Oxidative Stress Contributes to Slit Diaphragm Defects Caused by Disruption of Endocytosis.

Podocyte slit diaphragms are an important component of the glomerular filtration barrier. Podocyte injury frequently includes defects in slit diaphragms, and various mechanisms for these defects have been described, including altered endocytic trafficking of slit diaphragm proteins or oxidative stress. However, the potential relationship between endocytosis and oxidative stress in the context of slit diaphragm integrity has not been extensively considered. 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). 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. Oxidative stress is an important consequence of defective endocytosis and contributes to the defects in slit diaphragm integrity associated with disruption of endocytic trafficking.