Christopher Altmann

Interleukin-6 mediates lung injury following ischemic acute kidney injury or bilateral nephrectomy

Patients with acute kidney injury frequently have pulmonary complications. Similarly ischemic acute kidney injury or bilateral nephrectomy in rodents causes lung injury characterized by pulmonary edema, increased pulmonary capillary leak and interstitial leukocyte infiltration. Interleukin-6 is a pro-inflammatory cytokine that is increased in the serum of patients with acute kidney injury and predicts mortality. Here we found that lung neutrophil infiltration, myeloperoxidase activity, the neutrophil chemokines KC and MIP-2 and capillary leak all increased within 4 h following acute kidney injury in wild-type mice. These pathologic factors were reduced in interleukin-6-deficient mice following acute kidney injury or bilateral nephrectomy. The lungs of mutant mice had reduced KC but MIP-2 was similar to that of wild type mice. Wild-type mice, treated with an interleukin-6 inactivating antibody, had decreased lung myeloperoxidase activity and KC levels following acute kidney injury. Our study shows that interleukin-6 contributes to lung injury following acute kidney injury.

Serum Interleukin-6 and interleukin-8 are early biomarkers of acute kidney injury and predict prolonged mechanical ventilation in children undergoing cardiac surgery: a case-control study

IntroductionAcute kidney injury (AKI) is associated with high mortality rates. New biomarkers that can identify subjects with early AKI (before the increase in serum creatinine) are needed to facilitate appropriate treatment. The purpose of this study was to test the role of serum cytokines as biomarkers for AKI and prolonged mechanical ventilation.MethodsThis was a case-control study of children undergoing cardiac surgery. AKI was defined as a 50% increase in serum creatinine from baseline within 3 days. Levels of serum interleukin (IL)-1β, IL-5, IL-6, IL-8, IL-10, IL-17, interferon (IFN)-γ, tumor necrosis factor-α (TNF-α), granulocyte colony-stimulating factor (G-CSF), and granulocyte-macrophage colony-stimulating factor (GM-CSF) were measured using a bead-based multiplex cytokine kit in conjunction with flow-based protein detection and the Luminex LabMAP multiplex system in 18 cases and 21 controls. Levels of IL-6 and IL-8 were confirmed with single-analyte ELISA; IL-18 was also measured with single-analyte ELISA.ResultsIL-6 levels at 2 and 12 hours after cardiopulmonary bypass (CPB) and IL-8 levels at 2, 12 and 24 hours were associated with the development of AKI using the Wilcoxon rank-sum test and after adjustment for age, gender, race, and prior cardiac surgery in multivariate logistic regression analysis. In patients with AKI, IL-6 levels at 2 hours had excellent predictive value for prolonged mechanical ventilation (defined as mechanical ventilation for more than 24 hours postoperatively) by receiver operator curve (ROC) analysis, with an area under the ROC curve of 0.95. IL-8 levels at 2 hours had excellent predictive value for prolonged mechanical ventilation in all patients. Serum IL-18 levels were not different between those with and without AKI.ConclusionsSerum IL-6 and IL-8 values identify AKI early in patients undergoing CPB surgery. Furthermore, among patients with AKI, high IL-6 levels are associated with prolonged mechanical ventilation, suggesting that circulating cytokines in patients with AKI may have deleterious effects on other organs, including the lungs.

IL-33 Exacerbates Acute Kidney Injury

Inflammation contributes to the pathogenesis of acute kidney injury (AKI). IL-33 is a proinflammatory cytokine, but its role in AKI is unknown. Here we observed increased protein expression of full-length IL-33 in the kidney following induction of AKI with cisplatin. To determine whether IL-33 promotes injury, we administered soluble ST2 (sST2), a fusion protein that neutralizes IL-33 activity by acting as a decoy receptor. Compared with cisplatin-induced AKI in untreated mice, mice treated with sST2 had fewer CD4 T cells infiltrate the kidney, lower serum creatinine, and reduced acute tubular necrosis (ATN) and apoptosis. In contrast, administration of recombinant IL-33 (rIL-33) exacerbated cisplatin-induced AKI, measured by an increase in CD4 T cell infiltration, serum creatinine, ATN, and apoptosis; this did not occur in CD4-deficient mice, suggesting that CD4 T cells mediate the injurious effect of IL-33. Wildtype mice that received cisplatin and rIL-33 also had higher levels of the proinflammatory chemokine CXCL1, which CD T cells produce, in the kidney compared with CD4-deficient mice. Mice deficient in the CXCL1 receptor also had lower serum creatinine, ATN, and apoptosis than wildtype mice following cisplatin-induced AKI. Taken together, IL-33 promotes AKI through CD4 T cell-mediated production of CXCL1. These data suggest that inhibiting IL-33 or CXCL1 may have therapeutic potential in AKI.

