Patients and specimen collection

Peripheral blood samples of 15 patients diagnosed with MPO-ANCA-associated vasculitis [30, 31]at the Children's Hospital of Chongqing Medical University/The Second Affiliated Hospital of Chongqing Medical University from 2022 to 2023 were included for single-cell RNA sequencing. Peripheral blood samples from 3 age-matched, healthy children served as controls. The peripheral blood samples in this study were leftover from clinical testing. At the time of sampling, all patients had no serious infection or other diseases such as SLE. Additionally, single cell spatial transcriptome sequencing was conducted on remaining kidney biopsy samples from 3 untreated AAGN patients [32, 33] and three paracancerous kidney tissues used as controls, collected at the Children's Hospital of Chongqing Medical University (2023–2024). For immunological staining, remaining sections from kidney biopsies of patients with AAGN, IgAN, LN and IgAVN were collected, with sections from 2 paracancer patients and 2 MCD patients included as controls. Informed consent was obtained from all participants. The study adhered to the Declaration of Helsinki and received approval from the Ethics Committee of the Children's Hospital of Chongqing Medical University (NO.2022–149/NO.2024–189). Detailed sample collection and classification information were presented in Supplementary Fig. 1 and Tables 1, 2, 3, 4, 5 and 6. To clarify identification and readability, this article does not highlight that PR3 + AAGN all refers to MPO + patients.

Table 1 Baseline characteristics of MPO + AAGN patientsTable 2 Baseline characteristics of IgAVN patientsTable 3 Baseline characteristics of LN patientsTable 4 Baseline characteristics of IgAN patientsTable 5 Baseline characteristics of PR3 + AAGN patientsTable 6 Clinical characters of patients performing single-cell RNA sequencingRNA and spatial transcriptome sequencing

Peripheral blood samples (2 mL) underwent red blood cell lysis, and the resulting single-cell suspension was loaded onto a microfluidic device. Single-cell RNA sequencing (scRNA-seq) libraries were prepared using the GEXSCOPE Single-Cell RNA Library Kit (Singleron Biotechnologies, Nanjing, China), followed by RNA sequencing. For spatial transcriptome sequencing, renal biopsy tissues were washed with PBS, digested using GEXSCOPE® tissue dissociation solution (Singleron, Cat#1020012) at 37 °C for 20 min, then filtered and centrifuged. Tissue sections were processed and imaged using the 10X Genomics Visium Spatial Gene Expression Solution.

Immunofluorescence, immunohistochemistry and PAS staining analysis

To validate Mφ polarization via immunofluorescence (IF), tissue sections were incubated at 4℃ for 14 h with anti-CD163 (1:100, ab156769, Abcam, Cambridge, UK) and anti-CD86 (1:200, A1199, ABclonal, Wuhan, China) antibodies. For monocyte infiltration validation, sections were incubated with anti-CD14 antibody (1:100, 17000–1-AP, Proteintech, Wuhan, China) at 4℃ for 16 h. To verify endothelial cell apoptosis, tissue sections were incubated with anti-CD31 (1:100, A19014, ABclonal) and anti-ERG (1:50, sc-376293, Santa Cruz, Dallas, USA) antibodies at 4℃ for 14 h, followed by TUNEL staining at 37℃ for 50 min (One-step TUNEL In Situ Apoptosis Kit, E-CK-A325, Elabscience, Wuhan, China). For CXCR4 expression validation in Mφ, sections were incubated with anti-CXCR4 (5 μg/mL, ab124824, Abcam) and anti-CD68 (1:20, sc-20060, Santa Cruz) antibodies at 4℃ for 16 h. After a 1-h incubation with secondary antibody, slices were stained with DAPI (1:1000, C1002, Beyotime, Shanghai, China) and sealed with antifade mounting medium. Images were captured using a Nikon laser confocal microscope (A1R, Nikon, Tokyo, Japan). For immunohistochemistry (IHC), sections were incubated respectively with anti-TGF-β1 (1:500, ab215715, Abcam), anti-α-SMA (1:1000, ab5964, Abcam) and anti-CXCL12 (2 μg/mL, ab9797, Abcam) antibodies. PAS staining (G1281, Solarbio, Beijing, China) was applied to IHC sections with DAB color development (PV-9001, ZSGB-BIO, Beijing, China) and to IF sections for imaging. Images were captured using a digital pathology scanner (SQS-600P, Shengqiang Technology CO, Ltd, Shenzhen, China). Analysis of glomerular crescents were determined using ImageJ.

ELISA

The supernatant was centrifuged at 1000 g for 15 min, subpackaged, and frozen at −80℃. CXCL12 (CSB-EQ027490HU for human, CSB-E08729r for rat, Cusabio, Wuhan, China) and soluble CXCR4 (CSB-E12825 h for human, CSB-E12703r for rat, Cusabio) in serum and urine were measured with ELISA kits following the manufacturer’s instructions.

