Patients
Renal biopsy specimens of 60 patients with ANCA associated GN were collected from March 2014 to July 2018. Blood samples of 63 patients with ANCA associated GN were also collected from January 2021 to January 2022. They were tested positive for p-ANCA or c-ANCA by indirect immunofluorescence and had a positive test for MPO-ANCA or PR3-ANCA by antigen-specific enzyme-linked immunosorbent assay (ELISA) and all met the Chapel Hill Consensus Conference (CHCC) definition of AAV[11]. Disease activity in vasculitis patients was measured using the Birmingham Vasculitis Activity Score (BVAS) 2003[12]. Remission was defined as the absence of clinical signs of the disease activity, as indicated by a BVAS of 0. Patients with secondary vasculitis, co-existence of other autoimmune diseases were excluded. 37 blood samples of healthy people were collected as controls. Normal parts of 10 nephrectomized kidneys caused by renal carcinoma were obtained and confirmed as normal renal controls by using light microscopy, immunofluorescence and electron microscopy. All medical records were reviewed regarding demographic, clinical, biological, and histological findings. The study was approved by the ethics committee of West China Hospital of Sichuan University and the written informed consent was obtained from each participant.
Evaluation of renal histology
According to previous standardized protocol[13–15], the renal histology of patients with AAV-associated GN was assessed by lesions of glomeruli and tubulointerstitial compartment. The percentages of glomeruli with crescent, glomerulosclerosis and fibrinoid necrosis were recorded. The interstitial and tubular lesions were scored semi-quantitatively based on the percentage of the tubulointerstitial compartment that was affected: interstitial infiltrate (sores 0, 1, 2, 3 for 0, 0–20, 20–50 and > 50%, respectively), interstitial fibrosis (scores 0, 1, 2 for 0, 0–50% and > 50%, respectively) and tubular atrophy (scores 0, 1, 2 for 0, 0–50% and > 50%, respectively).
Evaluation of Leucocytes Surface MAC-1 and LFA-1 by Flow Cytometry
We investigated the surface expression of MAC-1 and LFA-1 on neutrophils, monocytes, and lymphocytes (T cells and B cells) using FACS analysis. FACS analysis of the cells in 150 µL of EDTA-anticoagulated blood samples was performed. Erythrocyte lysis buffer (555899, BD Biosciences, San Jose, CA, USA) was used to remove erythrocytes from the blood cells. For gating purposes, the neutrophil, monocytes, T cells and B cells population were separately verified by staining with an anti-CD16b-PE antibody (Clone CLB-gran11.5; BD Biosciences, San Jose, CA, USA), an anti-CD14-BUV395 antibody (Clone MPφ9; BD Biosciences, San Jose, CA, USA), an anti-CD3-APC-Cy7 antibody (Clone SK7; BD Biosciences, San Jose, CA, USA) and an anti-CD19-APC antibody (Clone HIB19; BD Biosciences, San Jose, CA, USA). To investigate LFA-1 and MAC-1on white blood cell surface, anti-CD11a- PE-Cy™7 antibody (Clone HI111; BD Biosciences, San Jose, CA, USA) and anti-CD11b-FITC antibody (Clone M1/70; BD Biosciences, San Jose, CA, USA) were used. Cell suspensions were analyzed by flow cytometry (BD Bioscience, FACSymphony A5). All white blood subpopulation cells were gated firstly using FSC-H/FSC-A parameters, then SSC-A/FSC-A parameters, and finally, on the basis of expression of their specific antibody separately. Manual data analysis including median fluorescence intensity, cell counts and cell percentage was performed using the FlowJo software.
