Abstract: TH-PO069

Par6β Interaction with Ephrin-B1 at the Slit Diaphragm Could Be a Differential Diagnostic Marker of Nephrotic Syndrome: Interaction of the Par-Complex Molecules with Ephrin-B1/Nephrin in Podocyte

Session Information

Category: Glomerular

  • 1002 Glomerular: Basic/Experimental Pathology

Authors

  • Takamura, Sayuri, Dept. Cell Biology, Kidney Research Center, Niigata University, Niigata, Japan
  • Fukusumi, Yoshiyasu, Dept. Cell Biology, Kidney Research Center, Niigata University, Niigata, Japan
  • Zhang, Ying, Dept. Cell Biology, Kidney Research Center, Niigata University, Niigata, Japan
  • Narita, Ichiei, Division of Clinical Nephrology and Rheumatology, Niigata University, Niigata, Japan
  • Kawachi, Hiroshi, Dept. Cell Biology, Kidney Research Center, Niigata University, Niigata, Japan
Background

Par-complex, Par3/Par6/Cdc-42/aPKC is reported to be a component of the slit diaphragm (SD) and to play a role in maintaining the podocyte function. However, the precise differential roles of the Par-complex molecules in podocyte are not fully understood. We have reported that ephrin-B1 is a novel component of the SD. Although ephrin-B1 is reported to control cell-cell junctions of vascular endothelial cells through Par-complex, no studies on the interaction of the Par-complex molecules with ephrin-B1 at the SD have not been analyzed.

Methods

The expressions of the Par-complex molecules in podocyte were analyzed in the normal adult and developing glomeruli and in the rat nephrotic models of MCNS and FSGS by immunofluorescence, Western blot and RT-PCR. The interactions of Par3 and Par6 with ephrin-B1 and nephrin were analyzed by the IP analyses with glomerular lysates and HEK-293 transfected cells. The expressions of the Par-complex molecules in the podocyte-specific ephrin-B1 conditional KO (CKO) mouse were analyzed.

Results

mRNA expressions of Par6β and Par3 decreased from the early phase in the MCNS model and the decreases were detected when proteinuria peaked in both models. mRNA of Par6α increased in both models. Western blot analyses showed Par6β decreased in both models and Par3 decreased in the MCNS model. Immunostainings of Par3 and Par6β clearly decreased in both models. The decrease of Par6β was more evident in the FSGS model than in the MCNS model (IF score: 2.1 in FSGS model vs control 5.0). By contrast Par3 staining decreased more evidently in the MCNS model. The Par6β staining in normal glomeruli was co-localized with ephrin-B1, whereas no co-localization of Par3 and ephrin-B1 was observed. Whereas, some portions of the Par6β staining were apart from nephrin, and the Par3 staining was almost co-stained with nephrin. The IP study showed Par6β was interacted with ephrin-B1. The expression of Par6β was altered in the ephrin-B1 CKO mice.

Conclusion

Altered expressions of Par3 and Par6β are involved in the pathogenesis of both nephrotic models, and these molecules could be differential diagnostic markers of nephrotic syndrome. Not Par3 but Par6β is highly associated with ephrin-B1 at the SD.