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Abstract: TH-OR056

Disruption of Endosomal Trafficking of Membrane Bound Nephrin Results in an Earlier Phenotype in Nephrin Deleted Adult Mice

Session Information

Category: Glomerular Diseases

  • 1202 Glomerular Diseases: Immunology and Inflammation

Authors

  • Verma, Rakesh, University of Michigan, Ann Arbor, Michigan, United States
  • Venkatareddy, Madhusudan M., University of Michigan, Ann Arbor, Michigan, United States
  • Cara-Fuentes, Gabriel M., University of Michigan, Ann Arbor, Michigan, United States
  • Patel, Sanjeevkumar R., University of Michigan Hospitals, Ann Arbor, Michigan, United States
  • Garg, Puneet, University of Michigan, Ann Arbor, Michigan, United States
Background

In a recent publication we reported that deletion of nephrin in an adult mature glomerulus results in FSGS. Following cre-induction a small fraction (10-15%) of nephrin remains at the membrane and is sufficient to maintain the slit diaphragm for 4-6 weeks. In vitro studies have suggested that membrane bound nephrin undergoes endocytic recycling. Vesicular trafficking is an important part of cellular development and growth, helping in maintaining a balance between synthesis, degradation and recycling of cellular components. Vps34, an important phosphoinositol kinase that generates PI3P, plays critical role in vesicular trafficking. We hypothesized that in our model recycling is responsible for maintenance of nephrin at the membrane following nephrin deletion.

Methods

Standard biochemical and cell biology techniques were used to analyze kidneys from knock out mice. Cre-loxP system was used to generate podocyte specific deletion of nephrin and vps34.

Results

Nephrin deleted adult animals have normal phenotype at 4-6 weeks age, when mice develop proteinuria. Vps34fl/fl, cre+ mice have normal phenotype at birth, develop massive proteinuria and accumulation of vesicles by 3 weeks of age and majority of them die by 9 weeks of age. Immunogold EM analysis reveals accumulation of Nephrin in vesicles suggesting involvement of Vps34-dependent endocytic pathway in nephrin’s maintenance at the slit diaphragm. In order to examine the role of endocytic recycling of nephrin at the membrane we generated a mouse model where both nephrin and vps34 are simultaneously deleted using tamoxifen in an adult mature mouse podocytes. These double knockout mice develop proteinuria at 1 week of age and die at 10-12 days indicating a earlier phenotype compared to deletion of vps34 or nephrin alone. Immunostaining revealed nephrin in vesicles that are EEA1 and Rab5 positive indicating nephrin localization in early endosomes and not in vesicles originating from the Golgi network.

Conclusion

We report an in vivo model that provides evidence for nephrin endocytosis and recycling at the membrane. A small fraction of nephrin is stably present at the membrane once production of new nephrin is stopped. Our data provides evidence of recycling of this nephrin from and to the membrane.

Funding

  • NIDDK Support