Abstract: FR-PO742
Impact of Glomerular Basement Membrane Stiffness on Podocyte Metabolism in Alport Syndrome
Session Information
- Glomerular Diseases: Mechanisms and Podocyte Biology
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
- Kim, Jin Ju, University of Miami Katz Family Division of Nephrology and Hypertension, Miami, Florida, United States
- Ficarella, Maria, University of Miami Katz Family Division of Nephrology and Hypertension, Miami, Florida, United States
- Molina David, Judith T., University of Miami Katz Family Division of Nephrology and Hypertension, Miami, Florida, United States
- Fontanesi, Flavia, University of Miami Miller School of Medicine Department of Biochemistry, Miami, Florida, United States
- Merscher, Sandra M., University of Miami Katz Family Division of Nephrology and Hypertension, Miami, Florida, United States
- Miner, Jeffrey H., Washington University School of Medicine, Division of Nephrology, St. Louis, Missouri, United States
- Fornoni, Alessia, University of Miami Katz Family Division of Nephrology and Hypertension, Miami, Florida, United States
Background
Cells utilize mechanotransduction to sense mechanical cues, which regulate behaviors like proliferation and apoptosis. These pathways are linked to metabolic processes, with ECM stiffness influencing cellular metabolism and cytoskeletal integrity. Stiff ECM promotes glycolysis,while soft ECM suppresses it, affecting glucose utilization. Key pathways involve phosphofructokinase 1(PFK1) in glycolysis and tripartite motif-containing protein 21(TRIM21) in protein degradation, with TRIM21 activity inhibited by actin-mediated sequestration. In Alport Syndrome(AS), mutations in type IV collagen genes lead to a softer, more degradable glomerular basement membrane(GBM) composed of α112 chains of collagen IV. This study investigates the impact of GBM stiffness on podocyte metabolism and structure, focusing on stressfiber formation and mitochondrial function.
Methods
Immortalized AS and WT podocytes were created by breeding Col4a3KO mice with H-2kb-tsA58 transgenic mice, then cultured on soft(1.5 kPa) and stiff(10-12 kPa) gelatin hydrogel matrices. Mitochondrial respiration was measured using a high-resolution respirometer. CPT1A, PFK1, and TRIM21 expression were assessed via western blot. Mitochondrial function was evaluated through ATP production and mitochondrial membrane potential(MMP) assays. Stress fiberformation and TRIM21 colocalization were analyzed using immunofluorescence microscopy.
Results
Fatty acid oxidation(FAO) was significantly increased in AS podocytes compared to WT podocytes(P<0.05), with higher CPT1 expression in AS podocytes. Actin cytoskeleton rearrangement was observed in WT podocytes on soft matrices compared to stiff matrices. Mitochondrial function was impaired in AS podocytes, as indicated by reduced ATP production(P<0.05) and decreased MMP(P<0.01). Decreased PFK1 and increased TRIM21 activity were observed in podocytes on soft matrices, suggesting suppressed glycolysis and enhanced protein degradation and stress response.
Conclusion
Changes in GBM stiffness in AS mice may lead to a metabolic shift in podocytes from glycolysis to fatty acid oxidation, reduced stress fibers,and impaired mitochondrial function. These findings underscore the importance of ECM stiffness and related mechanotransduction pathways in developing treatments for AS and potentially other glomerular diseases.
Funding
- NIDDK Support