Recombinant thrombomodulin prevents acute lung injury induced by renal ischemia-reperfusion injury

Abstract

Acute kidney injury (AKI) complicated by acute lung injury has a detrimental effect on mortality among critically ill patients. Recently, a renal ischemia-reperfusion (IR) model suggested the involvement of histones and neutrophil extracellular traps (NETs) in the development of distant lung injury after renal IR. Given that recombinant thrombomodulin (rTM) has anti-inflammatory roles by binding to circulating histones, we aimed to clarify its effect on distant lung injury induced by AKI in a murine bilateral renal IR model. Both pretreatment and delayed treatment with rTM significantly decreased pulmonary myeloperoxidase activity, but they did not affect renal dysfunction at 24 h after renal IR. Additionally, rTM mitigated the renal IR-augmented expression of proinflammatory cytokines (tumor necrosis factor-α, interleukin-6, and keratinocyte-derived chemokine), and vascular leakage, as well as the degree of lung damage. Intense histone accumulation and active NET formation occurred in both the kidneys and the lungs; however, rTM significantly decreased the histone and NET accumulation only in the lungs. Administration of rTM may have protective impact on the lungs after renal IR by blocking histone and NET accumulation in the lungs, although no protection was observed in the kidneys. Treatment with rTM may be an adjuvant strategy to attenuate distant lung injury complicating AKI.

Figure 1

Lung MPO activity and plasma BUN after AKI induced by IR insults. The association of rTM treatment with lung MPO activity and renal function was evaluated after ischemic AKI induced by bilateral renal pedicle clamping for 30 min. (a) Pretreatment dosage of rTM and lung MPO activity at 24 h after surgery (n = 4 in sham group, n = 6 in IR and IR + rTM groups). (b) Pretreatment dosage of rTM and plasma BUN elevation at 24 h after IR insults (n = 4 in sham group, n = 6 in IR and IR + rTM groups). (c) Effect of pretreatment with 10 mg/kg rTM on lung MPO activity in mice at 6 h and 24 h after surgery (n = 5 in 6 h group, n = 6 in 24 h group) and effect of delayed treatment (administration of 10 mg/kg rTM at 6 h after renal IR) at 24 h after surgery (n = 5 in delayed treatment group). (d) Effect of pretreatment with 10 mg/kg rTM on plasma BUN levels in the mice at 6 h and 24 h after renal IR (n = 5 in 6 h group, n = 6 in 24 h group) and effect of delayed treatment at 24 h after surgery (n = 5 in delayed treatment group). **P < 0.05 versus the IR group, ^#P < 0.05 versus sham. Abbreviations: AKI, acute kidney injury; BUN, blood urea nitrogen; IR, ischemia-reperfusion; MPO, myeloperoxidase; rTM, recombinant thrombomodulin.

Figure 2

Pulmonary and renal histology of renal IR-injured mice. The mice were injected with 10 mg/kg of rTM or vehicle solution at 30 min before surgery and subjected to bilateral renal pedicles clamping for 30 min followed by 24-h reperfusion. Representative images of lung sections (hematoxylin and eosin staining) from (a) the sham-operated, (b) IR, and (c) IR + rTM groups are shown. Scale bar = 50 µm. Pulmonary histology after renal IR is characterized by septal edema and neutrophil infiltration (arrows). (d) The number of neutrophils in the lung after surgery (n = 5 per group). Representative image of renal sections (hematoxylin and eosin staining) from (e) the sham-operated, (f) IR, and (g) IR + rTM groups are shown. Scale bar = 50 µm. Arrows (f,g) denote cast formation, and arrowheads (f and g) exhibit tubular dilation and loss of brush border. (h) Average score of tubular injury for respective animals was determined. n = 5 per group. *P < 0.05 versus respective control. Abbreviations: IR, ischemia-reperfusion; rTM, recombinant thrombomodulin.