Cell culture and stimulation

The endothelial cell line EA.hy926, the monocyte line THP-1, the tubular epithelial cell line HK-2 (Cell Bank/Stem Cell Bank, Chinese Academy of Sciences, Shanghai, China), the mesangial cell line HRMC and the podocyte line HPC (Procell, Wuhan, China) were respectively cultured in medium with 10% fetal bovine serum (FBS) and 1% penicillin–streptomycin (Gibco, Grand Island, NY, USA) at 37 °C with 5% CO2. EA.hy926, HRMC, HPC, HK-2, and THP-1 cells were starved in serum-free medium for 12 h, then stimulated with 10% serum form AAGN patients (mixed with 5 MPO + AAGN patients) for 3 days. THP-1 were also stimulated with PMA (150 ng/mL, Sigma-Aldrich, Darmstadt, Germany) for 48 h to induce adherent Mφs, followed by stimulation with 10% AAGN patient serum for 3 days for polarization induction experiments. The remaining serum samples from 5 healthy children were mixed for use as a control group stimulant.

Transwell assay

EA.hy926 (105/well) were seeded in 24-well plates with cell slides and complete medium. Once attached, they were starved in serum-free medium for 12 h, then exposed to AAGN patient or healthy control serum for 3 days. THP-1 cells were incubated in serum-free medium for 12 h, then added to the upper Transwell chambers (Corning, NY, USA) at 5*104/well. The upper chambers were placed in 24-well plates to co-culture with EA.hy926 for 8 h in fresh AAGN patient or healthy control serum medium. Cells were fixed with 4% paraformaldehyde, incubated overnight with anti-CD14 and anti-ERG antibodies (both 1:50), and imaged using a Nikon 90i upright fluorescence microscope (Tokyo, Japan).

CCK8 assay

EA.hy926 were seeded in 96-well plates and cultured in 10% AAGN patient serum once they adhered. Every 12 h, cells were incubated for 2 h in a CCK-8 reaction mixture (10μL CCK-8: 90μL serum-free medium) (CCK-8, TOPSCIENCE, Shanghai, China) before measuring the optical density (OD) value at 450 nm. THP-1 cells were cultured in 96-well plates and transformed into adherent Mφs using PMA. 10% AAGN patient serum medium containing LIT927 (a CXCL12 neutral ligand antagonist, Selleck, Shanghai, China) or AMD 3100 (a CXCR4 receptor inhibitor, Selleck) were added to culture cells for 3 days. The CCK-8 assay was conducted for 2 h to determine the optimal drug concentration for stimulating Mφs with AAGN patient serum in vitro, using DMSO as a control.

Cell apoptosis assay by flow cytometry

EA.hy926 cells were cultured in 6-well plates with 10% AAGN patient serum for 3 days. After trypsinization and a 2000 rpm centrifuge for 5 min, cells were washed with ice-cold PBS, and re-suspended in 300μL of 1 × Binding Buffer. They were subsequently incubated with 5μL of Annexin V-FITC (BD Pharmingen™ FITC Annexin V Apoptosis Detection Kit I, BD Biosciences, San Jose, USA) in the dark for 15 min, followed by 5μL of PI 5 min before analysis. Stained cells were examined using a FACSCanto plus flow cytometer (BD) and data were analyzed with FlowJo software.

Mφ efferocytosis

THP-1 induced Mφs, stimulated with AAGN patient serum for 3 days, were placed on cell slides for future use. Jurkat cells (Cell Bank/Stem Cell Bank, Chinese Academy of Sciences) were first exposed to 254 nm UV light for 15 min to induce apoptosis [34]. These apoptotic Jurkat cells were then labeled with 5μL of Vybrant™ DiD solution (Invitrogen, Carlsbad, USA) at a cell concentration of 2*107/mL for 10 min, and washed twice with PBS before being resuspended for later use. Mφs were co-incubated with stained Jurkat cells at a ratio of 1:5 for 2 h, then unengulfed cells were washed off by PBS. After fixation and anti-CD68 antibody incubation, images were collected using an upright fluorescence microscope.

siRNA knockdown

Small interfering RNAs (siRNAs) targeting CXCR4 and ELMO1 were designed and synthesized by Sangon Biotech (Shanghai, China). For each gene, three siRNA sequences were screened, and the one achieving the most efficient knockdown (> 50% reduction in target mRNA expression) was selected for subsequent experiments. The sequences used are listed in Table 7. Transfection was performed using Lipofectamine RNAiMAX (Thermo Scientific, Waltham, MA, USA) according to the manufacturer's protocol. THP-1 cells were cultured in serum-free medium for 12 h to induce a starvation state prior to transfection. siRNA was then added to initiate knockdown. After 12 h of transfection, the medium was replaced with fresh medium containing 10% FBS and cells were cultured for an additional 36 h, resulting in a total knockdown period of 48 h. Following knockdown, THP-1 cells were differentiated into Mφs. These Mφs were subsequently stimulated with serum from either patient or control groups. After stimulation, total RNA or protein was extracted for downstream analyses.

