Abstract: FR-PO242

Podocyte-Specific Loss of Huwe1 Causes Vacuolization and Glomerular Damage

Session Information

Category: Cell Biology

  • 202 Apoptosis, Proliferation, Autophagy, Cell Senescence, Cell Transformation

Authors

  • Volker, Linus A., University of Cologne, Cologne, Germany
  • Bertsch, Sabine, University of Cologne, Cologne, Germany
  • Dittrich, Sebastian, University of Cologne, Cologne, Germany
  • Rinschen, Markus M., University of Cologne, Cologne, Germany
  • Schermer, Bernhard, University of Cologne, Cologne, Germany
  • Benzing, Thomas, University of Cologne, Cologne, Germany
  • Höhne, Martin, University of Cologne, Cologne, Germany
Background

As terminally differentiated cells, podocytes rely on precise regulation of homeostasis and specific responses to cellular stress to forego cell damage and loss, which would ultimately lead to glomerular scarring and kidney disease. In an attempt to characterize the networks that regulate cell stress responses, we identified the E3-ubiquitin ligase Huwe1 via interaction screening. In other cell types, Huwe1 has been shown to be a regulator of a wide spectrum of intracellular signaling cascasdes such as MAPK/ERK, Wnt, DNA-damage and cell cycle controll.

Methods

We generated a podocyte-specific Huwe1-knockout mouse and a Huwe1-knockdown podocyte cell line to characterize the role of Huwe1 in podocyte stress response and homeostasis in vivo and in vitro.

Results

Huwe1-knockout mice were born at expected ratios. They showed signs of kidney disease beginning at 4 weeks of age. Affected mice weighed less than littermates, developed heavy proteinuria, and died prematurely of uremic complications. At ultrastructural level, we saw significant foot process effacement, podocyte vacuolization, and cell loss.
We employed biochemistry and mass spectrometry to elucidate the effect of Huwe1-knockdown on podocyte signaling. No direct interactors of Huwe1 could be identified, most likely due to the transient nature of interaction of E3-ubiquitin ligases. Mass spectrometry revealed a significant differential regulation of protein networks associated with lysosome function and DNA-damage signaling. Fittingly, podocyte vesicles stained positive for Lamp1 indicating a lysosomal origin. Biochemical analyses showed a marked downregulation of mTOR signaling as a putative effector of vesicle formation. Experiments to elucidate the role of Huwe1 in DNA-damage response in podocytes are in progress.

Conclusion

We identified the E3-ubiquitin ligase Huwe1 at the center of crucial signaling cascades as an indispensable regulator of podocyte homeostasis. Future research will be directed at identifying direct Huwe1-targets in podocytes and potential therapeutic exploitability

Funding

  • Private Foundation Support