- Patients
A total of 142 CKD patients including 57 men and 85 women aged from 40-76 were recruited from the Nephrology Department at Shanghai General Hospital between May 2018 and July 2019. In this study, 32 healthy patients with normal renal function were selected as the control group. All patients provided with the signed consent forms. And, studies were performed in accordance with the declaration of Helsinki. The patient diagnoses are summarized in Table 1.
- VSMCs culture and treatments
The VSMCs were isolated from the thoracic aorta of male Sprague-Dawley (SD) rats (n=5, 9-10 weeks) using the explants technique and cultured as previously described (16). Briefly, cultured in M199 (Gibco, NY, USA) medium containing 10% fetal bovine serum (FBS) (Gibco) and 2 ng/ml basic FGF and were maintained at 37 °C in the presence of 5% CO2. Complete medium replacement with fresh M199 medium was performed post 72 h. Within 2 weeks, cells emerged from the explant and confluency was achieved by 4 weeks. Smooth muscle identity was confirmed through α-smooth muscle actin (α-SMA) staining and the “hill and valley” growth patterning of the cells. Once the cells had undergone 5-8 passages, experiments were performed.
- Animal model
Mice with a C57BL/6J background (Jackson Laboratory, Sacramento, CA) were used to develop the 5/6 Nx model. Further, the mice were kept at 12 h light and 12 h dark cycle in a (19–21°C) temperature-maintained facility with standard rodent diet and access to drinking water. The 5/6 Nx model (CKD model, n = 18 was developed on 16-week-old mice by decapsulating the left kidney through flank incision and resection of the upper and lower poles. Post 1 week, another surgery, specifically by incision in the right flank the right kidney was removed entirely. Sham group (n = 18) mice underwent similar surgical procedure except for the removal of right kidney. Post 1-week recovery, mice from both sham and CKD group were randomly split into groups with either a normal (0.5%) phosphate diet (n = 9) or high (1.5%) phosphate diet (n = 9) for 12 weeks. Further, after 12 weeks, using computerized tail-cuff system, blood pressure of the mice was measured. Urine was also assessed using ELISA kits (Assay Designs, Ann Arbor, MI) for creatinine and protein levels. Mice were anesthetized with isoflu-rane, exsanguinated successfully, renal and artery tissues were removed for further processing. These tissues were either quickly fixed and processed using Mason’s staining or flash frozen using liquid nitrogen and stored at -80 °C for further analysis.
- Calcium deposition
To assess the deposited calcium, cells were initially decalcified for 24h using HCl (0.6 mol/L). Calcium concentration was measured by measuring the concentration of calcium in the HCL supernatant by atomic absorption spectroscopy and normalized to the protein content of cells measured using the BCA protein assay kit the BCA assay kit (Pierce, Rockford, IL) after solubilization in 0.1 M NaOH/ 0.1% sodium dodecyl sulfate.
- 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay
To determine cell viability, VSMCs were seeded at a density of 4×103 cells/well on a 96 well plate. The cells were allowed to grow for 48 h and were incubated with MTT solution (Sigma Chemical Co., USA) at a concentration of 5 mg/ml at 37 °C. After 4 h of incubation, the MTT solution was removed and 100 µl DMSO was added. To allow complete solubility of the intracellular purple crystals, cells were incubated in an orbital shaker with DMSO for 5 mins. Further at 570 nm, absorbance was measured using a microplate reader (Thermo, Rockford, IL). Further, cell viability percentage was calculated using the formula “OD of treatment group/OD of control group * 100”.
- Alizarin red staining
Calcification was determined using Alizarin red staining as previously described18, 19. Briefly, Alizarin S (100 mg) was dissolved in 10 mL of purified water and adjusted to pH 6.4 using a 0.1% KOH solution. Cells seeded onto 6 well plate were fixed using 70% ethanol and stained with Alizarin red. Further, the accumulated stain was dissolved using ethylpyridium chloride and thus obtained supernatant was measured at 550 nm using a microplate reader.
- Measurement of alkaline phosphatase (ALP) activity
Cells cultured on a 24 well plate were detached after reaching confluency and sonicated in the presence of 600 ul distilled water. Using a modified Lowry method, ALP activity was measured. Briefly, assay mixtures containing 0.1 M 2-amino-2-methyl-1-propanol, 8 mM p-nitrophenyl phosphate disodium, and 1 mM MgCl2, were added onto the cell homogenates. Post 4 mins incubation at 37 °C, 0.1 N NaOH was used to inhibit the reaction. Further at 405 nm, absorbance was read using a microplate reader. Measurements p-nitrophenol was used to prepare the standard curve. Further, normalization of the values was achieved using protein concentrations which were measured from the sonicated samples using BCA method20.
- Apoptosis assay
Apoptotic cell death was assessed by staining the cells with Annexin-V FITC and PI- staining. Based on the manufacturer’s protocols, both attached and free-floating cells were collected and stained with Annexin V-FITC antibody and PI. Further, these cells were quantified using flow cytometry21.
- Cell line and viral transduction
Overexpression of CKAP4 was performed using a lentivirus designed cDNA obtained from OriGene (Rockville, MD). 293A cells were used for production of CKAP4 containing lentivirus. To attain efficient transduction, a multiplicity of infection (MOI) of 100 was used.
