Fig 1: Analysis of IgG subclasses and complement deposition in experimental autoimmune MN.a mIgG subclass-specific anti-THSD7A antibody levels over time as measured by ELISA in controls (n = 3) and THSD7A-immunized mice (n = 10). Data are presented as mean and SEM. b Representative immunofluorescence stainings for mIgG1, mIgG2a, mIgG2b, and mIgG3 in controls (n = 5) and THSD7A-immunized mice (n = 10). Bars 20 µm. c Quantitative analysis (histological score) of glomerular IgG subclass deposition in control and THSD7A-immunized mice. Data are presented as mean and SEM. d Representative immunofluorescence staining of complement C1q in co-localization with mouse IgG (mIgG) in THSD7A-immunized mice (n = 4). Bar 20 µm. The lower image is an enlargement of the boxed area in the upper image. e Representative immunofluorescence stainings for complement C1q, C4d, CFB, CFH, C3, and C5b-9 in co-localization with wheat germ agglutinin (WGA) and MBL in co-localization with nephrin in control (n = 5) and THSD7A-immunized mice (n = 10). Bars 4 µm. f Quantitative analysis (histological score) of glomerular C5b-9 deposition in control (n = 5) and THSD7A-immunized mice (n = 10). Data are presented as mean and SEM. g Correlation analyses of the mIgG1, mIgG2a, mIgG2b and mIgG3 scores with the C5b-9 score and correlation of the cumulative mIgG2a, mIgG2b, and mIgG3 (referred to as mIgG2-3) score with the C5b-9 score in THSD7A-immunized mice (n = 10). ns, not significant. Spearman’s r correlation coefficient (two-tailed). h Correlation analysis of C5b-9 scores with individual albuminuria values as measured by the area under the curve (AUC) per week in THSD7A-immunized mice (n = 10). Spearman’s r correlation coefficient (two-tailed).
Fig 2: Complement is dominantly activated via the classical pathway in patients with MN.a Representative images of proximity ligation assays showing the alternative convertase C3bBb (left) and the classical/lectin convertase C4bC2b (right) performed on biopsies from patients with MN (n = 39). Bars 50 µm. b Detection of C3bBb, C4bC2b and IgGC1q using proximity ligation assays as well as of MBL/IgG in immunofluorescence and proximity ligation assay. c Representative images of proximity ligation assays showing the assembly of IgGC1q (left) and IgGMBL (right) performed on biopsies from patients with MN (n = 39 and n = 26 for IgGC1q and IgGMBL, respectively). Bars 50 µm. d Quantitative analysis of proximity ligation assay signals in biopsies from patients with MN and time point zero biopsies from renal transplant recipients. Data are presented as mean and SEM. C3bBb, *p = 0.0442; C4bC2b, ****p < 0.0001; IgGC1q, ***p = 0.0005; IgGMBL, ***p = 0.0003 (two-tailed Mann–Whitney test). e Representative immunohistochemical stainings for the IgG subclasses IgG1, IgG2, IgG3, and IgG4 in a biopsy sample from a patient with MN (n = 39). Bars 50 µm. f Quantitative analysis (histological score) of IgG subclass signals in 39 biopsies from patients with MN and 3 time point zero biopsies from renal transplant recipients. Data are presented as mean and SEM. g Detection of IgG1, IgG2, IgG3, and IgG4 as well as at least one of the C1q-binding subclasses IgG1, IgG2, or IgG3 in biopsies from patients with MN. h, i Correlation of the cumulative IgG1-3 score with IgGC1q proximity ligation assay signals (h) and IgGC1q signals with the C4bC2b signals (i). Spearman’s r correlation coefficient (two-tailed).
Fig 3: Complement components from all three complement activation pathways can be detected in biopsies from MN patients.a–d Representative immunofluorescence (IF) stainings (paraffin-embedded tissue) for C3b and CFB (a), C4b and C2 (b), MBL and IgG (c), and C1q and IgG (d) in co-localization with the glomerular basement membrane constituent collagen IV in biopsies from MN patients (n = 5) and controls (n = 4). The lower panels represent 5-fold enlargements of the boxed areas in the upper panels. Bars 50 µm. e Representative IF stainings (frozen tissue) for C1q in co-localization with collagen IV in biopsies from patients with MN (n = 6), patients with lupus nephritis (LN) class V (n = 2), and patients with minimal change disease (MCD, n = 2) using conventional indirect IF (upper panels) and IF after antigen retrieval with methanol and trypsin (lower panels). Panels on the right represent 5-fold enlargements of the boxed areas in the left panels. Bars 50 µm.
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