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Abstract: TH-PO1071

Rab11-Dependent Recycling Maintains Nephrin at the Slit Diaphragm

Session Information

Category: Glomerular Diseases

  • 1204 Podocyte Biology

Authors

  • Verma, Rakesh, University of Michigan, Ann Arbor, Michigan, United States
  • Cara-Fuentes, Gabriel M., University of Michigan, Ann Arbor, Michigan, United States
  • Garg, Puneet, University of Michigan, Ann Arbor, Michigan, United States
Background

A role of endocytosis for podocyte biology has previously been proposed. In humans, until recently no direct endosomal regulator had previously been implicated in nephrotic syndrome. Whole exome sequencing identified mutations in Rab5 and Rab11 regulatory proteins resulting in congenital nephrotic syndrome and FSGS. Rab5 and Rab11 are expressed ubiquitously, suggesting perturbation of trafficking of podocyte-specific proteins as one potential mechanism for podocyte-restricted phenotype. We reported deletion of nephrin in an adult mature glomerulus, a small fraction (10-15%) of nephrin remains at the membrane and is sufficient to maintain the slit diaphragm for 4-6 week. In vitro studies have suggested that membrane bound nephrin undergoes endocytic recycling. 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 knockout mice. Cre-loxP system was used to generate podocyte specific deletion of nephrin, VPS34 and Rab11.

Results

Combined deletion of nephrin and VPS34 results in an earlier phenotype compared to deletion of nephrin or vps34 alone. Immunogold EM analysis reveals accumulation of Nephrin in vesicles suggesting involvement of Vps34-dependent endocytic pathway in nephrin’s maintenance at the slit diaphragm. As VPS34-generated PI3P affects trafficking to early endosome as well as recycling endosomes we generated a mouse model where rab11a and rab11b are deleted in a podocyte-specific manner in an adult mice. Deletion of rab11b alone does not result in a phenotype, whereas combined deletion of rab11a and rab11b results in development of proteinuria at 4-6 week following deletion. Simultaneous deletion of nephrin/rab11a/rab11b results in an earlier phenotype where mice develop proteinuria at 1 week following deletion along with foot process spreading. Analysis of nephrin at the membrane indicates a faster decline following deletion of rab11a/b.

Conclusion

Simultaneous deletion of Rab11 and nephrin results in an earlier phenotype and shortens nephrin's half life at the membrane. Our model provides us with mechanistic insights into human mutations where perturbation of endosomal regulatory proteins result in proteinuric kidney disease.

Funding

  • NIDDK Support