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Abstract: FR-PO350

Uncovering the Podocyte Foot Process Proteome

Session Information

Category: Development‚ Stem Cells‚ and Regenerative Medicine

  • 500 Development‚ Stem Cells‚ and Regenerative Medicine

Authors

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

Podocyte foot process integrity is vital for kidney function and health. Disruptions to podocyte architecture, or effacement, is one of the most common clinical observation in kidney disease. However, the full complement of proteins responsible for maintaining podocyte foot process integrity remains unresolved.

Methods

The discovery of a proximity-dependent biotin identification (BioID) moiety that utilizes a promiscuous biotin ligase has opened new avenues to discover spatially localized proteomes. Podocin (Nphs2), localizing to the slit diaphragm, was employed as a handle to identify the in vivo proteome of the podocyte foot process via knock in of the BioID moiety into the murine Nphs2 locus (Podocin-BioID).

Results

We validated our Podocin-BioID model by assessing correct expression and localization of the fusion protein via western blot, immunofluorescence (IF), and localization to the slit diaphragm via electron microscopy. Subcutaneous injection of biotin into Podocin-BioID mice yields a significant enrichment of biotinylated proteins in podocytes. We isolated these biotinylated proteins and performed mass spectrometry (MS) analysis to identify proteins localized to the foot process. In silico analysis of the top proteins uncovered from MS identified ‘cell junctions’, ‘actin binding’, and ‘cytoskeleton organization’ as the top gene ontogeny terms. Our analyses uncovered a novel Immunoglobulin-like domain-containing receptor 2 (Ildr2) protein as highly expressed within the podocyte foot process. We confirmed Ildr2 expression in mouse podocytes by IF and in situ hybridization. Further, publicly available single cell RNA-seq databases detail conserved Ildr2 expression in human podocytes.

Conclusion

Ildr2 has documented expression in tricellular tight junctions and roles in immunomodulation with the ability to ameliorate autoimmune disease states in models of multiple sclerosis, type I diabetes, and rheumatoid arthritis. Yet the function of Ildr2 in podocyte development, integrity, and contribution to kidney immune cell modulation remains unknown. At large the podocytes’ role in immunomodulation remains a relatively unexplored niche which we aim to test through podocyte specific deletion of Ildr2. In summary our innovative strategy enabled the identification of novel components of the podocyte foot process proteome leading to a new set of biomarkers and candidates for renal disease therapeutics.

Funding

  • NIDDK Support