Nephrin-Ephrin-B1-NHERF2-Ezrin-Actin axis is critical in podocyte injury.

Abstract We reported ephrin-B1 is one of the critical components of slit diaphragm of kidney glomerular podocyte. However, the precise function of ephrin-B1 is unclear. To clarify the function of ephrin-B1, we explored the ephrin-B1-associated molecules. RNA-seq analysis suggested NHERF2, a scaffolding protein is associated with ephrin-B1. Thus, we focused on NHERF2, and the interaction of NHERF2 with ephrin-B1 and the role of NHERF2 in podocyte were investigated. NHERF2 is expressed at not only the apical area but also the slit diaphragm, and NHERF2 interacts with the nephrin–ephrin-B1 complex at slit diaphragm. The nephrin–ephrin-B1–NHERF2 complex interacts with ezrin that bound to F-actin. NHERF2 binds ephrin-B1 via the first PDZ domain, and binds podocalyxin via the second PDZ domain. Both in vitro analyses with HEK-293 cells and in vivo study with rat nephrotic model showed that if the slit diaphragm is stimulated and nephrin and ephrin-B1 are phosphorylated, NHERF2 and ezrin are de-phosphorylated, and the linkages of ephrin-B1–NHERF2 and NHERF2–ezrin are disrupted. It is conceivable that the linkage of nephrin–ephrin-B1–NHERF2–ezrin–actin is a novel critical axis in the podocyte. The phosphorylation of ephrin-B1 also disrupts the linkage of an apical transmembrane protein podocalyxin with NHERF2–ezrin–actin. The phosphorylation of ephrin-B1 and the consequent de-phosphorylation of NHERF2 are critical initiation events leading to podocyte injury.