- Small interfering RNA (siRNA) or small hairpin RNA (shRNA) transfection
NC-siRNA and CKAP4-siRNA were synthesized chemically at Suzhou GenePharma Co. Ltd. (Suzhou, China). Mouse CKAP4 gene was constructed into pcDNA3.1+HA vector by Life Technologies (Invitrogen, California, USA), and the empty vector served as the negative control. The cells were cultured to attain 70-80% confluency, after which cells were transfected either with, NC-siRNA or CKAP4-siRNA using Lipofectamine 2000 (Invitrogen, California, USA) based on the manufacturer’s instructions.
- Immunocytochemical staining
Firstly, cells were washed twice with PBS for 10 mins. Subsequently, they were fixed with 4% paraformaldehyde for 10 mins and permeabilized using 0.5% Triton-X in 0.1 M Tris-buffered saline (TBS; pH 7.6). Further, the cells were quickly washed with PBS. The cells were blocked 3% goat serum in TBS. Then, the cells were incubated with YAP (ab52771, Abcam) and MMP-2 (ab97779, Abcam) primary antibody diluted in TBS with normal goat serum (10%) for 1 h. Subsequently, the cells were incubated with the Alexa Red conjugated anti-mouse IgG (Molecular Probes) secondary antibody in TBS for 1 h. Nuclear staining was performed using Hoechst 33258. Subsequent imaging of the cells was performed usingLaser Scanningconfocal fluorescence microscope (Olympus FV1000S).
- Immunohistochemical analysis
Paraffin embedded tissue sections (1-2 µM) were used to perform immunohistochemical analysis. The sections were mounted onto SUPERFROST slides and incubated at 37 °C overnight. De-waxing in xylene and ethanol was followed by antigen-retrieval by boiling in microwave oven three times for 5 mins (pH 6.0, 0.94 ml Antigen Unmasking Solution (Vector Laboratories, Burlingame, CA, USA)/100 ml distilled water). Further, the slides were immersed in 3% hydrogen peroxide solution for 20 mins. Blocking was performed with 10% goat serum in PBS for 30 mins. Slides were incubated with primary antibodies against CKAP4 (ab84712, Abcam) overnight at 4°C which was subsequently followed by incubation with secondary antibody conjugated with hydrogen peroxidase for 30 mins. Visualization was achieved with the aid of 3-Amino-9-Ethylcarbazole (AEC) substrate chromogen (ADI-950-200-0003, WAKO) and utilized based on the manufacturer’s instructions. The sections were stained with hematoxylin and dehydrated through the treatment with gradient ethanol. Finally, the sections were mounted and visualized.
- Biochemical parameters
Serum creatinine and urea, urinary calcium and phosphorus concentrations were determined using an automatic analyzer system (Ilab 1800, Lexington, MA, USA). Serum FGF-23 (Immunotopics, San Clemente, CA, USA) and DKK1 levels (Enzo Life Sciences, Farmingdale, NY, USA) were determined by using commercial ELISA kits according to the manufacturer’s protocol.
- Western blot analysis
Cells were cultured in 6 well dishes for 48 h and the protein was extracted RIPA buffer containing 0.1% SDS . The extracted protein was quantified using BCA assay kit. 20 μg of protein denatured using Lamelli buffer were loaded and migrated using a 10-15% SDS-PAGE gel, transferred onto a nitrocellulose membrane. Blocking of the membranes were performed using 5% skim milk in PBST (PBS with 0.1% Tween-20) for 1 h. Further, the membranes were incubated with the same blocking buffer containing either of the primary antibodies; anti-CKAP4 (Abcam, ab84712, 1:1000), anti-YAP (Abcam, ab52771, 1:2000), anti-α-SMA (Abcam, ab32575, 1:1000), anti-RUNX2 (Abcam, ab236639, 1:1000), anti-Pit1 (Abcam, ab273048, 1:2000), anti-GAPDH (Abcam, ab9485, 1:2500), anti-MMP2 (Abcam, ab92536, 1:1000), and anti-SM22α (bioss, bs-2163R, 1:1000). The blots were incubated at 4°C overnight. Further, the blots were washed with PBST thrice and 5 mins each. The membranes were subsequently incubated with secondary antibody conjugated with horse-radish peroxidase for 1 h at RT. The membranes were visualized using chemiluminescent reagent in accordance with the manufacturer’s instructions. The quantitative results of western blot analysis were determined in Image J software (U. S. National Institutes of Health, Bethesda, MD, USA).
- QRT-PCR
The total RNA in samples were isolated using TRIZOL reagent (Invitrogen). After quantification, cDNA was synthesized using the First-strand cDNA synthesis kit (Invitrogen). A light cycler (Roche) and FastStart DNA Master Plus SYBR green I kit (Roche) was used to perform the quantitative PCR. Further, the expression was normalized with the values obtained from GAPDH. The primers used in the study are summarized in Table 2. PCR conditions used in this study is a as follows 95°C for 15 s, 57°C for 30 s, and 72°C for 1 min.
- Statistical analysis
Data presented in this study are representative of three independent experiments. The results are expressed as mean ± SD. The significance of the values was assessed using one-way analysis of variance (ANOVA) followed by Student’s T-test. All the statistical analysis in this study was carried out using SPSS 16.0 software. P < 0.05 was considered statistically significant.
Ethics Statement
The present study was approved by the local investigational review board and written informed consent was obtained from all participants.