Patient samples
Kidney biopsy samples were obtained from 15 inpatients diagnosed with HBV-GN and 50 inpatients without HBV-GN. HBV-GN was diagnosed based on the following criteria: (a) serum hepatitis B surface antigen (HBsAg) positivity, (b) glomerular nephritis, where secondary glomerular disorders and lupus nephritis were excluded, and (c) HBV DNA or HBV antigen positivity in renal samples. Patients were classified into four groups (groups I–IV), i.e., HBV-GN (Group I, n = 15), HBV-positive PGN (Group II, n = 20, PGN cases showing positive serum HBV DNA or HBsAg results), HBV-negative PGN (Group III, n = 15, PGN cases not affected by HBV), and normal control (n = 15, matched non-carcinoma kidney tissue samples). This study was conducted in accordance with the Second Declaration of Helsinki. Each patient provided informed consent.
Animals
Fourteen 6-week-old HBV-Tg C57BL/6 male mice (HBV mice) and age-matched WT C57BL/6 male mice (C57 mice) were obtained from Vitalstar Biotechnology Co., Ltd. (Beijing, China). Each mouse was raised in an SPF environment under a 12-hour/12-hour light/dark cycle at 22 °C. Animals were allowed ad libitum access to drinking water. Half of the HBV (n = 7) and C57 mice (n = 7) were randomly chosen to receive 5-azacytidine dissolved in PBS (5-Aza, Sigma-Aldrich, St. Louis, MO, USA) at 16 weeks of age. These mice were intraperitoneally injected with 5-Aza (1 mg/kg body weight) three times per week. The same volume of PBS was intraperitoneally injected into the control HBV (n = 7) and C57 mice (n = 7). After 8 weeks of treatment, 24 h urine, blood, and kidney samples were collected. All experiments involving animals were approved by the Animal Care and Use Committee of Shanghai General Hospital, Shanghai Jiaotong University School of Medicine. And we declared that each experiment was carried out according to the guidelines for the Care and Use of Laboratory Animals (NIH publication, Eighth edition, 2011) released by the National Institutes of Health.
Assessment of renal function
The total blood samples were centrifuged at 3500 × g for 15–30 min. Serum creatinine (Scr) levels were determined with an automatic biochemical analyzer. The 24 h urine samples were collected from metabolic cages, and urinary protein (Upro) was determined with an automatic biochemical analyzer.
Cell culture
Human tubular epithelial cells, i.e., HK-2 cells were obtained from the American Type Culture Collection (ATCC), and cultured in DMEM/F12 (Gibco, NY, USA) supplemented with 1% penicillin/streptomycin (Sigma-Aldrich, St. Louis, MO, USA) and 10% fetal bovine serum (FBS, Gibco, NY, USA) and incubated in a humid atmosphere of 5% CO2 at 37 °C. The conditionally immortalized human podocyte cell line demonstrating nephrin and podocin expression was kindly provided by Prof. Chuanming Hao (Huashan Hospital, Fudan University) and cultured as previously described [19]. Differentiated podocytes were cultured in RPMI-1640 medium (Gibco, NY, USA), containing 1% penicillin/streptomycin and 10% FBS.
Plasmid and RNA interference
Full-length sequences of the gene encoding the HBx (Gene ID: 944,566) were chemically synthesized, and these were then subcloned into a lentiviral expression vector (PGMLV-PA6) obtained from Genomeditech (Shanghai, China). The sequences of the DNMT1, VDR, and PTEN genes were obtained from GenBank. Genepharma Co., Ltd (Genepharma, Shanghai, China) designed and synthesized control siRNA and siRNA against DNMT1, VDR, PTEN. siSNAI1 was purchased from Santa Cruz (sc-38398, CA, USA) (Additional file 1: Table S1).
Transfection and stable cell line generation
Cells were seeded in 6-well plates (1 × 105/well), and when the cell density reached approximately 70–80%, siRNA or plasmids were transfected into cells using Lipofectamine 3000 (Invitrogen, Carlsbad, CA, USA) in accordance with the manufacturer’s instructions. HBx overexpression and control lentiviral vectors—containing a random sequences—were transfected into HEK 293 T cells. After 48 h of transfection, the medium containing the lentivirus was harvested and used for infecting the HK-2 cells and human podocytes, followed by a week of puromycin (2 µg/mL) selection to generate stably transfected cells. Cells with stable HBx expression were considered HBx-HK-2 cells or HBx-podocytes, whereas those with stable empty vector were used as negative control (NC) cells. The untreated cells were called control (Cont) cells.
