Our retrospective study was approved by the ethics committee of Jinan Infectious Disease Hospital (JCLL-2016-04). A total of 75 patients diagnosed with chronic nephritis, identified between 2008 and 2016 at Jinan Infectious Disease Hospital and Qilu Hospital of Shandong University Shandong, China, were included in the study. The experimental group consisted of 50 HBV-GN patients, the negative control group consisted of 25 CGN patients, CGN should be ruled out the following criteria:1) PCR detection of HBV-DNA and Elisa detection of HBV-associated antigen including HBsAg and HBeAg to test HBV infection; 2) PCR detection of EBV-DNA, HSV-DNA, HCVM-DNA and HPV-DNA to test the common ds-DNA virus infection; 3) with auto-immune disease. Each patient received kidney puncture biopsy under ultrasound guidance to attain renal tissue for diagnosis and subsequent research. Participation was dependent upon fulfillment of the following criteria:(1) patients must not use an immune agent or antiviral agent in the past 3 months; (2) patients must not have HAV, HCV, HDV, HEV or HIV co-infection; (3) patients must not have a history or current evidence of secondary glomerulonephritis; and (4) consent for participation must have been obtained from those who participated. The basic characteristics of patients are listed in Table 1.
Diagnosis of HBV-GN, CGN and pathological classification of HBV-GN
The diagnostic criteria used for CGN and HBV-GN were in accordance with the 2002 Kidney Disease Outcome Quality Initiative (K/DOQI), edited by the National Kidney Foundation (NKF) . The diagnosis of HBV-GN was confirmed by pathology. The pathological classification of and diagnostic criteria used for HBV-GN were in accordance with 1990 WHO classification criteria . Frozen slices from biopsies of the 50 HBV-GN patients were kept in a low-temperature freezer. Monoclonal goat-anti-human HBsAg and HBcAg antibodies were purchased from Dako (Denmark), antibodies against IgA, IgG, IgM, C3 and C1q complement component. Fluorescently-labeled IgA, IgG, IgM, C3 and C1q rabbit-anti-human antibodies were purchased from Dako. Immunohistochemical staining for HBsAg and HBcAg in renal biopsies was used or electron microscope detection for HBV to confirm the diagnosis (Fig. 1). For HBV-GN patients with undetectable HBsAg or HBcAg in renal tissue, HBV was detected using the JCM-6000 scanning electron microscope from Jeol. Ltd. (Japan). Sections from all biopsy specimens were stained routinely with hematoxylin and eosin (H&E), periodic acid-sliver methenamine (PASM), Masson’s trichrome.
Immunohistochemistry and scoring
Immunohistochemistry was carried out using standard techniques. Renal tissue specimens were first fixed in 10% formalin, then the tissue was cut, dehydrated, dipped in wax, embedded and sectioned. These sections were then placed on slides, baked, placed into xylene, cleared of the wax, rehydrated using graded ethanol and immersed in 0.3% hydrogen peroxide for 5 min to reduce non-specific background staining caused by endogenous peroxidase. The slides were then washed with PBS buffer three times for 5 min each, placed in citrate buffer solution at a pH of 6.0 and then into a high temperature pressure pot to recover the tissue antigen. After being heated, the slides were cooled and restored at room temperature, washed three more times in PBS buffer and incubated with IFI16 (ab55328, rabbit anti-human polyclonal antibody; Abcam, USA), caspase-1 (sc-56,063, mouse anti-human polyclonal anti- body; Santa Cruz Biotechnology Inc., USA) and IL-1β antibodies (ab2105, rabbit anti-human polyclonal anti-body; Abcam), respectively. The slides were then placed in a 4 °C refrigerator overnight. The next day, the slides were washed with PBS buffer three times, each time lasting longer than 5 minu, then incubated with the secondary antibody PV-9000 (universal antibody) at 37 °C for 10 min, washed with PBS buffer, and DAB staining was applied. The stain was terminated using running water, then the slides were washed with hydrochloric acid alcohol for differentiation. Lastly, the slides were washed with distilled water, cleared with xylene and mounted. Appearance of a tan stain in the cytoplasm signaled positive expression of the protein. After staining, scores were assigned based on stain intensity and percentage of positive cells as follows: For stain intensity, a score of 0 was given for no brown staining (i.e., no cells stained), 1 for light brown, 2 for brown and 3 for dark brown; for percentage of positive cells, a score of 0 was given for fewer than 5% positive cells, 1 for 5 to 30%, 2 for 30 to 60% and 3 for greater than 60%. Scores for stain intensity and percent positive were then added together, and a negative sign (−) was assigned for scores totaling 0, mildly positive (+) for scores between 1 and 3, moderately positive (++) for scores between 4 and 6 and strongly positive (+++) for scores greater than 7.
