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Abstract: TH-PO1072

Targeting Defective Trafficking of Slit Diaphragm Protein in INF2-Related Podocytopathy and FSGS

Session Information

Category: Glomerular Diseases

  • 1204 Podocyte Biology

Authors

  • Sun, Hua, Boston Children's Hospital, Boston, Massachusetts, United States
  • Pollak, Martin R., Beth Israel Deaconess Medical Center/Harvard Medical School, Boston, Massachusetts, United States
Background

Focal segmental glomerulosclerosis (FSGS) caused by mutations in INF2 is characterized by mistrafficked nephrin, which comprises the slit diaphragm (SD) of podocytes by its extracellular domain, suggesting INF2 plays an important role in maintaining the surface transport of INF2. This hypothesis is supported by a zebrafish model in which INF2 knockout led to glomerular dysplasia with mistrafficked nephrin, and INF2 R218Q knockin mice which showed defective recycling of nephrin and recovery of the SD following protamine perfusion.
This study is to delineate this newly recognized role of INF2 in regulating the trafficking of nephrin, and how it is disrupted by FSGS-causing mutations.

Methods

By performing a yeast 2 hybridization screen, I found the interaction of INF2 with signaling molecules involved in vesicle trafficking pathways. Their interactions with wildtype or FSGS-causing mutants of INF2 were analyzed using yeast mating and co-IP. Podocytes with INF2 knockout or R218Q knockin were treated with antagonists for these pathways, and the trafficking of nephrin was studied by surface biotinylation and fluorescent based trafficking assays, live cell imaging with analysis using KymographClear, KymographDirect and TrackMate software.

Results

Yeast mating and Co-IP demonstrated the interaction of INF2 with mDia (a Rho effector), Dynein light chain 1 (Dynll1), Nipsnap3a (a SNARE protein), molecules involved in cytoskeleton regulation and lipid raft dependent vesicle trafficking. The interactions were disrupted by FSGS-causing mutations of INF2, suggesting the dysregulated pathways by INF2 mutants contributed to the pathogenesis of FSGS. Podocytes with INF2 knockout or R218Q knockin showed impaired nephrin recycling, which was rescued by targeting these pathways using Rho inhibitor (C3 transferase), Dynein inhibitor (Ciliobrevin) or lipid raft sequestrator (Nystatin).

Conclusion

INF2 plays a key role in maintaining the functional trafficking of nephrin by modulating 1) Rho/mDia signaling that halts vesicle movement; 2) Dynein mediated retrograde transport; 3) Lipid raft dependent vesicle trafficking. This role of INF2 can be disrupted by FSGS-causing mutations, leading to mistrafficked nephrin and disintegrity of SD. The dissection of the dysregulated pathways underlying the mistrafficked nephrin will provide new therapeutic targets for INF2 related podocytopathy and FSGS.

Funding

  • NIDDK Support