RNA-sequencing and transcriptome analysis
NC cells transfected with siNC were used as negative controls. HBx-HK-2 cells or HBx-podocytes transfected with or without siDNMT1 were used to analyze the potential downstream targets of DNMT1. TRIzol (Invitrogen, Carlsbad, CA, USA) was used to extract total RNA, and libraries were established using the TruSeq Stranded mRNA LTSample Prep Kit (Illumina, San Diego, CA, USA) in accordance with the manufacturer’s instructions. The constructed libraries were then sequenced using the Illumina sequencing platform (Illumina HiSeq X Ten or HiSeqTM 2500), which produced paired-end reads of 125 bp/150 bp. Transcriptomic analysis and sequencing was outsourced to OE Biotech Co., Ltd. (Shanghai, China). P < 0.05, and fold change (FC) < 0.5 or FC > 2 indicated significant differential expression.
Immunofluorescence
After rinsing twice with PBS, cells were fixed for 30 min in 4% paraformaldehyde, permeabilized using 0.2% Triton X-100, and blocked using 0.5% bovine serum albumin (BSA) at room temperature. Thereafter, cells were probed overnight with anti-DNMT1 (#ab188453), anti-VDR (#ab89626), anti-PTEN (#ab137337) (Abcam, Cambridge, MA, UK), anti-DNMT1 (#sc-271,729), anti-SNAI1 (#sc-271,977), anti-nephrin (#sc-376,522) (Santa Cruz, CA, USA), anti-E-cadherin (#14,472), and anti-p-NF-κB P65 (#3033) (Cell Signaling Technology, Danvers, MA, USA), anti-HBx (#MA1-81021) (Thermo Fisher Scientific, Waltham, MA, USA) antibodies at 4 °C. This was followed by a 60 min incubation with fluorescence-conjugated secondary antibodies (Yeasen, Shanghai, China) in the dark at room temperature. Then, cells were stained with DAPI (Beyotime, Shanghai, China) for 10 min at room temperature.
Immunohistochemistry
Human and animal kidney samples were fixed with 4% paraformaldehyde, embedded in paraffin, and sliced into 4 μm sections. After blocking with 5% BSA at room temperature, the sections were probed overnight with antibodies against HBx (#ab235), IL-6 (#ab9324, #ab208113), DNMT1 (#ab188453) (Abcam, Cambridge, MA, UK), and TNF-α (#sc-52,746) (Santa Cruz, CA, USA) at 4 °C, followed by incubation with appropriate secondary antibodies. The sections were visualized using a light microscope (Leica, Heidelberg, Germany) and quantified using ImageJ.
Masson’s trichromatic staining
Kidney sections were prepared as described above and then dewaxed and hydrated. After rinsing with water, the sections were stained using reagents from a Masson Modified International Medical Equipment (IMEB) Stain Kit (IMEB, San Marcos, CA, USA), in accordance with the manufacturer’s instructions. Collagen deposition was imaged using a microscope (Leica, Heidelberg, Germany).
Quantitative real-time PCR
TRIzol was used to isolate the total RNA. Thereafter, 1 µg of total RNA was reverse transcribed into cDNA using the Toyobo reverse transcription system (Osaka, Japan). SYBR Green Premix (Takara, Otsu, Japan) was used to conduct quantitative real-time PCR (qRT-PCR) on an ABI 7700 Sequence Detector System (Applied Biosystems, USA). The expression of target genes was normalized to the expression of GAPDH. We used the 2−ΔΔCT method to determine the fold-change at the target gene level. Additional file 1: Table S2 displays all the primers used.