Detection of LFA-1 and MAC-1 expression in kidneys by immunohistochemistry
To clarify the expression of LFA-1 and MAC-1 in kidneys of patients with AAV, immunohistochemical staining was performed for CD11a and CD11b on 4µm paraffin sections of formaldehyde-fixed kidney tissue of patients with ANCA-associated GN as previously described[11]. Briefly, after deparaffinization, renal sections were immersed in 3% hydrogen peroxide for 30 min at room temperature. For antigen retrieval, the sections for CD11a and CD11b were heated respectively in citrate acid buffer (pH 6.0) and ethylenediamine tetraacetic acid (EDTA) buffer (pH 8.0) in an 800 W microwave oven twice with 8 min each time. Following antigen retrieval, tissue sections were blocked in PBS containing 5% normal bovine serum and incubated overnight at 4°C with rabbit anti-human CD11a polyclonal antibodies (Bioss, Beijing, China; bs-1804R) and rabbit anti-human CD11b monoclonal antibodies (Abcam, Cambridge, UK; ab1344). After washing with PBS, the tissue sections were incubated with secondary antibodies (goat anti-rabbit; Jackson ImmunoResearch, West Grove, PA, USA) for 30 min at 37°C. After washed by PBS, the sections were covered with fresh hydrogen peroxide plus 3–3’-diaminobenzidine tetrahydrochloride solution for 30 s. Finally, the sections were counterstained with hematoxylin and dehydrated through alcohol and xylene. Renal tissues from the normal part of nephrectomized kidneys due to renal carcinoma were used as normal controls. Tonsil slides were used as positive controls for CD11a and CD11b. As negative controls, the primary antibodies were substituted by normal rabbit IgG. The sections were examined by light microscopy.
Evaluation of immunohistochemical staining
All the images of the glomeruli and the tubulointerstitial compartment in slides of kidney sample from ANCA-associated GN patients were acquired, and at least 15 randomly images of the tubulointerstitial compartment in kidneys from the normal controls were taken for a semiquantitive assessment of immunohistochemical staining. The staining of CD11a and CD11b were evaluated by using Image Pro Plus analysis software version 6.0 for each image. Positive signals were quantified as the mean optical density (integrated option density/area).
Detection of LFA-1 and MAC-1 expression on various cell types
In order to determine on which cell type of kidney tissue LFA-1 and MAC-1 were expressed, we performed double staining of the two integrins and specific markers of various cell types, including glomerular intrinsic cells and infiltrating cells. The staining of glomerular intrinsic cells was as follows: glomerular endothelial cells were identified by immunofluorescence staining with primary antibodies against CD31 (R&D Systems, Minneapolis, MN, USA; AF3628), and mesangial cells were identified by staining with primary antibodies against α-smooth muscleactin (α-SMA; Abcam; ab5694) and podocytes were identified by staining with primary antibodies against podocalixin (R&D Systems; MAB1658). The staining of infiltrating cells was as follows: neutrophils were identified by immunofluorescence staining with antibodies against CD16b (Abcam, ab183354), lymphocytes were identified with antibodies against CD3 (Abcam, ab16669) and monocytes/ macrophages were identified with antibodies against CD68 (Abcam, ab955).
After the paraffin sections of kidney tissue were deparaffinized, rehydration, antigen retrieval, and bovine serum block was performed as mentioned above, we incubated the specimens overnight with the first primary antibody at 4°C. After washing with PBS, the corresponding secondary antibody was applied and incubated at 37 ℃ for 1 h. Sections were then incubated overnight with the second primary antibody at 4°C. After washing with PBS, the corresponding secondary antibody was applied and incubated at 37 ℃ for 1 h. The samples were washed with PBS, stained with 40,6-diamidino-2-phenylindole (DAPI) (Zhongshan Golden Bridge Biotechnology, Beijing, China) and mounted with cover clips. In negative controls, primary antibodies were replaced by PBS. Secondary antibodies (Jackson ImmunoResearch; no. 115-605-003, 115-025-003, 111-605-003, 111-025-003 and 305-025-003) matched with a corresponding primary antibody were used to display fluorescent signals. The representative images were captured with a Zeiss Axio Imager Z2 microscope (Zeiss, Jena, Germany). Images were exported from the ZEN 2012 (blue edition) microscopy software.
Statistics
For normally distributed data, quantitative parameters were presented as mean ± standard deviation and compared with t-test between groups. For non-normally distributed data, quantitative parameters were presented as median and interquartile range (IQR), and compared with the Mann-Whitney U test between groups. Categorical data was presented as the frequency (%). We analyzed the association between two continuous variables using Pearson’s correlation (for two parametric variables) or Spearman’s rank correlation (for two non-parametric variables, or one non-parametric variable with one parametric variable). Spearman’s correlation was used to analyse the association between two categorical variables. A P-value of 0.05 or less was considered statistically significant. All analyses were performed with SPSS version 22.0 (SPSS Inc., Chicago, IL, USA).