Mice and paricalcitol treatment
The experimental animals were approved by the Institutional Animal Care and Use Committee of Xian jiaotong University. Female MRL/lpr mice (7 weeks of age) and age- and sex-matched C57BL/6 mice were purchased from Model Animal Research Center Of Nanjing University (Nanjing, China). C57BL/6 mice were used as control group, lupus-prone MRL/lpr mice were used as mouse model of LN [21]. The experimental animals were placed in a special pathogen-free environment (24 ± 1 °C, 50 ± 5% relative humidity and normal 12-h light/12-h dark cycle) in the Animal Experiment Center of Xian jiaotong University. All the mice were kept in controlled conditions for one week before the experiment. After 1 week acclimatization, all the MRL/lpr mice were continuously administrated intraperitoneally for 8 weeks with VDR agonist paricalcitol (19-nor-1,25-dihydroxyvitamin D2, PAL, Abbott Laboratories, CA, USA, 300 ng/kg/mouse per dose, 5 times a week), the control groups were given equal dosage of saline. The amount of 24 h proteinuria were respectively obtained from mice using metabolic cages, and was assessed weekly, quantitative analysis of mouse urine protein was performed by bicinchoninic acid (BCA) protein assay kit (Thermo Scientific, MA, USA). All mice survived to the end of treatment, the mice were sacrificed at 0 week and 8 weeks after intervention (8 weeks old or 16 weeks old), respectively. Serum collected from mouse orbit was obtained by the centrifuge at 3000 rpm for 10 min at 4 °C, and stored at -20 °C for future ELISA use. Fresh Renal tissues were frozen at -80 °C for Western blot and PCR analysis. Renal tissues were fixed in 4% neutral-buffered formalin and embedded in paraffin for histopathological and immunohistochemistry analysis.
Cells Culture
Mouse renal tubular epithelial cells (mRTECs) were obtained from Jennio-bio (Guangzhou, China). Cells were cultured in Dulbecco's Modified Eagle Medium (DMEM) (Gibco, CA, USA) supplemented with 10% fetal bovine serum (FBS) (Gibco, CA, USA), 100 U/mL penicillin, and 100 mg/mL streptomycin at 37 °C and 5% CO2 in a humidified incubator after resuscitation. After starvation for 24 h, cells were stimulated with vehicle or PAL (0.2 ng/ml) for 24 h. In anti-dsDNA antibody stimulation experiment, mRTECs were treated with serum (the concentration of anti-dsDNA antibody was adjusted to 10 ng/ml) obtained from MRL/lpr mice with LN, for control group, mRTECs were treated with serum of same dilution obtained from normal C57BL/6 mice.
Plasmid Construction
VDR overexpression plasmid was constructed. In brief, specific primers used to amplify the coding region of mouse VDR (NM_009504.04) were Mus-VDR-Forward: 5’-CCCAAGCTTATGGAGGCAATGGCAGCCAG-3’ and Mus-VDR-Reverse: 5’-CCGCTCGAGTCAGGAGATCTCATTGCCGA-3’. The above PCR products were TA subcloned into pcDNA3.1-Hygro(+) vector. The inserts were inserted into the XhoI and Hind III sites of the vector, plasmids containing the insert were isolated for sequencing.
Cell Transfection
Cells were plated onto 6-well or 96-well plates at 50% confluence. Transient transfection of siRNA or plasmids of VDR was carried out using Lipofectamine 2000 reagent following the manufacturer’s procedure. Briefly, mRTEC cells were washed with serum-free medium and cultured in serum-free medium without antibiotics. The transfection complex (siRNA/plasmids and the transfection reagent mixture) were added to the medium in a drop-wise manner and mixed gently by rocking the media back and forth. After 4–6 h, the cell culture medium was changed back to DMEM containing serum and antibiotics and incubated at 37 °C for 48 h before proliferation assay, Western blot analysis, or PCR experiments.
The sequences of siRNA were as follows:
NFκB: sense: 5’-GCAGGUAUUUGACAUACUATT-3’;
anti-sense: 5’-UAGUAUGUCAAAUACCUGCTT-3’
NLRP3: sense: 5’- CCAACUGGUCAAGGAGCAUTT − 3’
anti-sense: 5’- AUGCUCCUUGACCAGUUGGTT − 3’
Negative control (NC): sense: 5’-UUCUCCGAACGUGUCACGUTT-3’;
anti-sense: 5’-ACGUGACACGUUCGGAGAATT-3’.
RT-PCR
Total RNA was extracted from Renal tissue or cultured cells using Trizol reagents (Sigma-Aldrich, USA). Reverse transcription was performed using Superscript III First-strand Synthesis System (Invitrogen, USA) and quantitative, real-time PCR with Power SYBR Green PCR Master Mix (Applied Biosystems, UK). The relative abundance of target mRNA was normalized to that of the GAPDH, by a comparative cycle threshold method (2−ΔΔCT). The primer sequences were as listed below:
mus GAPDH: Forward: 5’-ATGGGTGTGAACCACGAGA-3’, and Reverse: 5’-CAGGGATGATGTTCTGGGCA-3’.
mus NLRP3: Forward: 5’-CTGGTCTGCTGGATTGTGTG-3’, and Reverse: 5’-AGAGCCCCTGTAGGTAGTCA-3’.
mus caspase1: Forward: 5’-GGAGGGAATATGTGGGACCA-3’, and Reverse: 5’-TTGTTTCTCTCCACGGCATG-3’.
mus VDR: Forward: 5’-CTTTGACCGGAATGTGCCTC-3’, and Reverse: 5’- GTGATGCGGCAATCTCCATT-3’.
mus NF-κB: Forward: 5’-CACCGGATTGAAGAGAAGCG-3’, and Reverse: 5’-AAGTTGATGGTGCTGAGGGA-3’.
mus importin-4: Forward: 5’-AAGCTACTGGGCCTCCTTTT-3’, Reverse: 5’-AGGAAGCTGAAGAGGTGACC-3’.