Table 7 Sequence of siRNAsQuantitative real-time PCR

Total RNA of cells was extracted with a SteadyPure quick RNA extraction kit (Accurate Biology, Changsha, China), then reverse transcribed into cDNA using a HiScript III 1 st Strand cDNA Synthesis Kit (Vazyme, Nanjing, China). The cDNA was amplified with specific primers and the SYBR Green (Accurate Biology, Changsha, China). Primer sequences are listed in Table 8.

Table 8 Primer sequences for PCR analysesProtein extraction and western blotting

Cells were lysed by RIPA (NCM Biotech, Suzhou, China), and the supernatant was centrifuged at 14000 g for 15 min, mixed with loading buffer, and heated at 99 °C for 10 min. Proteins were separated by polyacrylamide gel electrophoresis, transferred to PVDF membranes, and incubated with primary and secondary antibodies. Bands were exposed using a SuperSignal™ West Femto Maximum Sensitivity Substrate kit (Thermo Scientific, Waltham, USA). Antibodies are listed in Table 9.

Table 9 Antibodies used for western blotting and co-immunoprecipitationCo-immunoprecipitation of CXCR4 and ELMO1

THP-1 induced Mφs, after 3 days of FBS/healthy controls/AAGN serum stimulation, were lysed by IP Lysis Buffer (Thermo Scientific). The lysate, containing 2 mg of protein, was incubated with specific primary antibodies for 12 h at 4 °C to form antigen–antibody complexes. These complexes were then combined with Protein A/G magnetic beads (Selleck) and mixed overnight at 4 °C. The resulting immunoprecipitates were washed and subjected to western blot analysis.

Cycloheximide chase assay to examine protein half-life

After transfecting THP-1 cells with siCXCR4, they were differentiated into Mφs by PMA and polarized with AAGN patient serum for 2 days. On the third day, CHX (50 μg/mL, Selleck) was added [35], and Mφs were collected at various intervals to analyze ELMO1 protein expression via western blotting.

Animal model and medical treatments

Four-week-old female WKY rats were obtained from Vital River Laboratory Animal Technology Co. Ltd in Beijing, China. To induce the experimental autoimmune vasculitis (EAV) model, 1600 μg/kg of human MPO (hMPO, Elastin Products Company, Owensville, USA) in Freund’s complete adjuvant were injected intramuscularly and subcutaneously into rats (n = 24), along with twice intraperitoneal injection of 1 μg Pertussis toxin (PTX, List Biological Labs, Campbell, USA) post immunization. Rats immunized with equivalent human serum albumin (HSA, Sigma-Aldrich) served as control groups (n = 8) [36, 37]. The EAV rat models was further divided into two groups to start drug treatment experiments: the day after the end of PTX injection (0 W groups) and the 15 th day after the end of PTX injection (3 W groups). EAV rats received daily intraperitoneal injections of either LIT927 (100 mg/kg) or AMD3100 (5 mg/kg) with 0.5% sodium carboxymethyl cellulose (CMC, Sigma-Aldrich), for 5 consecutive days each week [38]. EAV rats received 0.5%CMC as a negative control, while HSA-injected rats received the same CMC amount as a normal control. Weekly, their body weight was recorded, and urine was collected for hematuria and proteinuria tests. All rats were sacrificed 8 weeks after modeling by inhaling carbon dioxide. The study was approved by Laboratory Animal Ethics Committee of Children's Hospital of Chongqing Medical University (IACUC Issue NO: CHCMU-IACUC20240617004).

Urine analysis of EAV rats

Hematuria was semi-quantitatively evaluated (0–4) with a dipstick (Mission, Hangzhou, China), and proteinuria was assessed by an automatic urine biochemical analysis system (Cobas c701, Roche, Basel, Switzerland).

Renal function analysis of EAV rats

Serum was collected from the rats before sacrifice, centrifuged at 1000 × g for 15 min, and analyzed for creatinine (Scr) and urea nitrogen (BUN) using an automated blood biochemical system (Cobas 8000, Roche, Basel, Switzerland).

Histology staining of rats

Rat lung and kidney tissues were fixed with 4% paraformaldehyde, embedded in paraffin, and sectioned to 4 µm and 3 µm thicknesses respectively. A prussian blue staining kit (Solarbio) confirmed pulmonary hemorrhage in rats. Hemosiderin cell counts were averaged from three random visual fields per rat. Additionally, one lung was visually inspected for pulmonary vasculitis according to the amount of lung surface bleeding: 0 = no hemorrhage; 1 = a single hemorrhage; 2 = 2–5 hemorrhage; 3 = 6–12 hemorrhage and 4 ≥ 12 hemorrhage [37]. After PAS staining, the number of glomeruli in the section were counted, and the percentage of cellular crescents and cellular fibrous with fibrous crescents in all glomeruli was recorded.

Statistical analysis

All experiments were repeated at least three times, with results shown as mean ± SEM or median and interquartile range. Statistical analysis was performed using Graphpad Prism9 (GraphPad Software, La Jolla, CA, USA). Differences between two groups were assessed with Student’s t-test, while one-way ANOVA was used for multiple groups. Significance was set at P < 0.05, with levels indicated as *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001.