Figure 3 | Scientific Reports

Figure 3

From: Development of a new macrophage-specific TRAP mouse (MacTRAP) and definition of the renal macrophage translational signature

Figure 3The alternative text for this image may have been generated using AI.

Cell type-specific expression of eGFP-L10a in healthy and fibrotic MacTRAP kidneys and transgene responsiveness in vivo. (A) Immunofluorescent staining of healthy MacTRAP kidneys demonstrates cell type-specific expression of the eGFP-L10a transgene. EGFP-L10a+ cells co-stain with macrophage surface marker F4/80 and reside in the tubulointerstitium as highlighted by anti-laminin staining. Endothelial cells (anti-CD31+) of peritubular capillaries are closely associated with eGFP-L10a+ cells, but no co-staining is observed. Scale bar: 25 µm. (B) In fibrotic kidneys (7d UUO), eGFP-L10a+ cells remain confined to the tubulointerstitial compartment as illustrated by anti-laminin staining. Co-staining with other interstitial cell markers is minimal (pericyte marker PDGFRβ, myofibroblast marker αSMA) or completely absent (T cell marker CD3). Scale bar: 25 µm. (C) RT-qPCR analysis shows strong upregulation of eGFP-L10a in fibrotic kidneys (7d UUO) along with macrophage- and fibrosis-related markers compared to CLK kidneys, indicating responsiveness of transgene expression upon proinflammatory challenge (**p < 0.01); data are shown as mean ± SEM; n = 5. (D) Western Blot of protein lysates from 7d UUO MacTRAP kidneys indicates robust upregulation of the eGFP-L10a protein compared to CLK controls when probed with an anti-GFP antibody. A protein band was detected at ~50 kDa corresponding to the expected molecular weight of the eGFP-L10a fusion protein. GAPDH antibody was used as internal loading control (full-length blot is shown in Sup. Fig. S10). (E) Quantitative analysis of green fluorescent area in MacTRAP kidney sections shows a significant increase in eGFP-L10a signals at 7 days of UUO compared to CLK controls (5.2 fold, **p < 0.01); data are shown as mean ± SEM; n = 3. (F) Proportion of cells with double-positivity for eGFP-L10a and macrophage-marker F4/80; a total of 1205 cells were counted; n = 5 mice. Data are shown as mean ± SEM. (G) Quantification of additional co-staining. Co-staining of eGFP-L10a+ cells (green) for other cell markers (red) was minimal (yellow) or absent. Bars show proportions based on all identified cells; n = 3–5 mice.

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