Native HDL (Chemicon International, Inc., Billerica, MA, USA) was incubated with CuSO₄ (20 μmol/l final concentration) in 1 mol/l PBS pH 7.4 at a lipoprotein concentration of 1 mg protein/ml for 24 h at 37°C. Oxidation was terminated by the addition of ethylenediaminetetraacetic acid (EDTA) (200 μmol/l final concentration) and confirmed by measurements of thiobarbituric acid reactive substances (TBARS) and oxidized HDL was sterilized by passing it through a 0.22-μm filter, as previously described (14).

Human renal proximal tubule epithelial cells (HK-2) were purchased from the American Type Culture Collection (ATCC; Manassas, VA, USA) and cultured in keratinocyte serum-free medium (K-SFM) (Gibco, Grand Island, NY, USA) supplemented with 5 ng/ml epidermal growth factor (EGF) and 40 mg/ml of bovine extract (Gibco), as well as 100 U/ml of penicillin and 100 Ug/ml of streptomycin. The cells were placed in an atmosphere of 5% CO₂-95% air at 37°C, and passaged by trypsinization (0.25% trypsin, 0.02% EDTA) following the formation of a confluent monolayer and placed in a serum-free medium 24 h prior to stimulation. Oxidized HDL or native HDL was added to the medium at the indicated concentrations for 24 h, and used for various analyses.

The HK-2 cells were transfected with 100 nM of CD36 siRNA (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA) and the transfection procedure was performed using Lipofectamine 2000 (Invitrogen Life Technologies, Carlsbad, CA, USA) according to the manufacturer’s instructions. Briefly, 1 day prior to transfection, the cells were plated in growth medium without antibiotics so that they would be 50% confluent at the time of transfection. Subsequently, CD36 siRNA or scrambled control plasmid oligomer-Lipofectamine 2000 complexes were prepared, added to each well containing cells and medium and mixed gently by rocking the plate back and forth. The cells were incubated for 24 h and then treated with various doses of oxidized HDL or native HDL.

Measurement of intracellular ROS was based on the ROS-mediated conversion of non-fluorescent 2′,7′-dichlorodihydrofluorescein diacetate (DCFH-DA) into dichlorodihydrofluorescein (DCFH), as previously described (15,16). The fluorescence intensity reflects enhanced oxidative stress. Following treatment as described above, the HK-2 cells were incubated with DCFH-DA in medium at 37°C for 45 min and then washed with PBS. Following incubation, the DCFH fluorescence of the cells from each well was imaged under a fluorescence microscope and analyzed using AxioVision 4.5 software (Carl Zeiss, Jena, Germany).

Annexin V-fluorescein isothiocyanate/propidium iodide (FITC/PI) staining was used to detect the apoptosis induced by treatment with oxidized HDL in HK-2 cells. The HK-2 cells were harvested by centrifugation at 300 × g for 5 min, followed by 2 washes with cold PBS. The cells were stained using an Annexin V-FITC/PI staining kit (Roche Diagnostics, Indianapolis, IN, USA) according to the manufacturer’s instructions. The early stages of apoptosis were assayed using a flow cytometer (BD Biosciences, San Jose, CA, USA).

The migratory function of the HK-2 cells was measured using 24-well-modified Boyden chambers (Corning Life Sciences, Oneonta, NY, USA). Following transfection and treatment as described above, the cells (1×10⁴) were detached, resuspended and seeded onto the filters (8-μm pore size) in the top compartment of the chamber. Following incubation at 37°C for 24 h, the membrane was washed with PBS and fixed with 4% paraformaldehyde for 15 min at room temperature. Non-migrating cells were gently removed from the upper side of the Transwell, and the membrane of the Transwell filter was then stained using hexamethyl pararosaniline solution for a further 15 min, and the upper surface of the filters was carefully wiped with a cotton-tipped applicator. For quantification, the migrated cells were counted in 6 random microscopic fields (×40) in a blinded manner.

