Objective This pilot study assesses the degree of hemolysis induced by cardiopulmonary bypass (CPB) and determines its association with acute kidney injury (AKI) in pediatric patients. Further, it evaluates the degree to which the use of urinary biomarkers neutrophil gelatinase-associated lipocalin (NGAL) and cystatin C correlate with the presence of AKI and hemolysis following CPB. Design Prospective observational study Setting A 13-bed pediatric cardiac intensive care unit in a university hospital Patients Children undergoing cardiac surgery with the use of CPB Interventions None Measurements and Main Results Blood and urine samples were obtained at multiple time points before and after CPB. Hemolysis was assessed by measuring levels of plasma hemoglobin and haptoglobin. AKI was defined as a doubling in serum creatinine from preoperative baseline and by using the pediatric-modified RIFLE criteria. Urinary NGAL and Cystatin C levels were measured. A total of 40 patients (range: 3 days to 4.8 years) were enrolled. Plasma hemoglobin levels increased markedly on separation from CPB with a concurrent decrease in haptoglobin. This was associated with an increase in protein oxidation in the plasma. Hemolysis was more evident in younger patients with a longer duration of bypass and in those requiring a blood-primed circuit. 40% of patients had a doubling in serum creatinine and 88% of patients developed acute kidney injury when defined by the pediatric-modified RIFLE criteria. Controlling for CPB time, persistently elevated levels of plasma hemoglobin were associated with a 5 fold increase in AKI. Further, urinary NGAL measured 2 hours after separation from CPB was associated with AKI and with elevations in plasma hemoglobin. Conclusions CPB in pediatric patients results in significant hemolysis, which is associated with the development of AKI. The biomarker NGAL correlates with both AKI and hemolysis in this population.

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BackgroundHepcidin serves as a major regulator of systemic iron metabolism and immune function. Airway epithelial cells have an extensive interface with the environment, and so must be able to respond locally to the presence of particulates, infection, and inflammation. Therefore, we hypothesized that hepcidin is expressed in airway epithelial cells and is regulated by early phase cytokines.MethodsPrimary, differentiated human bronchial epithelial (NHBE) cells were used to assess hepcidin gene expression in response to IFN-γ, TNF-α, IL-1β, and IL-6, as well as to LPS + CD14. The role of the Janus Kinase-signal transducer and activator of transcription (JAK-STAT) pathway in IFN-γ-mediated hepcidin production was assessed by measuring JAK2 phophorylation and STAT1 nuclear translocation. Inductively coupled plasma mass spectroscopy (ICP-MS) was used to determine whether hepcidin altered iron transport in either NHBE cells or primary alveolar macrophages.ResultsWe demonstrate that differentiated human airway epithelial cells express hepcidin mRNA and that its expression is augmented in response to IFN-γ via activation of STAT1. However, while IFN-γ induced hepcidin gene expression, we were not able to demonstrate diminished expression of the iron export protein, ferroportin (Fpn), at the cell surface, or iron accumulation in airway epithelial in the presence of exogenous hepcidin.ConclusionThese data demonstrate that airway epithelial cells express hepcidin in the lung in response to IFN-γ. The presence of hepcidin in the airway does not appear to alter cellular iron transport, but may serve as a protective factor via its direct antimicrobial effects.

We tested the hypothesis that oxidative stress and biological effect after ozone (O3) exposure are dependent on changes in iron homeostasis. After O3 exposure, healthy volunteers demonstrated increased lavage concentrations of iron, transferrin, lactoferrin, and ferritin. In normal rats, alterations of iron metabolism after O3 exposure were immediate and preceded the inflammatory influx. To test for participation of this disruption in iron homeostasis in lung injury following O3 inhalation, we exposed Belgrade rats, which are functionally deficient in divalent metal transporter 1 (DMT1) as a means of iron uptake, and controls to O3. Iron homeostasis was disrupted to a greater extent and the extent of injury was greater in Belgrade rats than in control rats. Nonheme iron and ferritin concentrations were higher in human bronchial epithelial (HBE) cells exposed to O3 than in HBE cells exposed to filtered air. Aldehyde generation and IL-8 release by the HBE cells was also elevated following O3 exposure. Human embryonic kidney (HEK 293) cells with elevated expression of a DMT1 construct were exposed to filtered air and O3. With exposure to O3, elevated DMT1 expression diminished oxidative stress (i.e., aldehyde generation) and IL-8 release. We conclude that iron participates critically in the oxidative stress and biological effects after O3 exposure.

Hemolysis can occur as a consequence of extracorporeal membrane oxygenation (ECMO) and is associated with increased mortality and morbidity. Shear stress generated by flow through the circuit and oxygenator is believed to cause ECMO-induced hemolysis. We hypothesize that either a smaller dimension oxygenator or an in-line hemofilter will increase ECMO-associated hemolysis. Circuits were configured with a Quadrox-D Adult oxygenator (surface area 1.8 m2), Quadrox-iD Pediatric oxygenator (surface area 0.8 m2), or Quadrox-D Adult oxygenator with an in-line hemofilter (N=4) and ran for six hours. Samples were collected hourly from the ECMO circuit and a time-based hemolysis control. Plasma hemoglobin levels were assayed. Circuit-induced hemolysis at each time point was defined as the change in plasma hemoglobin standardized to the time-based hemolysis control. Plasma hemoglobin increased with the use of the smaller dimension pediatric oxygenator as compared to the adult oxygenator when controlling for ECMO run time (p=0.02). Further, there was a greater pressure gradient with the smaller dimension pediatric oxygenator (p<0.05). Plasma hemoglobin did not change with the addition of the in-line hemofilter. The use of a smaller dimension pediatric oxygenator resulted in greater hemolysis and a higher pressure gradient. This may indicate that increased shear forces augment ECMO-induced hemolysis.

Iron is essential for many aspects of cellular function. However, it also can generate oxygen-based free radicals that result in injury to biological molecules. For this reason, iron acquisition and distribution are tightly regulated. Constant exposure to the atmosphere results in significant exposure of the lungs to catalytically active iron. The lungs have a mechanism for detoxification to prevent associated generation of oxidative stress. Those same proteins that participate in iron uptake in the gut are also employed in the lung to transport iron intracellularly and sequester it in an inactive form within ferritin. The release of metal is expedited (as transferrin and ferritin) from lung tissue to the respiratory lining fluid for clearance by the mucocilliary pathway or to the reticuloendothelial system for long-term storage. This pathway is likely to be the major method for the control of oxidative stress presented to the respiratory tract.

Anion exchange protein 2 (AE2) is a membrane-bound protein that mediates chloride-bicarbonate exchange. In addition to regulating intracellular pH and cell volume, AE2 exports superoxide (O[Formula: see text]·) to the extracellular matrix in an HCO[Formula: see text]-dependent process. Given this ability to export O[Formula: see text]·, we hypothesized that expression of AE2 in the lung is regulated by oxidative stress. AE2 mRNA and protein expression was measured by RT-PCR and Western blot analysis, respectively, in differentiated human bronchial epithelial cells exposed to H2O2 (100 μM). Alterations in in vivo AE2 protein expression were evaluated in lung tissue of rats exposed to 70% O2. The role of transcription factor activator protein (AP)-1 in oxidant regulation of AE2 was evaluated by EMSA and by immunoblotting of nuclear phospho-c- jun. Results show increased AE2 mRNA and protein expression after oxidant exposure. This was preceded by transient increases in DNA binding of AE2-specific AP-1 and phosphorylation of c- jun. This study demonstrates that AE2 expression is regulated by oxidative stress in airway epithelial cells and that this regulation correlates with activation of AP-1.