Inhibition of Peptidyl Arginine Deiminase-4 Prevents Renal Ischemia-Reperfusion-Induced Remote Lung Injury

Abstract

Ischemia reperfusion (IR) can lead to acute kidney injury and can be complicated by acute lung injury, which is one of the leading causes of acute kidney injury-related death. Peptidyl arginine deiminase-4 (PAD4) is a member of the PAD enzyme family and plays a critical role in inflammatory reactions and neutrophil extracellular trap formation in a variety of pathological conditions. It has been reported that PAD4 inhibition can protect certain organs from ischemic injury. In this study, we aimed to understand the mode of action of PAD4 in renal ischemia-reperfusion-mediated acute lung injury. Bilateral renal pedicle occlusion was induced for 30 min followed by reperfusion for 24 h. A specific inhibitor of PAD4, GSK484, was delivered via intraperitoneal injection to alter the PAD4 activity. The pulmonary PAD4 expression, pulmonary impairment, neutrophil infiltration, Cit-H3 expression, neutrophil extracellular trap formation, inflammatory cytokine secretion, and pulmonary apoptosis were analyzed. We found that renal ischemia reperfusion was associated with pulmonary pathological changes and increases in neutrophil infiltration, neutrophil extracellular trap formation, and inflammatory cytokine secretion in the lungs of the recipient animals. Suppression of PAD4 by GSK484 reduced remote lung injury by mitigating neutrophil infiltration, neutrophil extracellular trap formation, apoptosis, and inflammatory factor secretion. Our findings demonstrate that specific PAD4 inhibition by GSK484 may be an effective strategy to attenuate distant lung injury complicating renal ischemia-reperfusion injury.

Figure 1

Kidney and pulmonary histopathological changes and lung PAD4 activity in renal ischemia-reperfusion- (IR-) treated mice. (a) Representative photographs of H&E-stained kidney samples from 24 h after renal IR injury (scale bar: 50 μm). (b, c) Determination of blood urea nitrogen (BUN) and creatinine levels. (d) Representative photographs of H&E-stained lung sections from 24 h after renal IR injury (scale bar: 50 μm). (e) Histological analysis of lung sections. (f) The PAD4 mRNA expression measured by quantitative RT-PCR. (g) The PAD4 expression evaluated by immunohistochemistry (scale bar: 50 μm). (h) Analysis of the PAD4 expression. The values are the means ± SEMs; n = 6; ^∗∗p < 0.01 and ^∗∗∗p < 0.001.

Figure 2

Effect of GSK484 on renal IR-induced pulmonary injury. (a) H&E staining of lung sections from different groups (scale bar: 50 μm). (b) Histological analysis of different conditions. (c) W/D weight ratio of pulmonary tissue. (d) Protein leakage in the bronchoalveolar lavage fluid (BALF). The values are means ± SEMs; n = 6; ^∗p < 0.05, ^∗∗p < 0.01, and ^∗∗∗p < 0.001.

Figure 3

Effect of GSK484 on the PAD4 expression and citH3 accumulation in the lung after renal IR. (a, b) Representative images of immunohistochemical staining and analysis of PAD4-positive cells in lung sections from different groups (scale bar: 50 μm). (c, d) Immunofluorescence images and analysis of H3cit-positive cells in the lung (scale bar: 20 μm). The data are the means ± SEMs; n = 6; ^∗∗p < 0.01 and ^∗∗∗p < 0.001.

Figure 4

Neutrophil extracellular traps (NETs) in the mouse lung following renal IR-induced pulmonary injury. (a) Representative confocal images of NETs demonstrated colocalization of NE and CitH3 with diffuse DAPI staining nuclei. NETs: neutrophil extracellular traps; CitH3: citrullinated histone H3 (scale bar: 10 μm). (b) Quantification of NETs in pulmonary sections from different groups. (c) Quantification of DNA/MPO levels in bronchoalveolar lavage fluid (BALF). The data are the means ± SEMs; n = 6; ^∗p < 0.05, ^∗∗p < 0.01, and ^∗∗∗p < 0.001.

Figure 5

Effect of GSK484 on pulmonary neutrophil infiltration after renal IR-induced pulmonary injury. (a) Immunohistochemical staining for LY6G was performed on lung sections from different treatment groups (scale bar: 50 μm). (b) Quantification of LY6G-positive cells. The values are presented as the means ± SEMs; n = 6; ^∗∗∗p < 0.001.

Figure 6

The influence of GSK484 on inflammatory cytokine secretion after renal IR injury. (a)–(c) Expression levels of IL-1β, IL-6, and TNF-α in the blood serum and (d)–(f) lung tissue homogenates. The values are presented as the means ± SEMs; n = 6; ^∗p < 0.05, ^∗∗p < 0.01, and ^∗∗∗p < 0.001.

Figure 7

Effect of GSK484 on apoptosis in the lungs after renal IR-induced lung injury. (a) TUNEL staining was performed on lung sections (scale bar: 50 μm). (b) Quantification of TUNEL-positive cells (n = 6). (c)–(e). Western blot analysis of the Bcl-2 and cleaved caspase-3 expression in the lungs (n = 3). The values are presented as the means ± SEMs. ^∗p < 0.05, ^∗∗p < 0.01, and ^∗∗p < 0.001.