Macrophage-to-Myofibroblast Transition Contributes to Interstitial Fibrosis in Chronic Renal Allograft Injury

Abstract

Interstitial fibrosis is an important contributor to graft loss in chronic renal allograft injury. Inflammatory macrophages are associated with fibrosis in renal allografts, but how these cells contribute to this damaging response is not clearly understood. Here, we investigated the role of macrophage-to-myofibroblast transition in interstitial fibrosis in human and experimental chronic renal allograft injury. In biopsy specimens from patients with active chronic allograft rejection, we identified cells undergoing macrophage-to-myofibroblast transition by the coexpression of macrophage (CD68) and myofibroblast (α-smooth muscle actin [α-SMA]) markers. CD68⁺/α-SMA⁺ cells accounted for approximately 50% of the myofibroblast population, and the number of these cells correlated with allograft function and the severity of interstitial fibrosis. Similarly, in C57BL/6J mice with a BALB/c renal allograft, cells coexpressing macrophage markers (CD68 or F4/80) and α-SMA composed a significant population in the interstitium of allografts undergoing chronic rejection. Fate-mapping in Lyz2-Cre/Rosa26-Tomato mice showed that approximately half of α-SMA⁺ myofibroblasts in renal allografts originated from recipient bone marrow-derived macrophages. Knockout of Smad3 protected against interstitial fibrosis in renal allografts and substantially reduced the number of macrophage-to-myofibroblast transition cells. Furthermore, the majority of macrophage-to-myofibroblast transition cells in human and experimental renal allograft rejection coexpressed the M2-type macrophage marker CD206, and this expression was considerably reduced in Smad3-knockout recipients. In conclusion, our studies indicate that macrophage-to-myofibroblast transition contributes to interstitial fibrosis in chronic renal allograft injury. Moreover, the transition of bone marrow-derived M2-type macrophages to myofibroblasts in the renal allograft is regulated via a Smad3-dependent mechanism.

Keywords: M2 macrophage; Smad3; chronic allograft rejection; interstitial fibrosis; lineage tracing; macrophage myofibroblast transition.

Graphical abstract

Figure 1.

MMT cells in patients with chronic active renal transplantation rejection. Two-color immunofluorescence identifies MMT cells that coexpress macrophage (CD68, green) and myofibroblast (α-SMA, red) markers in biopsy tissues. (A) Allograft immediately after transplantation (control). (B) Acute rejection. (C) Chronic active rejection. Examples of CD68⁺ α-SMA⁺ MMT cells are indicated by arrows and insert shows a high-power view of a single MMT cell. (D) Chronic allograft injury with no evidence of active rejection. Nuclei were stained with DAPI in blue. (E) Quantification of the number of CD68⁺ cells, α-SMA⁺ cells, and CD68⁺ α-SMA⁺ cells. Data are mean±SEM. ***P<0.001 versus acute rejection. (F–I) Correlation analysis of cell populations and allograft function in patients with chronic active transplant rejection. SCr, serum creatinine.

Figure 2.

MMT cells produce collagen in human chronic active renal allograft rejection. (A) Three-color confocal microscopy identifies cells coexpressing CD68 (green), α-SMA (red), and collagen I (blue). One example of collagen-producing MMT is shown in high power in the insert. (B) Z-stack images illustrate the coexpression of CD68 (green), α-SMA (red), and collagen I (blue) in an MMT cell, which is also shown in the Supplemental Material.

Figure 3.

