autophagic machinery, the present study determined whether the renoprotective function of CHBP is mediated by autophagy in HK-2 cells after H₂O₂ treatment. Results demonstrated that beclin-1 and LC3 A/B-II/I expression levels were significantly enhanced, and the expression of p62 following CHBP pretreatment was reduced much more compared with H₂O₂ alone (Fig. 5A). However, the expression of p-mTOR Ser2481 following CHBP pretreatment was increased more than that of H₂O₂ alone. By contrast, p-mTOR Ser2448 levels were enhanced in HK-2 cells after H₂O₂ stimulation, and were significantly reduced by CHBP pretreatment (Fig. 5B). These results suggested that CHBP pretreatment enhances autophagy in HK-2 cells under oxidative stress.