Interleukin-18 binding protein transgenic mice are protected against ischemic acute kidney injury

IL-18 function is neutralized in IL-18 binding protein transgenic (IL-18BP Tg) mice. First, we determined whether IL-18BP Tg mice are protected against ischemic acute kidney injury (AKI). Ischemic AKI was induced by bilateral renal pedicle clamping. IL-18BP Tg mice were functionally and histologically protected against ischemic AKI as determined by blood urea nitrogen, serum creatinine, and acute tubular necrosis score. We have demonstrated that the injurious effect of IL-18 in the kidney is independent of neutrophils and lymphocytes. Thus the effect of IL-18 inhibition on renal macrophage infiltration was determined. The number of macrophages was significantly reduced in IL-18BP Tg compared with wild-type kidneys. To determine the cytokines and chemokines that are dependent on IL-18, we performed flow cytometry based assays. Multiple chemokines/cytokines, IL-3, IL-6, IL-15, IL-18, leukemia inhibitory factor, macrophage colony-stimulating factor, macrophage inflammatory protein-2, granulocyte-macrophage colony-stimulating factor, and monocyte chemotactic protein-1 were significantly increased in AKI vs. sham kidneys. Only CXCL1 (also known as KC or IL-8) was significantly increased in AKI vs. sham kidneys and significantly reduced in IL-18BP Tg AKI vs. wild-type AKI kidneys. To determine whether macrophages are the source of CXCL1 in the kidney, we depleted macrophages with liposomal encapsulated clodronate. CXCL1 was significantly decreased in macrophage-depleted vs. control AKI mice. In summary, in ischemic AKI in mice, 1) IL-18BP Tg mice are functionally and histologically protected, 2) macrophage infiltration in the kidney and CXCL1 are significantly reduced in IL-18BP Tg mice, and 3) macrophage depletion significantly reduces CXCL1 in the kidney. In conclusion, protection against ischemic AKI in IL-18BP Tg mice is associated with less macrophage infiltration and less production of CXCL1 in the kidney.

Urine interleukin-6 is an early biomarker of acute kidney injury in children undergoing cardiac surgery

IntroductionInterleukin-6 (IL-6) is a proinflammatory cytokine that increases early in the serum of patients with acute kidney injury (AKI). The aim of this study was to determine whether urine IL-6 is an early biomarker of AKI and determine the source of urine IL-6. Numerous proteins, including cytokines, are filtered by the glomerulus and then endocytosed and metabolized by the proximal tubule. Since proximal tubule injury is a hallmark of AKI, we hypothesized that urine IL-6 would increase in AKI due to impaired proximal tubule metabolism of filtered IL-6.MethodsUrine was collected in 25 consecutive pediatric patients undergoing cardiac bypass surgery (CPB). AKI was defined as a 50% increase in serum creatinine at 24 hours (RIFLE (Risk, Injury, Failure, Loss, End stage), R). Mouse models of AKI and freshly isolated proximal tubules were also studied.ResultsUrine IL-6 increased at six hours in patients with AKI versus no AKI (X2 = 8.1750; P < 0.0042). Urine IL-6 > 75 pg/mg identified AKI with a sensitivity of 88%. To assess whether increased urine IL-6 occurs in functional versus structural renal failure, mouse models of pre-renal azotemia after furosemide injection (no tubular injury), ischemic AKI (tubular injury) and cisplatin AKI (tubular injury) were studied. Urine IL-6 did not significantly increase in pre-renal azotemia but did increase in ischemic and cisplatin AKI. To determine if circulating IL-6 appears in the urine in AKI, recombinant human (h)IL-6 was injected intravenously and urine collected for one hour; urine hIL-6 increased in ischemic AKI, but not pre-renal azotemia. To determine the effect of AKI on circulating IL-6, serum hIL-6 was determined one hour post-intravenous injection and was increased in ischemic AKI, but not pre-renal azotemia. To directly examine IL-6 metabolism, hIL-6 was added to the media of normal and hypoxic isolated proximal tubules; hIL-6 was reduced in the media of normal versus injured hypoxic proximal tubules.ConclusionsUrine IL-6 increases early in patients with AKI. Animal studies demonstrate that failure of proximal tubule metabolism of IL-6 results in increased serum and urine IL-6. Impaired IL-6 metabolism leading to increased serum IL-6 may contribute to the deleterious systemic effects and increased mortality associated with AKI.