Fate-mapping identifies a bone marrow macrophage origin for myofibroblasts in mouse chronic renal allograft rejection. Kidney allografts were transplanted into Lyz2-Cre/Rosa26-Tomato recipient mice and examined 28 days later. (A) Confocal microscopy shows that most F4/80⁺ macrophages (blue) express the Tomato marker, confirming their origin from recipient bone marrow macrophages. In addition, F4/80⁺Tomato⁺ cells can be seen coexpressing α-SMA (green), indicating MMT (insert shows high power of an MMT cell). (B) Identification of Tomato⁺ cells expressing α-SMA (green) and collagen I (blue). Insert shows an example of a Tomato⁺α-SMA⁺collagen I⁺ MMT cell. (C) Z-stack images show individual MMT cells coexpressing Tomato (red), α-SMA (green), and collagen (blue) in the rejecting allograft. Note that an elongated Tomato⁺ F4/80⁺α-SMA⁺ MMT cell with rich submembranous bundles of α-SMA+ actin filaments is clearly shown, which is further illustrated in the Supplemental Material. (D) Two-color flow cytometry analysis shows most Tomato⁺ cells in the rejecting allograft coexpress the CD68 macrophage marker. In addition, 90% of the CD68⁺α-SMA⁺ cells in the rejecting allograft express Tomato, demonstrating a bone marrow macrophage origin. Graph shows quantification (mean±SEM) of these populations from a group of six transplant recipients.

Figure 4.

Deletion of Smad3 in the recipient alleviates interstitial fibrosis in mouse chronic renal allograft rejection. Kidney allografts were transplanted into Smad3 WT or Smad3 KO recipient mice and examined 28 days later. An isograft group was used as a control. (A) Masson trichrome stain. (B) Immunohistochemistry stain for collagen I deposition. (C) Immunohistochemistry stain for F4/80⁺ macrophage infiltration. Graphs show quantification of the area of collagen staining using Masson trichrome and collagen I staining and the number of F4/80⁺ macrophages. Data are mean±SEM for groups of 6–8 mice. *P<0.05, ***P<0.001 versus isograft control; ^###P<0.001 versus Smad3 WT mice.

Figure 5.

Deletion of Smad3 in the recipient inhibits fibrosis in mouse chronic renal allograft rejection. Kidney allografts were transplanted into Smad3 WT or Smad3 KO recipient mice and examined 28 days later. An isograft group was used as a control. (A) α-SMA protein levels in renal allografts shown by Western blotting and α-SMA mRNA levels shown by real-time PCR. (B) Collagen I protein levels in renal allografts shown by Western blotting and collagen I mRNA levels shown by real-time PCR. Data are mean±SEM for groups of 6–8 mice. *P<0.05, ***P<0.001 versus isograft controls; ^#P<0.05, ^##P<0.01, ^###P<0.001 versus Smad3 WT mice.

Figure 6.

Deletion of Smad3 in the recipient inhibits MMT in mouse chronic renal allograft rejection. Kidney allografts were transplanted into Smad3 WT or Smad3 KO recipient mice and examined 28 days later. (A) Immunofluorescence shows many F4/80⁺ α-SMA⁺ MMT cells in renal allografts from Smad3 WT recipients. This was substantially inhibited in Smad3 KO recipients. (B) Three-color confocal microscopy identifies MMT cells in an allograft from a Smad3 WT recipient on the basis of coexpression of F4/80 (green), α-SMA (red), and collagen I (blue) as indicated by arrows. (C) Z-stack image shows collagen-producing MMT cells, which is also further illustrated in the Supplemental Material. Note that a CD68⁺α-SMA⁺ collagen I⁺ MMT cell is indicated by arrow. (D) Quantitative data for counting of the F4/80⁺, α-SMA⁺, and F4/80⁺α-SMA⁺ cell populations. Data are mean±SEM for groups of 6–8 mice. ^##P<0.01, ^###P<0.001 versus Smad3 WT mice.

Figure 7.

Flow cytometric analysis shows that deletion of Smad3 from recipient mice inhibits collagen-producing MMT cells in grafted kidneys with chronic renal allograft rejection. Kidney allografts were transplanted into Smad3 WT or Smad3 KO recipient mice and examined 28 days later. An isograft group was used as a control. (A) Analysis of macrophage (CD68) and myofibroblast (α-SMA) antigen expression identifies CD68⁺α-SMA⁺ MMT cells in renal allografts in Smad3 WT or KO recipients. (B) Identification of CD68⁺collagen I⁺ MMT cells in renal allografts in Smad3 WT or KO recipients. (C–F) Quantitative analysis of MMT cells as a percentage of the total α-SMA⁺ (C), collagen I⁺ (D), or CD68⁺ macrophage (E and F) populations. Data are mean±SEM for groups of six mice. ^#P<0.05, ^##P<0.01, ^###P<0.001 versus Smad3 WT mice.

