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Abstract: SA-PO740

Nephrin-Ephrin-B1-Par6 Complex Is Crucial for Slit Diaphragm in Podocytes: Ephrin-B1 Suppresses Tight Junction Formation by Interfering With Par-6-Cdc42 Binding

Session Information

Category: Glomerular Diseases

  • 1304 Glomerular Diseases: Podocyte Biology

Authors

  • Fukusumi, Yoshiyasu, Department of Cell Biology, Kidney Research Center, Niigata University, Niigata, 9518510, Japan
  • Zhang, Ying, Department of Cell Biology, Kidney Research Center, Niigata University, Niigata, 9518510, Japan
  • Yasuda, Hidenori, Department of Cell Biology, Kidney Research Center, Niigata University, Niigata, 9518510, Japan
  • Kawachi, Hiroshi, Department of Cell Biology, Kidney Research Center, Niigata University, Niigata, 9518510, Japan
Background

We have reported that ephrin-B1 interacts with nephrin, a key molecule of slit diaphragm at the extra-cellar domain (Fukusumi et al. JASN 2018) and interacts with Par-6, a member of the Par polarity complex, at the cytoplasmic domain in podocyte (Takamura et al. Am J Pathol 2021). However, the precise role of the ephrin-B1-Par-6 interaction in the slit diaphragm is unclear. The binding of Cdc42 with Par-6 is understood to be essential for tight junction formation. Converting to slit diaphragm from pre-mature junctions is an important step in podocyte differentiation, and it is reported that the transition of slit diaphragm to tight junction appears in many nephrotic conditions. Thus, we hypothesize that ephrin-B1-Par-6 interaction contributes to the maintenance of the slit diaphragm by interfering with the binding of Par-6-Cdc42.

Methods

To verify the hypothesis, the interactions of ephrin-B1, Par-6 and Cdc42 were analyzed with the HEK cell expression system. The alterations of the interactions in podocyte injury models were investigated with dual-labeling immunofluorescence study.

Results

The interaction of Par-6 and Cdc42 was detected with immunoprecipitation techniques with HEK cells transfected with these molecules. However, if ephrin-B1 was co-transfected, Par-6 was dissociated from Cdc42 and interacted with ephrin-B1. Then, if ephrin-B1 was phosphorylated by the treatment of Eph-Fc, the interaction of Par-6 with ephrin-B1 was disrupted and Par-6 interacted with Cdc42. In anti-nephrin antibody-induced nephropathy, a rat nephrotic model, not only nephrin but also ephrin-B1 was phosphorylated, and ephrin-B1 was dissociated from Par-6.

Conclusion

Ephrin-B1-Par6 interaction interferes with the Par-6-Cdc42 interaction, and consequently suppresses tight junction formation. The interaction of Par-6 with ephrin-B1 instead of Cdc42 is a critical step for converting the slit diaphragm from the tight junction. In injured podocytes, ephrin-B1 is phosphorylated and dissociated from Par-6, and Par-6 interacts with Cdc42 instead of ephrin-B1. It is conceivable that dissociation of ephrin-B1 from Par-6 is one of the critical events in podocyte injury. The stabilization of ephrin-B1-Par-6 interaction could be a novel therapeutic approach for nephrotic syndrome.

Funding

  • Government Support – Non-U.S.