Angiotensin-converting enzyme 2 protects from severe acute lung failure

Yumiko Imai, Keiji Kuba, Shuan Rao, Yi Huan, Feng Guo, Bin Guan, Peng Yang, Renu Sarao, Teiji Wada, Howard Leong-Poi, Michael A Crackower, Akiyoshi Fukamizu, Chi-Chung Hui, Lutz Hein, Stefan Uhlig, Arthur S Slutsky, Chengyu Jiang, Josef M Penninger, Yumiko Imai, Keiji Kuba, Shuan Rao, Yi Huan, Feng Guo, Bin Guan, Peng Yang, Renu Sarao, Teiji Wada, Howard Leong-Poi, Michael A Crackower, Akiyoshi Fukamizu, Chi-Chung Hui, Lutz Hein, Stefan Uhlig, Arthur S Slutsky, Chengyu Jiang, Josef M Penninger

Abstract

Acute respiratory distress syndrome (ARDS), the most severe form of acute lung injury, is a devastating clinical syndrome with a high mortality rate (30-60%) (refs 1-3). Predisposing factors for ARDS are diverse and include sepsis, aspiration, pneumonias and infections with the severe acute respiratory syndrome (SARS) coronavirus. At present, there are no effective drugs for improving the clinical outcome of ARDS. Angiotensin-converting enzyme (ACE) and ACE2 are homologues with different key functions in the renin-angiotensin system. ACE cleaves angiotensin I to generate angiotensin II, whereas ACE2 inactivates angiotensin II and is a negative regulator of the system. ACE2 has also recently been identified as a potential SARS virus receptor and is expressed in lungs. Here we report that ACE2 and the angiotensin II type 2 receptor (AT2) protect mice from severe acute lung injury induced by acid aspiration or sepsis. However, other components of the renin-angiotensin system, including ACE, angiotensin II and the angiotensin II type 1a receptor (AT1a), promote disease pathogenesis, induce lung oedemas and impair lung function. We show that mice deficient for Ace show markedly improved disease, and also that recombinant ACE2 can protect mice from severe acute lung injury. Our data identify a critical function for ACE2 in acute lung injury, pointing to a possible therapy for a syndrome affecting millions of people worldwide every year.

Conflict of interest statement

J.M.P. declares personal financial interests.

