HK-2 cells were obtained from the Kidney Disease Laboratory of Second Xiangya Hospital of Central South University (Changsha, China). The cells were cultured at 37°C in a humidified atmosphere containing 5% CO₂ in Dulbecco's modified Eagle's medium (DMEM)/F12 (Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA) supplemented with 10% fetal bovine serum (FBS; Gibco; Thermo Fisher Scientific, Inc.) and antibiotics (100 IU/ml penicillin and 100 µg/ml streptomycin). Iohexol was purchased from Nycomed China (Shanghai, China) and 3-methyladenine (3-MA) was purchased from Sigma-Aldrich; Merck KGaA (Darmstadt, Germany).

HK-2 cells were incubated in FBS-free DMEM/F12 for 12 h. The establishment of a damaged cells model was performed as previously reported (11). 3-MA (10 mM) was used to inhibit the formation of autophagosomes. The cells were divided into the following four groups, all of which were cultured at 37°C: Control group, in which cells were cultured in DMEM/F12 supplemented with 1% FBS; CM group, in which cells were treated with 200 mg iodine/ml iohexol for 6 h; 3-MA group, in which cells were treated with 10 mM 3-MA for 30 min; and CM + 3-MA group, in which cells were pretreated with 10 mM 3-MA for 30 min prior to treatment with 200 mg iodine/ml iohexol for 6 h.

HK-2 cells were cultured in 6-well plates at a density of 1-5×10⁵/ml, and were treated as aforementioned. The cells were digested and collected routinely. Subsequently, the cells were suspended and fixed with 2.5% glutaraldehyde in 0.1 mM PBS (pH 7.4) at 4°C for 2 h. After washing twice with PBS, cells underwent conventional dehydration, osmosis, embedding, sectioning and staining, as previously described (12). Cell ultrastructure was observed under a Philips 300 electron microscope (Philips Healthcare, Amsterdam, The Netherlands). TEM was used to detect the effects of iohexol on HK-2 cell autophagy.

Cells were cultured on polylysine-coated glass slides (Sigma-Aldrich; Merck KGaA) in 6-well plates at a density of 1–5×10⁵/ml, fixed in 4% paraformaldehyde for 15 min, washed with PBS and permeabilized with 0.1% Triton-X-100 (Sigma-Aldrich) for 5 min, all at room temperature. Subsequently, the cells were incubated with LC3-II primary antibody for 1 h at room temperature (1:1,000; cat. no. L8918; Sigma-Aldrich; Merck KGaA), followed by incubation with a secondary antibody [fluorescein isothiocyanate (FITC)-conjugated; 1:100; cat. no. KC-RB-095; KangCheng Biotech, Shanghai, China] at room temperature for 1 h. Cells were then incubated in the dark with DAPI (Invitrogen; Thermo Fisher Scientific, Inc.) for 5 min. After washing with PBS, glass slides were sealed with a seal sheet containing a fluorescence-quenching agent. A laser-scanning confocal fluorescence microscope (Olympus BX60; Olympus Corporation, Tokyo, Japan) was used to capture images.

Cells were lysed with cell lysis buffer (radioimmunoprecipitation assay buffer: phenylmethylsulfonyl fluoride, 100:1; Beyotime Institutes of Biotechnology, Shanghai, China) on ice for 30 min and were centrifuged at 8,500 × g for 5 min at 4°C. The protein concentration was determined using the Bicinchoninic Acid Protein Assay Reagent kit (Beyotime Institutes of Biotechnology). Proteins (20 µg) were separated by 10% SDS-PAGE and were transferred to polyvinylidene fluoride membranes (EMD Millipore, Billerica, MA, USA). The membranes were blocked with calf serum (Gibco; Thermo Fisher Scientific, Inc.) for 1 h at room temperature and incubated with anti-LC3-II (1:1,000; cat. no. L8918; Sigma-Aldrich; Merck KGaA), anti-Atg7 (1:200; cat. no. A2856; Sigma-Aldrich; Merck KGaA) and anti-β-actin antibodies (1:2,000; cat. no. sc-7210; Santa Cruz Biotechnology, Inc., Dallas, TX, USA) overnight at 4°C, followed by incubation with a secondary antibody (1:2,000; cat. no. A0545; Sigma-Aldrich; Merck KGaA) for 1 h at room temperature. Enhanced chemiluminescent reagent (EMD Millipore) was used for chemiluminescence detection. The Kodak Gel Logic 200 Imaging System (Kodak, Rochester, NY, USA) was used to analyze the results.