Total RNA was extracted using the RNase mini kit (Invitrogen Life Technologies) and was reverse-transcribed. The primer sequences were as follows: tumor necrosis factor-α (TNF-α) forward, 5′-TGCTT GTTCCTCAGCCTCTT; and reverse, 5′-GGTTTGCTACAA CATGGGCT; monocyte chemoattractant protein-1 (MCP-1) forward, 5′-CCCCAGTCACCTGCTGTTAT; and reverse, 5′-AGATCTCCTTGGCCACAATG; regulated upon activation normal T cell expressed and secreted (RANTES) forward, 5′-GAAGGAAGTCAGCATGCCTC; and reverse, 5′-AGCC GATTTTTCATGTTTGC; CD36 forward, 5′-GAGAGCCT GTGCCTCATTTC; and reverse, 5′-GACTGGCTCCAGAGT CTTGC; 18S forward, 5′-CGCACGGCCGGTACAGTGAA; and reverse, 5′-GGGAGAGGAGCGAGCGACCA. Real-time PCR was performed using SYBR-Green PCR Master mix (Toyobo Co., Ltd., Osaka, Japan) and Rotor-Gene-3000. A real-time PCR system (Corbett Research Pty, Ltd., Sydney, Australia) was used according to the manufacturer’s instructions. In brief, the PCR amplification reaction mixture (25 μl) contained 1 μl cDNA, 0.4 mM sense and antisense primer and 12.5 μl SYBR-Green I. After the initial denaturation at 95°C for 10 min, the reaction was cycled 35 times. Each cycle consisted of denaturation at 95°C for 30 sec, primer annealing at 60°C for 30 sec and primer extension at 72°C for 30 sec. The results are presented as the relative expression of TNF-α, MCP-1, RANTES and CD36 normalized to the expression of 18S.

The protein levels of TNF-α, MCP-1 and RANTES under the different experimental conditions were determined in the cell supernatants using commercial ELISA kits (R&D Systems, Inc., Minneapolis, MN, USA) according to the manufacturer’s instructions, and each protein level was normalized with the cell numbers, as previously described (17).

The HK-2 cells were washed twice with cold PBS and lysed in protein extract buffer (1 ml protein extract buffer with 10 μl mixture of protease inhibitors and 10 μl 100-mM PMSF) at 4°C for 30 min. The lysates were centrifuged at 14,000 × g and 4°C for 10 min. The supernatant was collected and the concentration of total soluble protein was quantified using the BCA protein assay kit (Pierce Biotechnology, Inc., Rockford, IL, USA). The extracts were employed for immunoblot analysis. Cellular proteins were electrophoresed through a 10% SDS-PAGE gel before transferring to PVDF membranes. After blocking for 1 h at room temperature in blocking buffer (1% gelatin in PBS with 0.05% Tween-20), the membranes were incubated for 16 h with monoclonal mouse anti-human β-actin antibody (1:1,000; Santa Cruz Biotechnology); monoclonal mouse anti-human CD36 antibody (1:1,000; Abcam, Cambridge, UK), monoclonal mouse anti-human poly(ADP-ribose) polymerase (PARP) antibody, polyclonal rabbit anti-human Src antibody, polyclonal rabbit anti-human phosphorylated (phospho)-Src (tyr416) antibody, polyclonal rabbit anti-human phospho-Src (tyr527) antibody, monoclonal mouse anti-human extracellular regulated kinase (ERK)1/2 antibody, monoclonal mouse anti-human phospho-ERK1/2 antibody, monoclonal mouse anti-human p38 antibody, monoclonal mouse anti-human phospho-p38 antibody, monoclonal mouse anti-human c-Jun N-terminal kinase (JNK) antibody, monoclonal mouse anti-human phospho-JNK antibody, monoclonal mouse anti-human p65 antibody and monoclonal mouse anti-human phospho-p65 antibody (1:1,000; Cell Signaling Technology, Danvers, MA, USA) in PBS Tween-20. The membranes were washed and incubated for 1 h at room temperature with a secondary antibody for 1 h at room temperature and then visualized by enhanced chemiluminescence detection reagents. Relative intensities of the protein bands were analyzed using ImageJ software.

The results are expressed as the means ± standard deviation (SD) from at least 3 experiments. Significance was determined by analysis of variance (ANOVA) and a t-test using StatView 4.0. Differences with P-values <0.05 were considered statistically significant.

In order to elucidate the role of CD36 in the impairment of cellular function induced by oxidized HDL, we transfected the cells with CD36 siRNA. As shown in Fig. 1, 100 nM CD36 siRNA effectively suppressed CD36 mRNA (approximately 80% inhibition) and protein expression (approximately 90% inhibition) in the HK-2 cells. Hence, CD36 siRNA was applied in the following experiments.

