Tubular Injury Biomarkers to Detect Gentamicin-Induced Acute Kidney Injury in the Neonatal Intensive Care Unit.

OBJECTIVE: We evaluated whether urinary excretion of tubular injury markers could be useful for early detection of gentamicin (GM)-induced renal damage in neonates. STUDY DESIGN: We conducted a prospective, observational trial in neonates admitted to the neonatal intensive care unit (26 GM treated, 20 control). Kidney injury molecule-1 (KIM-1), neutrophil gelatinase-associated lipocalin (NGAL), N-acetyl-ß-D-glucosaminidase (NAG), and π- and α-glutathione-S-transferase (GSTP1-1 and GSTA1-1) were measured every 2 hours during admission and compared with serum creatinine (sCr) and urine output. RESULTS: Nine neonates developed AKI during the course of the study. The peak in excretion of urinary biomarkers preceded the peak in sCr (p < 0.0001). GM administration resulted in a more pronounced increase of sCr compared with control (13 [12-28] vs. 10 µmol/L [8.5-17]; p < 0.05). The urinary excretion of NAG (178 [104-698] vs. 32 ng/mol Cr [9-82]; p < 0.001) and NGAL (569 [168-1,681] vs. 222 ng/mol Cr [90-497]; p < 0.05) was higher in the GM group compared with control and preceded the peak of sCr and urine output decrease. CONCLUSION: GM administration to neonates is associated with renal damage reflected by a more pronounced increase in sCr preceded by urinary excretion of biomarkers. Urinary biomarkers may be useful for earlier identification of renal injury in neonates.

Circulating angiopoietin-2 levels in the course of septic shock: relation with fluid balance, pulmonary dysfunction and mortality.

PURPOSE: To investigate whether angiopoietin-2, von Willebrand factor (VWF) and angiopoietin-1 relate to surrogate indicators of vascular permeability, pulmonary dysfunction and intensive care unit (ICU) mortality throughout the course of septic shock. METHODS: In 50 consecutive mechanically ventilated septic shock patients, plasma angiopoietin-2, VWF and angiopoietin-1 levels and fluid balance, partial pressure of oxygen/inspiratory oxygen fraction and the oxygenation index as indicators of vascular permeability and pulmonary dysfunction, respectively, were measured until day 28. RESULTS: Angiopoietin-2 positively related to the fluid balance and pulmonary dysfunction, was higher in non-survivors than in survivors and independently predicted non-survival throughout the course of septic shock. VWF inversely related to the fluid balance and pulmonary dysfunction throughout the course of septic shock, was comparable between survivors and non-survivors and predicted non-survival on day 0 only. Angiopoietin-1 positively related to pulmonary dysfunction throughout the course, but did not differ between survivors and non-survivors. CONCLUSIONS: In contrast to VWF, plasma angiopoietin-2 positively relates to fluid balance, pulmonary dysfunction and mortality throughout the course of septic shock, in line with a suggested mediator role of the protein.

Development of endotoxin tolerance in humans in vivo.

OBJECTIVE: Endotoxin (lipopolysaccharide [LPS]) tolerance is characterized by a reduced responsiveness to a subsequent LPS challenge. In animal and human in vitro experiments, LPS tolerance is associated with an attenuated response of proinflammatory cytokines and an enhanced production of anti-inflammatory cytokines. It is unclear if this mechanism accounts for the development of LPS tolerance in humans in vivo. DESIGN: Clinical experimental study. SETTING: Intensive care research unit. PATIENTS: Fourteen healthy male volunteers. INTERVENTIONS: Intravenous injections of 2 ng/kg/day Escherichia coli LPS on 5 consecutive days. MEASUREMENTS AND MAIN RESULTS: Symptom scores, vital signs, leukocyte (elastase) and endothelial cell activation (von Willebrand factor [vWF]), and circulating cytokine levels. On day 1, the symptom score increased to 6.1 +/- 3.1, temperature to 37.8 +/- 0.4 degrees C, heart rate to 103 +/- 6/min (p < 0.0001 for all parameters) compared with 0.3 +/- 0.6, 36.2 +/- 0.5 degrees C, 79 +/- 4/min on day 5, respectively (p < 0.0001 between days 1 and 5). On day 1, elastase, vWF, and all cytokine levels increased significantly (p < 0.001 for all, except transforming growth factor (TGF)-beta, p = 0.02), whereas on day 5, this increase was significantly attenuated (p < 0.001) for elastase (61% +/- 6%), vWF (68% +/- 5%), tumor necrosis factor (97% +/- 3%), interleukin (IL)-6 (88% +/- 8%), IL-10 (87% +/- 7%), and IL-1ra (93% +/- 9% p = 0.018) but not for TGF-beta (5% +/- 22% p = 0.22). The tumor necrosis factor-alpha/IL-10 ratio showed an initial proinflammatory phase, followed by an anti-inflammatory phase on the first day. The proinflammatory phase was attenuated with 95% +/- 2%, whereas the reduction of the anti-inflammatory phase, without TGF-beta levels, was 99% +/- 1% on day 5 (p = 0.13 between phases). CONCLUSIONS: Endotoxin tolerance developed during five consecutive LPS administrations as demonstrated by the attenuated release of proinflammatory cytokines on the fifth day and was associated with less leukocyte and endothelial activation. In contrast to animal and human in vitro data, the attenuated response was not limited to the proinflammatory response, as a similar reduction in the anti-inflammatory cytokines was observed, with the exception of TGF-beta.