HK-2 cells were cultured in 24-well plates at a density of 1–5×10⁵/ml. Once they reached 70–80% confluence, the cells were incubated with serum-free medium for 12 h and treated with iohexol and 3-MA as indicated. Cell injury was assessed using a lactate dehydrogenase (LDH) assay (Beyotime Institutes of Biotechnology) and an automatic biochemistry analyzer (Abbott Laboratories, Chicago, IL, USA).

An MTT assay (Sigma-Aldrich; Merck KGaA) was used to assess the viability and proliferation of HK-2 cells (4). Briefly, confluent HK-2 monolayers were incubated for 6 h with control medium, 200 mg/ml iohexol, or 200 mg/ml iohexol + 10 mM 3-MA. Subsequently, 20 µl (5 mg/ml) MTT was added to the wells and was incubated for 4 h at 37°C, after which 150 µl dimethyl sulfoxide was added and oscillated for 10 min. Cell viability was measured using a Labsystems Wellscan MK2 (Thermo Fisher Scientific, Inc.) at a 490-nm wavelength.

Cell apoptosis was detected with an Annexin V FITC-conjugated/propidium iodide (PI) apoptosis kit (Nanjing Keygen Biotech Co., Ltd., Nanjing, China) using flow cytometry. Cells were seeded in a 6-well plate at 2×10⁵ cells/well. Once the cells reached 70–80% confluence, they were incubated with serum-free medium for 12 h and treated with iohexol and 3-MA as aforementioned. Subsequently, the cells were collected and incubated with 5 µl Annexin V-FITC and 5 µl PI (50 mg/ml) for 15 min in the dark at room temperature. The cells were immediately analyzed using a flow cytometer (BD Biosciences, San Jose, CA, USA).

SPSS software (version 17.0; SPSS, Inc., Chicago, IL, USA) was used to conduct statistical analysis. Data are presented as the mean ± standard deviation from 6 independent experiments. For comparisons between any two groups, an unpaired Student's t-test was performed. Comparisons between data from more than two groups were assessed by one-way analysis of variance followed by Fisher's least significant difference test for post hoc comparisons. P<0.05 was considered to indicate statistically significant difference.

The present study used TEM to detect the effects of iohexol on autophagy. As presented in Fig. 1A, in the control group, the HK-2 cells exhibited normal cytoplasm with no autophagosomal formation. However, a marked accumulation of autophagosomes was observed in the iohexol-treated group; cytoplasmic material and/or membrane vesicles were encapsulated in vacuoles (red arrows; Fig. 1B).

LC3 is a representative autophagosome marker, which is essential for the formation of autophagosomes. During autophagy, the cytosolic form of LC3 (LC3-I) is converted to LC3-II by Atg7 (E1-like enzyme) and Atg3 (E2-like enzyme) (8). In the present study, the results of an immunofluorescence assay demonstrated that LC3-II expression was evidently upregulated in the cytoplasm by iohexol (Fig. 2A). Furthermore, these observations were confirmed by western blot analysis. As shown in Fig. 2B, LC3-II and Atg7 expression were markedly upregulated by iohexol (200 mg iodine/ml) compared with the control group (P<0.05). These results indicated that iohexol may increase the levels of autophagy in HK-2 cells.

3-MA, which is an inhibitor of phosphatidylinositol 3-kinase, was used to inhibit the initiation of autophagosome formation. As presented in Fig. 3, 3-MA not only affected the expression levels of LC3-II and Atg7 in HK-2 cells compared with the control group, but also reversed iohexol-induced upregulation of LC3-II and Atg7 expression (Fig. 3).

As presented in Fig. 4 and Table I, the cytotoxicity of iohexol was evaluated by MTT assay and LDH release. LDH levels were significantly increased (P<0.05) and cell viability was significantly decreased in the iohexol-treated group compared with the control group, which was further enhanced by 3-MA.

The role of autophagy in iohexol-induced HK-2 cell apoptosis was analyzed (Figs. 5 and 6). As shown in Figs. 5B and 6, iohexol significantly increased apoptosis of HK-2 cells compared with the control group. Treatment with 3-MA alone did not affect cell apoptosis (Figs. 5C and 6). However, cell apoptosis was significantly increased in the iohexol + 3-MA group compared with the iohexol group (Figs. 5D and 6).