Extracranial organ dysfunction (EOD) is a typical feature of critically ill patients with traumatic brain injury (TBI), potential candidates for organ donation. EOD is associated with worse outcome and with systemic inflammation leading to subclinical acute kidney injury (AKI). The aim of this study was to evaluate AKI in TBI also studying the effects of patients' plasma on kidney tubular epithelial cells (TEC). We enrolled 22 TBI patients recording APACHE II and RIFLE. Plasma and urine samples were drawn to evaluate plasma levels of inflammatory cytokines (IL-1beta, IL-6, IL-8, TNF-alpha, TNF-RI, TNF-RII), Neutrophil Gelatinase-Associated Lipocalin (pNGAL, Alere Inc., San Diego, CA) and urine immunoelectrophoresis for Retinol Binding Protein (RBP) and alpha1-Microglobulin (a1-M). In vitro, we studied the effects of patients' plasma on TEC evaluating: a) neutrophil adhesion; b) cell polarity (trans-epithelial electrical resistance-TEER); c) apoptosis (TUNEL and caspase assays); d) gene and protein expression of NGAL, ZO-1 and megalin. TBI patients' characteristics: age 40±19 yr, GCS 5±3, APACHE II 18±13, elevated levels of inflammatory cytokines: significant correlation between IL-6 and APACHE II (p=0.032), duration of endocranic hypertension (p <0.0001) or of hypoxia (p=0.042) and between TNF-RII and duration of endocranic hypertension (p =0.045) or of hypoxia (p=0.013). One TBI patient was included in RIFLE criteria for AKI. A significant increase of pNGAL levels (199±84 ng/ml) and a 5-to-7-fold increase of urine RBP and a1-M was observed. TBI plasma increased neutrophil adhesion to TEC, altered cell polarity and triggered apoptosis. TBI plasma-induced TEC dysfunction was confirmed by down-regulation of ZO-1/megalin and by increase of gene and protein NGAL expression. In TBI patients, the severity of primary insult (APACHE II) and the duration of secondary insult (endocranic hypertension, hypoxia) lead to systemic inflammation and subclinical AKI. Tubular injury may be ascribed to circulating pro-apoptotic mediators and may facilitate delayed graft function after kidney transplantation.

Objectives: Lung ischemia-reperfusion injury is the main cause of primary graft dysfunction after lung transplantation and results in increased morbidity and mortality. Fas-mediated apoptosis is one of the pathologic mechanisms involved in the development of ischemia-reperfusion injury. We hypothesized that the inhibition of Fas gene expression in lungs by intratracheal administration of small interfering RNA could reduce lung ischemia-reperfusion injury in an ex vivo model reproducing the procedural sequence of lung transplantation.Design: Prospective, randomized, controlled experimental study.Setting: University research laboratory.Subjects: C57/BL6 mice weighing 28-30 g.Interventions: Ischemia-reperfusion injury was induced in lungs isolated from mice, 48 hours after treatment with intratracheal small interfering RNA targeting Fas, control small interfering RNA, or vehicle. Isolated lungs were exposed to 6 hours of cold ischemia (4 degrees C), followed by 2 hours of warm (37 degrees C) reperfusion with a solution containing 10% of fresh whole blood and mechanical ventilation with constant low driving pressure.Measurements and Main Results: Fas gene expression was significantly silenced at the level of messenger RNA and protein after ischemia-reperfusion in lungs treated with small interfering RNA targeting Fas compared with lungs treated with control small interfering RNA or vehicle. Silencing of Fas gene expression resulted in reduced edema formation (bronchoalveolar lavage protein concentration and lung histology) and improvement in lung compliance. These effects were associated with a significant reduction of pulmonary cell apoptosis of lungs treated with small interfering RNA targeting Fas, which did not affect cytokine release and neutrophil infiltration.Conclusions: Fas expression silencing in the lung by small interfering RNA is effective against ischemia-reperfusion injury. This approach represents a potential innovative strategy of organ preservation before lung transplantation.