The HK-2 cells were co-incubated with various concentrations of oxidized HDL (0, 10, 20 and 50 μg/ml), HDL (50 μg/ml) or oxidized HDL (50 μg/ml) plus CD36 siRNA (100 nM). Oxidized HDL has been shown to induce oxidative stress in human umbilical vein endothelial cells by promoting the generation of intracellular ROS (18). As shown in Fig. 2, oxidized HDL increased the generation of ROS in the HK-2 cells in a concentration-dependent manner and this effect was markedly attenuated by CD36 siRNA, while native HDL had no effect on ROS production.

Real-time reverse transcription PCR revealed that the mRNA expression of TNF-α, MCP-1 and RANTES markedly increased in a concentration-dependent manner following exposure to oxidized HDL for 24 h, and this effect was markedly inhibited by treatment with CD36 siRNA, while native HDL had no effect on the expression levels of these inflammatory factors, apart from TNF-α (Fig. 3A–C). ELISA revealed that the TNF-α, MCP-1 and RANTES protein levels in the conditioned culture medium were altered in a pattern similar to that of the mRNA expression (Fig. 3D–F).

Early apoptosis of the HK-2 cells was detected using an Annexin V-FITC kit. To exclude dead cells, only the Annexin V-positive and PI-negative cells were counted. As shown in Fig. 4A–E, oxidized HDL markedly promoted the early apoptosis of the HK-2 cells compared with the control group. This effect was markedly inhibited by treatment with CD36 siRNA; native HDL also attenuated the early apoptosis of the cells compared with the control group.

In order to further confirm that the cell death induced by oxidized HDL was primarily caused by apoptosis, the cleavage of the caspase substrate, PARP, which is considered a biochemical hallmark of apoptosis, was investigated by western blot anaysis. The results revealed that the expression of cleaved PARP increased as the concentration of oxidized HDL increased from 10 to 50 μg/ml, and this increment was reduced following the transfection of the cells with CD36 siRNA (Fig. 4F and G).

The migration of the HK-2 cells was inhibited by oxidized HDL in a dose-dependent manner, and this effect was markedly attenuated by transfection with CD36 siRNA. There was no significant difference between the control group and the native HDL-treated group (Fig. 5).

The Src family of protein tyrosine kinases is important in the regulation of the growth and differentiation of eukaryotic cells (19). Src activity can be regulated by tyrosine phosphorylation at 2 sites, with opposing effects. The phosphorylation of tyr416 in the activation loop of the kinase domain upregulates enzyme activity, while the phosphorylation of tyr527 in the carboxy-terminal tail triggers enzyme inactivation (20). Our results revealed that the phosphorylation of the Src-family kinase tyr527 was downregulated by oxidized HDL in a dose-dependent manner, and this effect was eliminated by treatment with CD36 siRNA, while the incubation of the HK-2 cells with oxidized HDL did not affect the expression of phospho-Src (tyr416) (Fig. 6).

MAPK mainly consists of 3 different pathways (p38/MAPK, ERK/MAPK and JNK/MAPK) linking growth, differentiation, proliferation and apoptotic signals with transcription in the nucleus (21). In the present study, the expression of MAPK family proteins was detected by western blot analysis in order to determine whether it was affected by oxidized HDL. The incubation of HK-2 cells with oxidized HDL increased the phosphorylation of p38, JNK and ERK in a dose-dependent manner. Pre-treatment with CD36 siRNA partially attenuated the upregulation of phosphorylated p38, JNK and ERK by oxidized HDL (Fig. 7).

The activation of the transcription factor, NF-κB, is considered to be a vital signaling factor for apoptosis, ROS generation, and in particular, inflammatory responses in HK-2 cells (22). The phosphorylation of the p65 unit of NF-κB has been reported to initiate its activation and plays an important role in regulating the specificity of NF-κB-dependent gene expression (23). In this study, to investigate whether NF-κB activation is involved in the effects of oxidized HDL, the HK-2 cells were pre-incubated with various concentrations of oxidized HDL or HDL, as already mentioned in the previous sections. The results of western blot analysis indicated that oxidized HDL induced the phosphorylation of p65 and this effect was attenuated by CD36 siRNA (Fig. 8).