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

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.

Splenectomy exacerbates lung injury after ischemic acute kidney injury in mice

Patients with acute kidney injury (AKI) have increased serum proinflammatory cytokines and an increased occurrence of respiratory complications. The aim of the present study was to examine the effect of renal and extrarenal cytokine production on AKI-mediated lung injury in mice. C57Bl/6 mice underwent sham surgery, splenectomy, ischemic AKI, or ischemic AKI with splenectomy and kidney, spleen, and liver cytokine mRNA, serum cytokines, and lung injury were examined. The proinflammatory cytokines IL-6, CXCL1, IL-1β, and TNF-α were increased in the kidney, spleen, and liver within 6 h of ischemic AKI. Since splenic proinflammatory cytokines were increased, we hypothesized that splenectomy would protect against AKI-mediated lung injury. On the contrary, splenectomy with AKI resulted in increased serum IL-6 and worse lung injury as judged by increased lung capillary leak, higher lung myeloperoxidase activity, and higher lung CXCL1 vs. AKI alone. Splenectomy itself was not associated with increased serum IL-6 or lung injury vs. sham. To investigate the mechanism of the increased proinflammatory response, splenic production of the anti-inflammatory cytokine IL-10 was determined and was markedly upregulated. To confirm that splenic IL-10 downregulates the proinflammatory response of AKI, IL-10 was administered to splenectomized mice with AKI, which reduced serum IL-6 and improved lung injury. Our data demonstrate that AKI in the absence of a counter anti-inflammatory response by splenic IL-10 production results in an exuberant proinflammatory response and lung injury.

Acute lung injury and acute kidney injury are established by four hours in experimental sepsis and are improved with pre, but not post, sepsis administration of TNF-α antibodies.

Introduction Acute kidney injury (AKI) and acute lung injury (ALI) are serious complications of sepsis. AKI is often viewed as a late complication of sepsis. Notably, the onset of AKI relative to ALI is unclear as routine measures of kidney function (BUN and creatinine) are insensitive and increase late. In this study, we hypothesized that AKI and ALI would occur simultaneously due to a shared pathophysiology (i.e., TNF-α mediated systemic inflammatory response syndrome [SIRS]), but that sensitive markers of kidney function would be required to identify AKI. Methods Sepsis was induced in adult male C57B/6 mice with 5 different one time doses of intraperitoneal (IP) endotoxin (LPS) (0.00001, 0.0001, 0.001, 0.01, or 0.25 mg) or cecal ligation and puncture (CLP). SIRS was assessed by serum proinflammatory cytokines (TNF-α, IL-1β, CXCL1, IL-6), ALI was assessed by lung inflammation (lung myeloperoxidase [MPO] activity), and AKI was assessed by serum creatinine, BUN, and glomerular filtration rate (GFR) (by FITC-labeled inulin clearance) at 4 hours. 20 µgs of TNF-α antibody (Ab) or vehicle were injected IP 2 hours before or 2 hours after IP LPS. Results Serum cytokines increased with all 5 doses of LPS; AKI and ALI were detected within 4 hours of IP LPS or CLP, using sensitive markers of GFR and lung inflammation, respectively. Notably, creatinine did not increase with any dose; BUN increased with 0.01 and 0.25 mg. Remarkably, GFR was reduced 50% in the 0.001 mg LPS dose, demonstrating that dramatic loss of kidney function can occur in sepsis without a change in BUN or creatinine. Prophylactic TNF-α Ab reduced serum cytokines, lung MPO activity, and BUN; however, post-sepsis administration had no effect. Conclusions ALI and AKI occur together early in the course of sepsis and TNF-α plays a role in the early pathogenesis of both.

