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Abstract: FR-PO766

Synaptopodin-Enhanced Podocytes Perpendicular Force Resistance In Vivo and In Vitro

Session Information

Category: Glomerular Diseases

  • 1401 Glomerular Diseases: Mechanisms, including Podocyte Biology

Authors

  • Qu, Chengqing, Washington University in St Louis, St Louis, Missouri, United States
  • Jiang, Shumeng, Washington University in St Louis, St Louis, Missouri, United States
  • Miner, Jeffrey H., Washington University in St Louis School of Medicine, St Louis, Missouri, United States
  • Genin, Guy M., Washington University in St Louis, St Louis, Missouri, United States
  • Suleiman, Hani, Washington University in St Louis School of Medicine, St Louis, Missouri, United States
Background

Focal adhesions are able to resist shear forces in almost all types of cells. However, kidney podocytes face a unique challenge as they must also withstand stress that is perpendicular to their basement membrane. We hypothesis that synaptopodin, a specialized actin-binding protein that is highly expressed in podocytes, acts as a linker to facilitate the resistance of focal adhesions to these perpendicular forces.

Methods

We developed an in vitro system to study the impact of perpendicular forces on cells cultured on hydrogels with defined stiffness and coated with Laminin a5b2g1. By applying centrifugal forces, this setup was able to mimic the perpendicular forces experienced by podocytes in their natural glomerular microenvironment. We used primary podocytes taken from both wild-type control (WT) mice as well as mice lacking synaptopodin (Synpo KO). We compared these cells to osteosarcoma cells (U2OS cells), a bone cancer cell that also highly express synaptopodin. We assessed the cellular responses to the centrifugal forces using morphological analysis as well as immunostaining for integrins, synaptopodin, a-actinin-4, myosin IIA and actin. We also used AAV8-TBG-Ren1 virus to increase the blood pressure in vivo and further increased the detaching force on the podocytes from the basement membrane.

Results

The WT primary podocytes spread and form a continuous skirt of integrins at their periphery. The continuous integrin is colocalized with synaptopodin and maintains the sarcomere like structure (SLS) at the periphery. However, Synpo KO primary podocytes do not exhibit this continuous integrin b1 pattern at the cell periphery. Instead, Synpo KO cells show a diffuse pattern and are likely to detach. After the injection of AAV8-TBG-Ren1virus in WT and Synpo KO mice, we observed the severe proteinuria with Albumin/Creatinine ratio (ACR) measurement.

Conclusion

We discovered a mechanical role for synaptopodin in podocytes. When centrifugal force is applied, WT primary podocytes can withstand these perpendicular forces by significantly rearranging their focal adhesions. A deficiency in synaptopodin weakens the podocytes’ ability to respond to these forces and is linked to increased detachment. This function of synaptopodin may play a crucial role in podocyte adhesion to the basement membrane during their responses to elevated stresses and injury.