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Abstract: FR-PO719

Oxidative Stress Is a Mediator of Slit Diaphragm Defects Initiated by Disruption of Endocytosis

Session Information

Category: Glomerular Diseases

  • 1304 Glomerular Diseases: Podocyte Biology


  • Poulton, John S., University of North Carolina System, Chapel Hill, North Carolina, United States
  • Xi, Gang, University of North Carolina System, Chapel Hill, North Carolina, United States

Studies in podocytes and fly nephrocytes suggest that disruption of endocytosis leads to loss of slit diaphragms. Previous studies indicate that slit diaphragm integrity also requires redox balance. Therefore, we hypothesized that disrupting endocytosis may lead to oxidative stress, which in turn perturbs slit diaphragm integrity.


Genes were knocked down or overexpressed in Drosophila nephrocytes using the Gal4-UAS system. ROS levels were measured using a ROS reporter (GstD>GFP) or DHE dye. To assess slit diaphragm integrity, localization of the slit diaphragm protein ZO-1 was determined by confocal microscopy. In cultured human podocytes, endocytosis was disrupted by Dynasore, an inhibitor of Dynamin. ROS levels were determined using DHE or Nrf2 levels. Slit diaphragm integrity was indirectly assessed using Western Blot and immunoprecipitation to examine Nephrin's association with ZO-1 or Podocin.


In fly nephrocytes, knockdown of regulators of endocytosis (e.g., Rab5, Cubn, Amn, Dlg) all led to significant loss of slit diaphragms, as previously reported. Depletion of these proteins also led to increased oxidative stress and activation of the Nrf2 antioxidant response pathway. Overexpression of the Nrf2 targets and antioxidant enzymes G6PD or Catalase significantly restored slit diaphragm integrity in these cells (as indicated by correct surface localization of ZO-1). Conversely, knockdown of G6PD or Nrf2 significantly enhanced the loss of slit diaphragms in Dlg knockdown cells.
Dynasore treatment in human podoctes induced ROS generation within hours, evidenced by DHE staining and Nrf-2 activation. Long-term (e.g., over 6 hr) Dynasore treatment disrupted Nephrin/ZO-1 and Nephrin/Podocin associations, as well as ZO-1 plasma membrane localization. Similar results were obtained when high dose or long-term treatment of hydrogen peroxide was applied. We are currently investigating whether antioxidant reagents, such as Catalase, can rescue the alterations in Nephrin's protein interactions during long-term Dynasore treatment.


Our data indicate that disruption of endocytosis leads to increased ROS levels, which disrupt slit diaphragm integrity. Thus, in addition to its direct role in the trafficking of slit diaphragm proteins, endocytosis also affects slit diaphragm integrity by promoting redox balance.