Abstract: TH-PO0274
A Human Mesangial-Cell Platform to Evaluate C3 Deposition and Complement Inhibition in C3 Glomerulopathy
Session Information
- Glomerular Diseases: Cell Biology
October 22, 2026 | Location: Exhibit Hall A, Convention Center
Abstract Time: 10:00 AM - 12:00 PM
Category: Glomerular Diseases
- 1401 Glomerular Diseases: Mechanisms, including Podocyte Biology
Authors
- Snyder, Madeline, Immunoregulation Section, National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK), Bethesda, Maryland, United States
- Tajmul, Md, Immunoregulation Section, National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK), Bethesda, Maryland, United States
- Portilla, Didier, Center for Immunity, Inflammation and Regenerative Medicine (CIIR), Division of Nephrology, Department of Medicine, University of Virginia, Charlottesville, Virginia, United States
- Kolev, Martin, Apellis Pharmaceuticals Inc, Waltham, Massachusetts, United States
- Afzali, Behdad, Immunoregulation Section, National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK), Bethesda, Maryland, United States
Background
Complement activation converges on C3, whose cleavage and downstream proteolytic processing generate bioactive fragments including C3a, C3b, iC3b, C3c, C3dg, and C3d that mediate inflammation, opsonization, and receptor signaling. C3 glomerulopathy (C3G) is driven by dysregulated alternative-pathway activation and glomerular accumulation of C3 fragments. Although complement inhibitors are emerging therapies for C3G, their effects on C3 deposition, clearance, and downstream processing remain incompletely defined. This gap is compounded by the broad reactivity of many commercial anti-C3 antibodies and by the lack of simple human glomerular-cell systems for pharmacologic testing. We aim to establish an in vitro system to evaluate the effects of complement inhibitors on C3 fragment deposition, clearance, and downstream processing, leveraging a combinatorial antibody detection approach.
Methods
Antibodies were tested by Western blot and direct ELISA against recombinant complement C3, C3a, C3b, iC3b, C3c, and C3d, with bovine serum albumin as a negative control. Epitope recognition and binding affinity were assessed for individual antibodies and antibody combinations. Immortalized human mesangial cells were cultured according to established protocols. Twenty-four hours prior to imaging, cells were supplemented with 10% C3/C5-depleted human serum and 1 mg/mL purified human C3. C3 deposition was assessed by confocal microscopy.
Results
Individual anti-C3 antibodies showed broad reactivity across multiple C3 fragments, indicating that single-antibody staining is insufficient to assign deposited C3 species. In contrast, staining with combinations of anti-C3 antibodies distinguished C3d-containing from C3c-containing fragments. Direct ELISA demonstrated nanomolar binding affinities, but affinity did not predict fragment specificity. In parallel, immortalized human mesangial cells provided a tractable glomerular-cell platform in which C3 deposition was detectable under C3-supplemented conditions.
Conclusion
We developed and characterized a human mesangial-cell assay, paired with combinatorial C3-fragment antibody detection, to study C3 deposition and processing relevant to C3G. This platform will enable pharmacologic testing of complement inhibitors to determine how pathway blockade alters C3 deposition, clearance, and fragment composition on glomerular cells.
Funding
- NIDDK Support – Apellis Pharmaceuticals