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Kidney Week

Abstract: TH-PO792

The E3-Ubiquitin Ligase HUWE1 Is a Central Regulator in Podocyte Homeostasis

Session Information

Category: Genetic Diseases of the Kidneys

  • 1002 Genetic Diseases of the Kidneys: Non-Cystic

Authors

  • Brand, Marie, University of Cologne, Cologne, Germany
  • Höhne, Martin, University Hospital Cologne, Cologne, Germany
  • Schermer, Bernhard, University Hospital Cologne, Cologne, Germany
  • Benzing, Thomas, University of Cologne, Cologne, Germany
  • Volker, Linus A., University Hospital of Cologne, Cologne, Germany
Background

As terminally differentiated cells, podocytes depend on precise regulation of protein turnover and response to cellular stress in order to preserve homeostasis. Disruption of these processes can lead to podocyte damage and loss and subsequently to glomerular scarring and kidney disease.
Ubiquitination is a posttranslational modification that targets proteins not only to proteasomal degradation, but also to different cellular pathways such as DNA repair, cell cycle regulation, and apoptosis. The HECT type E3-ubiquitin ligase Huwe1 has been shown to be a regulator of various intracellular signaling cascades in different cell types. To characterize the effects of Huwe-mediated ubiquitination in podocyte homeostasis in vivo and in vitro, we generated a podocyte-specific Huwe1-knockout mouse and HUWE1-deficient human podocyte cell lines.

Methods

To phenotype podocyte-specific Huwe1-knockout mice, we analyzed the urinary albumin/creatinine ratio and performed immunohistochemistry and electron microscopy. To elucidate the molecular effects of HUWE1 knockout on podocyte signaling, we generated a CRISPR/Cas9-mediated HUWE1-deficient human podocyte cell line and analyzed protein expression by mass spectrometry and RNA sequencing.
To investigate HUWE1 specific alterations of ubiquitination, mass spectrometry after enrichment of ubiquitination sites was performed.

Results


Podocyte-specific loss of Huwe1 in mice caused kidney disease beginning at 5 weeks of age. Affected mice developed massive proteinuria and died prematurely of uremic complications. At the ultrastructural level, we saw extensive foot process effacement, podocyte vacuolization, and cell loss.
Proteome and transcriptome analysis of HUWE1-knockout cells revealed significant differential regulation in a number of relevant pathways such as cell division, mitochondrial metabolism and autophagy. Analysis of the ubiquitome is ongoing.

Conclusion


The E3-ubiquitin ligase Huwe1 is pivotal for podocyte function and overall survival in mice. We identified HUWE1 as a central regulator of a multitude of cellular pathways to preserve human podocyte signaling and homeostasis. To address the complexity of affected cellular systems within the podocyte, a systems biology approach is required in order to develop new diagnostic and therapeutic strategies.

Funding

  • Private Foundation Support