(A) Expression of individual canonical podocyte genes from mRNA-Seq data of young mice (Y, gray bars), aged mice injected with IgG2a (red bars), and aged mice injected with anti–PD-1 antibody (αPD1, blue bars). The levels of all genes were lower in aged IgG2a-injected mice compared with young mice, and all except Ptpro were increased in aPD1ab-injected mice. TPM, transcripts per million. (B–M) Protein validation of select genes by immunostaining. Representative immunoperoxidase staining for nephrin (B–D, brown), immunofluorescent staining for synaptopodin (F–H, green), and immunoperoxidase staining for VEGFA (J–L, black). The box in each panel shows an example of a glomerulus. Bar graphs show the quantification of nephrin (E), synaptopodin (I), and VEGFA (M) staining with each circle representing an individual mouse. Compared with young mice (gray bars), immunostaining was lower for each in aged IgG2a-injected mice (red bars) but was higher in aged aPD1ab-injected mice (blue bars). (N) VIPER (virtual inference of protein activity by enriched regulon) analysis of transcription factor activity. The top 10 transcription factors impacted by aPD1ab treatment are shown in the third column, along with their significance (first column), representative activity (second column), conferred activity (fourth column), and expression (fifth column), with red showing increased and blue decreased levels/activities. The conferred activity of Hnf1b, Nr2f6, Mbd3, and Tada2b increased, with increased expression. Zfp39, Sall1, and Sox7 activity was decreased despite higher expression levels in aPD1ab-injected mice. While the lower activity of Ets1 and the lower activity of Tcf4 were independent of their expression levels, the lower activity of Zeb2 correlated with its lower expression. Scale bars represent 50 μm (B–D and J–L) and 100 μm (F–H). Statistical analysis was performed by t test.