Protein isolation and western-blotting
RIPA buffer (Thermo Fisher Scientific, Waltham, MA, USA), supplemented with phosphatase/protease inhibitors (Thermo Fisher Scientific, Waltham, MA, USA), was used to lyse cells and tissues. Proteins were collected after centrifuging the lysate at 15,000 × g for 15 min. Equivalent amounts of protein were loaded into each well of 6–12% SDS-PAGE gels, and then transferred onto a PVDF membrane (Thermo Fisher Scientific, Waltham, MA, USA). Thereafter, the membranes were blocked in 5% skim milk for 1–2 h at room temperature, followed by overnight incubation at 4 °C with primary antibodies, i.e., anti-HBx (#ab235), anti-DNMT1 (#ab188453), anti-DNMT3a (#ab188470), anti-DNMT3b (#ab2851), anti-ZO1 (#ab216880), anti-VDR (#ab3508), anti-PTEN (#ab137337), anti-podocin (#ab181143), anti-fibronectin (#ab2413), anti-IL-6 (#ab9324, #ab208113)(Abcam, Cambridge, MA, UK), anti-p-NF-κB P65 (#3033), anti-NF-κB P65 (#8242), anti-p-IκBα (#2859), anti-IκBα (#4814), anti-E-cadherin (#14,472), anti-vimentin (#5741), anti-p-mTOR (#5536), anti-mTOR (#2983), anti-p-Akt (#5012), anti-Akt (#4691), anti-β-actin (#3700), and anti-GAPDH (#5174) (Cell Signaling Technology, Danvers, MA, USA), and anti-SNAI1 (#sc-271,977), anti-nephrin (#sc-376,522) and anti-TNF-α (#sc-52,746) (Santa Cruz, CA, USA). This was followed by a 2 h incubation with horseradish peroxidase-conjugated secondary anti-rabbit IgG (#7074) and anti-mouse IgG (#7076) (Cell Signaling Technology, Danvers, MA, USA) under room temperatures. ECL (Millipore, Billerica, MA, USA) was used to visualize and analyze the protein bands.
Enzyme-linked immunosorbent assay
Commercial enzyme-linked immunosorbent assay (ELISA) kits (R&D, Minneapolis, MN, USA) were used to measure TNF-α and IL-6 levels in the supernatant, in accordance with the manufacturers’ instructions. TNF-α and IL-6 levels were analyzed based on OD450.
Bisulfite-sequencing PCR
Total genomic DNA was isolated from HK-2 cells and human podocytes. After bisulfite treatment, 1 µg genomic DNA was modified using the EpiTect Bisulfite Kit (Qiagen, Germantown, MD, USA), followed by PCR amplification using Bisulfite-sequencing PCR (BSP) forward and reverse primers (Additional file 1: Table S3). After PCR amplification procedure, the PCR products corresponding to the CpG islands of the PTEN and VDR promoters (351 base pairs and 399 base pairs) were subjected to gel purification and insertion into the pMD18-T vector. Ten clones were chosen for sequencing after transformation into TOP10 (DL1010) competent cells.
Dual-luciferase reporter assay
Cells were seeded into 24-well plates, followed by co-transfection with pcDNA3.1/Flag-SNAI1, pGL4.10-VDR luciferase promoter-reporter plasmid (wild-type or mutant plasmid), and a plasmid encoding Renilla luciferase (pRL-TK plasmid, OBiO Technology, Shanghai, China). After 48 h, cells were harvested, and Renilla and firefly luciferase activities were assessed—using the Dual-Luciferase Reporter Assay System purchased from Promega (Madison, WI, USA)—based on a previous description [20]. The firefly luciferase to Renilla luciferase ratio was calculated.
Nuclear extract preparation
Nuclear extracts were prepared using a nuclear protein extraction kit (Beyotime, Shanghai, China) in accordance with the manufacturer’s instruction. Briefly, cells were rinsed twice with PBS and resuspended in reagent A to extract the cytoplasmic protein fraction. After extraction of cytoplasmic proteins, the pellets were resuspended in 50 µL nuclear protein extraction reagent. After agitation at 4 °C and centrifugation at 12,000×g for 10 min, the supernatant containing the nuclear proteins was harvested. Nuclear extracts were used for further analysis or stored at − 80 °C.
DNMT1 activity assay
Ten to twenty micrograms of the nuclear extract was harvested, and DNMT1 activity was evaluated using the DNMT1 assay kit (Abcam, Cambridge, MA, UK) in accordance with the manufacturer’s protocol. DNMT activity based on OD450 values was measured using a microplate reader.
Statistical analysis
Each experiment was performed with three or more independent replicates. All data are shown as the mean ± SD and were compared using one-way ANOVA or Student’s t-test. GraphPad Prism 7.0 (GraphPad Software, Inc., Jolla, CA, USA) was used for graphical visualization and statistical analyses. Significance was set at P < 0.05.