Angiotensin-converting enzyme 2 activation attenuates inflammation and oxidative stress in brain death donor followed by rat lung transplantation

Abstract

Brain death (BD) provides most of the donor organs destined for lung transplantation (LTx). However, the organs may be affected by inflammatory and oxidative processes. Based on this, we hypothesize that the angiotensin-converting enzyme 2 (ACE2) activation can reduce the lung injury associated with LTx. 3 h after BD induction, rats were injected with saline (BD group) or an ACE2 activator (ACE2a group; 15 mg/kg^-1) and kept on mechanical ventilation for additional 3 h. A third group included a control ventilation (Control group) prior to transplant. After BD protocol, left LTx were performed, followed by 2 h-reperfusion. ACE2 activation was associated with better oxygenation after BD management (p = 0.01), attenuating edema (p = 0.05) followed by the reduction in tissue resistance (p = 0.01) and increase of respiratory compliance (p = 0.02). Nrf2 expression was also upregulated in the ACE2a group (p = 0.03). After transplantation, ACE2a group showed lower levels of TNF-α (p = 0.02), IL-6 (p = 0.001), IL-1β (p = 0.01), ROS (p = 0.004) and MDA (p = 0.002), in addition to higher CAT activity (p = 0.04). In conclusion, our study suggests that ACE2 activation improves anti-inflammatory and antioxidant activity in a model of LTx.

Keywords: Angiotensin-converting enzyme 2; Brain death; Donor management; Ischemia–reperfusion injury; Lung transplantation.

Fig. 1

Graphic schema of experimental design. ( Donor Phase ) BD was established model through an intracranial balloon inflation using a reproducible method. 3 h after BD induction, animals were randomized into 2 different groups. Rats were injected i.p. with 0.9% saline (BD group; 0.1 mL/100 g) or a single dose of ACE2 activator diminazene aceturate (ACE2a group; 15 mg/kg^-1) and kept on mechanical ventilation for additional 3 h. A third group included a control ventilation (CTR—Control group) prior to transplant. ( Transplant phase ) After each BD protocol, left lung transplants were performed, followed by 2 h reperfusion.

Fig. 2

Brain death-induced lung injury results in the deterioration of donor lung function. (A) Mean arterial pressure (MAP) recorded at different time points (baseline to 6 h) in rats submitted to brain death (BD). * means the significant difference between Control and BD groups during hypertensive response to balloon inflation (Time 0—Gray area). Two-way ANOVA testing was performed followed by Student–Newman–Keuls method of multiple comparisons. Brackets on the right represents the p-value comparing the overall average pressure between the groups measured through one-way ANOVA. (B) P/F ratio (as an oxygenation index). The values for all of the measurements are expressed as the means ± SEM (n = 7–8 per group). One-way ANOVA testing was performed followed by Tukey method of multiple comparisons.

Fig. 3

ACE2 activation during the brain death donor’s management improves graft quality. (A) Left, compliance of the respiratory system; Right, tissue damping. (B) Angiotensin-converting enzyme 2 (ACE2) activity in the control, BD and ACE2a group. (C) Donor wet-to-dry weight ratio. (D) Donor perivascular edema. (E) Photomicrographs of lung sections stained with hematoxylin and eosin (× 40 magnification; scale bar 20 µm) in the control, BD and ACE2a group. (F,G) Fold increase in Nuclear factor erythroid 2-related factor 2 (Nrf2) expression (× 40 magnification; scale bar 1000 µm). The values for all of the measurements are expressed as the means ± SEM (n = 5—8 per group). One-way ANOVA testing was performed followed by Tukey’s multiple comparison or Kruskal–Wallis’ test method of multiple comparisons, when appropriate.

Fig. 4

Previous ACE2 activation during BD management did not translate to better post-transplant graft function. (A) Post-transplant wet-to-dry weight ratio. (B) Photomicrographs of lung sections stained with hematoxylin and eosin (× 40 magnification; scale bar 20 µm) in the control, BD and ACE2a group after reperfusion. (C) Macroscopic view of lung grafts after reperfusion. The values for all of the measurements are expressed as the means ± SEM (n = 4–8 per group). One-way ANOVA testing was performed followed by Tukey’s multiple comparison or Kruskal–Wallis’ test method of multiple comparisons, when appropriate.

Fig. 5

ACE2 activation attenuates proinflammatory responses associated with post-reperfusion lung injury. (A) Left, TNF-α levels; Middle, IL-6 levels; Right, IL-1β levels after reperfusion. (B) Post-transplant Tissue Ang II. The values for all of the measurements are expressed as the means ± SEM (n = 4—6 per group). One-way ANOVA testing was performed followed by Tukey’s multiple comparison.

Fig. 6

Previous ACE2 during BD management modulates redox imbalance after reperfusion. (A) Post-transplant tissue reactive oxygen species (ROS). (B) Post-transplant tissue malondialdehyde (MDA). (C) Post-transplant tissue catalase (CAT) activity. (D) Post-transplant tissue superoxide dismutase (SOD) activity.