Macrophage Wnt7b is critical for kidney repair and regeneration

Abstract

Macrophages are required for tissue homeostasis through their role in regulation of the immune response and the resolution of injury. Here we show, using the kidney as a model, that the Wnt pathway ligand Wnt7b is produced by macrophages to stimulate repair and regeneration. When macrophages are inducibly ablated from the injured kidney, the canonical Wnt pathway response in kidney epithelial cells is reduced. Furthermore, when Wnt7b is somatically deleted in macrophages, repair of injury is greatly diminished. Finally, injection of the Wnt pathway regulator Dkk2 enhances the repair process and suggests a therapeutic option. Because Wnt7b is known to stimulate epithelial responses during kidney development, these findings suggest that macrophages are able to rapidly invade an injured tissue and reestablish a developmental program that is beneficial for repair and regeneration.

Fig. 1.

Injured proximal tubule epithelial cells are Wnt-pathway-responsive, and inflammatory macrophages are a source of Wnt ligands in the kidney during repair following injury. (A–C) Photomicrographs of whole-mount X-gal-stained BATgal kidneys indicating Wnt pathway responses. (D and E) Sections from day 5 injured kidneys from BATgal mice labeled for nuclei (blue), Lacz (green), the proximal epithelial tubule marker LTL (D), or the macrophage marker F4/80 (E). Epithelial cells but not macrophages are Lacz-positive. (F–H) X-gal staining for Wnt signaling activity in Axin2^+/LacZ kidneys. In F, arrowheads indicate epithelial cells of flattened morphology typical of severe injury that contrasts with cuboidal morphology shown by arrowheads in uninjured kidney (G). Injured tubules contain necrotic debris (F, nec), whereas uninjured tubules do not (G, asterisks). X-gal staining is prominent in epithelial cells of Axin2^+/LacZ kidneys (H and I, arrowheads). (I and J) Sections from day 5 injured kidneys from Axin2^+/LacZ mice labeled for nuclei (blue), Lacz (green), the fibroblast marker α-SMA (I), or the macrophage marker F4/80 (J). In I, Lacz-positive fibroblasts are seen (arrows). (K) Semiquantitative RT-PCR for transcript levels of Wnt-signaling-pathway ligands and receptors in whole kidney or purified cell types (C, normal control; I, d5 postinjury; P, peripheral blood monocytes). Red arrowheads indicate up-regulated transcripts. All studies were repeated at least three times and gave comparable results. *P < 0.05. (Scale bars, 50 μm.)

Fig. 2.

Macrophages are a source of Wnts and mediate Wnt responses in regeneration of the kidney epithelium. (A–D) Relative luciferase activity (RLA) from STF cells expressing Lrp5 or Lrp6 with Fzd3, Fzd4, or Fzd7, induced by coculture with: (A–C) d5 post-I/R kidney macrophages (I/R Mφ) compared with autologous peripheral blood monocytes (PBM) and inhibited (C) by recombinant Dkk1; or (D) coculture with bone marrow macrophages (Mφ-CON) or Wnt7b-expressing bone marrow macrophages and inhibited by the addition of Dkk1-expressing 293T cells. (E–L) PAS-stained kidney sections and F4/80 immunofluorescence confocal images of kidney sections from mice with and without conditional macrophage ablation during recovery from injury (asterisks, regenerating tubules; arrowheads, injured flattened epithelia; nec, necrotic debris). (M–O) Quantification of macrophages, active Wnt signaling (X-gal staining) in kidney cortex and medulla, and tubule injury d6 after injury. (P) Kidney function testing (plasma creatinine levels) in cohorts of mice (n = 6/group) with or without macrophage ablation from d3 to d6. Normal recovery is prevented by ablation. P < 0.05. n = 5 or 6/group. (Scale bars, 50 μm.)

Fig. 3.

Mutation of Frizzled4 or coreceptors Lrp5 and Lrp6 prevents normal repair and regeneration of the kidney following ischemia reperfusion injury. (A and B) Lacz staining in d5 post-IRI kidney showing restriction of Fzd4 expression to epithelial tubules and (C) low- (top left) and high-power views (split color panels) showing Fzd4 receptor expression is not present in F4/80+ macrophages in postinjury kidneys. (D–G) PAS-stained sections of normal (D and F) and d5 post-IRI kidneys (E and G) showing persistence of epithelial injury in mice lacking Fzd4 (Fzd4^LacZ/LacZ). (H–L) Quantification of inflammation, injury, and repair parameters 5d post-kidney-IRI injury in Fzd4^LacZ/LacZ or littermate control mice. (M–R) PAS-stained sections of kidneys. (S and T) Quantification of inflammation, injury, and repair parameters in WT or Lrp5^+/lacz; Lrp6^+/lacz kidneys. P < 0.05. n = 5 or 6/group. (Scale bars, 50 μm.) Asterisks, regenerating tubules; arrowheads, injured flattened epithelia; nec, necrotic debris.

Fig. 4.

Somatic mutation of Wnt7b in macrophages prevents normal repair and regeneration of the kidney following ischemia reperfusion injury. (A and B) Genomic map and PCR products (tail or macrophage genomic DNA) showing the third exon of Wnt7b is deleted in monocytes/macrophages of mice with the LoxP-flanked conditional allele Wnt7b^C3 and the transgene Csf1r-icre. WT allele, 153 bp; C3 allele, 200 bp; ΔC3 allele, no product. (C–E) PAS-stained sections of the outer cortex of Csf1R-icre; Wnt7b^C3/− kidneys or control kidneys (Csf1R-icre; Wnt7b^C3/+ and Wnt7b^C3/−) 7d following injury. (F–J) Quantification of inflammation, injury, and repair parameters 7d postinjury of Csf1R-icre; Wnt7b^C3/− or control mice (Csf1R-icre; Wnt7b^C3/+ and Wnt7b^C3/−). (K and L) Immunofluorescence images of kidneys showing dissolution of epithelial basement membrane (arrowheads) and quantification of dissolution in control and experimental (Csf1R-icre; Wnt7b^C3/−) mice. (M–O) Immunofluorescence images of experimental postinjury kidneys showing the presence of markers of the cell cycle in epithelial cells and quantification of epithelial cells entering G1/S or in G2M phase of the cell cycle. P < 0.05. n = 5 or 6/group. (Scale bars, 50 μm.)

Fig. 5.

Dickkopf-2 promotes repair in the post-ischemia reperfusion injury kidney. (A–D) PAS-stained sections of kidneys. (E) Coomassie-stained polyacrylamide gel showing purified Dkk2-C2 (D2C2). (F and G) Graphs showing quantified injury and repair parameters in kidneys treated with vehicle or Dkk2. (H) Graph showing plasma creatinine levels in mice treated with Dkk2 from d1 post-I/R onward. (I) Western blot (upper) for pLrp6 (210 kDa) and loading control (β-actin 45 kDa) in kidney cortex from mice on d5 post-I/R treated with Dkk2-C2 or vehicle. (J) RT-PCRs for Kremen-1 and Kremen-2 in purified d5 post-I/R kidney macrophages (Mφ) (I) compared with autologous peripheral blood monocytes (P) (upper) and d5 post-IRI proximal tubule epithelial cells (PTEC) (I) or epithelial cells from control kidneys (C). *P < 0.05. n = 6/group. (Scale bars, 50 μm.)