Patients and sample collection

According to the diagnostic criteria for SLE and the level of proteinuria at enrollment (> 0.5 g/24 h), kidney biopsies, and blood and urine samples were collected from untreated patients with LN (n = 23) at Fuyang Hospital of Anhui Medical University. Patients aged < 18 years or > 75 years, or patients with hypertension, diabetes mellitus, possible hepatitis B infection, tumors, pregnancy, or other types of kidney-related diseases were not included in this study. Normal control kidney tissue (n = 14), blood, and urine samples were obtained from deceased patients who had diseases other than kidney-related diseases at Fuyang Hospital of Anhui Medical University. The relevant information on patients with LN and controls is listed in Table 1. Institutional ethics committee approval was obtained. Written informed consent was acquired from all participants or their relatives.

Table 1 Patient demographics for the studyMice

Seven-week-old female MRL/lpr mice and wild-type (WT) C57BL/6 J mice were purchased from the Jackson Laboratory (Sacramento, CA, USA) and housed under specific-pathogen-free (SPF) conditions with a 12-h light/dark cycle at 25 ± 1 °C with water and food available. All procedures were conducted following the Guide for the Care and Use of Laboratory Animals published by National Institutes of Health (NIH). All animal study protocols were approved by the animal care and use committee of Anhui Medical University. All mice were acclimatized in a rearing room for 1 week before the start of the experiment.

The mice were divided into six groups: the WT (C57BL/6 J mice), MRL/lpr (MRL/lpr mice), MRL/lpr + short hairpin RNA (sh)-negative control (NC) (injected with adenoviral vectors containing sh-NC), MRL/lpr + sh-CEBPB (injected with adenoviral vectors containing sh-CEBPB), MRL/lpr + sh-CEBPB + overexpression (oe)-NC (injected with adenoviral vectors containing sh-CEBPB + oe-NC), and MRL/lpr + sh-CEBPB + oe-BZW1 groups (injected with adenoviral vectors containing sh-CEBPB + oe-BZW1). A single intravenous (IV) injection of 109 particles of the corresponding plasmid-encapsulated adenovirus vector was administered to the mice during the first week of the experiment, and the packaging and preparation of the adenovirus vector was performed by VectorBuilder (Guangzhou, Guangdong, China). When the mice grew to 24 weeks of age, orbital blood was collected, and 24-h urine was collected in a metabolic cage. After an intraperitoneal injection of 150 mg/kg sodium pentobarbital for euthanasia, the kidney tissue was collected from the mice.

Biochemical tests

The Creatinine Assay kit (C011-2-1, JianCheng, Nanjing, Jiangsu, China) and Urine Protein Test kit (C035-2-1, JianCheng) were used to analyze serum and urine creatinine and protein levels in mice. The protein-to-creatinine ratio in mice urine was also measured to assess changes in mouse renal function.

Immunofluorescence

After the euthanasia of mice, kidney tissues were removed, fixed in formaldehyde, and made into paraffin section (4-µm). The sections were deparaffinized in xylene I and xylene II for 15 min each, and rehydrated in ethanol (100%, 95%, 90%, 80%, and 70%) for 5 min, respectively, followed by antigen retrieval using the ethylenediaminetetraacetic acid buffer. The sections were blocked with goat serum for 30 min to remove endogenous non-specific binding sites and cultured with the primary IgG antibody (Abcam, Cambridge, UK) at 4 °C overnight and with fluorescein isothiocyanate (FITC)-coupled goat anti-rabbit secondary antibody (1:1000, ab6717, Abcam) for 1 h at room temperature in the dark. The sections were counter-stained with 4,6-diamidino-2-phenylindole (DAPI), which was applied to visualize the nuclei. The average fluorescence intensity of IgG in the mouse kidney tissues from at least five fields of view was observed under a microscope (200×, Olympus IX71, Olympus Optical Co., Ltd., Tokyo, Japan) after sealing the sections using an antifluorescence-quenching sealing solution.

