Parabiosis reveals leukocyte dynamics in the kidney

Abstract

The immune cellular compartment of the kidney is involved in organ development and homeostasis, as well as in many pathological conditions. Little is known about the mechanisms that drive intrarenal immune responses in the presence of renal tubular and interstitial cell death. However, it is known that tissue-resident leukocytes have the potential to have distinct roles compared with circulating cells. We used a parabiosis model in C57BL/6 CD45 congenic and green fluorescent protein transgenic mice to better understand the dynamics of immune cells in the kidney. We found F4/80^Hi intrarenal macrophages exhibit minimal exchange with the peripheral circulation in two models of parabiosis, whether mice were attached for 4 or 16 weeks. Other intrarenal inflammatory cells demonstrate near total exchange with the circulating immune cell pool in healthy kidneys, indicating that innate and adaptive immune cells extensively traffic through the kidney interstitium during normal physiology. Neutrophils, dendritic cells, F4/80^Low macrophages, T cells, B cells, and NK cells are renewed from the circulating immune cell pool. However, a fraction of double-negative T (CD4^- CD8^-) and NKT cells are long-lived or tissue resident. This study provides direct evidence of leukocyte sub-populations that are resident in the renal tissue, cells which demonstrate minimal to no exchange with the peripheral blood. In addition, the data demonstrate continual exchange of other sub-populations through uninflamed tissue.

Figure 1

Validation of the parabiosis model for study of renal inflammation. (a) Schematic representing animal husbandry and age at which mice undergo the parabiosis procedure. 28-day parabiosis period is shown. (b) Development of CD45 parabiosis chimeras over time. Percent chimerism among blood leukocytes before parabiosis and after 14 and 28 days of parabiosis. Mean ± SEM, n = 6 pairs (6 mice per group). (c) Gating strategy employed for identifying host and non-host chimeric viable leukocytes in the kidney of CD45 parabiosis chimeras. Circled kidneys represent sample used to generate histograms shown to the right. (d) Representative flow cytometry histograms for resolving host and non-host chimeric leukocytes in the kidney spleen and blood. Percent gated is shown for each region. (e) Photomicrograph of periodic acid-Schiff stained transverse section of 28-day parabiont kidney. Scale bars 1 mm (left panel) and 20 μm (right panel). (f) Serum creatinine from unattached controls or CD45 parabionts after 28 days of parabiosis. Mean ± SEM, n = 4 – 7 for unattached controls, n = 16 pairs for 28 days parabiosis.

Figure 2

Distribution of leukocyte proportions in 4-week and 16-week CD45 congenic parabiotic chimeras and unattached controls, calculated as a percentage of viable CD45⁺ leukocytes. (a) Kidney and (b) spleen are shown. Abbreviated cell phenotypes with color coding are included. See detailed phenotypes in Table 1. Mean ± SEM, n = 6 pairs for CD45 congenic parabiotic chimeras, n = 2 for each type of unattached control. Two-way ANOVA, Sidak’s multiple comparisons test; *p < 0.05 for proportion of given cell type after 16 weeks parabiosis vs same cell type after 4 weeks parabiosis. PMN = neutrophil, DC = dendritic cell, NK = natural killer.

Figure 3

CD45⁺ leukocyte percent chimerism in kidney, spleen, and blood after 4 weeks and 16 weeks of parabiosis. (a and b) Percent chimerism among CD45⁺ leukocytes in kidney, spleen, and blood from parabiotic chimeras; (a) eGFP system, (b) CD45 system. Each dot represents the mean of two measurements from an individual mouse. Data are from 7 independent experiments. Mean ± SEM, n = 7 pairs for eGFP-4 weeks, n = 3 pairs for eGFP-16 weeks, n = 6 pairs for CD45-4 weeks, n = 3 pairs for CD45-16 weeks. ND = no data.

Figure 4

Method for identifying renal myeloid-lineage inflammatory cells. Renal mononuclear phagocyte populations were identified based on previously published phenotypes. Flow histograms were plotted on (a) CD11b vs CD11c (see Kawakami et al. 2013) and (b) F4/80 vs CD11c (see Cao et al. 2015). (c) Flow cytometry gating strategy employed for identifying myeloid lineage cells, including PMN, F4/80^Hi macrophages, F4/80^Low macrophages, and DC in the kidney. Percent gated is shown for each region. (d) One parameter flow cytometry histograms demonstrate the surface phenotype of renal mononuclear phagocyte subsets. Gray histograms are mixed isotype and fluorescence minus one-stained controls from kidney single cell suspensions for the given cell type. Histograms are representative of 3 independent experiments. PMN = neutrophil, Mac = macrophage, DC = dendritic cell.

Figure 5

Percent chimerism for myeloid lineage cells from kidney, spleen, and blood of CD45 parabionts. (a) Representative flow cytometry histograms of chimeric populations of renal mononuclear phagocytes. Quantification of percent chimerism for (b) PMN, (c) F4/80^Hi macrophages, (d) F4/80^Low macrophages, and (e) CD11b⁺ and CD11b⁻ DC. Mean ± SEM, n = 12 for CD45-4 week (6 pairs), n = 6 for CD45-16 week (3 pairs), n = 14 for eGFP-4 week (7 pairs), and n = 3 for eGFP-16 week (3 pairs). Two-way ANOVA, Sidak’s multiple comparisons test, comparing kidney versus spleen for each cell type at each time point, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. PMN = neutrophil, Mac = macrophage, DC = dendritic cell, ND = no data.

Figure 6

Gating strategy for kidney lymphoid lineage cells from quiescent CD45 parabionts, including B, NK, NKT, T, CD4 and CD8 T cells, and DNTL. Percent gated is shown for each region. (b–i) Percent chimerism for lymphoid lineage cells from kidney, spleen, and blood of CD45 parabionts. (b) Representative flow cytometry histograms of chimeric populations of lymphocytes observed in the kidney. Quantification of percent chimerism for (c) T cells, (d) CD4, (e) CD8, (f) double negative T lymphocytes, (g) NK cells, (h) NKT cells, and (i) B cells from kidney, spleen, and blood of CD45 parabionts. Mean ± SEM, n = 12 for 4w and n = 6 for 16w. Two-way ANOVA, Sidak’s test for multiple comparisons, comparing kidney versus spleen for each cell type at each time point, **p < 0.01, ***p < 0.001. ND = no data. NK = natural killer, DNTL = double negative T lymphocyte.