Figure 1

Renal macrophages comprise of long-lived kidney-resident macrophages and monocyte-derived CD11c^hi and CD11c^lo macrophages. (A) Workflow of the experiment. Mouse kidneys were enzyme-digested, percoll separated and stained for lineage and macrophage markers. After removing the lineage positive cells, three populations of macrophages were identified and flow sorted in the RNA lysis buffer and subjected to transcriptional profiling by RNA-sequencing. (B) Live, Lineage^negCD45⁺ were gated as F4/80⁺CD64^+/lo macrophages while non-macrophage population is CD45⁺11b/c^negCD64^negF4/80^neg. We classified kidney macrophages as CD11c^hiMϕ (CD11b^hiCD11c^hi), CD11c^hiMϕ (CD11b^hiCD11c^lo-neg), and Kidney-resident macrophages (KRM) (CD11c^IntCD11b^Int). Overlay of CD11c^hiMϕ (red), CD11c^loMϕ (blue) and KRM (orange) gated on Ly6c vs FCRIV, Cx3cr1 vs MerTK, and SSA vs CD45. KRM are Ly6c⁻FcrIV⁺MerTK⁺Cx3cr1⁺MHCII⁺CD45^int while the non-KRM CD11c^loMϕ are FcrIV⁺MerTK⁺Cx3cr1⁺MHCII⁺Ly6c^hiCD45^int-hi and CD11c^hiMϕ are FcrIV⁺MerTK⁺Cx3cr1⁺MHCII⁺Ly6c^loCD45^hi. (C) Fate-mapping studies using Cx3cr1^CreER+/−Rosa26^+/− mice demonstrates >80% of tdTomato⁺ cells gated as KRM. Live, Lineage^negCD45⁺ were gated as F4/80⁺tdTomato⁺ that were then gated as CD11b vs CD11c to identify KRM. (D) CD45.1 and CD45.2 congenic mice analyzed on 10 weeks of parabiosis. Histograms represents percentage of partner-derived cells in the kidney. n = 4 parabionts. Bars indicate mean value ± SEM. Symbols represent individual mice. (E-G) Transcriptional differences detected by RNA-Seq. (E) Comparison of gene expression between KRM and CD11c^hiMϕ (above), CD11c^loMϕ (below) displayed as volcano plots of individual genes, where fold-change between populations is plotted on the x-axis and significance on the y-axis. Genes upregulated >2-fold are colored in red and genes downregulated >2-fold in blue. 1257 genes are differentially expressed between KRM and CD11c^hiMϕ, 649 are up- and 608 down-regulated; and 2674 genes are differentially expressed between KRM and CD11c^hiMϕ, 1386 are up- and 1288 down-regulated. (F) Selected genes reflecting tissue resident status and upregulated in KRM, and (G) inflammatory genes downregulated in KRM, are presented as heatmaps with hierarchical clustering. Mean values per macrophage populations are shown. The z-score-based color-scale shows gene expression standard deviations below (blue) or above (red) the population mean. Data is representative of n = 4 independent experiments with at least n = 3 mice per group (KRM, n = 4, CD11c^hiMϕ, n = 3, CD11c^loMϕ, n = 3; DEGs: fold change >2, P < 0.05).Kidney image cropped and adopted from openclipart.org (https://openclipart.org/detail/28929/kidneyreins) and mouse images adopted from (https://openclipart.org/detail/174870/mouse and https://openclipart.org/detail/17558/simple-cartoon-mouse).