Expression of Nephrin and Podocin in Cells and Interaction between Them in vitro and in vivo

Objective To study the cellular distribution and interaction of Nephrin and Podocin.Methods Plasmids containing Nephrin and Podocin were constructed and transfected to COS-7 cells.Double immunofluorescence was performed to detect Nephrin and Podocin mole-cules plasma expressing.A laser scanning confocal microscopy was applied to study the distributions pattern of Nephrin and Podocin.Gluta-thione-S-transferase(GST)-Nephrin and GST-Podocin fusion protein were expressed in competent cell.Co-immunoprecipitation and GST pull-down assay were performed to study the interaction of Nephrin and Podocin in vitro and in vivo.Results Nephrin and Podocin colocallized in COS-7 cell.A substantial portion of Nephrin and Podocin was colocalized on the intracellular vesicle-like structures as well as the edges.Nephrin and Podocin interact with each other via C-terminals in vitro and in vivo.The interaction sites were C-terminals of each other,in detail,1 134-1 241 amino acids of Nephrin and 262-383 amino acids of Podocin interacted in the cytoplasma.Conclusion Physically interaction of Nephrin and Podocin may play key roles in maintaining the structural and functional integration of slit diaphgram.