Western blot
Western Blots were performed to quantify the expression of GAPDH (rabbit, 1:1000, Goodhere Biological Technology, AB-P-R 001, Hangzhou, China), Caspase1 (rabbit, 1:1000, proteintech, 22915-1-AP, Wuhan, China), VDR (mouse, 1:500, Santa Cruz, SC-13133, Texas, USA), NF-κB (mouse, 1:500, Santa Cruz, SC-166588, Texas, USA), NLRP3 (rabbit, 1:300, Novus, NBP2-12446, CO, USA), Importin4 (rabbit, 1:10000, abcam, ab181046, MA, USA). Briefly, protein samples were loaded in equal amounts into individual wells of separated on a SDS-PAGE gel and subsequently blotted onto a nitrocellulose membrane (Millipore, MA, USA), and nonspecific sites on the membrane were blocked with 5% dried milk in Tris-Buffered Saline containing 0.05% Tween-20 (TBST buffer) for X-ray film. The membranes were incubated overnight at 4 °C with optimized dilutions of primary antibody as mentioned above. After the overnight incubation, membranes were washed with TBST buffer and re-incubated with a goat anti-rabbit or mouse IgG-HRP secondary antibody. The membranes were washed with TBST buffer and reacted with the electrochemiluminescence (ECL) substrate (Thermo, MA, USA) and exposed to X-ray film.
Enzyme-linked Immunosorbent Assay (elisa)
The concentrations of IL-1β, IL-18 and anti-dsDNA antibody in serum of mice or cell supernatant were measured using mouse IL-1β (Elabscience Biotechnology, E-EL-M0038c, Wuhan, China), IL-18 (Elabscience Biotechnology, E-EL-M0730c, Wuhan, China) and dsDNA (CUSABIO, CSB-E11194m,Wuhan, China) ELISA kits following the manufacturer’s protocol.
Immunohistochemistry
Protein expression levels were detected in paraffin-embedded lung sections using the Importin4 (1:50 dilution), NF-κB (1:200 dilution), NLRP3 (1:150 dilution), VDR (1:2000 dilution), Caspase1 (1:200 dilution) antibodies, which were visualized using HRP-conjugated goat anti-rabbit/anti-mouse secondary antibody. Slides were counterstained with hematoxylin, dehydrated and mounted.
Histopathologic Analysis
Renal tissues were collected and fixed in 10% neutral buffered formalin usually for 48–96 hours. Tissues were then routinely processed, embedded, sectioned (∼4 µm), and stained with routine H&E stains for general examination. Periodic acid Schiff (PAS) stains were selected for observing renal glomerular basement membrane and the mesangial area.
Flow Cytometry
Apoptosis of cells was determined by flow cytometric analysis using the AnnexinV-APC/7-AAD Assay kit (BD Biosciences, CA, USA) according to the manufacturer’s instructions. Briefly treated mRTEC cells were harvested with trypsin, washed in cold PBS twice, and resuspended in binding buffer. A volume of 100 µL of the solution was transferred to a 5 mL culture tube and 5 µL AnnexinV-APC and 5 µL 7-AAD were added and incubated at room temperature for 15 minutes in the dark. Then, 400 µL of binding buffer was added to the culture tube. Samples were analyzed using CytoFLEX (Beckman Coulter, CA, USA) within 1hour after staining. Apoptotic cells were defined as AnnexinV-APC-positive, 7-AAD-negative cells.
Co-Immunoprecipitation (Co-IP)
Cells were collected and lysed with lysis buffer on ice for 10–15 min and centrifuged at 10000 rpm at 4 °C for 10 min. The supernatant fractions were collected and incubated with appropriate antibody at 4 °C overnight and precipitated with protein A/G-agarose beads (Santa Cruz Biotechnology, sc-2003, Texas, USA) for another 3–6 h at 4 °C. The beads were washed with the lysis buffer 3 times by centrifugation at 3000 rpm for 5 min at 4 °C. The immunoprecipitated proteins were separated by SDS-PAGE, and western blotting was performed as previously described. The primary antibodies were as follows: anti-importin 4 (rabbit, abcam, Ab181046, MA, USA), anti-NF-κB (mouse, Santa Cruz, SC-166588, Texas, USA), anti-NLRP3 (mouse, Santa Cruz, SC-13133, Texas, USA). The second antibodies were HRP-goat anti-rabbit secondary antibody (BOSTER biological technology, BA1054, Wuhan, China) and HRP-goat anti-mouse secondary antibody (BOSTER biological technology, BA1051, Wuhan, China).
Statistical analysis
Data were presented as means ± SEM. Comparison between the groups of data was evaluated using the Student’s unpaired t-test. For multiple comparisons, one-way ANOVA was used with a Bonferroni post hoc test. A p value < 0.05 was considered statistically significant.