Preparation of COM crystals
We prepared crystals as described previously. COM crystals formed immediately as we mixed equal volumes of 10 mM CaCl2 and 10 mM sodium oxalate at approximately 26°C and after approximately 3 days at 4°C. Deionized water was used to wash the COM, which was then dried at 62°C. Fourier transform infrared spectroscopy was used to confirm that the crystals were COM. A stock solution was created with 5 mg/ml COM in sterile phosphate-buffered saline (PBS). The COM was evenly distributed in a monolayer (67 μg of crystals/cm2 of cells) and settled on the cells under the force of gravity. PBS was added to ensure that the volume was the same for each group.
Human kidney epithelial cells (HK-2 cells) were maintained in culture in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 1% antibiotics and 10% foetal bovine serum (FBS) in a humidified atmosphere of 95% O2/5% CO2 at 37°C.
Protein extraction, two-dimensional gel electrophoresis (2-DE) and staining
After cultivating HK-2 cells with or without COM (67 μg of crystals/cm2 of cells) for 24 h, we harvested the cells into tubes containing 0.5 M EDTA in PBS and incubated at 4°C for 30 min before removing the adherent crystals. Then, the cells were washed with PBS to remove EDTA, resuspended in lysis buffer, and incubated at 4°C for 30 min. The suspensions were then centrifuged to remove insoluble debris and particulate matter.
A total of 200 mg of protein derived from each sample was mixed with a rehydration buffer (8 M urea, 2% CHAPS, 18 mM DTT, 0.5% IPG buffer, trace bromophenol blue) to obtain a final volume of 200 ml per sample. The samples were then rehydrated onto individual Immobiline DryStrips (7 cm long, nonlinear pH gradient of 3–10; GE Healthcare, Uppsala, Sweden) for 16 h at room temperature. Isoelectric focusing (IEF) was performed at 500 V for 1 h, 1,000 V for 1 h, and 8,000 V for 10 h to reach a total of approximately 60–80 kVh. After IEF, the strips were placed in an equilibration buffer (130 mM DTT, 112 mM Tris-base, 6 M urea, 30% glycerol, 4% SDS, and 0.002% bromophenol blue) for 20 min and then incubated with a similar buffer (in which 130 mM DTT was replaced with 135 mM iodoacetamide) for 20 min. The proteins on the equilibrated strips were further separated on 12.5% polyacrylamide gels at 120 V for approximately 1.5 h. The proteins were detected by a modified silver-staining method compatible with MS analysis.
Image analysis and protein identification by ESI-MS/MS
We analysed differences in protein levels among samples with Image Master software. Following spot detection, a matched set including all three batches of gels was built. A reference gel was selected from the control gels, and unmatched spots were added to the reference gel. Normalization was based on the total spot density.
We excised differentially expressed protein spots from the preparative gels, destained them and digested them overnight. For protein identification, a Finnigan LTQ mass spectrometer coupled with a Surveyor HPLC system (ThermoQuest) was used to analyse the extracted peptides. Briefly, we separated the protein digests with Microcore RP columns (C18 0.15 mm*150 mm; ThermoHypersil, San Jose, CA, USA). Solvent A was 0.1% v/v formic acid, and solvent B was 0.1% v/v formic acid in 100% v/v acetonitrile (can). The gradient was held at 2% solvent B for 20 min and increased linearly to 98% solvent B in 1.5 h. The peptides were eluted from C18 microcapillary columns (120 μl per min) and electrosprayed directly into an LCQ-Deca mass spectrometer (with a spray voltage of 3.0 kV and a capillary temperature of 180°C). The full scan ranged from M/Z 400 to 2,000. Protein identification using the MS/MS raw data was performed with SEQUEST software based on the Swiss-Prot database. Both y ions and b ions were included in the database search. The results of protein identification were filtered by the DelCn (≥0.1) and Xcorr (1+≥1.9, 2+≥2.2, 3+≥3.75) values. The NCBI and ExPASy protein databases were used to determine the main functions of all the identified proteins.
Annexin V binding analysis
A FITC-labelled annexin V staining assay was used to measure the PS exposure of HK-2 cells. Cells were seeded in 6-well plates, grown to approximately 80% confluence (67 μg of crystals/cm2 of cells) and treated with COM crystals with or without the CaSR activator GdCl3 (300 μM) for 30 min or the CaSR inhibitor NPS2390 (10 μM) for 60 min. After the reaction, the cells were harvested and labelled with annexin V-FITC in Annexin V binding buffer (BD Pharmingen™). We used a FACSAria™ flow cytometer with an excitation wavelength of 488 nm and an emission wavelength of 530 nm to evaluate the annexin V fluorescence of the cells after 15 min of incubation in the dark. The percentage of annexin V-positive cells was used to measure PS exposure.
Measurement of MDA, LDH, and SOD levels
The levels of MDA, SOD, and LDH were measured using a commercial kit according to the manufacturer’s instructions.
