Fig 2: Generation of a knock-in Nphs2 BioID2 mouse line via CRISPR/Cas9 genome editing of the Nphs2 locus. (A) Schematic of the CRISPR/Cas9 genome editing strategy utilized to generate the Nphs2 BioID2 mouse line. A small guide RNA (sgRNA), targeting the stop codon in exon 8 of Nphs2 (purple arrowhead), was combined with a donor vector containing the knock-in cassette (zoom view) to induce homologous recombination and integrate the BioID2 moiety containing a 13x glycine/serine (G/S) linker, biotin ligase, and HA tag into the Nphs2 locus. (B) Genotyping strategy (top panel). Wild-type (a single band at 264 base pairs (bp)), heterozygous (two bands; one at wild-type size of 264 bp and a second that amplifies the BioID2 linker region giving a band at 480 bp), or homozygous (a single band at the 480 bp). (C) Genotyping of 73 embryos at E18.5-P0 verifies a relative Mendelian ratio of genotypes being recovered (25:50:25). (D) Immunofluorescence analysis of 8–10-week-old adult mice injected with biotin shows an enrichment of streptavidin within the glomerulus of Nphs2 BioID2 mice and absence of streptavidin signal in control littermate mice. The HA signal from the BioID2 moiety closely overlaps with that of streptavidin (merged image). Wt1 (cyan) is utilized as a podocyte marker to delineate the glomerular boundaries. Scale bar: 50 µm.

Fig 3: Ildr2 is detected in developing nephron structures and shows increased signal in glomeruli of aged mice and humans compared to that in the glomeruli of young mice and humans. (A) Proteomic profiling of two aged (~2 years; 108 week old) male Nphs2 BioID2/+ mice compared to 8–10 week-old Nphs2 BioID2/+ mice. Table lists three significantly increased proteins, podocin, and two decreased proteins. All proteins were detected with at least two razor unique peptides. (B) Micrographs of E15.5 kidney sections showing Ildr2 (magenta) in developing nephron structures (dotted outline) of mice (Mm; Mus musculus) with podocytes denoted by the Wt1 (green). Scale bar: 20 µm. (C) Micrographs of sections from young (~30-year-old) and aged (91-year-old) human (Hs; Homo sapiens) kidneys tissue coimmunostained for ILDR2 (magenta) and podocin (cyan). Scale bar: 50 µm.

Fig 4: Biotin-administered Nphs2 BioID2/+ mice show an enrichment of biotinylated proteins specifically within the glomeruli. (A) Lysates from biotin-injected Nphs2 BioID2/+ and wild-type age-matched 8–10-week-old littermate controls were subjected to protein separation, Western blot, and probed for streptavidin. (B) Streptavidin isolates blotted with an anti-HA antibody exhibit robust and specific detection of the HA signal only within the Nphs2 BioID2/+ sample (arrowhead). (C) Streptavidin isolates probed for podocin show two specific bands for both BioID2-tagged podocin (arrowhead) and endogenous podocin (asterisk). (D) Micrographs of P0 kidney sections showing DAPI (gray) and streptavidin (magenta) labeling. Pregnant dams were injected with 5 mg/kg of biotin from E11.5 to E18.5. Arrowheads show glomeruli labeled with streptavidin. Scale bar: 75 µm.