Chemicals

l-Ascorbic acid, β-glycerophosphate, probenecid, probucol, N-acetylcysteine (NAC), and IS were all obtained from Sigma (St. Louis, MO, USA). All cell culture media and supplements were from Hyclone (Logan, UT, USA). Reagents for reverse transcription and those for real-time PCR reactions were from Toyobo (Osaka, Japan). Anti-Bax, Anti-Bcl-2, and anti-p53 mouse monoclonal antibodies were purchased from Santa Cruz (Santa Cruz, CA, USA). Secondary goat anti-rabbit IgG was obtained from Thermo Fisher Scientific (Rockford, USA). The assay kit for caspase-3/7 activity was purchased from Promega (Mannheim, Germany).

Cells and osteogenic induction

Newborn mouse calvaria-derived MC3T3-E1 subclone 14 pre-osteoblastic cells (ATCC, USA) were cultured in α-MEM medium (Hyclone) supplemented with 10% fetal bovine serum (Hyclone), 100 U/mL penicillin, and 100 mg/mL streptomycin (Hyclone) at 37°C in an atmosphere with 100% humidity and 5% CO₂. Osteoblast differentiation was induced by the addition of 10 mM β-glycerophosphate, as described previously [11].

Cell viability

Cell viability was assessed using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, as described previously [12]. MC3T3-E1 cells were incubated in osteogenic induction medium with or without IS at 37°C for 72 h. After the cells were lysed with DMSO solution, the optical density was measured at 590 nm using the optical density at 630 nm as reference (VICTOR™X3; PerkinElmer, USA).

Bone differentiation

Alkaline phosphatase (ALP) activity was measured in cells treated with 0–1.5 mM IS and in control cells incubated for 3, 5, 7, and 10 d. Cells were washed with PBS and were lysed with a solution containing 0.1% Triton X-100 at the same time as the cellular alkaline phosphatase activity and cell protein content were determined. The enzymatic reaction was started by the addition of 50 μL of substrate/buffer mixture (equal volumes of p-nitrophenol phosphate substrate [N 1891; Sigma Chemicals, St. Louis, MO] and alkaline buffer solution [A9226; Sigma Chemicals]). After 30 min of incubation at 37°C, the reaction was stopped by adding an equal volume of 0.05 M NaOH. The lysate from the wells was collected into individual Eppendorf tubes and vortexed. The ALP activity was determined colorimetrically at 405 nm using p-nitrophenol (PNP) standards (0–50 nmol, N7660; Sigma Chemicals). The protein concentration in the lysate was determined using the Bradford assay. ALP activity is expressed as nanomoles of PNP released per milligram of protein.

Assessment of cellular oxidative stress

Production of intracellular reactive oxygen species was detected using the nonfluorescent cell-permeating compound, 2′-7′-dichlorofluorescein diacetate (DCF-DA). DCF-DA is hydrolyzed by intracellular esterases, and is then oxidized by reactive oxygen species (ROS) to a fluorescent compound, 2′-7′-dichlorofluorescein (DCF). After treatment with 0–1.5 mM IS, MC3T3-E1 cells were treated with DCF-DA (10 μM) for 30 min at 37°C. Following DCF-DA exposure, the cells were rinsed and then scraped into PBS with 0.2% Triton X-100. Fluorescence was measured with a plate reader (VICTOR™X3) with excitation at 485 nm and emission at 535 nm.

Flow cytometry analysis of apoptosis

Quantification of cells undergoing programmed cell death was conducted using an annexin V-propidium iodide apoptosis kit (Invitrogen). Analyzed cells were washed once in phosphate-buffered saline and resuspended in the binding buffer provided. Annexin V (Alexa 488-conjugated) and propidium iodide were added and incubated for 15 min at room temperature in the dark. The cells were analyzed using a FACS Calibur flow cytometer and CellQuest software.

Apoptosis measurement: caspase-3/7 activity, and immunoblot assay for apoptosis-related factors p53, Bcl-2, and Bax

Caspase-3/7 activity was detected using a Caspase-Glo 3/7 Assay system (Promega) after preincubating the MC3T3-E1 cells (2 × 10⁵/96-well plate), followed by treatment with various IS concentrations (control, 0.5 mM, 1 mM) for 3, 6, 9, 12, and 24 h. The background luminescence associated with the cell culture and assay reagent (blank reaction) was subtracted from the experimental values. The activity of caspase-3/7 is presented as the mean value of triplets for the given cells. The intensity of the emitted fluorescence was determined at a wavelength of 521 nm with the use of luminometry (VICTOR™X3).

The immunoblot assay was conducted as follows. After stimulation, cells were washed once with phosphate-buffered saline and lysed with radioimmunoprecipitation assay (RIPA) lysis buffer (ROCKLAND, USA) and placed on ice for 30 min. Total cell extracts were centrifuged at 14 000 g (for 20 min at 4°C), and protein-containing supernatants were collected. Equal amounts of proteins (40 μg) were resolved by sodium dodecyl sulfate–polyacrylamide gel electrophoresis, transferred to a nitrocellulose membrane, and immunoblotted with specific antibodies against Bax, Bcl-2, and p53. Secondary antibodies were obtained from Thermo Fisher Scientific. Equal loading was confirmed using a β-actin antibody. Protein expression levels were quantified using a densitometer (ChemiDoc™ XPS + with Image Lab™ Software, Bio-Rad). The data are represented as the ratio of expression of the target protein to that of β-actin.

