Conclusion
Oxidative stress is an important consequence of defective endocytosis and contributes to the defects in slit diaphragm integrity associated with disruption of endocytic trafficking.
Methods
To examine the potential relationships between endocytosis, oxidative stress, and slit diaphragm integrity, we induced genetic or pharmacological disruption of endocytosis in Drosophila nephrocytes (the insect orthologue of podocytes) and cultured human podocytes. We then employed immunofluorescence microscopy to analyze protein localization and levels, and to quantify signal from reactive oxygen species (ROS) dyes. Immunoprecipitation from podocyte cell lysates was used to examine effects on slit diaphragm protein complex formation (i.e., nephrin/podocin and nephrin/ZO-1).
Results
Disruption of endocytosis in nephrocytes and podocytes led to slit diaphragm defects, elevated levels of ROS (oxidative stress), and activation of the nuclear factor erythroid 2-related factor 2 (Nrf2) antioxidant pathway. In nephrocytes with defective endocytosis, perturbation of Nrf2 signaling exacerbated slit diaphragm defects. Conversely, overexpression of Nrf2 target genes catalase or glucose-6-phosphate dehydrogenase (G6PD) significantly ameliorated slit diaphragm defects caused by disruption of endocytosis.