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Introduction

Aristolochic acid (AA) is a component present in Chinese herbs (for example Asarum and Aristolochia) from remedies for the treatment of arthritis pain, coughs and gastrointestinal symptoms (1–4). Previous studies have indicated that AA can lead to renal injury (5,6) and this finding has led to further studies (7,8). Previous studies have indicated that renal damage from renal cell death and renal fibrosis is associated with AA treatment (9,10).

AA-induced oxidative stress may serve an important role in the development of renal injury (11–13). Previous studies have demonstrated that oxidative stress causes lipid peroxidation, DNA damage and protein peroxidation, and results in cell damage (14–16). O2− and H2O2 are key reactive oxygen species (ROS) identified in cells (17,18). Normally, O2− and H2O2 are produced in the mitochondria via electron transport chain (19,20) and these ROS are removed by cellular superoxide dismutase (SOD), glutathione peroxidase (Gpx) and catalase (CAT) (21–23). However, various toxins also induce O2− and H2O2 production (24–26). The excessive O2− and H2O2 lead to cell injury (27,28) and it has additionally been reported that AA-induced H2O2 leads to renal damage (29).

Various studies have demonstrated that oxidative stress can induce cell apoptosis or cell necrosis (30–32), and consequently AA-induced oxidative stress can cause apoptosis or necrosis of renal cells (29,33–35). Concerning apoptosis, caspase-dependent and caspase-independent pathways have been reported previously (36,37). Although certain mechanisms of AA-induced cell death remain unclear, the caspase activation may be associated with AA-induced apoptosis (38,39). Previous studies indicated that AA can activate caspase-9 and caspase-3 leading to cell apoptosis (40–42).

The isoforms of vitamin E consist of α-tocopherol, β-tocopherol, δ-tocotrienol and γ-tocotrienol (43). Among them, α-tocopherol possesses anti-oxidative activities and has been used in a clinical setting (44,45). In addition, previous studies have suggested that α-tocopherol can inhibit renal fibrosis (46,47). Due to the fact that AA-induced renal injury was associated with oxidative damage and fibrotic renal injury (9,11–13), the effects of α-tocopherol on AA-induced renal cell cytotoxicity were studied. The results of the present study demonstrated that α-tocopherol can inhibit the H2O2 level and caspase-3 activities to attenuate renal tubular epithelial cell death under AA treatment.

Materials and methods

Materials

The MTT assay kit was obtained from Bio Basic Canada, Inc. (Markham, OT, Canada). Vitamin E (α-tocopherol), luminol, lucigenin, tubulin polyclonal antibody and Hoechst 33342 were obtained from Sigma-Aldrich (Merck KGaA, Darmstadt, Germany). Caspase-3 and cleaved caspase-3 polyclonal antibodies were obtained from Cell Signaling Technology, Inc. (9662; 1:1,000; Danvers, MA, USA). Fetal bovine serum, DMEM, non-essential amino acid, L-glutamine, and penicillin/streptomycin were obtained from Gibco (Thermo Fisher Scientific, Inc., Waltham, MA, USA).

Cell culture

Rat renal tubular epithelial cells (NRK-52E) were obtained from the Bioresource Collection and Research Center (Shin Chu, Taiwan). NRK-52E cells were cultured with DMEM medium containing 10% fetal bovine serum, 2 mM L-glutamine, 100 IU/ml penicillin/streptomycin and 0.1 mM non-essential amino acids. Cells were maintained in a humidified atmosphere containing 5% CO2 at 37°C.

ROS detection

H2O2 and O2− levels were measured by using the lucigenin-amplified chemiluminescence method (48,49). The culture supernatant (200 µl) were added with 0.2 mmol/l of luminol solution (100 µl) and measured subsequently by using a chemiluminescence analyzing system (CLA-FSI; Tohoko Electronic Industrial Co., Ltd., Sendai, Japan) for the determination of H2O2 levels. The samples (200 µl) were treated with 0.1 mmol/l lucigenin solution (200 µl) and then O2− levels were measured using the CLA-FSI chemiluminescence analyzing system.

