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. 2019 Jan 24;4(2):e125503.
doi: 10.1172/jci.insight.125503. Online ahead of print.

Resident Macrophages Reprogram Toward a Developmental State After Acute Kidney Injury

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Free PMC article

Resident Macrophages Reprogram Toward a Developmental State After Acute Kidney Injury

Jeremie M Lever et al. JCI Insight. .
Free PMC article

Abstract

Acute kidney injury (AKI) is a devastating clinical condition affecting at least two-thirds of critically ill patients, and, among these patients, it is associated with a greater than 60% risk of mortality. Kidney mononuclear phagocytes (MPs) are implicated in pathogenesis and healing in mouse models of AKI and, thus, have been the subject of investigation as potential targets for clinical intervention. We have determined that, after injury, F4/80hi-expressing kidney-resident macrophages (KRMs) are a distinct cellular subpopulation that does not differentiate from nonresident infiltrating MPs. However, if KRMs are depleted using polyinosinic/polycytidylic acid (poly I:C), they can be reconstituted from bone marrow-derived precursors. Further, KRMs lack major histocompatibility complex class II (MHCII) expression before P7 but upregulate it over the next 14 days. This MHCII- KRM phenotype reappears after injury. RNA sequencing shows that injury causes transcriptional reprogramming of KRMs such that they more closely resemble that found at P7. KRMs after injury are also enriched in Wingless-type MMTV integration site family (Wnt) signaling, indicating that a pathway vital for mouse and human kidney development is active. These data indicate that mechanisms involved in kidney development may be functioning after injury in KRMs.

Keywords: Immunology; Innate immunity; MHC class 2; Macrophages; Nephrology.

Conflict of interest statement

Conflict of interest: MM reports grants and consulting fees from Otsuka Pharmaceutical and Sanofi. AA serves as a consultant for DynaMed and is on the advisory board of Goldilocks Therapeutics.

