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Abstract: PO1966

A Novel In Vivo Approach to Capture the Podocyte Foot Process Proteome

Session Information

  • Podocyte Biology
    October 22, 2020 | Location: On-Demand
    Abstract Time: 10:00 AM - 12:00 PM

Category: Glomerular Diseases

  • 1204 Podocyte Biology

Authors

  • O'Brien, Lori L., University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, United States
  • Gerlach, Gary F., University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, United States
Background

Podocytes are an extraordinary cell of the kidney filtration system with their tentacle-like foot processes flowing out from each cell body and interdigitating with neighboring processes. Proper kidney function relies on these cells and the complex architecture created by the interdigitating processes. They are the most critical component of the glomerular filter. Podocytes are injured and their integrity is compromised in the majority of kidney diseases leading to progressive proteinuria. However, we know little about the full complement of proteins localized to the foot process and how they change with disease.

Methods


We have developed a novel genetic mouse model capable of generating a spatially restricted, real-time, in vivo proteome. Recently, proximity labeling techniques have been developed to provide snapshots of spatially localized proteomes. The BioID method utilizes a promiscuous bacterial biotin ligase flexibly linked to a target protein of interest to biotinylate proteins within the vicinity. We have adapted this approach to identify the podocyte foot process proteome. Using podocin as a handle, we have modified the Nphs2 (podocin) locus to link the mutated, promiscuous BirA biotin ligase to podocin (Nphs2BioID). A flexible, 13x linker allows for a generous proximity around podocin, thereby capturing the broad proteome of the podocyte foot process.

Results

We have obtained viable Nphs2BioID/+ animals. Utilizing immunstaining, we have confirmed the proper expression and localization of the podocin-BioID. The HA-tagged BirA ligase appended to podocin colocalized with podocin and other foot process proteins. To test it's functionally, we injected biotin dialy for 7 days. This produced an increase of biotinylated proteins in Nphs2BioID/+ podocytes versus wild type biotin injected controls or uninjected Nphs2BioID/+ mice. We have affinity purified the biotinylated proteins from glomerular isolations and are currently performing proteomic analyses.

Conclusion

We have generated the first of it's kind in vivo mouse model to specifically identify the spatially localized proteome of the podocyte foot process. Our proteomics results will provide unprecedented insights into the make-up of this highly specialized and critical structure.

Funding

  • NIDDK Support