We provide direct evidence that PAN-induced inactivation of RhoA in human cultured podocytes is associated with aberrant remodeling of the actin cytoskeleton and consecutive podocyte dysfunction. EV ameliorated these effects by preventing the inhibition of RhoA signaling involving ROCK and MLC. These observations might prompt further studies of mTOR inhibitors in proteinuric disease. Prior to pharmacological treatments, we investigated the cellular morphology and cytoskeletal organization of cultured human podocytes in detail. Differentiated podocytes typically display a rather non-polarized phenotype in 2D with peripheral cell-cell contacts forming the basis for the functional glomerular filtration barrier in vivo. Confocal microscopy of the actin cytoskeleton in untreated individual podocytes revealed a dense meshwork of linear actin stress fibers in the cell center spanning the entire cell. In addition, we observed distinct actin rich regions in central areas of the cell body reminiscent of small dynamic protrusions. Indeed, in phase contrast live-cell experiments we were able to detect multiple transient protrusions in individual cells revealing the highly dynamic Cycloheximide Small Molecules inhibitor behavior of the cytoskeleton in differentiated podocytes. In addition to the actin structures, focal adhesions were visualized using antibody staining against the adaptor protein paxillin. As expected, focal adhesions were associated with the ends of stress fibers connecting the cytoskeleton with the substrate. However, in contrast to other cell types such as fibroblasts, focal adhesion localization was not strictly restricted to these distal regions but was also detected along the entire length of stress fibers. Studies of human podocytes indicated direct effects of mTOR inhibitors on the podocyte cytoskeleton besides their previously suggested immunosuppressive actions. In proteinuric animal models, the application of the mTOR inhibitor EV was demonstrated to significantly reduce proteinuria. To study the molecular effects of EV in podocyte injury in detail, we applied puromycin aminonucleoside as a well-recognized in vitro model. As expected, treatment with PAN caused strong morphological and cytoskeletal defects. Overall, treated cells were significantly smaller and often adopted a frontto- back polarized shape reminiscent of migratory fibroblasts. More prominently, central stress fibers were diminished significantly after exposure to PAN. Instead, we noticed substantial accumulation of thin and less organized actin fibers at the cell periphery. When exposed to EV along with PAN, cell body size enlarged and CHIR-99021 moa number of podocytes with central stress fibers increased significantly. In addition, cells displayed a less aberrant organization of stress fibers as compared to PAN alone. Interestingly, EV alone did not affect podocyte morphology or the actin cytoskeleton suggesting that the compound might specifically act on signaling pathways altered in podocyte damage. As we observed substantial podocyte loss following PAN treatment, we subsequently tested whether this massive decrease in cell numbers was due to apoptosis. DNA fragmentation was quantified by Hoechst-staining in human podocytes after exposure to PAN for 48 h. In agreement with recent data PAN treatment led to a significant induction of apoptosis as the number of cells with fragmented DNA increased dramatically. Combining PAN treatment with EV led to significant increase in cell number paralleled by reduced apoptosis as compared to PAN treatment alone, approximating the basal apoptosis rate of control cells.