Heparanase mediates renal dysfunction during early sepsis in mice

Heparanase, a heparan sulfate‐specific glucuronidase, mediates the onset of pulmonary neutrophil adhesion and inflammatory lung injury during early sepsis. We hypothesized that glomerular heparanase is similarly activated during sepsis and contributes to septic acute kidney injury (AKI). We induced polymicrobial sepsis in mice using cecal ligation and puncture (CLP) in the presence or absence of competitive heparanase inhibitors (heparin or nonanticoagulant N‐desulfated re‐N‐acetylated heparin [NAH]). Four hours after surgery, we collected serum and urine for measurement of renal function and systemic inflammation, invasively determined systemic hemodynamics, harvested kidneys for histology/protein/mRNA, and/or measured glomerular filtration by inulin clearance. CLP‐treated mice demonstrated early activation of glomerular heparanase with coincident loss of glomerular filtration, as indicated by a >twofold increase in blood urea nitrogen (BUN) and a >50% decrease in inulin clearance (P < 0.05) in comparison to sham mice. Administration of heparanase inhibitors 2 h prior to CLP attenuated sepsis‐induced loss of glomerular filtration rate, demonstrating that heparanase activation contributes to early septic renal dysfunction. Glomerular heparanase activation was not associated with renal neutrophil influx or altered vascular permeability, in marked contrast to previously described effects of pulmonary heparanase on neutrophilic lung injury during sepsis. CLP induction of renal inflammatory gene (IL‐6, TNF‐α, IL‐1β) expression was attenuated by NAH pretreatment. While serum inflammatory indices (KC, IL‐6, TNF‐α, IL‐1β) were not impacted by NAH pretreatment, heparanase inhibition attenuated the CLP‐induced increase in serum IL‐10. These findings demonstrate that glomerular heparanase is active during sepsis and contributes to septic renal dysfunction via mechanisms disparate from heparanase‐mediated lung injury.

Macrophages mediate lung inflammation in a mouse model of ischemic acute kidney injury.

Serum IL-6 is increased in acute kidney injury (AKI) and inhibition of IL-6 reduces AKI-mediated lung inflammation. We hypothesized that circulating monocytes produce IL-6 and that alveolar macrophages mediate lung inflammation after AKI via chemokine (CXCL1) production. To investigate systemic and alveolar macrophages in lung injury after AKI, sham operation or 22 min of renal pedicle clamping (AKI) was performed in three experimental settings: 1) systemic macrophage depletion via diphtheria toxin (DT) injection to CD11b-DTR transgenic mice, 2) DT injection to wild-type mice, and 3) alveolar macrophage depletion via intratracheal (IT) liposome-encapsulated clodronate (LEC) administration to wild-type mice. In mice with AKI and systemic macrophage depletion (CD11b-DTR transgenic administered DT) vs. vehicle-treated AKI, blood monocytes and lung interstitial macrophages were reduced, renal function was similar, serum IL-6 was increased, lung inflammation was improved, lung CXCL1 was reduced, and lung capillary leak was increased. In wild-type mice with AKI administered DT vs. vehicle, serum IL-6 was increased. In mice with AKI and alveolar macrophage depletion (IT-LEC) vs. AKI with normal alveolar macrophage content, blood monocytes and lung interstitial macrophages were similar, alveolar macrophages were reduced, renal function was similar, lung inflammation was improved, lung CXCL1 was reduced, and lung capillary leak was increased. In conclusion, administration of DT in AKI is proinflammatory, limiting the use of the DTR-transgenic model to study systemic effects of AKI. Mice with AKI and either systemic mononuclear phagocyte depletion or alveolar macrophage depletion had reduced lung inflammation and lung CXCL1, but increased lung capillary leak; thus, mononuclear phagocytes mediate lung inflammation, but they protect against lung capillary leak after ischemic AKI. Since macrophage activation and chemokine production are key events in the development of acute lung injury (ALI), these data provide further evidence that AKI may cause ALI.