Neutrophil elastase contributes to acute lung injury induced by bilateral nephrectomy

Abstract

Acute kidney injury (AKI) is a serious problem in critically ill patients of intensive care units. It has been reported previously that AKI can induce acute lung injury (ALI), as well as cause injuries to other remote organs, including the lungs. Patients with AKI complicated by ALI show remarkably high mortality. ALI is characterized by neutrophil infiltration into the lung. Neutrophil elastase (NE) is a key enzyme for tissue injury caused by activated neutrophils, such as occurs in ALI. Therefore, this study investigated the role of NE in AKI-induced ALI using a specific NE inhibitor, sivelestat sodium hydrate (ONO-5046), in a mouse bilateral nephrectomy model. Bilateral nephrectomy showed not only a remarkable increase in blood urea nitrogen levels, but also demonstrated neutrophil infiltration into the lung, increased pulmonary inflammatory cytokine expression [interleukin-6, neutrophil chemokine keratinocyte-derived chemokine, and tumor necrosis factor-α], and protein leakage with early increases in both systemic and pulmonary NE activity. ONO-5046 treatment reduced NE activity and improved these pulmonary inflammatory responses. Additionally, ONO-5046-treated animals had longer survival times. These data demonstrate that increasing NE activity induces pulmonary inflammatory damage in a bilateral nephrectomy model. Blockade of NE activity will be a useful therapeutic strategy for ALI complications in AKI patients.

Figure 1

Protocol of ONO-5046 treatment. Acute lung injury was induced by bilateral nephrectomy. The experimental design of the BNx model and ONO-5046 treatment is shown. ONO-5046 treatment was performed 11 hours before surgery and every 11 hours after until death. Animals were killed 6 hours and 24 hours for blood and lung tissue sampling except survival analysis.

Figure 2

Neutrophil infiltration in ALI induced by BNx. Bilateral nephrectomy induced neutrophil infiltration in the lung. ONO-5046 treatment significantly reduced neutrophil numbers in the lung tissue. A–C: Neutrophil infiltration at 6 and 24 hours after the surgery was observed by Giemsa stain (arrows). Original magnification, ×400. Scale bar = 50 μm. D: The neutrophil count in the lung was decreased significantly by ONO-5046 (ONO) treatment (n = 5 to 6 in each group; the number of neutrophils was determined in 10 randomly selected nonoverlapping fields in each section of the individual mouse lung.). *P < 0.001 versus BNx+saline, ***P < 0.05 versus sham.

Figure 3

BALF cell count and protein concentration in ALI induced by BNx. BALF obtained at 6 hours and 24 hours after bilateral nephrectomy was analyzed for evaluating pulmonary inflammation and vascular leakage. A: No significant difference was found in BALF cell count among the groups at 6 hours and 24 hours (n = 5 to 6 in each group). B: BALF protein concentration was increased at 24 hours and ONO-5046 (ONO) treatment significantly decreased BALF protein at 24 hours (n = 5 to 6 in each group). *P < 0.001 versus BNx+saline.

Figure 4

Effect of ONO-5046 on survival. Animals started to die 24 hours after bilateral nephrectomy and ONO-5046 (ONO) treatment showed a significant improvement of the survival after BNx compared with saline injection (P < 0.05, by log-rank test). Closed circles indicate the BNx+saline group (n = 16) and open circles ONO-5046 treatment group (n = 14).

Figure 5

Liver injury in ALI induced by BNx. Liver injury was observed after bilateral nephrectomy. Plasma aspartate aminotransferase (A) and alanine aminotransferase (B) levels were significantly increased after the surgery. ONO-5046 (ONO) treatment did not improve liver injury (n = 7 in each group). *P < 0.001 versus sham.

Figure 6

NE activity after AKI by BNx. To demonstrate the mechanism of protective effect of ONO-5046, lung (A) and plasma (B) NE activity after BNx were examined. BNx increased pulmonary and plasma NE activity at 6 hours and ONO-5046 (ONO) treatment significantly decreased it (n = 5 to 6 in each group). *P < 0.001, **P < 0.01 versus BNx+saline.

Figure 7

Pulmonary NE expression in ALI induced by BNx. A: The amount of NE protein was measured by Western blot analysis of lung homogenates. B: Densitometric analysis of immunoreactive bands compared with the density of β-actin demonstrated NE protein amount was significantly increased in the BNx+saline group, and attenuated by ONO-5046 (ONO) treatment (n = 6 in each group). *P < 0.05 versus BNx+saline.

Figure 8

Inflammatory cytokine mRNA expression in ALI induced by BNx. Inflammatory responses induced by BNx were evaluated by several inflammatory cytokine expressions. mRNA expressions of IL-6 (A), KC (B), and TNF-α (C) in the lung were increased by BNx and ONO-5046 (ONO) treatment remarkably reduced these inflammatory cytokine expressions (n = 5 to 7 in each group). *P < 0.001, **P < 0.01, ***P < 0.05 versus BNx+saline.