Figure 3

Proinflammatory gene mRNA expression in the lungs and kidneys after renal IR. Bilateral renal IR model mice (30-min ischemia followed by 24 hours of reperfusion) were pretreated with saline (IR group) or 10 mg/kg of rTM (IR + rTM group). Pulmonary mRNA expressions of (a) TNF-α, (b) IL-6, and (c) KC were shown. The mRNA expressions of (d) TNF-α, (e) IL-6, (f) KC, and (g) NGAL were also evaluated in the IR-injured kidney. n = 5 per group. *P < 0.05 versus respective control. Abbreviations: IL, interleukin; IR, ischemia-reperfusion; KC, keratinocyte-derived chemokine; mRNA, messenger RNA; NGAL, neutrophil gelatinase-associated lipocalin; rTM, recombinant thrombomodulin; TNF-α, tumor necrosis factor-α.

Figure 4

Vascular permeability in the lungs and kidneys after bilateral renal IR. EBD contents were determined in the lung and kidney at 24 h after bilateral renal IR. The mice were pretreated with saline (IR group) or 10 mg/kg of rTM (IR + rTM group). n = 4 in sham group; n = 6 in IR and IR + rTM groups. *P < 0.05 versus respective control. Abbreviations: EBD, Evans blue dye; IR, ischemia-reperfusion; rTM, recombinant thrombomodulin.

Figure 5

Accumulation of histones in the lung and kidney after renal IR. Histone levels in (a) the lung, (b) kidney, (c) plasma were evaluated after 30-min ischemia followed by 24 -h reperfusion. The mice were pretreated with saline (IR group) or 10 mg/kg of rTM (IR + rTM group). n = 6 per group. *P < 0.05 versus respective control. Abbreviations: AU, absorbance unit; IR, ischemia-reperfusion; rTM, recombinant thrombomodulin.

Figure 6

NET formation in the lungs and kidneys after renal IR. Bilateral renal IR model mice (30-min ischemia followed by 24 hours of reperfusion) were pretreated with saline (IR group) or 10 mg/kg of rTM (IR + rTM group). (a) Representative images of NETs in the lung and kidney are shown with positive staining for CitH3 (green), Ly6G/C (red), and DNA (blue). The colocalization of CitH3, Ly6G/C and DNA indicates NET formation. The impact of rTM pretreatment on NET formation in the lung and kidney were assessed by (b) an immunofluorescence assay and (c) quantitative analysis of NETs at 24 h after bilateral renal IR. Scale bar, 25 µm. n = 7 per group. *P < 0.05 versus respective control. Abbreviations: CitH3, citrullinated histone H3; IR, ischemia-reperfusion; NET, neutrophil extracellular trap; rTM, recombinant thrombomodulin.

Figure 7

Histone levels in the lungs and plasma after renal IR. The effect of renal IR and rTM pretreatment on histone contents in (a) the lung and (b) plasma were evaluated at 6 h and 24 h following bilateral renal IR. (n = 6 per group) (c) Pulmonary neutrophils and histones at 6 h and 24 h after bilateral renal IR were exhibited by immunostaining of Ly6G/C (neutrophil marker) and CitH3. (d) Ly6G/C- and CitH3-positive area were also calculated at 6 h and 24 h after surgery. Scale bar, 25 µm. n = 7 per group. ^#P < 0.05 versus sham. *P < 0.05 between the IR and IR + rTM group. **P < 0.05 between 6 h and 24 h. Abbreviations: AU, absorbance unit; CitH3, citrullinated histone H3; IR, ischemia-reperfusion; rTM, recombinant thrombomodulin.

Figure 8

Plasma HMGB1 levels in mice after renal IR. The plasma levels of HMGB1 were measured at 6 h and 24 h after bilateral renal IR. The mice were pretreated with saline (IR group) or 10 mg/kg of rTM (IR + rTM group). n = 6 per group. *P < 0.05 versus respective control. Abbreviations: HMGB1, high morbidity group box-1; IR, ischemia-reperfusion; rTM, recombinant thrombomodulin.