Patients and specimens
A total of 8 patients with stage 5 chronic kidney disease in the department of nephrology and 8 patients who underwent postoperative renal transplantation re-examination in the department of the Center of Organ Transplantation during the same period were selected at the Second Xiangya Hospital, Central South University. Meanwhile, 8 healthy age paired adult volunteers in the physical examination centre were recruited in this study. They were divided into three groups: ESRD, RTR, and normal healthy controls (Nor). The inclusion criteria were as follows: chronic kidney disease patients with the glomerular filtration rate (GFR) less than 15 ml/min/1.73 m2 of body-surface areas were selected as the ESRD group. Patients with renal transplantation due to ESRD but whose renal function was normal were enrolled into the RTR group. The healthy volunteers with normal renal function and without other diseases such as a malignant tumour, hypertension, diabetes, coronary heart disease, and so on were enrolled into the Normal group. The clinical parameters of all the participants in this study are presented in Table 1.
Coronary artery calcification measurement
Coronary artery calcification scores (CACS) were calculated using the Siemens Somatom Definition computed tomographic multi-layer spiral scanner (Germany) and the calcification of coronary arteries was quantified via Agaston and analysed by Siemens CaScoring software (syngo. via, Siemens Healthcare GmbH). The width of the detector was 128×0.6 mm and the scan thickness was 1.5 mm simultaneously over 50-70 images of fault scans. A total coronary artery calcification score was generated by using the Agatston method, which has been described before.[30] The vessels evaluated included the left main coronary artery (LM), left anterior descending coronary artery (LAD), left circumflex (CX), and the right coronary artery (RCA). The measure of the area of calcification lesions times a fixed coefficient (the maximum pixel density decision) and the total score of the calcification of all faults was termed the CACS for the patient.
Exosome extraction
Blood was obtained from patients with ESRD, patients with RTR, and the Normal group, respectively. The blood was centrifuged at 3000×g for 20 min and the supernatant plasma was subjected to the ExoQuick-TC Exosome Precipitation Solution Kit (System Biosciences, USA) for exosomes extraction. Briefly, 250 ul of plasma was added to 63ul ExoQuick Exosomes Precipitation Reagent and fully mixed. After the mixture had been incubated at 4 ℃ for 30 min, it was centrifuged at 1500× g for 30 min at room temperature, and the yellow or white precipitate in the bottom of the EP tube was the exosomes. Then, the supernatant was removed by aspiration and the exosome pellets were centrifuged again at 1500× g for 5 min. Finally, the exosome pellets were resuspended in 200 μL PBS. The protein content of exosomes was determined by the BCA kit (Beyotime Biotechnology, Shanghai, China).
Identification of exosomes
The morphology of plasma exosomes was detected by a transmission electron microscope. Briefly, exosomes were fixed with equal volumes of 1% phosphotungstic acid (pH 7.4). A 10μL sample was loaded onto a bronze net with film after washing and holding at room temperature (RT) for 10 min. Then, 10μL of phosphotungstic acid staining solution was added to negatively stain, and it was observed under a Hitachi H-7650 transmission electron microscope (Hitachi, Tokyo, Japan). A particle and molecular size analyser (Zetasizer Nano ZS; Malvern Instruments) was used to measure the size distribution of the exosomes. The expression of exosomal surface markers, including CD9 (ab92726, abcam, USA), CD63 (ab68418, abcam, USA), and CD81 (ab79559, abcam, USA), were analysed by Western Blot analysis.
VSMCs uptake exosomes
The VSMCs were isolated from 6–8 week-old male C57/BL mice as described before [31]. VSMCs were cultured in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% FBS (Gibco BRL Co. USA), penicillin (100 U/mL), and streptomycin (100 μg/mL) at 37 °C in a humidified atmosphere of 5% CO2. VSMCs were treated with 50μg of ESRD-Ex, RTR-Ex, and Nor-Exo, and the calcification levels were evaluated. Exosomes were labelled with PKH26 (MINI26, Sigma, USA) according to the manufacturer’s instructions. Briefly, 4µl of PKH26 fluorescent solution was dissolved in 1ml of diluting solution C, and then the mixed solution was added to 40 μl exosomes. After stopping the reaction by using 5% BSA, the mixture was ultracentrifugated at 100,000 × g for 70 minutes and the labelled exosomes were incubated with VSMCs at 37 °C for 12h. After fixing the cells with 4% paraformaldehyde for 30 minutes at RT and washing the cells 3 times with PBS, DAPI (Invitrogen, Carlsbad, USA) was added for 5 minutes. After washing with PBS 3 times, the staining signals were analysed with a fluorescence microscope (DMI6000B, Leica, Germany).