Cell survival rates determination

The cell survival rates were determined using the MTT assay kit according to the manufacturer's instructions. In brief, NRK-52E cells were cultured into 96-well plates at a density of 8×103 cells/well and incubated for 24 h in 100 µl DMEM medium. The suitable concentration and optimum exposure time of AAI were determined as 5, 10, 20 and 100 µM at 6 h time intervals. Cells were treated with MTT assay kit for 3 h at 37°C and were measured at 570 nm absorbance using a Multiskan™ FC microplate photometer (Molecular Devices, Inc., Sunnyvale, CA, USA). The cell survival rate was calculated as the following formula: Optical density (OD) 570 experimental group/OD 570 control group ×100%.

Observation of apoptotic features

Apoptotic features containing DNA fragmentation and nuclear condensation were observed by using Hoechst 33342 (23491-52-3; Sigma-Aldrich; Merck KGaA) nuclear staining (49,50). Control and experimental cells were treated with Hoechst 33342 (10 µg/ml) at 37°C for 10 min. DNA fragmentation and nuclear condensation were observed under an Olympus DP71 fluorescence microscope (excitation, 352 nm; emission, 450 nm; Olympus Corporation, Tokyo, Japan).

Western blotting

Cells were treated with radioimmunoprecipitation assay buffer (20–188; EMD Millipore, Billerica, MA, USA). Following 10 min centrifugation (16,000 × g) at 4°C, proteins were obtained from the supernatant layer and the concentration was determined by using the protein assay kit (23200; Thermo Fischer Scientific, Inc.). Equal quantities of samples were separated on a 13.3% SDS-PAGE (Bio-Rad Laboratories, Inc., Hercules, CA, USA), and then transferred onto polyvinylidene difluoride membranes (EMD Millipore). The membranes were blocked with 5% milk for 2 h at room temperature. Next, the membranes were washed with phosphate-buffered saline (PBS) then incubated with the primary antibodies for 4 h. Following that, membranes were washed with PBS and treated with anti-rabbit-horseradish peroxidase secondary antibodies (NA934; 1:1,000 Amersham; GE Healthcare Life Sciences, Chalfont, UK) for 1 h at room temperature. Finally, proteins were observed by using Western Lightning Chemiluminescence Reagent Plus (PerkinElmer, Inc., Waltham, MA, USA).

Statistical analysis

Student's t-test and two-way analysis of variance were utilized for the analysis of the data using SPSS version 18.0 (SPSS, Inc., Chicago, IL, USA). Values are expressed as the mean ± standard error. P<0.05 was considered statistically significant different between values.

Results

Increases of H2O2 and O2- levels by different concentrations of AA treatment

Previous studies have demonstrated that AA induced ROS generation in renal tubular cells (13,51). H2O2 and O2−, two major types of ROS, were measured in AA-treated renal tubular cells. Experimental results indicated that H2O2 levels were increased dose-dependently in the AA-treated cells (Fig. 1A). Compared with H2O2 levels, O2− levels were increased only at the 100 µM AA treatment however not at 20–50 µM AA concentrations (Fig. 1B). The data suggested low-dose AA (5–20 µM) can induce increases in H2O2 levels, but not O2− levels. Additionally, high-dose AA (100 µM) can induce increases of H2O2 and O2− levels.

Figure 1. H2O2 and O2− levels. (A) H2O2 levels and (B) O2− levels were measured in control group (0 µM AA) and AA-treated groups (5–100 µM AA). The levels were measured following 2 h treatment. Data from four independent experiments were presented as the mean ± standard deviation. *P<0.05, vs. the control group (0 µM AA). AA, aristolochic acid.

AA decreased cell survival rates in dose- and time- dependent manners

In order to determine the cytotoxic effects on AA-treated renal tubular cells, various concentrations (5–100 µM) of AA were studied. As presented in Fig. 2, the cell survival rates were below 50% at 100 µM AA (6 h), 20 µM AA (12 h), 10 µM AA (24 h) and 5 µM AA (48 h) treatment. These results demonstrated AA-induced cell cytotoxicity was dose- and time-dependent.