Histological analysis

Paraffin-embedded mouse kidney tissues were cut into 3-μm thick paraffin sections. After deparaffinization and rehydration, the histopathological conditions of mouse kidney tissues and the degree of periodic acid–Schiff (PAS)-positive deposition were analyzed using the hematoxylin–eosin (HE) Staining Kit (G1120, Beijing Solarbio Life Sciences Co., Ltd., Beijing, China) and Glycogen PAS Staining Kit (G1281, Solarbio), respectively.

For pathologic scoring of HE staining, uninformed semiquantitative assessment of each mouse kidney tissue was performed by two experienced pathologists according to a previous report [13]. For glomerulonephritis: 0, normal; 1, a mild to moderate increase in cellularity with mesangial proliferation; 2, a moderate increase in cellularity with endocapillary and mesangial proliferation, increased matrix, and/or karyorrhexis; and 3, a marked increase in cellularity with endocapillary proliferation, crescent formation, and/or necrosis, and/or sclerosis.

Uninformed semiquantitative assessment of PAS staining of each mouse kidney tissue was performed by two experienced pathologists according to the relevant scoring criteria [14]. In brief, 0, a healthy condition; 1, mild focal disease; 2, a moderate focal disease; and 3–4, diffuse disease.

Immunohistochemistry

Paraffin-embedded sections of mouse kidney tissues were deparaffinized and rehydrated, immersed in citrate buffer, then subjected to a heat-mediated antigen retrieval procedure and hydrogen peroxide to remove endogenous peroxidase. After sealing the non-specific binding sites using goat serum, the sections were incubated with CEBPB (1:50, PA5-120,052, Thermo Fisher Scientific Inc., Waltham, MA, USA) and CD68 (1:100, ab283654, Abcam), F4/80 (1:500, 25514, Cell Signaling Technologies, Beverly, MA, USA) at 4 °C overnight and with horseradish peroxidase (HRP)-coupled goat anti-rabbit secondary antibody (1:1000, ab6721, Abcam) for 120 min at room temperature. For this procedure, 3,3′-diaminobenzidine (DAB) was used for color development and hematoxylin for nuclei counter-staining. Finally, the sections were dehydrated and cleared in ethanol and xylene, respectively, and sealed with neutral gum. The number of positive cells was observed under the Olympus IX71 microscope, and the positive rate was calculated.

Dual-immunofluorescence

To assess the changes in the expression of CEBPB and BZW1 in macrophages of mouse kidney tissues, we used dual-immunofluorescence to label the macrophage marker F4/80 with CEBPB or BZW1, respectively. Mouse kidney tissue sections from each group were incubated in xylene I and xylene II for 15 min each for deparaffinization, followed by incubation in 100%, 95%, 90%, 80%, and 70% ethanol for 5 min for rehydration, respectively. After antigen retrieval of the sections in ethylenediaminetetraacetic acid buffer, the sections were incubated in goat serum for 30 min to remove endogenous non-specific binding sites. The sections were incubated overnight at 4 °C with the following primary antibodies (all from Thermo Fisher): F4/80 (1:100, 14-4801-82), CEBPB (1:100, MA5-32252), or BZW1 (1:200, PA5-35860). This was followed by incubation with goat anti-rat secondary antibody with Alexa Fluor® 488 (1:200, ab150157, Abcam) or goat anti-rabbit secondary antibody with Alexa Fluor® 647 (1:200, ab150079, Abcam), respectively, for 1 h at room temperature. Nuclei were counterstained with DAPI, and images were acquired on the Olympus IX71 microscope.

