Fig. 3

Macrophage autophagy-related 5 (Atg5) deletion protects against kidney fibrosis after I/R injury. (A) Serum creatinine and BUN levels of MΦ atg5^−/− and wild type (WT) mice. Mice underwent 31-min unilateral renal ischemia (UI31R) surgery, the right kidney was removed after 13 days of left renal reperfusion, and blood samples were collected 1 day later. Data are presented as the mean ± SEM. ** P < 0.01 (n = 6 per group). Unpaired, two-tailed Student’s t test. (B-K) WT and MΦ atg5^−/− mice underwent 31-min unilateral renal I/R and were sacrificed at 2 or 4 weeks. Representative images and positive area rate of Sirius red (B and C) and Masson (B and D) staining of MΦ atg5^−/− mouse kidney cortex sections showing reduced renal tubulointerstitial fibrosis. (E) Representative fibrosis immunohistochemical staining images showing decreased renal fibrosis in MΦ atg5^−/− mice. Renal expression of fibronectin 1 (FN1) (F), vimentin (G), and α-SMA (H), a marker of myofibroblasts. Scale bars: 50 μm. Data are presented as the mean ± SEM. ** P < 0.01, ***P < 0.001, ****P < 0.0001 (n = 4 per group). One-way ANOVA with Tukey’s multiple-comparison test (C, D, F, G, H). (I) Flow cytometric analysis showing the number of insulted renal macrophages (F4/80⁺ CD11b⁺ CD11c⁻ CD45⁺ cells) and the M1/M2 ratio of macrophages in MΦ atg5^−/− and WT mice on day 28 after ischemic injury. Data are presented as the mean ± SEM. ** P < 0.01, ***P < 0.001 (n = 4 per group). Unpaired, two-tailed Student’s t test. (J and K) The mRNA levels of profibrotic and fibrotic collagen 1a1 and α-smooth muscle actin (α-SMA) in insulted left kidneys of MΦ atg5^−/− and WT mice. Data are presented as the mean ± SEM. ****P < 0.0001 (collagen 1a1 groups, n = 6 each; α-SMA groups, n = 4 each). Unpaired, two-tailed Student’s t test