Figure 2. Cell survival rates. The renal tubular cells were treated with 5–100 µM AA for 48 h. Data from four independent experiments are presented as the mean ± standard deviation. AA, aristolochic acid.

Apoptotic characteristics in AA-treated renal tubular cells

Cell death can be described as apoptosis or necrosis (52,53). Apoptotic cells can be removed by macrophages in order to prevent serious inflammatory responses (54,55), and previous studies have indicated that nuclear condensation and DNA fragmentation are key apoptotic characteristics (49,56). In the present study, the cell nuclei were observed by Hoechst 33342 staining (49,50). As presented in Fig. 3, compared with control cell, the nuclear condensation and DNA fragmentation were identified in AA-treated renal tubular cells. The results indicated that AA-induced cell death was associated with the apoptotic death pathway.

Figure 3. Nuclear condensation and DNA fragmentation. (A) Control group. (B) AA-treated group. Renal tubular cells were treated with 10 µM AA for 24 h, nuclear condensation and DNA fragmentation (yellow arrows) were observed in the AA-treated cells under a phase-contrast microscope at magnifications of ×400. AA, aristolochic acid.

α-tocopherol attenuated H2O2 levels and increased cell survival in AA-treated cells

The antioxidant stress activities of vitamin E (α-tocopherol) has been demonstrated in clinical cases (44,45). Due to the fact that AA elevated H2O2 levels (Fig. 1A), it was investigated whether α-tocopherol could inhibit H2O2 in AA-treated cells with various concentrations of α-tocopherol (5, 10, 20 and 100 µM). The data indicated that α-tocopherol attenuated AA-induced H2O2 levels (Fig. 4). Notably, it was observed that 10 µM α-tocopherol appeared to have a more marked effect on AA-induced H2O2 compared with other concentrations. Subsequently, the effects of α-tocopherol on AA-induced renal cell death were investigated. As presented in Fig. 5, the cell survival rates were increased in AA-treated renal tubular cells undergoing 10 or 20 µM α-tocopherol treatment. These results first demonstrated that α-tocopherol attenuated AA-induced H2O2 levels and increased cell survival of AA-treated cells.

Figure 4. H2O2 levels. H2O2 levels were measured in 10 µM AA-treated groups (0 µM α-tocopherol vehicle) and 10 µM AA + 5–100 µM α-tocopherol treated group. The levels were measured following 2 h treatment. Data from four independent experiments were presented as the mean ± standard deviation. *P<0.05, vs. the AA alone-treated group (0 µM α-tocopherol cotreatment). AA, aristolochic acid.

Figure 5. Cell survival rates. The renal tubular cells were treated with 10 µM AA + 0, 10 and 20 µM α-tocopherol treatment, respectively. At 24 h the survival rates were calculated. Data from four independent experiments are presented as the mean ± standard deviation. **P<0.001, vs. the 10 µM AA alone-treated group. AA, aristolochic acid.

α-tocopherol reduced AA-activated caspase-3

Caspase-3 activation is associated with the apoptotic death pathway (40–42). Due to the fact that apoptotic characteristics were predominantly identified in AA-treated renal tubular cells (Fig. 3B), whether AA could activate caspase-3 was investigated. As presented in Fig. 6, compared with the control group (lane 1), the level of cleaved caspase-3 was markedly increased in the AA-treated group (lane 2). AA was identified to induce caspase-3 activity and the effect of α-tocopherol on AA-induced caspase-3 was further determined. AA was reported to decrease mitochondrial membrane depolarization and to lead to an increase of caspase-3 (42). The results demonstrated that cleaved caspase-3 levels were reduced in the AA plus α-tocopherol group (lane 5 and 6) compared with the AA treatment group (lane 2). Due to the fact that AA-induced H2O2 levels are also reduced in the AA + α-tocopherol group (Fig. 4), this suggested that α-tocopherol attenuation of AA-induced cell cytotoxicity may be associated with caspase-3 activity from the reduce of H2O2 levels.