Figures

Figure 1
Figure 1. F4/80hi kidney macrophages are minimally replaced by precursors from the blood after IR-AKI.
(A) Schematic representation of AKI in parabiosis model. One animal in a pair of CD45 congenic parabiotic chimeras underwent bilateral ischemia/reperfusion AKI (IR-AKI), and tissues were studied at 1, 3, and 14 days after injury. IR, injured; UPM, uninjured pair member. (B) Gating strategy and naming convention for kidney mononuclear phagocytes (MPs), including R2 kidney-resident macrophages (KRMs) and R1-infiltrative MPs. (CG) Total absolute numbers (cells/g tissue) and percentage of chimerism for kidney (C) neutrophils (PMN), (D) R1a Ly6Chi, (E) R1b Ly6CInt, (F) R1c Ly6Clo, and (G) R2 F4/80hi MPs after IR-AKI at days 1, 3, and 14. Mean ± SEM, n = 4–7 (≥4 pairs), 2-way ANOVA with Tukey’s post-test, *P < 0.05 for IR vs. Sham, #P < 0.05 for IR vs. UPM. (H) Mander’s overlap coefficients (percentage overlapping pixels) were plotted for CD45 allotypes overlapping with F4/80+ pixels. (I) Representative colocalization of CD45 allotypes with F4/80+ kidney MPs by confocal microscopy of transverse sections from injured kidneys in the medullae and cortices. Macrophages expressing CD45.2 (arrow) or CD45.1 (arrowheads) are indicated. Scale bar: 100 μm; 20 μm (insets). Representative of 3 biologic replicates per treatment group from 2 independent experiments.
Figure 2
Figure 2. Fcgr4hiFcgr1+ kidney MPs are F4/80hi KRMs, and these cells upregulate MerTK, Fcgr1, and Fcgr2/3 in response to AKI.
(A–D) Kidney mononuclear phagocyte (MP) flow cytometry measurements of mean fluorescence intensity (MFI) for R2 F4/80hi kidney-resident macrophages (KRMs), R1-infiltrative MPs, R1c Ly6Clo infiltrative MPs, and lymphoid lineage cells (CD3, CD19, NK1.1). Mean ± SEM, n = 6 per group from 2 independent experiments, 2-way ANOVA, *P < 0.05 for IR vs. Sham (Sidak’s post-test), #P < 0.05 for R2 vs. R1, R1c, and lymph (Tukey’s post-test). (A) MerTK, (B) Fcgr1 (CD64), (C) Fcgr2/3 (CD16/CD32), (D) Fcgr4 (CD16.2). (E) Representative 2-parameter flow histograms of a gating strategy for R2 KRMs and R1-infiltrative MPs that depends on Fcgr4 and Fcgr1. Values are percentage gated. Representative of n = 6 per group from 2 independent experiments. Lymph, lymphoid lineage cells (CD3, CD19, NK1.1); MerTK, Mer tyrosine kinase; Fcgr, Fc γ receptor.
Figure 3
Figure 3. Kidney F4/80lo mononuclear phagocytes are derived from the peripheral circulation and turn over within 14 days.
(A) Schematic representation of parabiosis separation experiment of steady-state parabiotic chimeras. 10-week-old CD45 congenic mice (CD45.1 and CD45.2) were parabiotically attached for 4 weeks. The pairs were separated and blood was drawn at the time of separation and then 3, 7, and 14 days after separation. On day 14, kidneys were harvested and immune cell chimerism was studied. (B) The percentage of chimerism in peripheral blood of parabiotic chimeras at time points after separation. Mean ± SEM, n = 8 (4 pairs). Statistical comparison: day 3 Ly6Chi vs. Ly6Clo, 2-way ANOVA with Tukey’s post-test, *P < 0.05. (C) The percentage of chimerism for kidney CD45+ leukocytes, intrarenal neutrophils (PMN), and kidney MPs 14 days after separation. Mean ± SEM.
Figure 4
Figure 4. Kidney-resident macrophages are replaced by bone marrow–derived precursors only after the niche is opened.
(A) Schematic of Cre induction and Myb gene deletion in Mx1CreMybfl/fl mice, either followed by CD45.1 congenic bone marrow transplant (BMT), further followed by a 3-month period before endpoint analysis (B and C), or by tissue harvest to study effects of poly I:C (pIC) on kidney MPs (E and F). (B) Percentage of chimerism (%CD45.1+) of kidney PMNs and MPs 3 months following BMT. Mean ± SEM. (C) Representative contour flow cytometry plots of chimerism 3 months after BMT for PMN, R1a, R1c, and lymphoid lineage cells (CD3, CD19, NK1.1), and R2. The red gate encloses chimeric bone marrow–derived cells. The black gate encloses host-derived, nonchimeric cells. Values are percent gated. Representative of n = 4. (D) Representative contour flow cytometry plot of chimerism for R2 from AKI in parabiosis kidney 14 days after injury. The red gate encloses chimeric blood-borne cells. The black gate encloses host-derived, nonchimeric cells. Values are percentage gated. Representative of n = 7; 3 independent experiments. (E and F) Proportions and absolute numbers, normalized to whole organ preparations, of neutrophils (PMN) and R2 or R1 kidney MPs following either saline vehicle (Sal) or pIC in Cre+ or Cre mice. Mean ± SEM, 2-way ANOVA with Tukey’s post-test, *P < 0.05.