Flow cytometry

The peritoneum was removed from freshly isolated mouse kidneys, which were then chopped for homogenization. The homogenized kidney tissue fragments were detached in type I collagenase for 30 min to obtain a single-cell suspension, which was filtered through a 70-μm cell filter. The filtered cell suspension was centrifuged at 300 g for 2 min at 4 °C, and the supernatant was removed. The cells were incubated with erythrocyte lysis buffer (C3702, Beyotime Biotechnology Co., Ltd., Shanghai, China) for 2 min at room temperature to remove erythrocytes, centrifuged at 1000 g for 5 min at 4 °C, washed with phosphate-buffered saline (PBS) containing 1% bovine serum albumin (BSA) and 0.1% sodium azide, and then filtered through a 40-μm cell filter. The final single cell suspension was stained with APC-CD11b (1:50, ab25482, Abcam) and FITC-F4/80 (1:50, ab60343, Abcam) antibodies for 20 min at 4 °C after being blocked by CD16/CD32 antibodies and. FlowJo software was used to analyze the percentage of macrophages in the single-cell suspension of mouse kidney tissues.

Enzyme-linked immunosorbent assay (ELISA)

LBIS® Anti-dsDNA-Mouse ELISA Kit (631-02699, Amresco, Radnor, PA, USA), Mouse C3 ELISA Kit (ab263884, Abcam), Mouse IL-1 beta/IL-1F2 Quantikine ELISA Kit (MLB00C, R&D Systems, Minneapolis, MN, USA), Mouse IL-6 Quantikine ELISA Kit (M6000B, R&D Systems), and TNF-α (Mouse) ELISA Kit (K1051, BioVision, Inc., Exton, PA, USA) were used.

Bone marrow-derived macrophages (BMDM) extraction and in vitro stimulation

The femur and tibia were isolated from 6 to 8-week-old C57BL/6 J mice. After cutting the bone, the bone marrow was flushed into Dulbecco’s modified Eagle’s medium (DMEM) containing 2% fetal bovine serum and macrophage colony-stimulating factor. After day 5 of culture, the cells were separated from the culture dish with Tris-buffered saline containing 5 mM EDTA, re-suspended in fresh DMEM, and seeded in 12-well plates.

For in vitro stimulation of BMDM, ovalbumin (Ova), or Ova-IC stimulation was used for 6 h. The BMDM were treated with 5 mM of 2-DG (HY-13966, MedChemExpress, Monmouth Junction, NJ, USA) for 45 min, 10 nmol/μL of 4-PBA (HY-A0281, MedChemExpress) for 1 h, and 1 μM of ISRIB (HY-12495, MedChemExpress) for 4 h to inhibit glycolysis, ER stress, and eIF2α, respectively.

Transfection

BMDM were seeded into 12-well plates at 3 × 105 cells/well. When the cells grew to 80% confluence, sh-CEBPB, overexpression (oe)-BZW1, and control plasmid (VectorBuilder) were transfected into BMDM using Lipofectamine 2000, respectively. After 24 h, the medium was replaced with a fresh medium, and the transfection efficiency was verified by reverse transcription quantitative polymerase chain reaction (RT–qPCR) after 48 h of further incubation.

Measurements of metabolic changes

In the Seahorse XFe96 analyzer (Agilent Technologies, Santa Clara, CA, USA), the treated BMDM were seeded in XF96 microtiter plates. Extracellular acidification rate (ECAR) and oxygen consumption rate (OCR) were measured 3–4 times at baseline, and kinetics were recorded for the indicated times after treatment with different concentrations of glucose or inhibitors (oligomycin, FCCP, rotenone, and antimycin A) to calculate ECAR to OCR ratios.

Glucose uptake assay

Briefly, BMDM were seeded in a 96-well plate and incubated overnight at 37 °C. The cells were starved in serum-free medium for 2 h and incubated with 100 μL Krebs–Ringer phosphate HEPES buffer containing 2% BSA for 40 min to remove endogenous glucose and with 10 μL 2-deoxyglucose (10 mM) for 20 min. The cells were collected with extraction buffer and processed to assay glucose uptake capacity. The glucose uptake level was assessed by measuring the optical density (OD) value in a microplate reader.