Cell lines and reagents
The human glomerular mesangial (HGM) cell line and HEK-293 T cell line used in this study were purchased from the cell bank of the Chinese academy of sciences (Shanghai, China) and cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% FBS (Life Technologies, Carlsbad, CA, USA), ampicillin and streptomycin at 37 °C, 5% CO2 conditions. HBV expression plasmids were constructed with a pcDNA3.0 vector. The 1.1-fold over length HBV genome was cloned into the pcDNA3 vector to generate pcDNA3.0–1.1HBVDNA, 1.1HBV as the expression gene and ampicillin resistance for antibiotic selection (Amresco, Penn- sylvania, USA). We designed three kinds of IFI16-siRNAs for this experiment, which were synthesized by Gene Pharma (Shanghai, China). the siRNA was synthesized accordingly:
siRNA #1: GGAAGUGGAUGCUACUUCAdTdT;
siRNA #2: GGAAUAUGAUAGUCUCCUAdTdT;
siRNA #3: GGAAGUGGAUGCUACUUCAdTdT;
siRNA negative control: GAUGAGAUUAGAUACUCUCdTdT.
IFI16-siRNA#2 was selected as the most effective silencer compared with the others, which was used for the following experiment (Fig. 4A). IFI16, Caspase-1 and IL- 1β antibodies were obtained from Cell Signaling Tech Abcam (Cambridge, MA, USA).
HGM cell line and HEK-293 T cell line were seeded into 12-well plates, then the cells were transfected with either pcDNA3.0–1.1HBVDNA- IFI16 and negative control for overexpression studies, or with siIFI16 and a scrambled siRNA for knockdown studies. Lipofectamine 2000 (Invitrogen) was used according to the manufacturer’s instructions with minor modifications for transfection studies. IFI16 overexpression and knockdown were confirmed by qRT-PCR and Western blot 48 h post transfection.
According to the manufacturer’s instructions, the whole cell protein extracts were prepared and were separated using 10% sodium dodecyl sulphate polyacrylamide gel electrophoresis. Proteins were then transferred to a polyvinylidene difluoride membrane (Millipore, Bed- ford, MA, USA), according to the instruction manual. Filters were blocked overnight in 5% w/v low- fat dry milk in 10 mmol/L Tris- HCl, pH 7.5, 0.1 mol/L NaCl and 0.1% Tween- 20 and incubated with primary antibodies overnight at 4 °C. After washing with TBST buffer, the blots were then incubated with HRP- conjugated secondary antibody for 2 h at room temperature. After washing with TBST buffer, Immunoreactive bands were visualized using the ECL-Plus reagent (Millipore, Billerica, MA, USA). GAPDH was used as the loading control in the Western blotting.
RNA isolation and qRT-PCR
Total RNA was isolated using Trizol RNA reagent (Invitrogen, California, USA). Quantitative real- time PCR was performed, and the expression levels of IFI16, caspase-1 and Il-1ß mRNA were normalized to GAPDH for gene expression. The primers are listed in Table 2.
The SPSS program (version 19.0) and GraphPad Prism (version 8.0) were used for analysis. Measurement data was described as mean ± standard deviation. Background factors were compared using Student’s-test (numerical data) or the Chisquare test (categorical data). Spearman’s two-tailed test was used for correlation analysis, and differences were regarded as significant if the p value was less than 0.05 on either side.