Fig. 2: FMO3 knockout protects mouse renal function after I/R.

a In WT and Fmo3^−/− mice, the mRNA levels of Fmo3 in kidney tissues were analyzed by RT-qPCR, n = 10 per group. Data are presented as mean ± SEM. ***P < 0.001 (Student’s t-test). b Representative immunoblotting images showing protein levels of FMO3 in WT and Fmo3^−/− mice kidneys (upper panel) and livers (lower panel). c Serum TMAO levels of WT and Fmo3^−/− mice tested before I/R, n = 6 per group. Data are presented as mean ± SEM. ***P < 0.001 (Student’s t-test). d Schematics of the experiment. Renal I/R surgery including the removal of the contralateral kidney was performed in WT (C57BL/6 N) and Fmo3^−/− mice. Serum samples were collected at 24 h, 48 h, and 72 h after I/R. Renal tissue samples were collected at 48 h after I/R for histology of AKI and 4 weeks after I/R for observation of chronic injury. The image was created with figdraw.com and released under a license: PPTTY74a57. e Kaplan–Meier curve of WT and Fmo3^−/− mice after renal I/R. All the Fmo3^−/− mice survived one month after I/R surgery, whereas only 15 (46.9%) mice in the WT group survived 8 days. WT group, n = 32; Fmo3^−/− group, n = 20, P < 0.001 (Log-rank (Mantel–Cox) test). f Renal function (serum creatinine and BUN) of WT and Fmo3^−/− mice was measured at 24 h, 48 h, and 72 h after I/R, n = 4–8 per group. Data are represented as mean ± SEM. Fmo3^−/− I/R vs WT I/R, **P < 0.01; ***P < 0.001 (Student’s t-test). g Relative mRNA level of KIM-1 (Havcr1) at 48 h post-I/R, n = 5 per group. Data are presented as mean ± SEM. **P < 0.01; ***P < 0.001 (one-way ANOVA with post hoc Tukey test). h Representative histopathological images of Periodic acid-Schiff (PAS) staining of kidneys from mice sacrificed at 48 h post-reperfusion and from sham-operated animals. Black arrows indicate the disappeared brush edge of renal tubules, tubular epithelial cell necrosis, and detachment from the basement membranes in WT mice after I/R injury. Red arrows indicate swollen tubules and renal casts. Scale bar, 50 μm. i Renal tubular injury score evaluated at 48 h post-I/R in mice. The tubular injury was defined as tubular dilation, tubular atrophy, tubular atrophy, tubular cast formation, sloughing of tubular epithelial cells, or loss of the brush border. Tubular injury scores (0–5) were valued and quantified according to histopathological images of kidneys from four groups, n = 5 per group. Data are presented as mean ± SEM. **P < 0.01; ***P < 0.001 (one-way ANOVA with post hoc Tukey test). j Evaluation of apoptotic nephrocytes in kidneys stained with TUNEL staining (red) at 48 h. Scale bar, 100 μm. k Quantification of the average number of apoptotic cells per field at 48 h after I/R, n = 5 per group. ***P < 0.001 (one-way ANOVA with post hoc Tukey test). WT wild type, I/R ischemia-reperfusion injury, BUN blood urea nitrogen, TUNEL TdT-mediated dUTP-biotin nick end labeling.