Fig 1.
hAAT (80 mg/kg/day; i.p.) treatment improves renal function after I/R Injury (IRI).
A. Plasma urea levels at day 7 pre-op and at different time-points after IRI. B. Plasma creatinine levels at day 7 pre-op and at different time-points after IRI. Grey boxes represent the control group treated with hAlb. White boxes represent the group treated with hAAT. Two-way analysis of variance (ANOVA) followed by Bonferroni post-test. **P<0.01 (n = 6–8 animals per group).
Fig 2.
hAAT (80 mg/kg/day; i.p.) treatment decreases acute tubular necrosis after I/R Injury (IRI).
A-F. PAS-stained frontal sections of mouse kidney: A, D PAS-stained frontal sections of naïve mouse kidney without any histological damage and with the intact brush border in the proximal tubules. B, C representative images of ischemic kidney of control mouse at different time-points after IRI. E, F representative images of ischemic kidney of mouse treated with hAAT at different time-points after IRI. C = cortex; CMJ = cortico-medullary junction; M = medullar region; PT = proximal tubules; G = glomeruli. The solid arrow indicates areas of cast formation. Open arrows indicate debris deposition in the tubular lumen. Scale bar = 100 μm (original magnification x100). G. Kidney histological damage score. The percentage of damaged tubules in 3 different areas of the kidney was estimated using a 5-point scale. Grey boxes represent the control group treated with hAlb. White boxes represent the group treated with hAAT. Two-way analysis of variance (ANOVA) followed by Bonferroni post-test: *P<0.05. Mann-Whitney U test (two-tailed) to analyze the effect of treatment at each time-point. #P<0.05 (n = 6–8 animals per group).
Fig 3.
hAAT (80 mg/kg/day; i.p.) treatment ameliorates AKI after I/R Injury (IRI).
A. Plasma NGAL levels at day 7 pre-op and at different time-points after IRI. B. Urine NGAL levels at day 7 pre-op and at different time-points after IRI. C. Urine KIM-1 levels at day 7 pre-op and at different time-points after IRI. Grey boxes represent the control group treated with hAlb. White boxes represent the group treated with hAAT. Two-way analysis of variance (ANOVA) followed by Bonferroni post-test: *P<0.05, ***P<0.001. Mann-Whitney U test (two-tailed) to analyze the effect of treatment at each time-point. #P<0.05, ##P<0.01 (n = 6–8 animals per group).
Fig 4.
Effect of hAAT (80 mg/kg/day; i.p.) treatment on renal leukocyte influx at different time-points after I/R Injury (IRI).
A, B. Immunofluorescence analysis of granulocytes in ischemic kidneys. Kidney sections were stained with a specific anti-Gr-1 antibody. A representative image of ischemic control mouse kidney at day 1 after IRI. B representative image of ischemic mouse kidney treated with hAAT at day 1 after IRI. Scale bar = 50 μm (original magnification x200). D, E. Immunofluorescence analysis of macrophages in ischemic kidneys. Kidney sections were stained with a specific anti-CD68 antibody. D representative image of ischemic kidney of a control mouse at day 1 after IRI. E representative image of ischemic kidney of a mouse treated with hAAT at day 1 after IRI. Scale bar = 20 μm (original magnification x400). Solid arrows indicate intertubular cell infiltration. The open arrow indicates cellular influx in the glomeruli. C. Gr-1+ cells were counted in non-overlapping fields and subsequently averaged. F. Macrophage staining was scored semi-quantitatively on a scale from 0 to 5 based on the extent of CD68 immunofluorescence staining. Grey boxes represent the control group treated with hAlb. White boxes represent the group treated with hAAT (dark green = autofluorescence of the tubuli; red = agrin; bright green = immune cells). Mann-Whitney U test (two-tailed) to analyze the effect of treatment at each time-point. #P<0.05 (n = 6–8 animals per group).
Fig 5.
Mouse anti-hAAT antibody formation and its correlation with urine levels of KIM-1 during the recovery phase of renal I/R Injury (IRI).
A. Mouse anti-hAAT antibody levels in plasma were measured before surgery (pre-op) and at different time-points after IRI. One-way analysis of variance (ANOVA) followed by Dunnett’s post-test. ***P<0.001 (n = 4–8 animals). B. Correlation between protein levels of KIM-1 in urine and mouse anti-hAAT antibodies in serum at day 15 post-op. Spearman´s correlation. *P<0.05 (n = 6 animals).