Although podocyte injury has been correlated with glomerular disease progression, the mechanisms underlying podocyte response to stress
in vivo remain poorly understood. As a terminally differentiated kidney visceral epithelial cell, the podocyte lines the glomerular capillary tuft and serves as the final barrier to protein loss
in vivo. Podocyte response to injury has been previously characterized by F-Actin rearrangement, effacement of podocyte foot processes, and detachment from the glomerulus. However, the signaling cascades underlying these phenotypes have yet to be fully elucidated. My study identified YAP as a potential biomarker for disease progression in Diabetic Nephropathy, and characterized podocyte stress response in three different stress models
in vitro. This data shows that reduced percentage of nuclear YAP within the glomerulus correlates with proteinuria, or protein loss in the urine, which is the clinical hallmark of glomerular disease. I show that exposure to three independent glomerular injury models: Puromycin Aminonucleoside, hypoxia, and high glucose, all result in cytoplasmic translocation of YAP in human podocytes
in vitro, and reduced total protein levels of YAP. It was previously unknown whether podocyte injury is reversible. These data show recovery of a fully differentiated phenotype in vitro following five-day washout of injury conditions. Mechanical forces have previously been shown to alter podocyte adhesion
in vitro, however, podocyte cellular forces had been previously uncharacterized. Continuous imaging using Elastic Resonator Interference Stress Microscopy (ERISM) demonstrated that in a PAN model of glomerular injury, podocyte cellular forces are lost, and can be partially recovered following washout. During stress-response, YAP is regulated at the level of nuclear retention, as YAP translocation preceded alterations in stress fiber integrity in a setting of non-muscle myosin inhibition with Blebbistatin. I further show that the podocyte response to mechanical environment is YAP-mediated, and that YAP over-expression is protective of cells cultured on a 1kPa soft substrate. Finally, the E3 ubiquitin ligase ITCH, which is upstream of YAP in the Hippo signaling pathway, is shown to be a potential mediator of podocyte cellular adhesion in vitro. Collectively, the evidence herein demonstrates that the podocyte response to stress may be YAP-mediated, and indicates a potential role for YAP in protecting against injury signaling cascades
in vivo.