Figure 3

Fate mapping identifies a macrophage origin of MMT cells during renal fibrosis in the obstructed kidney. Groups of eight LysM-Cre/Rosa26-tdTomato transgenic and littermate wild-type mice underwent unilateral ureteric obstruction (UUO) or sham surgery and were killed 7 days later. (a) Results of confocal microscopy show co-localization of the Tomato reporter (red) with F4/80⁺ (Green) macrophages, whereas α-SMA-expressing vascular smooth muscle cells (v) and tubular epithelial cells lack Tomato expression in the sham-operated kidney. The number of F4/80⁺ macrophages is significantly increased in the day 7 UUO kidney in which more than 90% of macrophage co-express the Tomato reporter. MMT cells derived from the macrophage lineage in the fibrotic kidney are identified as Tomato⁺F4/80⁺α-SMA⁺ cells. An example of a Tomato⁺F4/80⁺α-SMA⁺ MMT cell (arrow) is shown in the lower panel. (b) Quantitative two-color flow cytometric analysis shows that more than 90% of F4/80+ cells express the Tomato reporter gene in both normal and UUO kidney. (c) Quantification data of flow cytometry shows that nearly 80% of α-SMA⁺ myofibroblasts co-express F4/80 in both day 3 and 7 UUO kidney, indicating that MMT cells (F4/80⁺α-SMA⁺ cells) account for the majority of the total α-SMA⁺ myofibroblast population. (d) Three-color flow cytometric analysis of cells isolated from the UUO kidney in which α-SMA⁺Tomato⁺ double-stained cells are then analyzed for expression of the F4/80 antigen. The majority of α-SMA⁺ myofibroblasts (65±3.9%) co-express both the Tomato transgene and the F4/80 antigen, indicating their origin in the macrophage lineage. Data represent results from eight animals. Scale bar, 20 μM