Chronic Activation of the Renin-Angiotensin System Induces Lung Fibrosis

Abstract

Pulmonary fibrosis is a serious lung disorder that can lead to respiratory failure. Here we show that transgenic mice expressing active renin from the liver (RenTgMK) developed progressive pulmonary fibrosis leading to impaired pulmonary function. Histological analyses revealed a marked increase in extracellular matrix (ECM) deposition and decrease in alveolar size in the lungs of RenTgMK mice compared to wild-type (WT) littermates, accompanied with increased expression of ECM proteins and fibrogenic factors. The increase in lung fibrosis led to a substantial decrease in respiratory system compliance. Two-week treatment with aliskiren (renin inhibitor) or losartan (AT1 antagonist) ameliorated pulmonary ECM deposition, blocked the induction of ECM proteins and fibrogenic factors and improved respiratory compliance in RenTgMK mice, confirming a critical role of the renin-Ang II-AT1 cascade in promoting pulmonary fibrogenesis. However, when RenTgMK mice were treated with hydralazine (a smooth muscle relaxant), the blood pressure was normalized but the lung fibrotic abnormalities, fibrogenic gene induction and pulmonary elasticity were not corrected. Moreover, intratracheal instillation of lipopolysaccharide induced more severe lung injury in RenTgMK mice compared to WT littermates. These observations demonstrate that the renin-angiotensin system is a key mediator of lung fibrosis, and its pro-fibrotic effect is independent of blood pressure.

Figure 1. RenTgMK mice develop progressive lung fibrosis.

RenTgMK and WT littermates were analyzed at 2, 6 and 10 months of age as indicated. (A) H&E staining of lung structure. Magnification 200x; (B) Mean chord length; (C) Volume fraction of septal tissue area; (D) Volume fraction of alveolar air space; (E) Volume fraction of ductal air space; (F) Masson’s trichrome staining. Magnification 200x; (G) ECM deposition area (%) estimated from Masson’s trichrome staining; (H) Dynamic respiratory system compliance during tidal ventilation at increasing tidal volumes at 2 months of age; (I) Respiratory system compliance at 2 months of age vs. tidal volume, normalized to the value at 10 ml/kg in percentage calculation. *P < 0.05; **P < 0.01; ***P < 0.001. n = 4–5 mice in each group. Vol, volume; m, month.

Figure 2. Increased production of extracellular matrix protein and pro-fibrotic factors in RenTgMK mice.

(A) Lung immunostaining with anti-fibronectin antibody. Magnification 200x upper panels and 400x lower panels; (B–E) Western blot analysis of fibronectin (B), TGF-β1 (C), TGF-β3 (D) and α-SMA (E). (F) Densitometric quantitation of these proteins. **P < 0.01; ***P < 0.001 vs. WT. All gels were run under the same experimental condition, and gel images are cropped for concise presentation. All uncut gel images are provided in Supplementary Information.

Figure 3. Lung fibrosis is blocked by renin inhibition but not by blood pressure reduction.

RenTgMK mice were treated with hydralazine (20 mg/kg) or aliskiren (20 mg/kg) for two weeks. WT and RenTgMK controls were treated with PBS. (A) Systolic, diastolic and mean aortic blood pressure in these four groups of mice. *P < 0.05, **P < 0.01, ***P < 0.001. (B) Systolic and diastolic right ventricular blood pressure in WT and RenTgMK mice. (C) H&E staining (upper panels) and Masson’s trichrome staining (lower panels) of lung structure in these four groups of mice. Representative images are shown. These images are not sequential slices. (D) Mean chord length; (E) Volume fraction of septal tissue space; (F) ECM deposition area (%) estimated from Masson’s trichrome staining; (G) Respiratory system compliance during tidal ventilation at increasing tidal volumes; *P < 0.05, **P < 0.01, ***P < 0.001; (H) Normalized dynamic respiratory system compliance vs. tidal volume, as in Fig. 1I; *P < 0.05, **P < 0.01 vs. RenTg or RenTg + Hyd. n = 5–6 mice in each group.

Figure 4. Effects of aliskiren and hydralazine on the expression of ECM proteins and pro-fibrotic factors.

(A) Real time PCR quantitation of ECM proteins and pro-fibrotic factors; *P < 0.05, **P < 0.01, ***P < 0.001. (B,C) Western blot analysis and densitometric quantitation of fibronectin; **P < 0.01, ***P < 0.001. All gels were run under the same experimental condition, and gel images are cropped for concise presentation.

Figure 5. Lung fibrosis is blocked by inhibition of AT1 receptor signaling. RenTgMK and WT littermates were treated with losartan for two weeks before sacrifice for lung analysis.

(A) H&E staining (upper panels) and Masson’s trichrome staining (lower panels). Magnification 200x. Representative images are shown. These images are not sequential slices. (B) Mean chord length; (C) Volume fraction of septal area (%); (D) ECM deposition (%); *P < 0.05; ***P < 0.001. (E,F) Western blot analysis of TGF-β1, TGF-β3 and α-SMA (E) and densitometry quantitation (F). *P < 0.05; n = 4–5. All gels were run under the same experimental condition, and gel images are cropped for concise presentation. (G) Normalized dynamic respiratory system compliance vs. tidal volume, as in Fig. 1I. *P < 0.05, **P < 0.01 vs. RenTg; n = 4–5 in each group.

Figure 6. RenTgMK mice are more susceptible to LPS-induced acute lung injury.

RenTgMK mice and WT littermates were treated with PBS or LPS (20 mg/kg) intratracheally. Lung injury analyses were performed 24 hours after LPS treatment. (A) Lung appearance after Evans blue dye injection; (B) Evans blue permeability assay. OD, optical density; (C) Wet to dry lung weight ratio; (D) H&E stained lung histology from mice treated with LPS; (E) Cell number in broncho-alveolar lavage (BAL) fluid; (F) Protein concentration in BAL fluid. *P < 0.05, **P < 0.01, ***P < 0.001 vs. PBS-treated in the same group; ^#P < 0.05, ^##P < 0.01 vs. WT of the same treatment. n = 4–6 mice in each group.