Abstract: PO0612
Uncovering the Podocyte Foot Process Proteome
Session Information
- Development, Stem Cells, and Regenerative Medicine
November 04, 2021 | Location: On-Demand, Virtual Only
Abstract Time: 10:00 AM - 12:00 PM
Category: Development, Stem Cells, and Regenerative Medicine
- 500 Development, Stem Cells, and Regenerative Medicine
Authors
- Gerlach, Gary F., University of North Carolina at Chapel Hill School of Medicine, Chapel Hill, North Carolina, United States
- O'Brien, Lori L., University of North Carolina at Chapel Hill School of Medicine, 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 observations in kidney disease. However, the full complement of players responsible for maintaining podocyte foot process integrity is still unknown. The membranous cellular environment and specialized junctional complexes have previously hindered their isolation and testing.
Methods
The discovery of a proximity-dependent biotin identification (BioID) moiety that utilizes a promiscuous biotin ligase has opened new avenues to generate spatially localized proteomes. Podocin (Nphs2) localizes to the slit diaphragm and is one of the most abundant foot process proteins. Therefore, we developed a novel genetic mouse model via knock in of the BioID moiety to the Nphs2 locus (podocin-BioID) to identify the in vivo proteome of the podocyte foot process localized within the vicinity of podocin.
Results
We validated our transgenic podocin-BioID model by assessing correct expression and localization of the fusion protein via western blot, immunofluorescence (IF), and electron microscopy (EM). Injection of podocin-BioID mice with excess biotin leads to the significant biotinylation of proteins within podocytes. We isolated the biotinylated proteins and performed mass spectrometry analyses (MS) to uncover novel proteins localized to the foot process. In silico analysis of the top proteins uncovered from MS identified ‘cell junctions’, ‘adherence’, and ‘adhesion’ as the top gene ontogeny terms. One novel candidate we uncovered is the Immunoglobulin-like domain-containing receptor 2 (Ildr2) protein. We confirmed Ildr2 is expressed in mouse podocytes by immunofluorescence and utilized publicly available single cell RNA-seq data to confirm its restricted, conserved expression in both mouse and human podocytes.
Conclusion
Current efforts are aimed at knocking out Ildr2 specifically in the podocytes of mice and zebrafish to assess the functional significance of Ildr2 in podocyte foot process integrity. These biorthogonal assays have allowed us to identify and interrogate novel components of the foot process proteome leading to a new set of potential players and biomarkers for kidney disease.
Funding
- NIDDK Support