Circulating IL-6 mediates lung injury via CXCL1 production after acute kidney injury in mice

Abstract

Serum IL-6 is increased in patients with acute kidney injury (AKI) and is associated with prolonged mechanical ventilation and increased mortality. Inhibition of IL-6 in mice with AKI reduces lung injury associated with a reduction in the chemokine CXCL1 and lung neutrophils. Whether circulating IL-6 or locally produced lung IL-6 mediates lung injury after AKI is unknown. We hypothesized that circulating IL-6 mediates lung injury after AKI by increasing lung endothelial CXCL1 production and subsequent neutrophil infiltration. To test the role of circulating IL-6 in AKI-mediated lung injury, recombinant murine IL-6 was administered to IL-6-deficient mice. To test the role of CXCL1 in AKI-mediated lung injury, CXCL1 was inhibited by use of CXCR2-deficient mice and anti-CXCL1 antibodies in mice with ischemic AKI or bilateral nephrectomy. Injection of recombinant IL-6 to IL-6-deficient mice with AKI increased lung CXCL1 and lung neutrophils. Lung endothelial CXCL1 was increased after AKI. CXCR2-deficient and CXCL1 antibody-treated mice with ischemic AKI or bilateral nephrectomy had reduced lung neutrophil content. In summary, we demonstrate for the first time that circulating IL-6 is a mediator of lung inflammation and injury after AKI. Since serum IL-6 is increased in patients with either AKI or acute lung injury and predicts prolonged mechanical ventilation and increased mortality in both conditions, our data suggest that serum IL-6 is not simply a biomarker of poor outcomes but a pathogenic mediator of lung injury.

Fig. 1.

Serum and lung IL-6 after acute kidney injury (AKI). Serum (ELISA) and lung IL-6 (ELISA and mRNA) were determined 2 h after sham operation (Sham), bilateral nephrectomy (BNx), or ischemic AKI (AKI). Left: serum IL-6 was increased 2 h after BNx and ischemic AKI vs. sham operation (n = 4). Lung IL-6 as judged by ELISA (n = 4; middle) did not increase after BNx or ischemic AKI. Right: mRNA for IL-6 did not significantly increase after BNx (n = 8), but it was slightly increased after ischemic AKI (n = 8) vs. sham operation (n = 3).

Fig. 2.

Administration of intravenous IL-6 to IL-6-deficient mice with ischemic AKI. Two hundred nanograms of recombinant IL-6 or vehicle were given intravenously every hour for 3 h after ischemic AKI to IL-6-deficient mice, and serum IL-6, serum CXCL1, lung CXCL1, and lung myeloperoxidase (MPO) activity were determined at 4 h (n = 9–10). Serum IL-6 (A), serum CXCL1 (B), lung CXCL1 (C), and lung MPO activity (D) were all increased after intravenous administration of IL-6 vs. vehicle.

Fig. 3.

Pulmonary endothelial cell production of CXCL1 in ischemic AKI. Immunofluorescence was performed on lungs 4 h after sham operation or ischemic AKI and labeled for CXCL1 (green), von Willebrand Factor (red), and DAPI (blue; n = 3). A: no CXCL1 was seen after sham operation. B and C: after ischemic AKI, CXCL1 was found in the lungs (green) and colocalization (yellow) was found between CXCL1 and von Willebrand Factor (white arrows); 2 separate lung fields are shown (B and C). Blue: Dapi (nuclei); red: Von Willebrand Factor (endothelial cells); green: CXCL1; yellow: endothelial cell and CXCL1 colocalization.

Fig. 4.

IL-6-mediated production of CXCL1 by endothelial cells, in vitro. Recombinant murine vehicle vs. IL-6 + soluble IL-6 receptor (sIL-6R; IL-6 + sIL-6R) were added to cultured MS1 endothelial cells and CXCL1 was determined in the media and cells after 4 h (n = 10). A: media CXCL1 increased with IL-6 + sIL-6R. B: cell CXCL1 was similar in vehicle-treated and IL-6 + sIL-6R.

Fig. 5.

Lung inflammation in CXCR2-deficient mice with ischemic AKI or BNx. Lung MPO activity and lung CXCL1 were determined 4 h after ischemic AKI (AKI) or BNx in CXCR2-deficient (−/−) mice (n = 6–8). A, B: lung MPO was significantly reduced in CXCR2 −/− mice, compared with wild-type littermates (WT), after both BNx and ischemic AKI. C, D: lung CXCL1 was significantly reduced in CXCR2 −/− mice, compared with WT littermates, after both ischemic AKI and BNx.

Fig. 6.

Lung inflammation and lung capillary leak in CXCL1 antibody-treated mice with ischemic AKI or BNx. Lung MPO activity, lung CXCL1, and lung Evan's blue dye accumulation were assessed 4 h after ischemic AKI (AKI) or BNx in mice treated with vehicle (Veh; rabbit IgG) or CXCL1 antibody (Ab). A: lung MPO activity was reduced in CXCL1 antibody-treated mice with either ischemic AKI or BNx vs. vehicle-treated (n = 9). B: lung CXCL1 was similar in vehicle-treated and CXCL1 antibody-treated mice with either ischemic AKI or BNx (n = 8). C: lung Evan's blue dye was accumulation (lung capillary leak) was reduced in CXCL1 antibody-treated mice with ischemic AKI, but not with BNx vs. vehicle-treated ischemic AKI or BNx, respectively (n = 5–8).

Fig. 7.

Renal function and renal injury in CXCL1-inhibited mice after ischemic AKI and BNx. Renal function was assessed by serum creatinine and blood urea nitrogen (BUN) 4 h after ischemic AKI (AKI) or BNx in WT, CXCR2-deficient (−/−) mice, vehicle-treated (Veh), or CXCL1 antibody-treated (Ab) mice with ischemic AKI or BNx. Renal injury was assessed at 4 h by acute tubular necrosis score (ATN) on kidney histology in WT, CXCR2 −/−, vehicle-treated, and Ab-treated mice with ischemic AKI. Serum creatinine (A) and BUN (B) were similar in WT and CXCR2 −/− mice with AKI or BNx (n = 9–12). Serum creatinine (C) and BUN (D) were similar in Veh and CXCL1 Ab-treated mice with AKI or BNx (n = 9–12). E: ATN scores were similar in WT, CXCR2 −/−, Veh-treated, and CXCL1 Ab-treated mice with ischemic AKI (n = 4–9).

Fig. 8.

Serum IL-6 in CXCL1-inhibited mice after AKI. Serum IL-6 was measured 4 h after ischemic AKI (AKI) or BNx in WT, CXCR2-deficient (−/−) mice, Veh-treated, or CXCL1 Ab-treated mice. A: serum IL-6 was similar in WT and CXCR2 −/− mice with AKI (n = 11) or BNx (n = 7). B: serum IL-6 was similar in Veh and CXCL1 Ab-treated mice with AKI (n = 11) or BNx (n = 7–10).