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Abstract: TH-PO984

Identification of Cytoplasmic Proteins Associated with Ephrin-B1 at the Slit Diaphragm of Podocyte: PDZ Proteins, Par6, Par3 and NHERF2 Are Associated with Ephrin-B1

Session Information

Category: Pathology and Lab Medicine

  • 1501 Pathology and Lab Medicine: Basic

Authors

  • Fukusumi, Yoshiyasu, Department of Cell Biology, Kidney Research Center, Niigata University Graduate School of Medical and Dental Sciences, Niigata, Japan
  • Takamura, Sayuri, Department of Cell Biology, Kidney Research Center, Niigata University Graduate School of Medical and Dental Sciences, Niigata, Japan
  • Zhang, Ying, Department of Cell Biology, Kidney Research Center, Niigata University Graduate School of Medical and Dental Sciences, Niigata, Japan
  • Yasuda, Hidenori, Department of Cell Biology, Kidney Research Center, Niigata University Graduate School of Medical and Dental Sciences, 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 recently reported ephrin-B1 interacts with nephrin via their extracellular domains in cis form and plays an essential role at the slit diaphragm. We also reported the phosphorylated ephrin-B1 activates the JNK signaling independently of nephrin phosphorylation (JASN 2018). Because ephrin-B1 has a PDZ binding motif in its C-terminus, it is estimated that PDZ proteins interact with ephrin-B1. However, the cytoplasmic proteins interacted with ephrin-B1 in podocyte and the precise signaling pathways activated by the phosphorylated ephrin-B1 remain unclear.

Methods

To identify the proteins associated with ephrin-B1, we performed the gene expression profiling of glomerular sample of the podocyte-specific ephrin-B1 knockout (KO) mice. We found PDZ proteins, Par6, Par3 and NHERF2 were evidently downregulated, but the expressions of other PDZ proteins, ZO-1, MAGI1, CASK, which are already reported to be expressed at the slit diaphragm, were not changed. In this study the expression of Par6, Par3 and NHERF2 and their interaction with ephrin-B1 were analyzed.

Results

The decrease in mRNA expressions of Par6, Par3 and NHERF2 were confirmed with real-time RT-PCR (Par6, 20 ± 11%, p<0.01, Par3, 14 ± 9% to control, p<0.05, NHERF2, 17 ± 6%, p<0.001). The immuno-staining intensity of these proteins in glomeruli was decreased and their staining pattern evidently changed in the KO mice. Dual labeling analyses showed Par6, Par3 and NHERF2 were colocalized with ephrin-B1 in normal glomeruli. The immunostaining of these molecules as well as ephrin-B1 were clearly decreased in the nephrotic state caused by PAN injection. The remaining Par3 and Par6 in this proteinuric state were dissociated from ephrin-B1. By contrast, the remaining NHERF2 was still colocalized with the remaining ephrin-B1. Immunoprecipitation assay showed Par3 interacted with both ephrin-B1 and nephrin, and Par6 interacted with ephrin-B1 but not with nephrin. It is also observed that the phosphorylation of ephrin-B1 inhibited its interaction with Par6 and Par3.

Conclusion

Par6, Par3 and NHERF2 interact with ephrin-B1 and may contribute to the maintenance of the structure and the function of slit diaphragm cooperated with ephrin-B1.

Funding

  • Government Support - Non-U.S.