HK-2 cell labelling with nitrobenzoxadiazole (NBD)-labelled PS (NBD-PS)
PS transmembrane bidirectional movement was observed with NBD-PS (Avanti® Polar Lipids), a fluorescent analogue of phosphatidylserine. NBD-PS dissolved in chloroform was dried under nitrogen and solubilized in absolute ethanol (6 nmol for 2× 107 cells). We labelled 2 groups of cells. In one group, called group A, we inserted NBD-PS into the outer plasma membrane[18, 19]; in the other group, group B, we inserted NBD-PS into the inner plasma membrane. We prevented internal movement in group A by cooling the cells to 2°C. Then, an ethanolic solution of NBD-PS was added to a suitable volume of cell suspension (approximately 2 × 107 cells per ml) in mPBS, and the mixture was vortexed. Non-inserted NBD-PS was removed by washing the cells with ice-cold mPBS after incubation on ice for 15 min. The group B cell suspensions were incubated with NBD-PS in mPBS at 37°C for 1 h with 5 mM diisopropyl fluorophosphate (Sigma-Aldrich) to prevent NBD-PS degradation. Redistribution of NBD-PS to the cytoplasmic membrane leaflet was mediated by APLT, which mediates the localization of PS and phosphatidylethanolamine to the inner leaflet of the plasma membrane by rapidly transporting these substances from the outer to the inner leaflet. Any NBD-PS remaining in the outer leaflet was removed by washing the cells 3 times with mPBS containing BSA (1% w/v).
Fluorescence quenching and PS translocation analysis
McIntyre and Sleight used dithionite to quench the fluorescence of superficial NBD-PS. To use a similar approach, we first incubated cells with NBD-PS-labelled outer leaflets in mPBS with 5 mM diisopropyl fluorophosphate to evaluate PS inward movement. NBD-PS inward transmembrane movement was initiated by incubating the cells with prewarmed mPBS containing COM crystals (67 μg of crystals/cm2 of cells) and the CaSR activator GdCl3 (300 µM, 30 min) or the CaSR inhibitor NPS2390 (10 µM, 1 h) at 37°C. Every 10 min, we removed aliquots of the cell suspension and then measured the fluorescence intensity with an RF-5000 spectrofluorophotometer (Shimadzu, Kyoto, Japan) at a wavelength of 530 nm with a 10 nm slit width and an excitation wavelength of 470 nm with a 5 nm slit width. The average fluorescence (FT) during the first 50 seconds was recorded. Dithionite was then added from a stock solution of 1 M sodium dithionite freshly dissolved in 1 M Tris (pH 10) to a final concentration of 25 mM to quench NBD fluorescence on the cell surface. The average fluorescence intensity decrease (FD) over a period of 240 seconds was recorded. The cell membrane was permeabilized with 1% (w/v) Triton X-100, the NBD fluorescence of the inner leaflet of the membrane was quenched with dithionite, and the fluorescence intensity was recorded for 50 seconds (F0). The inner leaflet percentage was calculated with the following formula: inner leaflet percentage=100 [(FD−F0)/(FT−F0)]. Cells with NBD-PS-labelled inner leaflets were incubated in mPBS containing 1% BSA with COM crystals (67 μg of crystals/cm2 of cells) or with COM and GdCl3 or NPS2390 to evaluate PS outward movement. Every 10 min, we removed aliquots of the cell suspension and calculated the outer leaflet percentage with the following equation: outer leaflet percentage=100 [(FT−FD)/(FT−F0)].
Western blot analysis
We prepared total proteins according to the kit manufacturer’s instructions. A Bradford protein assay was performed to determine the supernatant protein concentration with BSA as the reference standard. We blotted all of the proteins (20 μg) onto a nitrocellulose membrane in transfer buffer at 100 V for 1 h in a water-cooled apparatus after electrophoresis in Tris–glycine electrophoresis buffer with standard 10% SDS-PAGE. The membrane was blocked in TBS-T buffer with 5% skimmed milk at 37°C for 1 h and then incubated overnight at 4°C with anti-CaSR antibodies (1:2,500), APLT antibodies (1:1,500), and antibodies against SOD, NOX, Bcl-2, Bax, pro-caspase-9, caspase-9, pro-caspase-8, cleaved-caspase-8, cleaved-caspase-3 and cytochrome C. TBS-T was used to wash the membrane 3 times. The membrane was then incubated with alkaline phosphatase-conjugated anti-IgG antibodies diluted 1:1,000 in TBS-T for 1 h at room temperature. Western Blue Stabilized Substrate for Alkaline Phosphatase was used to detect antibody–antigen complexes. A Bio-Rad ChemiDocTM EQ densitometer and Bio-Rad Quantity One software were used to evaluate the protein band densities.
Crystal adhesion assay.
One of the methods used before was performed to carry out crystal adhesion experiments. The cells were randomly divided into 4 groups after culture for 24 hours: (1) control group: cells were cultured in DMEM at 37°C; (2) COM group: cells were incubated with crystals (67 µg/cm2 of cells); (3) COM+GdCl3: GdCl3 (300 µM) was added to growth medium for half an hour, and then crystals were added; (4) COM+NPS2390 group: After NPS2390 (10 µM) was added to growth medium for 1 hour, the crystals were also added. After cells were treated with crystals for 6, 12, 24, and 48 hours, the unbound crystals were removed and collected. They were counted in 5 randomized high-power fields (HPFs) under a phase contrast microscope. This experiment was carried out in triplicate.
Quantitative data are presented as the mean ± SEM. All statistical analyses were performed using SPSS software version 13.0. ANOVA was used to analyse differences among multiple groups, and t tests were used to compare 2 groups. A 2-sided p<0.05 was considered to indicate statistical significance.