Abstract: TH-PO752
Podocyte Injury in Human Primary Membranous Nephropathy: Evidence Supporting a Role for Complement
Session Information
- Glomerular Diseases: Podocyte Biology - I
November 02, 2023 | Location: Exhibit Hall, Pennsylvania Convention Center
Abstract Time: 10:00 AM - 12:00 PM
Category: Glomerular Diseases
- 1403 Podocyte Biology
Authors
- Bruno, Valentina, The Hospital for Sick Children, Toronto, Ontario, Canada
- Ortiz-Sandoval, Carolina G., The Hospital for Sick Children, Toronto, Ontario, Canada
- Moran, Sarah Margaret, University College Cork, Cork, Cork, Ireland
- Cattran, Daniel C., Toronto General Research Institute, Toronto, Ontario, Canada
- Licht, Christoph, The Hospital for Sick Children, Toronto, Ontario, Canada
Background
Primary membranous nephropathy (PMN) is the leading cause of nephrotic syndrome in adults and a common cause of end-stage kidney disease (ESKD). The Heymann’s nephritis rat model of PMN shows that proteinuria is complement-mediated. However, the pathogenetic role of complement in human PMN remains unclear. We aim to demonstrate that complement activation can have both structural and functional effects on podocytes.
Methods
An in-vitro model of immortalized human podocytes (from Moin Saleem, Bristol, UK) was used for all the experiments. Cells were exposed to 25% serum of 19 PMN patients (from the Toronto Glomerulonephritis Registry) . Complement deposition was detected by immunofluorescence (IF). As positive control, cells were pre-sensitized with anti-CD59 and exposed to 25% normal human serum (NHS) to induce complement activation. Changes in intracellular calcium were monitored using a fluorescent dye (Fluo 8-AM), acquiring images every 20 seconds (up to 10 minutes) by confocal microscopy. Calcium effects on mitochondrial membrane potential were measured by flow cytometry. Intracellular adenosine triphosphate (ATP) changes were analyzed by bioluminescence. Actin cytoskeleton re-arrangements were evaluated by IF. Wound healing assays were performed to study functional effects on cell migration.
Results
Incubation with 25% PMN serum led to deposition of both C3b and C5b9 on the cell surface, which was significantly higher compared to controls (p < 0.05). Complement activation induced a significant rise in the intracellular calcium levels. Loss of mitochondrial membrane potential was also observed, together with intracellular ATP decrease, disruption of the actin cytoskeleton and impaired cell migration. Effects of both structure and function of podocytes were reversed by inhibition of the terminal complement pathway.
Conclusion
Complement is active in PMN, leading to both structural and functional effects on podocytes. Effects can be reversed by inhibition of the terminal complement pathway. Further studies are needed to fully understand the consequences of complement activation on the podocyte energy machinery and the rationale for the use of complement inhibitors in PMN. Our research may identify novel molecular treatment targets with the potential of improved patient outcomes and quality of life.