Mouse B lymphocytes (WEHI 231) were purchased from the Cell Bank of the Chinese Academy of Sciences and maintained in DMEM medium (Gibco, NY, USA) supplemented with 10% fetal bovine serum (FBS, Gibco) at 37°C in a humidified atmosphere containing 5% CO2. The WEHI 231 cells were passaged every 2–3 days.
Immortalized mouse podocytes (MPC5) (gifted by Professor Xu Ning from Lund University, Sweden) were maintained in RPMI-1640 medium (Gibco) supplemented with 10% FBS, 2 mM glutamine (Sigma, MO, USA) and 10 g/mL interferon-gamma (IFN-γ, Sigma) at 33°C in a humidified atmosphere containing 5% CO2. Cell were passaged every 2–3 days. The MPC5 cells were differentiated at 37°C in the absence of IFN-γ for 10 days[10, 11].
B lymphocyte supernatant groups
B lymphocytes were divided into three groups as follows. Normal control group: cells were centrifuged at 1,000×g for 10 min to remove particles and polymer. Phorbol myristate acetate (PMA) intervention group: cells were exposed to 100 ng/mL PMA for 24 h, followed by centrifugation at 1,000×g for 10 min to remove particles and polymer. ACTH4-10 intervention group: cells were cultured in the presence of ACTH4-10 at various concentrations (1 µg/L, 10 µg/L and 100 µg/L) for 1 h, followed by centrifugation at 1,000×g for 10 min to remove particles and polymer. PMA was obtained from Sigma Chemicals (MO, USA). ACTH4-10 was supplied by Cayman Chemical Company (MI, USA).
All podocytes were randomly divided into the following groups: normal control group, ADR group, the supernatant (1 µg/L, 10 µg/L and 100 µg/L ACTH4-10)+ADR groups, ACTH4-10 (10 µg/L)+ ADR group, and the supernatant (10 µg/L ACTH4-10)+ anti-IL-10R+ADR group. Briefly, 3 mL cell culture supernatant of B lymphocytes stimulated with different concentrations of ACTH4-10 was added to the culture medium of podocytes. Moreover, equal volumes of supernatants were added to culture medium of normal control and ADR groups. The normal control group received no treatment. The model of podocyte injury was established by exposing cells to ADR (1 mol/L) for 24 h, and the ACTH4-10 (10 µg/L)+ADR group was intervened with 10 µg/L ACTH4-10 for 1 h prior to podocyte injury. In the supernatant (1 µg/L, 10 µg/L and 100 µg/L ACTH4-10)+ADR groups, the cell culture supernatant of B lymphocytes stimulated with different concentrations of ACTH4-10 (1 µg/L, 10 µg/L and 100 µg/L) was added to the culture medium of podocytes for 1 h prior to podocyte injury. According to previous studies, anti-IL-10R was added at a concentration of 10 µg/mL for 1 h. In the supernatant (10 µg/L ACTH4-10)+anti-IL-10R+ADR group, the supernatant (10 µg/L ACTH4-10) as well as anti-IL-10R and ADR of above-mentioned concentrations were added to the culture medium of podocytes. Anti-IL-10R was obtained from R&D Company (MI, USA). ADR was purchased from Cayman Chemical Company (MI, USA).
Enzyme-linked immunosorbent assay (ELISA)
The levels of IL-4 and IL-10 were determined by commercially available ELISA kits (Arigo biolaboratories, Taiwan, China) according to the manufacturer’s instructions. Total cell lysates were prepared, and the protein concentration was adjusted using lysis buffer. To detect IL-4 level in the supernatant, cells were cultured in 6-well plates. Subsequently, cells were cultured in 1 mL of serum-free medium containing proteinase inhibitors. The medium was then harvested and centrifuged, and 100 µL supernatant was subjected to ELISA. Cells were counted to determine the amount of IL-4 secreted per cell.
Podocyte proliferation assay
Cell viability was determined using Cell Counting Kit-8 (CCK-8, MedChem Express, USA) according to the manufacturer’s instructions. The podocytes of the normal control and experimental groups were seeded into 96-well plates at a density of 1~5 * 104 cells/mL. Three replicates were set for each group. In addition, 20 µL CCK-8 reagent was added into each well, followed by incubation at 37°C for 2 h, and then the absorbance value of each well at a wavelength of 450 nm was measured and recorded. The cell growth curve was drawn accordingly.
TUNEL apoptosis assay
The level of apoptosis was determined by terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick end-labeling (TUNEL) assay using the In Situ Cell Death Detection Kit (Roche Diagnostics, Mannheim, Germany) following the manufacture’s protocol. Total cells were visualized for DAPI staining, and apoptotic cells were visualized for FITC staining using a laser-scanning confocal microscope (Zeiss).
Quantitative polymerase chain reaction (qPCR)
qPCR was carried out as described previously[15, 16]. Total RNA was isolated using Trizol Reagent (Invitro-gen, Carlsbad, CA, USA). Equal amounts of RNA (1 µg) were reversely transcribed into cDNA using Revert Aid First StrandcDNA synthesis kit (Fermentas). qPCR was performed on an ABI 7500 system (Applied Biosystems, Foster City, CA) using mouse IL-10R primers (forward: 5′-AGGCAGAGGCAGCAGGCCCAGCAGAATGCT-3′; reverse: 5′-TGGAGCCTGGCTAGCTGGTCACAGTAGGTCT-3′), nepherin primers(forward: 5′-TCGGGTTACATTCCACAGCT-3′; reverse: 5′-GAGTTCATGGGAGAGCAAGT-3′), podcocin primers(forward: 5′-TGGCAGCCTCACATCCTTAA-3′; reverse: 5′-TCCAAAGCCATCCAGTTCCT-3′), 18S primers (forward: 5′-TCAACACGGGAAACCTCAC-3′; reverse: 5′-CGCTCCACCAACTAAGAAC-3′) and SYBR®green PCRMaster Mix (Invitrogen). The relative expression of IL-10R was calculated using the 2-ΔΔCt method, and 18S was selected as a housekeeping gene for qPCR.
Cells were washed with PBS and lysed with RIPA buffer. Protein concentration was determined by BCA protein assay kit (Pierce, Rockford, IL, USA). Protein were separated by 10% sodium dodecyl sulfate-polyacrylamide gels(SDS-PAGE) and transferred onto nitrocellulose membranes. Then, membranes were blocked in 5% (w/v) non-fat dry milk powder in 0.1% Tris buffered saline/Tween 20 (TBST) for 1 h. Membranes were washed wirh TBST and then incubated with primary antibodies at optimized dilutions at 4°C overnight. After that, membranes were washed with TBST again and incubated with secondary antibodies for 1h. Membranes were visualized using ECL chemiluminescence (Thermo Company, West Chester, PA, USA). The band densities were analyzed with Image J software (National Institute of Health, USA). Primary antibodies were puechased form TIANGEN(Beijing, China).
Normally distributed data (Shapiro-Wilks test) were expressed as mean ± standard deviation. Statistical analysis was performed with GraphPad Prism 5 software (GraphPad Software, CA, USA) using Student’s t-test or one-way analysis of variance, followed by a post-hoc Tukey‘s test where applicable. A p value less than 0.05 was considered as statistically signiﬁcant. All experiments were performed at least for three times.