Figure 6. Caspase-3 activation by western blotting. The caspase-3 activity was analyzed at 24 h in the control (lane 1), 10 µM AA (lane 2), 10 µM α-tocopherol (lane 3), 20 µM α-tocopherol (lane 4), 10 µM AA + 10 µM α-tocopherol (lane 5), and 10 µM AA + 20 µM α-tocopherol (lane 6) cells. AA, aristolochic acid.

Discussion

The results of the current study indicated that AA causes increases in H2O2 levels and a reduction in cell survival rates in renal tubular cells and α-tocopherol (10 and 20 µM) attenuated AA-induced H2O2 levels and inhibited AA-induced cytotoxicity in these cells. These data suggested that AA-induced cell cytotoxicity may be associated with H2O2 levels. By contrast, α-tocopherol could not inhibit AA-induced cytotoxicity under high-dose (100 µM) AA treatment (data not shown), however it effectively ameliorated AA-induced cytotoxicity under low-dose (5–20 µM) AA treatment. This suggested that α-tocopherol ameliorated AA-induced renal cell damage was dependent on AA dosage. Furthermore, high-dose AA alone elevated both H2O2 and O2− levels. Therefore, this may partially explain why α-tocopherol could not inhibit high-dose AA-induced cell cytotoxicity.

CAT, Gpx and SOD are major cellular antioxidant enzyme systems (57,58). CAT is a tetrameric iron-porphyrin protein in peroxisomes that converts H2O2 to H2O and O2. CAT and Cu/Zn-SOD are expressed constitutively, whereas Mn-SOD expression within the mitochondria is induced by oxidative stress. GSH is a sulfhydryl peptide that may directly react with O2− or N2− containing free radicals, or is able to donate electrons in the enzymatic dismutation of H2O2 to H2O and O2 by GPx (59,60). Cellular CAT and Gpx can remove H2O2, whereas SOD removes O2− (61). In the present study, the data indicated that AA induced increases in H2O2 in a dose-dependent manner, however the 100 µM AA alone was capable of increasing O2− levels. This result suggested that low-dose AA may influence the activity of CAT and Gpx while high-dose AA may influence CAT, Gpx and SOD activities. However, further studies are required to confirm this hypothesis.

Studies have indicated that antioxidant can attenuate AA-induced renal damage (11,29,59) and it has been additionally demonstrated that vitamin C attenuated AA-induced renal damage (29). However, the effect of antioxidant α-tocopherol on AA-induced renal damage remains to be reported. The current study demonstrated that α-tocopherol ameliorated AA-induced renal cell cytotoxicity.

Vitamin C and α-tocopherol are common antioxidants and have been used in clinical cases (44,45,62,63). Vitamin C and α-tocopherol both have antioxidant activities, however their antioxidant mechanisms differ (64–66). These studies indicated that α-tocopherol (lipid-soluble material) is able to pass through the cell membrane and catch the free radicals to protect the cell from oxidative damage. However, vitamin C (water-soluble material) cannot pass through cell membrane to remove free radicals directly. Based on the results of a previous study (29) and the current study, it is suggested that both vitamin C and α-tocopherol scavenge H2O2 produced by AA-treated renal cells, leading to an increase of survival rate. It was suggested that AA-induced H2O2 existed not only in the cytosol however additionally in the cell membrane. In addition, another key antioxidant function of vitamin C is converting the oxidized α-tocopherol radical back to α-tocopherol (62). It was suggested that the combination of vitamin C and α-tocopherol may be more powerful for protection of AA-induced renal damage. In patients with coronary artery bypass surgery, vitamin cocktail (ascorbic acid and α-tocopherol) effectively attenuated oxidative stress than control (44). In summary, the results demonstrated that α-tocopherol attenuates AA-induced H2O2 and caspase-3 and ameliorated AA-induced renal cell cytotoxicity.

Acknowledgements

The present study was supported by grants from the Ministry of Science and Technology, Taiwan (grant nos. MOST103 2320-B-039-052-MY3 and MOST104-2321-B-039-005) and Ministry of Health and Welfare (MOHW104-TDU-B-212-124-002).

References