Adhesion, morphology, and structure of murine podocytes on varying substrate stiffness

Glomerular podocytes are epithelial cells that are attached to outer glomerular basement memberane (GBM) by foot processes, and blood filtration occurs through podocytes, GBM, and endothelial cells. Podocytes are under constant mechanical stress due to their location around outside of glomerular capillaries, which can be associated with glomerular hypertension. It is important for podocytes to maintain their mechanical integrity, since podocyte adhesion to GBM is crucial to prevent podocyte loss, detachment, and associated alteration in cell adhesive properties, and further progression of glomerular disease. In this study, we examined the role of stiffness in podocyte function with hypothesis that increasing substrate stiffness would promote development of cell structural features that are associated with stronger adhesion. In order to test this, polyacrylamide substrates with different stiffness ranged from 3750 Pa to 152600 Pa were generated and immortalized mouse podocytes were cultured on these substrates. Then we measured how substrate stiffness affects cell morphology and several structural proteins distribution. We found that the size and the number of attached cells increased with longer actin filaments as stiffness of substrate increased. Since proteinuria or glomerulosclerosis can be associated with podocyte actin cytoskeleton defect, we suggest podocytes in a "softer" environment are vulnerable to glomerular diseases, since stress fibers were shorter and less organized as substrates decreased stiffness. Our results relating to the presence and distribution of certain proteins in cells were somewhat inconclusive, since intensity of synaptopodin and vinculin did not correspond to the changes of stiffness, due to the possibility of other underway mechanisms that interfere with podocyte adhesion. There was no clear relationship between YAP and the changes of substrate stiffness, and one possible explanation could be due to the optical irregularities in the substrate. Overall, this study was able to show that increased substrate stiffness promoted cell structural feature development in podocytes. However, further studies are needed to better understand how changes in substrate mechanical properties can affect structural protein distribution in these cells