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Abstract: FR-PO937

Membrane-Associated Guanylate Kinase Inverted 2 Stabilizes Glomerular Filtration Barrier via the PDZ Domain

Session Information

Category: Glomerular Diseases

  • 1204 Podocyte Biology

Authors

  • Yamada, Hiroyuki, Chiba University Graduate School of Medicine, Chiba, Japan
  • Makino, Shinichi, Chiba University Graduate School of Medicine, Chiba, Japan
  • Yanagita, Motoko, Graduate School of Medicine, Kyoto University, Kyoto, Japan
  • Asanuma, Katsuhiko, Chiba University Graduate School of Medicine, Chiba, Japan
Background

Slit-diaphragm (SD) of podocytes plays a crucial role as a final barrier of glomerular filtration. The main components of SD, such as Nephrin and Neph1, are indispensable in preventing progression of glomerulosclerosis. However, it remains unknown what is important for protecting these main components. Here, we demonstrate that Membrane-associated guanylate kinase inverted 2 (MAGI-2) functions as a critical scaffold protein for maintaining the SD components.

Methods

We examined the immunofluorescence intensity of MAGI-2 in human biopsy sample. We analyzed the phenotype of podocyte specific MAGI-2 knock out (KO) mice. Additionally, using piggy-Bac transposon and CRISPR-Cas9 system, we analyzed the function of MAGI-2 in cultured MAGI-2 overexpression podocytes and ZO-1 KO podocytes. Next, using biochemical assay, we investigated which domain of MAGI-2 is necessary for the binding among these molecules.

Results

In the immunofluorescence of human biopsy sample, MAGI-2 is downregulated in glomerular diseases such as focal segmental global sclerosis or IgA nephropathy. Actually, podocyte specific MAGI-2 KO mice also significantly exhibited glomerulosclerosis and the reduced expression of Nephrin and Neph1. Cultured MAGI-2 overexpression podocytes showed the colocalization of MAGI-2 and Nephrin in cell-cell contact, while Nephrin was not expressed in cellular edge of cultured control podocytes. Additionally, although ZO-1 deletion undermines the Neph1 expression in cultured podocytes, the transfection of MAGI-2 in the ZO-1 deleted cells could retrieve the reduced Neph1 expression. Biochemical assay demonstrated that MAGI-2 binds to Nephrin and Neph1 through different PDZ binding domains.

Conclusion

These results showed that MAGI-2 is crucial for maintaining the key components of SD such as Nephrin and Neph1. Moreover, they also showed that MAGI-2 could localize these main components to intracellular compartments through different PDZ domains. Therefore, the MAGI-2 protection strategy could be a new route to the new drug development against glomerular diseases.