Kidney Week

Abstract: FR-PO750

Molecular Mapping of Injury-Induced Contractile Actomyosin Phenotypes in Podocytes Using Expansion Microscopy

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

• Puapatanakul, Pongpratch, Washington University in St Louis School of Medicine, St Louis, Missouri, United States

Pongpratch Puapatanakul, MD, MS, MSc

• Yaseen Alsabbagh, Dema, Washington University in St Louis School of Medicine, St Louis, Missouri, United States

Dema Yaseen Alsabbagh, MD

• Langner, Ewa, Washington University in St Louis School of Medicine, St Louis, Missouri, United States

Ewa Langner, PhD

• Mahjoub, Moe, Washington University in St Louis School of Medicine, St Louis, Missouri, United States

Moe Mahjoub, PhD

• Miner, Jeffrey H., Washington University in St Louis School of Medicine, St Louis, Missouri, United States

Jeffrey H. Miner, PhD, FASN

• Genin, Guy M., Washington University in St Louis, St Louis, Missouri, United States

Guy M. Genin, PhD

• Suleiman, Hani, Washington University in St Louis School of Medicine, St Louis, Missouri, United States

Hani Suleiman, MD, PhD

Background

The reorganization of actin cytoskeletons into sarcomere-like structures (SLSs), characterized by the periodic intertwining of synaptopodin and myosin IIA, has been previously observed in injured podocytes both in vivo and in vitro.

Methods

To further investigate SLSs in three dimensions at super-resolution, we employed Ultrastructure Expansion Microscopy (U-ExM) on kidney tissues. Deparaffinized 5-µm kidney sections from wild-type mice and mice with nephrotic syndrome (Cd2ap-/-, adriamycin nephropathy) were anchored and embedded in acrylamide-rich hydrogels, denatured at 95C, expanded isotropically in water, and immunolabeled with relevant antibodies. The expanded samples were then imaged in z-stacks using an Airyscan confocal microscope.

Results

U-ExM enabled en-face visualization of individual foot process boundaries by staining for nephrin, a major slit diaphragm protein. In healthy podocytes, actin and the actin-binding protein synaptopodin appeared in longitudinal bundles in foot processes, while myosin IIA was present in cell bodies and major processes, but absent from foot processes. In both injury models, there was a significant increase in foot process width, indicating foot process effacement. Within the widened foot processes, actin cables were disrupted and reorganized into antiparallel bands, alternating with myosin IIA that relocated from the major processes, forming SLSs (Fig.1A). Tropomyosin 1/2 and Caldesmon, important components of actomyosin bundles in smooth muscle and non-muscle cells, were found only in mesangial cells in healthy controls, but were present in the effaced foot processes as part of SLSs, in locations corresponding to myosin IIA and actin, respectively (Fig.1B-C).

Conclusion

Injured podocytes establish SLSs with a molecular composition similar to muscle sarcomeres, suggesting this contractile machinery is an adaptive response against detachment, opening avenues for future therapeutic interventions aimed at preventing podocyte loss and progression to kidney failure.

Funding

• NIDDK Support