Alizarin Red S staining
VSMCs were co-incubated with β-glycerophosphate (β-GP) and ESRD-Ex, RTR-Ex or Nor-Ex, respectively, for 21 days, and the Alizarin Red S staining was done as before [31]. The cells were fixed and then stained with 1% (pH 4.2) Alizarin Red S for about 10 minutes. The mineralized nodules were assessed and photographed with a microscope. To quantify calcium levels, the cells were washed with PBS and decalcified with 0.6 N HCl for 24 h. Calcium content was determined by measuring the concentration of calcium in the HCl supernatant by atomic absorption spectroscopy. Following decalcification, the cells were washed three times with PBS and solubilized with 0.1 N NaOH/0.1% SDS. The protein content was measured with a BCA protein assay (Beyotime Biotechnology, Shanghai, China). The calcium content of the cell layer was normalized to the protein content.
Western Blot analysis
The expression of proteins was determined by Western Blot as previously described [3]. Briefly, the concentration of protein was detected using a BCA kit (Beyotime, Shanghai, China). 30μg of total protein was loaded onto 8% or 12% SDS-PAGE gel and transferred to PVDF membranes. The membranes were incubated with primary antibody at 4 °C for over 12h after blocking with 5% non-fat milk for 1h. Subsequently, HRP-conjugated goat–anti-rabbit (sc-2004, 1:5000, Santa Cruz) or HRP-conjugated goat–anti-mouse (sc-2005, 1:5000, Santa Cruz) secondary antibodies were used to incubate with the membrane at RT for 1h. The immunoreactive bands were visualized using the ECL Plus Western Blot Detection Kit (Amersham Biosciences U.K. Ltd). The relative expression levels of proteins were normalized to the intensity of the β-actin band. Primary antibodies including CD9 (ab92726, 1:1,000, Abcam), CD63 (ab68418, 1:1,000, Abcam), CD81 (ab79559, 1:1,000, Abcam), Runx2 (ab76956; 1:1,000, Abcam), Fetuin-A (16571-1-AP, 1:1000, Proteintech), MGP (10734-1-AP, 1:1000, Proteintech), Annexin-A2 (11256-1-AP, 1:1000, Proteintech), BMP-2 (ab214821, 1:1000, Abcam), Rankl (23408-1-AP, 1:500, Proteintech), and β-actin (ab6276, 1:3000, Abcam).
ELISA analysis
The contents of Fetuin-A (ab108855), MGP (CSB-E09714h), Annexin-A2 (CSB-E12156h), BMP-2 (CSB-E04507h), and Rankl (ab213841) were detected using an ELISA kit according to the manufacturer’s instructions. Briefly, 0.1 ml sample or standard were added into each hole of the reaction plate and incubated at 37 °C for 2h. After discarding the liquid, 0.1 ml of biotin labelled antibody working liquid was added and put on the new plate paste, continuing to incubate at 37 ℃ for another 30 min. Then the liquid was discarded again and washed 3 times. 100 ul working liquid was added, and it was covered with the new post and incubated at 37℃ for 1h. The liquid was dropped and washed 5 times. Finally, 90 ul of substrate solution was added to each hole and coloration was done at 37 ℃ for 15-30 min away from light. Then the OD value (Optical Density) of each hole was measured with a microplate at the wavelength of 450 nm within 5 min after termination of the reaction.
Statistical analysis
The data are presented as means ± standard deviation (SD), and they were analysed with GraphPad Prism software (GraphPad Prism version 6.0). The Student’s t-test was used to compare normally distributed data between two different groups, while one-way analysis of variance (ANOVA) together with a Tukey's post hoc test was used for multiple groups. A level of p<0.05 was considered statistically significant. All experiments were repeated at least three times, and representative images are shown in the figures.