Figures

Figure 1. Loss of ACE2 worsens acid…
Figure 1. Loss of ACE2 worsens acid aspiration-induced acute lung injury.
a, Lung elastance after acid or saline treatment in wild type (WT) and Ace2 knockout (Ace2 KO) mice (n = 10 for acid-treated groups, n = 6 for saline-treated groups). P < 0.05 for the whole time course comparing acid-treated WT and Ace2 knockout mice. b, Partial pressure of oxygen in arterial blood (paO2) in acid-induced acute lung injury. c, Wet-to-dry weight ratios of lungs 3 h after acid injury. Single asterisk, P < 0.05; double asterisk, P < 0.01. d, Lung histopathology. Note the enhanced hyaline membrane formation, inflammatory cell infiltration and lung oedema in acid-treated Ace2 knockout mice (H&E staining, ×200). e, ACE and ACE2 protein expression in total lysates from control lungs and lungs 3 h after acid injury. Error bars indicate s.e.m.
Figure 2. ACE2 controls acute lung failure.
Figure 2. ACE2 controls acute lung failure.
a, Lung elastance after acute lung injury in WT and Ace2 knockout (KO) mice induced by caecal ligation perforation (CLP). Eighteen hours after sham or CLP surgery, animals received mechanical ventilation for 6 h (n = 10 in CLP-treated groups, n = 6 in sham-treated groups). As 8/10 CLP-treated Ace2 knockout mice died at 4–4.5 h, only data up to 4 h are shown. CLP-treated Ace2 knockout mice had significantly higher elastance than CLP-treated WT mice (P < 0.01). b, c, Wet-to-dry weight ratios of lungs (b) and lung histopathology (c) in sham or CLP-treated WT and Ace2 knockout mice determined after 4 h of ventilation. Asterisk denotes a significant difference (P < 0.05) between CLP-treated WT and Ace2 knockout mice. Note the enhanced lung oedema and inflammatory infiltrates in Ace2 knockout mice (H&E staining, ×200). d, e, Lung elastance (d) and wet-to-dry weight ratios (e) after acid or saline instillation of Ace2 knockout mice injected intraperitoneally with recombinant human ACE2 protein (rhuACE2; 0.1 mg kg-1), mutant rhuACE2 (mut-rhuACE2; 0.1 mg kg-1) or vehicle (n = 6 per group). Asterisk denotes a significant difference (P < 0.05) comparing rhuACE2-treated Ace2 knockout mice with mut-rhuACE2-treated and vehicle-treated Ace2 knockout mice at 3 h. f, Lung elastance after acid instillation in WT mice treated with rhuACE2 protein (0.1 mg kg-1), mut-rhuACE2 protein (0.1 mg kg-1) or vehicle (n = 6–8 per group). Asterisk denotes a significant difference (P < 0.05) between WT mice treated with rhuACE2 and mut-rhuACE2 or with vehicle at 3 h. Errors bars indicate s.e.m.
Figure 3. ACE deficiency reduces the severity…
Figure 3. ACE deficiency reduces the severity of acute lung injury.
a, Schematic diagram of the renin–angiotensin system. b, Lung levels of AngII in control and acid-treated WT and Ace2 knockout (KO) mice determined at 3 h by enzyme immunoassay (n = 3–5 per group). Asterisk denotes a significant difference (P < 0.05) between acid-treated WT and Ace2 knockout mice. c, Lung elastance after acid instillation in Ace+/+ (WT), Ace+/- and Ace-/- mice (n = 4–6 mice per group). Asterisk denotes a significant difference (P < 0.05) comparing Ace+/+ with Ace+/- and Ace-/- mice at 3 h. d, e, Lung elastance (d) and wet-to-dry lung weight ratios (e) in acid- or saline-treated Ace+/+Ace2 KO, Ace+/-Ace2 KO, Ace-/-Ace2 KO and WT mice (n = 5 per group). Asterisk denotes a significant difference (P < 0.05) comparing Ace2 KO with WT, Ace+/-Ace2 KO or Ace-/-Ace2 KO mice 3 h after acid-treatment. f, Lung histopathology. Severe lung interstitial oedema and leukocyte infiltration in Ace2 KO mice are attenuated by homozygous (Ace-/-) or heterozygous (Ace+/-) mutations of Ace (H&E staining, ×200). Error bars indicate s.e.m.
Figure 4. The AngII receptor AT 1…
Figure 4. The AngII receptor AT1a controls acute lung injury severity and pulmonary vascular permeability.
a, Lung elastance measurements in Agtr1a-/- mice, Agtr2-/y mice and WT mice after acid aspiration (n = 4–6 per group). All acid-treated Agtr2-/y mice died after 2 h. There is a significant difference (P < 0.01) between acid-treated WT and acid-treated Agtr1a-/- mice over the whole time course. Double asterisk denotes a significant difference (P < 0.01) between WT and Agtr2-/y mice at 2 h. b, Lung elastance measurements in Ace2 knockout mice treated with vehicle or inhibitors to AT1 (Losartan, 15 mg kg-1) or AT2 (PD123.319, 15 mg kg-1) after acid or saline instillation (see Methods, n = 4–6 per group). Double asterisk denotes a significant difference (P < 0.01) comparing Ace2 knockout mice treated with AT1 inhibitor with vehicle or AT2 inhibitor treatment at 3 h. c, Pulmonary vascular permeability as determined by intravenous injection of Evans Blue. Extravascular Evans Blue in lungs was measured in WT and Ace2 knockout mice 3 h after acid injury (n = 5 per group). Double asterisk denotes a significant difference (P < 0.01) between acid-treated WT and Ace2 knockout mice. d, Representative images of Evans Blue-injected lungs of WT and Ace2 knockout mice 3 h after acid aspiration. e, Extravascular Evans Blue in lungs of WT and Agtr1a-/- mice 3 h after acid injury (n = 5 per group). Asterisk denotes a significant difference (P < 0.05) between acid-treated WT and Agtr1a-/- mice at 3 h. Error bars indicate s.e.m.

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