Human Alpha-1-Antitrypsin (hAAT) therapy reduces renal dysfunction and acute tubular necrosis in a murine model of bilateral kidney ischemia-reperfusion injury

Abstract

Several lines of evidence have demonstrated the anti-inflammatory and cytoprotective effects of alpha-1-antitrypsin (AAT), the major serum serine protease inhibitor. The aim of the present study was to investigate the effects of human AAT (hAAT) monotherapy during the early and recovery phase of ischemia-induced acute kidney injury. Mild renal ischemia-reperfusion (I/R) injury was induced in male C57Bl/6 mice by bilateral clamping of the renal artery and vein for 20 min. hAAT (80 mg/kg, Prolastin®) was administered daily intraperitoneally (i.p.) from day -1 until day 7 after surgery. Control animals received the same amount of human serum albumin (hAlb). Plasma, urine and kidneys were collected at 2h, 1, 2, 3, 8 and 15 days after reperfusion for histological and biochemical analysis. hAAT partially preserved renal function and tubular integrity after induction of bilateral kidney I/R injury, which was accompanied with reduced renal influx of macrophages and a significant decrease of neutrophil gelatinase-associated lipocalin (NGAL) protein levels in urine and plasma. During the recovery phase, hAAT significantly decreased kidney injury molecule-1 (KIM-1) protein levels in urine but showed no significant effect on renal fibrosis. Although the observed effect size of hAAT administration was limited and therefore the clinical relevance of our findings should be evaluated carefully, these data support the potential of this natural protein to ameliorate ischemic and inflammatory conditions.

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).