Abstract: FR-PO822
Identification of the Localization of the Pathogenic IgA Autoantibodies on Mesangial Cells in IgA Nephropathy
Session Information
- Glomerular Diseases: Inflammation and Immunology
October 25, 2024 | Location: Exhibit Hall, Convention Center
Abstract Time: 10:00 AM - 12:00 PM
Category: Glomerular Diseases
- 1401 Glomerular Diseases: Mechanisms, including Podocyte Biology
Authors
- Mori, Kazuaki, Department of Nephrology, Juntendo University Faculty of Medicine, Bunkyo, Tokyo, Japan
- Nihei, Yoshihito, Department of Nephrology, Juntendo University Faculty of Medicine, Bunkyo, Tokyo, Japan
- Suzuki, Hitoshi, Department of Nephrology, Juntendo University Faculty of Medicine, Bunkyo, Tokyo, Japan
- Suzuki, Yusuke, Department of Nephrology, Juntendo University Faculty of Medicine, Bunkyo, Tokyo, Japan
Group or Team Name
- Dept of Nephrology, Juntendo University Faculty of Medicine.
Background
IgA nephropathy (IgAN) has been considered as an immune complex-mediated disease, and its possible existence of autoantigens has not been clarified. Recently, we discovered IgA autoantibodies specific to mesangial cells (MCs) in the sera of its patients. We identified two autoantigens targeted by these IgA (β2-spectrin and CBX3), both of which are originally known for their intracellular existence. Although in vivo studies with monoclonal antibodies against the autoantigens revealed their selective expressions on MC surfaces, their mechanisms have not been elucidated. To investigate them, we aimed to establish methods to visualize the MC-surface expression of these autoantigens in vitro.
Methods
For immunofluorescence microscopy (IFM), human primary MCs (HMCs) were cultured on cover glasses for 24 hours. The HMCs were fixed with methanol for intracellular protein staining or 4% paraformaldehyde (4%PFA) for cell-surface protein staining and stained with anti-β2 spectrin or CBX3 antibody and with DAPI. For immune electron microscopy (IEM), 24h-cultured HMCs were fixed with 4%PFA and stained with anti-β2 spectrin or CBX3 antibody and then with colloidal gold. To perform transmission electron microscopy, these cells were fixed with 2.5% glutaraldehyde in 0.1M phosphate buffer and then with 2% OsO4 in the same buffer followed by dehydration with a graded series of ethanol and embedded in Epok-812. Ultrathin sections were removed, and uranyl acetate and lead citrate were applied.
Results
By IFM analyses after application of methanol on the HMCs, we detected the intracellular expression of β2 spectrin and CBX3. And then by IFM analyses after application of 4%PFA on the HMCs, cell-surface β2 spectrin and CBX3 were detected. To elucidate their locations either on the cell surfaces or inside of the cells, we utilized IEM and clearly visualized their cell-surface locations with continuous cell membranes.
Conclusion
We identified the localization of β2 spectrin and CBX3 on the surfaces of HMCs by both IFM and IEM. We will utilize these methods to investigate the mechanisms of the MC-surface expression of each of these autoantigens and their relations with clinical features of IgAN.