Figure 5
Figure 5. Resident macrophages in developing kidneys undergo an MHCII phenotype switch.
(A) Representative 2-parameter flow histograms plotted on CD11b and F4/80 of CD45+ lymphoidlineageGr-1 kidney mononuclear phagocytes (MPs) through development from E16.5 to P28. R2 F4/80hi kidney-resident macrophages (KRMs) are further studied for CD11c and MHCII. (B) Proportion of R2 F4/80hi KRMs expressing MHCII protein at various stages during development from E14.5 to P28. Mean ± SEM, n = 3–6. (C) t-Distributed stochastic neighbor embedding (tSNE) of physical characteristics and surface phenotype of CD45+ lymphoid lineageGr-1 kidney MPs with color backgating at various stages of development from E16.5 to P28. Lymphoid lineage includes CD3, CD19, and NK1.1.
Figure 6
Figure 6. Kidney-resident macrophages downregulate MHCII after injury.
(A) The percentage of R2 kidney-resident macrophages (KRM) positive for MHCII expression in sham and injured (20 minutes bilateral ischemia/reperfusion) mice at varying time points after injury. Mean ± SEM, n = 5–9 from 2 independent experiments, 1-way ANOVA with Sidak’s post-test compared with sham control, *P < 0.05. (B) Flow cytometry 2-parameter histograms gated on R2 KRMs demonstrating MHCII and CD11c expression 6 days after injury compared with sham control. Values are percentage gated. (C) From AKI in parabiosis mice, percentage of chimerism for PMNs, R2 MHCII+, and R2 MHCII in sham and 3 days after injury. Mean ± SEM, n = 5–10 from 3 independent experiments. (D and E) Absolute numbers (D) and percentage of EdU+ (E) for R2 KRMs, further subset into MHCII+ and MHCII, indicating changes in cell numbers and proliferative activity during period of 3–4 days after injury (EdU treatment at day 3, endpoint at day 4). Mean ± SEM, n = 5–7 from 2 independent experiments, 2-way ANOVA with Tukey’s post-test compared with sham control, *P < 0.05.
Figure 7
Figure 7. AKI-responsive kidney-resident macrophages utilize developmental transcriptional programming during healing.
(A) Unsupervised 3-dimensional principal component analysis (PCA) using normalized counts from RNAseq analysis, with spheroids designated according to k-means clustering. RNA was isolated from sorted cells from P7 mice (sex indiscriminate), from adult male quiescent mice, or from adult male mice 6 days after injury. Independent biologic replicates were projected onto each individual principal component axis. (B) Hierarchical clustering and heatmap visualization of DEGs (P7 vs. Qui, P < 0.01), illustrating the treatment condition (injured vs. quiescent), MHCII expression status, and cellular origin. Expression levels represented as relative Z-score, with yellow indicating increased expression and blue decreased expression. (C) Three-way Venn diagram depicting the overlap in DEGs for MHCII+ AKI vs. Quiescent, MHCII AKI vs. Quiescent, and P7 vs. Quiescent (P < 0.01). (D) RNAseq normalized counts for MHCII and invariant chain gene transcripts in sorted kidney MPs. Mean ± SEM, n = 3 per group. MHCII, major histocompatibility complex class II.
Figure 8
Figure 8. Wnt/b-catenin canonical signaling is enriched in injury-responsive and P7 kidney-resident macrophages.
(A) Wnt/β-catenin signaling pathway based on Ingenuity Pathway Analysis (IPA), with overlaid differential gene expression based on MHCII AKI vs. Quiescent (FDR-adjusted P < 0.05). (B–D) Real-time PCR analysis of sorted kidney MPs for mRNA expression of (B) Wnt4, (C) Fzd1, and (D) Lrp6. Mean ± SEM, n = 3–8, 1-way ANOVA with Dunnett’s post-test comparing each group vs. Qui or R1c, *P < 0.05. (E) Normalized counts from RNAseq for genes associated with canonical Wnt signaling activation downstream of surface receptor binding of Wnt ligands as visualized in A. Expression data displayed for Src, Hdac1, Axin2, Gja1, Tcf4, and Jun. Mean ± SEM, n = 3 per group. Gene symbols are from Mouse Genome Informatics via IPA.
Figure 9
Figure 9. A lineage model for prohealing mononuclear phagocytes in AKI.
Schematic representation of lineage independence of kidney-resident macrophages (KRMs) from infiltrative mononuclear phagocytes (MPs) after AKI. KRMs show a phenotypic developmental switch for MHCII expression, and MHCII KRMs reappear in the setting of injury, reproducing transcriptional profiles present in these cells during nephrogenesis. Unique compared with infiltrative MPs, KRMs display activation of downstream canonical Wnt signaling in response to injury.

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