Western blot

Mouse kidney tissues or BMDM were lysed on ice using radioimmunoprecipitation assay (RIPA) lysis buffer, and the supernatant was collected by centrifugation. The protein concentration was examined using the bicinchoninic acid (BCA) method. The protein samples were heated in a water bath, and gel electrophoresis was performed using sodium dodecyl sulfate–polyacrylamide gel electrophoresis (PAGE), followed by transferring the proteins to polyvinylidene difluoride membranes. After blocking with skimmed milk powder for 2 h, the membranes were incubated with primary antibodies at 4 °C overnight. The blots were incubated with the HRP-coupled goat anti-rabbit secondary antibody (1:2000, ab6721, Abcam) for 1 h at room temperature. The signals were visualized using the ECL Western Blotting Substrate (PE0010, Solarbio) and analyzed using Image Pro-Plus 6.0. The primary antibodies were as follows: HK2 (1:1000, 2867, Cell Signaling Technologies), LDHA (1:1000, ab52488, Abcam), BZW1 (1:1000, GTX120426, GeneTex, Inc., Alton Pkwy Irvine, CA, USA), eIF2α (1:1000, 5324, Cell Signaling Technologies), p-eIF2α (1:1000, 3398, Cell Signaling Technologies), CHOP (1:1000, PA5-104528, Thermo Fisher), GRP78 (1:500, PA1-014A, Thermo Fisher), and β-actin (1:200, ab115777, Abcam).

Reverse transcription–quantitative PCR (RT–qPCR)

Total RNA was extracted from kidney tissues of mice or clinical patients with LN and BMDM using TRIzol reagent and measured for RNA concentration and purity using a spectrophotometer, followed by reverse transcription using TransScript® Reverse Transcriptase [M-MLV, RNaseH-] (AT101-02, Transgene Biotech, Beijing, China). The cDNA was prepared by reaction, followed by qPCR using TransStart® Green qPCR SuperMix (AQ101-01, Transgen). All results were normalized against β-actin expression, and all fold changes were determined using the 2−ΔΔCT method. The primers are listed in Table 2.

Table 2 Primers used for RT–qPCRChromatin immunoprecipitation (ChIP)–qPCR

Pierce™ Agarose ChIP Kit (26156, Thermo Fisher) was used as per the kit’s instructions to analyze the binding relationship between CEBPB and BZW1. Briefly, BMDM were cross-linked with 1% formaldehyde, and cross-linking was blocked by glycine. The chromatin/DNA complexes were sheared by ultrasonic fragmentation, and the fragmented fractions were incubated with magnetic beads and IP grade CEBPB antibody (1:100, GTX15050, GeneTex) overnight at 4 °C. The elution buffer was used to elute the immunoprecipitated complexes from the magnetic beads before incubation with NaCl at 65 °C for decrosslinking. The cross-linked DNA was purified and extracted for analysis by qPCR after the completion of the crosslinking. qPCR was performed using the following primers: 5′-GCCAGCTCTCAGTTTTGCAT-3′ and 5′-CCGTTCTTTGCATCTCCCAC-3′.

Dual-luciferase reporter assay

In the University of California Santa Cruz (UCSC) database (http://genome.ucsc.edu/index.html), we obtained the promoter fragment of BZW1 and cloned the fragment of the BZW1 promoter into the pGL3-basic firefly luciferase reporter vector (E1571, Promega Corporation, Madison, WI, USA) using restriction endonuclease. The reporter vector was transfected into BMDM overexpressing CEBPB using Lipofectamine 2000. After 48 h of incubation, luciferase reporter gene activity was measured using the Dual-Luciferase® Reporter Assay System (E1910, Promega).

Statistics

Results were expressed as mean ± standard deviation (SD). Statistical significance was calculated by performing an unpaired t-test, one-way ANOVA, or two-way ANOVA, followed by Tukey's post-test, as appropriate. A p-value of < 0.05 was considered statistically significant. GraphPad Software (8.0, San Diego, CA, USA) was used for the statistical analyses. The in vitro experiments were repeated at least three times.