RNA isolation, cDNA synthesis, and PCR analysis

Total RNA was isolated using an RNeasy Mini Kit (QIAGEN, Tokyo, Japan) according to the manufacturer’s instructions. Total RNA (1 μg) was used as the template for cDNA synthesis in a 50-μL reaction mixture using a reverse transcriptase-PCR kit (TOYOBO) according to the manufacturer’s instructions. Real-time PCR was performed on a CFX96™ (BIO-RAD). The PCR reactions consisted of Power SYBR Green PCR Master Mix (Applied Biosystems, UK), 0.1 mM (10 pM) specific primers, and 50 ng of cDNA. The primer sequences, designed with Beacon Designer 7.6 software (Bio-Rad), were as follows: mouse osteonectin, 5′-TCTCAACAAACAAATCAGGGAT-3′ and 5′-TGGCAGCACATTCATCTATG-3′; collagen 1, 5′-ATCACCAAACTCAGAAGATGTAG-3′ and 5′-CAGGAAGTCCAGGCTGTC-3′; organic anion transport 1 (OAT1), 5-ATG CCT ATC CAC ACC CGT GC-3 and 5-GGC AAA GCT AGT GGC AAA CC-3); OAT3, 5-CAG TCT TCA TGG CAG GTA TAC TGG-3 and 5-CTG TAG CCA GCG CCA CTG AG-3; and GAPDH, 5′-CAAGAAGGTGGTGAAGCA-3′ and 5′-TGTTGAAGTCGCAGGAGA-3′.

Statistical analysis

All results are expressed as the mean ± standard error of the mean (SEM) values. The mean values of the groups were compared by analysis of variance, and a P-value <0.05 was considered significant.

Effect of indoxyl sulfate on cell viability in the MC3T3-E1 cell line

To determine cytotoxicity, the effect of IS on the cell proliferation of MC3E3-T1 was studied using an MTT assay. As shown in Figure 1, IS, at the concentration range of 0.1–1.5 mM, inhibited cell proliferation at 72 h.

Gene expression of OATs in the MC3T3-E1 cell line

Because other studies have shown that IS is transported into osteoblasts and renal tubule cells via OAT, gene expression of OAT-1 and OAT-3 was investigated by real time PCR using RNA extracts. The expression of OAT-3 was relatively higher than that of OAT-1 in the MC3T3-E1 cell line (Figure 2A). After confirming the expression of the OAT gene in the MC3T3-E1 cell line, we investigated whether blocking OAT could prevent IS toxicity. To confirm the role of OAT in MC3T3-E1 cells, probenecid, a transporter inhibitor, was added to the cells during pretreatment with 1 mmol/L of IS. Blocking OAT in MC3T3-E1 cells improved cell survival (Figure 2B). ROS production was inhibited by probenecid. The effect obtained was similar to that obtained on pretreatment with the antioxidants NAC (500 μM) and probucol (62.5 μM) (Figure 2C). Probenecid works by interfering with the OAT in the kidneys, which blocks the efflux of IS in cells. Probucol is a phenolic lipid-lowering agent with antioxidant and anti-inflammatory properties. N-acetylcysteine (NAC) is the precursor of l-cysteine and therefore of reduced glutathione and has been widely used as an antioxidant.

Intracellular oxidative stress

As shown in Figure 3, IS increased cellular oxidative stress in a concentration-dependent manner. Addition of antioxidants or the OAT inhibitor suppressed free radical production (Figure 2C, Figure 3).

Inhibition of osteoblast differentiation by IS

To determine the differentiation of the pre-osteoblast cell line, ALP activity was measured in osteogenic induction medium with or without IS. As shown in Figure 4, ALP activity was suppressed above 1 mM IS. Collagen 1 and osteonectin were produced only in differentiated osteoblasts. To determine whether the osteoblasts had differentiated, the expression of collagen 1 and osteonectin mRNA was analyzed using real-time PCR. At 5 d, the production of collagen 1 and osteonectin mRNA was significantly inhibited by the addition of IS, as shown in Figure 5.

Apoptosis induction by IS

To determine whether IS induces apoptosis, the cells were incubated with different concentrations of IS for 12 h, stained, and subjected to Fluorescence-activated cell sorting (FACS) analysis to measure apoptosis and necrosis. IS increased the proportion of apoptotic cells, particularly in osteoblasts (Figure 6). To elucidate the role of caspases in osteoblasts, we first examined the activity of the executioner caspase-3/7 in response to IS in osteoblasts. The activity of caspases was determined using fluorometric peptide substrates specific to caspase. As shown in Figure 7, the activity of caspase-3/7 peaked at 6 h of incubation with IS (1.0 mM). Caspase-dependent apoptosis thus appears to be involved in IS-induced osteoblast toxicity. To determine which apoptosis-related factors may be acting upstream of caspase activation, the expression of p53, Bcl2, and Bax was measured after the addition of IS (1.0 mM). IS increased the expression of Bax and p53, which play a role in apoptosis. However, Bcl-2 was not influenced by IS (1.0 mM) at 1, 3, and 6 h (Figure 8).