Introduction:
IgA nephropathy (IgAN) has been considered as an immune-complex-mediated disease but its pathology has not been fully elucidated. We recently identified mesangial-cell-specific autoantigens (β2 spectrin and CBX3) of the disease (Nihei Y et al. Sci Adv. 2023 Mar 22;9(12), Higashiyama M et al. Life Sci Alliance. 2024 Feb 8;7(4)). Both of the autoantigens are originally known for their intracellular existence, and careful examinations are required to detect them on mesangial surfaces. In present study, we utilized immunofluorescent microscopy (IFM) and immune electron microscopy (IEM) to examine their expression on human primary mesangial cells (HMCs).
Methods:
For IFM, cells were cultured on cover glasses for 48 hours. They were fixed with either 2% paraformaldehyde (PFA) (for cell-surface staining) ± 0.4%TritonX-100 (for intracellular staining) or methanol alone (for intranuclear staining) and stained with anti-β2 spectrin or CBX3 antibody and with DAPI. For IEM, 48-hour-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:
We firstly examined whether our IFM method differentiates intracellular and extracellular antigens. In our previous studies, we found by flow cytometry that 20-30% of HEK293T cells express β2 spectrin and almost none of them express CBX3 on their surfaces. We performed the method with HEK293T cells and around 25% or very few of the 2%PFA-fixed cells were positively immunostained with anti-β2-spectrin or CBX3 antibody respectively. All the cells were also positively immunostained with anti-β2-spectrin or CBX3 antibody after 2%PFA plus 0.4%TritonX-100 treatment or methanol treatment, which verifies that our antibodies work with their antigens. These data certify that our method allows antibodies to immunostain cell-surface proteins. We then immunostained HMCs with this method and detected the expression of β2 spectrin and CBX3 on their surfaces. To further confirm the results, we performed IEM and proved their expression on the surface of HMCs.
Conclusions:
We have established the immunostaining methods to detect cell-surface proteins and proved the surface expression of the IgAN autoantigens (β2 spectrin and CBX3) on HMCs in vitro. We will utilize these methods to investigate the mechanisms of their MC-surface expression and their clinical influence on IgAN.
The content presented in this abstract was originally submitted in Kidney Week 2024. I declare that re-submitting the abstract is not prohibited by the organizers of the original meeting.
I have no potential conflict of interest to disclose.
I did not use generative AI and AI-assisted technologies in the writing process.