Figure 8.

The majority of MMT cells in human chronic active transplant rejection have an M2 phenotype. (A and B) Immunofluorescence shows many CD206⁺CD68⁺ M2 cells but few iNOS⁺CD68⁺ M1 cells in human chronic active transplant rejection. (C) Quantitative analysis of CD206⁺CD68⁺ cells and iNOS⁺CD68⁺ cells as a percentage of total CD68⁺ cells in human chronic active transplant rejection. (D and E) Two-color immunofluorescence shows that many α-SMA⁺ myofibroblasts (red) coexpress CD206 (M2 marker), but lack expression of iNOS (M1 marker) in human chronic active transplant rejection. Insert shows an example of a CD206⁺F4/80⁺α-SMA⁺ MMT cell (D). (F) Quantitative analysis of CD206⁺α-SMA⁺ cells and iNOS⁺α-SMA⁺ cells as a percentage of total α-SMA⁺ cells in human chronic active transplant rejection. Data are mean±SEM for six patients with chronic active allograft rejection. ^###P<0.001 versus CD206⁺CD68⁺ cells/CD68⁺ cells; ***P<0.001 versus CD206⁺α-SMA⁺ cells/α-SMA⁺ cells.

Figure 9.

Renal allografts in Smad3-deficient mice have reduced M2 macrophage infiltration. Kidney allografts were transplanted into Smad3 WT or Smad3 KO recipient mice and examined 28 days later. (A) Immunofluorescence staining for total macrophages (CD68, green) and the M1 marker (iNOS). (B) Immunofluorescence staining for total macrophages (CD68, green) and the M2 marker (CD206). (C and D) Quantitative data analysis of the number of M1 (iNOS⁺CD68⁺) and M2 (CD206⁺CD68⁺) macrophages in renal allografts from Smad3 WT and Smad3 KO recipients. Data are mean±SEM. ^##P<0.01, ^###P<0.001 versus Smad3 WT mice.

Figure 10.

The majority of MMT cells in allografts in Smad3 WT recipients have an M2 phenotype. Kidney allografts were transplanted into Smad3 WT or Smad3 KO recipient mice and examined 28 days later. Immunofluorescence staining shows that (A) many α-SMA⁺ myofibroblasts (red) coexpress CD206 (M2 marker), but (B) lack expression of iNOS (M1 marker). (C) Quantitative analysis of CD206⁺α-SMA⁺ cells and iNOS⁺α-SMA⁺ cells as a percentage of total α-SMA⁺ cells in renal allografts from Smad3 WT and Smad3 KO recipients. Data are mean±SEM. ^##P<0.01, ^###P<0.001 versus Smad3 WT mice.

Figure 11.

Analysis of macrophage phenotype in grafted kidneys with chronic renal allograft rejection in Smad3 WT or KO recipient mice. Kidney allografts were transplanted into Smad3 WT or Smad3 KO recipient mice and examined 28 days later. An isograft group was used as a control. (A) Two-color flow cytometry analysis identifies F4/80⁺α-SMA⁺ MMT cells followed by analysis of CD206 expression by the gated MMT cells. Graph shows a significant reduction in the percentage of MMT cells expressing CD206 in renal allografts in Smad3 KO recipient mice. (B) Real-time PCR analysis of RNA extracted from whole renal allograft tissue for M1 macrophage markers (iNOS, MCP-1, IL-1β, and TNF-α). (C) Real-time PCR analysis for M2 macrophage markers (CD206, arginase-1, TGF-β1, and IL-10). Data are mean±SEM for groups of six mice. **P<0.01, ***P<0.001 versus isograft controls; ^#P<0.05, ^##P<0.01 versus Smad3 WT mice.