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Abstract: FR-PO936

NHERF2 Interacts with Ephrin-B1 at the Slit Diaphragm: NHERF2 Bridges Podocalyxin and Slit Diaphragm Components

Session Information

Category: Glomerular Diseases

  • 1204 Podocyte Biology

Authors

  • Fukusumi, Yoshiyasu, Department of Cell Biology, Kidney Research Center, Niigata University, Niigata, Japan
  • Zhang, Ying, Department of Cell Biology, Kidney Research Center, Niigata University, Niigata, Japan
  • Yasuda, Hidenori, Kidney Research Center, Niigata University, Niigata, Japan
  • Kawachi, Hiroshi, Department of Cell Biology, Kidney Research Center, Niigata University Graduate School of Medical and Dental Sciences, Niigata, Japan
Background

We have reported ephrin-B1 is a novel component of the slit diaphragm (SD). Ephrin-B1 interacts with nephrin via their extracellular domains and plays an essential role in maintaining the barrier function of SD (JASN 29, 2018). We reported NHERF2, a scaffold protein possessing two PDZ domains, was downregulated in the glomeruli of the podocyte-specific ephrin-B1 KO mice, which suggesting NHERF2 is associated with ephrin-B1 (ASN 2018). It is reported that NHERF2 binds to podocalyxin at the second PDZ domain and plays the pivotal role in maintaining actin cytoskeleton by phosphorylating ezrin in podocytes. However, the precise localization of NHERF2 and its interaction with ephrin-B1 are unclear.

Methods

The interaction of NHERF2 with ephrin-B1 and nephrin was analyzed by the immunoprecipitation (IP) analyses with glomerular lysates and HEK293 transfected cells. The expressions of these molecules in glomeruli of normal rat and rat with nephropathy induced by the anti-nephrin antibody were analyzed by immunofluorescence.

Results

NHERF2 band was detected in the precipitates with anti-nephrin antibody by IP assay with normal rat glomerular lysate, which indicating NHERF2 is a member of the SD complex. The IP assay with the HEK cell expression system showed NHERF2 directly interacted with ephrin-B1 at the first PDZ domain and did not interacted with nephrin. The analyses with the HEK cells triple transfected with NHERF2, ephrin-B1 and nephrin showed that the anti-nephrin antibody binding phosphorylated not only nephrin but also ephrin-B1, and that the phosphorylated ephrin-B1 did not interacted with NHERF2. Dual-labeling analyses showed NHERF2 was detected not only apical area but also SD area, and a portion of NHERF2 was colocalized with ephrin-B1 in normal glomeruli. Ephrin-B1 was phosphorylated and dissociated from NHERF2 in the nephrotic state caused by the anti-nephrin antibody injection. The immunostaining of NHERF2 and ezrin as well as ephrin-B1 and nephrin were clearly decreased.

Conclusion

NHERF2 interacts with ephrin-B1 via its first PDZ domain and with podocalyxin via its second PDZ domain, and bridges podocalyxin, an apical surface protein and ephrin-B1, an SD component. The bridging structure was disrupted by the nephrin-mediated signal. The disruption is one of the critical mechanisms of podocyte injury.

Funding

  • Government Support - Non-U.S.