Supernatants were analyzed for protein content using a Bio-Rad DC protein assay kit (Hercules, CA)

Supernatants were analyzed for protein content using a Bio-Rad DC protein assay kit (Hercules, CA). 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 experienced 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 usually 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. Keywords:acute kidney injury, lung neutrophils, lung injury acute kidney injury(AKI) complicates up to 20% of hospital admissions (31) and 3050% of admissions to the ICU (29). Survival is decreased in AKI and critically ill patients with AKI have mortality rates in excess of 50% (6). Patients with AKI have a high incidence of systemic complications, particularly respiratory failure (5), which may contribute to increased mortality. Patients with AKI have prolonged ventilator time and increased total wean time (32). When AKI and respiratory DG051 failure occur in conjunction, the mortality rate is usually 6080% (5,32). We (13,15) and others (7,9,10,12,14,17,23,26) showed that AKI in animals leads to lung injury, characterized by neutrophil infiltration, capillary leak, and increased chemokine production (13). The proinflammatory cytokines IL-6 and IL-8 are elevated early in the serum of patients with AKI and are associated with prolonged mechanical ventilation (18,19). This raises the possibility that IL-6 and IL-8 contribute to lung injury in AKI, in addition to being early biomarkers. Mice also have increased serum IL-6 and CXCL1 (murine analog of IL-8) after AKI (13) and we showed that inhibition of IL-6 reduces lung injury in mice with AKI that is associated with reduced lung CXCL1 and lung neutrophils (15). Thus, these data suggest that IL-6 mediates lung injury in AKI via upregulation of lung CXCL1, causing subsequent neutrophil infiltration. Whether the protection against lung injury is due to inhibition of circulating IL-6 action at the lung or due to inhibition of IL-6 produced in the lung and acting locally in the lung is not known. The role of circulating vs. local IL-6 and the role of CXCL1 in AKI-mediated lung injury are investigated the present study. In the present study, we hypothesized that circulating IL-6 contributes to AKI-mediated lung neutrophil accumulation and lung injury via upregulation of lung CXCL1. To test the role Rabbit polyclonal to Wee1 of circulating IL-6 in AKI-mediated lung injury, recombinant murine IL-6 was administered to IL-6-deficient mice with ischemic AKI and lung CXCL1 and lung neutrophil content were examined; to test the role of CXCL1 in AKI-mediated lung injury, CXCL1 function DG051 was inhibited by use of CXCR2-deficient mice and anti-CXCL1 antibodies in mice with ischemic AKI or bilateral nephrectomy. == METHODS == == == DG051 == Animals. == Eight- to ten-week-old C57BL/6 mice and CXCR2-deficient mice (C57BL/6 background; Jackson Laboratories, Bar Harbor, ME) that weighed 20 to 25 g were used. Mice were maintained on a standard diet, and water was freely available. All experiments were conducted with adherence to the National Institutes of HealthGuide for the Care and Use of Laboratory Animals. The animal protocol was approved by the Animal Care and Use Committee of the University DG051 or college of Colorado, Denver. == Surgical protocol. == Surgical procedures were performed on three groups of mice:1) sham operation,2) ischemic AKI, and3) bilateral nephrectomy. For all those procedures, mice were anesthetized with intraperitoneal avertin (2,2,2-tribromoethanol; Aldrich, Milwaukee, WI), a midline incision was made, and the renal pedicles were identified. In the ischemic AKI group, both renal pedicles were clamped for 22 min. After clamp removal, kidneys were observed for restoration of blood flow by the return to initial color. The stomach was closed in one layer. Sham surgery consisted of the same process except that clamps were not applied. In the bilateral nephrectomy model, both renal pedicles were tied off with suture and then slice distal to the suture. The ureters were pinched off with forceps, and the kidneys were removed. Mice were weighed before and after procedures. == CXCL1 inhibition. == CXCL1 functions.