Alkaline phosphatase treatment improves renal function in severe sepsis or septic shock patients.

OBJECTIVE: Alkaline phosphatase (AP) attenuates inflammatory responses by lipopolysaccharide detoxification and may prevent organ damage during sepsis. To investigate the effect of AP in patients with severe sepsis or septic shock on acute kidney injury. DESIGN AND SETTING: A multicenter double-blind, randomized, placebo-controlled phase IIa study (2:1 ratio). PATIENTS: Thirty-six intensive care unit patients (20 men/16 women, mean age 58 +/- 3 years) with a proven or suspected Gram-negative bacterial infection, >or=2 systemic inflammatory response syndrome criteria (<24 hours), and <12 hours end-organ dysfunction onset were included. INTERVENTION: An initial bolus intravenous injection (67.5 U/kg body weight) over 10 minutes of AP or placebo, followed by continuous infusion (132.5 U/kg) over the following 23 hours and 50 minutes. MEASUREMENTS AND MAIN RESULTS: Median plasma creatinine levels declined significantly from 91 (73-138) to 70 (60-92) micromol/L only after AP treatment. Pathophysiology of nitric oxide (NO) production and subsequent renal damage were assessed in a subgroup of 15 patients. A 42-fold induction (vs. healthy subjects) in renal inducible NO synthase expression was reduced by 80% +/- 5% after AP treatment. In AP-treated patients, the increase in cumulative urinary NO metabolite excretion was attenuated, whereas the opposite occurred after placebo. Reduced excretion of NO metabolites correlated with the proximal tubule injury marker glutathione S-transferase A1-1 in urine, which decreased by 70 (50-80)% in AP-treated patients compared with an increase by 200 (45-525)% in placebo-treated patients. CONCLUSIONS: In severe sepsis and septic shock, infusion of AP inhibits the upregulation of renal inducible NO synthase, leading to subsequent reduced NO metabolite production, and attenuated tubular enzymuria. This mechanism may account for the observed improvement in renal function.

In vivo evidence for nitric oxide-mediated calcium-activated potassium-channel activation during human endotoxemia.

BACKGROUND: During septic shock, the vasoconstrictor response to norepinephrine is seriously blunted. Animal experiments suggest that hyperpolarization of smooth muscle cells by opening of potassium (K) channels underlies this phenomenon. In the present study, we examined whether K-channel blockers and/or nitric oxide (NO) synthase inhibition could restore norepinephrine sensitivity during experimental human endotoxemia. METHODS AND RESULTS: Volunteers received 2 ng/kg Escherichia coli endotoxin intravenously. Forearm blood flow (FBF) was measured with venous occlusion plethysmography. Infusion of 4 dose steps of norepinephrine into the brachial artery decreased the FBF ratio (ratio of FBF in the experimental arm to FBF in the control arm) to 84 +/- 4%, 70 +/- 4%, 55 +/- 4%, and 38 +/- 4% (mean +/- SEM) of its baseline value. After endotoxin administration, norepinephrine-induced vasoconstriction was attenuated (FBF ratio, 101 +/- 4%, 92 +/- 4%, 83 +/- 6%, and 56 +/- 7%; n = 30; P = 0.0018; pooled data). Intrabrachial infusion of the K-channel blocker tetraethylammonium (TEA) completely restored the vasoconstrictor response to norepinephrine from 104 +/- 5%, 93 +/- 7%, 93 +/- 12%, and 69 +/- 12% to 89 +/- 9%, 73 +/- 4%, 59 +/- 5%, and 46 +/- 8% (n = 6; P = 0.045). Other K-channel blockers did not affect the response to norepinephrine. The NO synthase inhibitor N(G)-monomethyl-l-arginine (L-NMMA; 0.2 mg x min(-1) x dL(-1) intra-arterially) also restored the norepinephrine sensitivity. In the presence of L-NMMA, TEA did not have an additional effect on the norepinephrine-induced vasoconstriction (n = 6; P = 0.9). CONCLUSIONS: The K-channel blocker TEA restores the attenuated vasoconstrictor response to norepinephrine during experimental human endotoxemia. Coadministration of L-NMMA abolishes this potentiating effect of TEA, suggesting that NO mediates the endotoxin-induced effect on vascular K channels. In the absence of an effect of the selective adenosine triphosphate-dependent K-channel blocker tolbutamide, we conclude that the blunting effect of endotoxin on norepinephrine-induced vasoconstriction is caused by NO-mediated activation of calcium-activated K channels in the vascular wall.

Upregulation of renal inducible nitric oxide synthase during human endotoxemia and sepsis is associated with proximal tubule injury.

The incidence and the mortality of septic acute kidney injury are high, partly because the pathogenesis of sepsis-induced renal dysfunction is not clear. The objective of this study was to investigate the upregulation of renal inducible nitric oxide synthase (iNOS) in human endotoxemia and sepsis and the effect of NO on tubular integrity. Septic patients and endotoxemia that was induced by a bolus injection of 2 ng/kg Escherichia coli LPS in human volunteers were studied. In addition, the effect of co-administration of the selective iNOS inhibitor aminoguanidine was evaluated. The urinary excretion of the cytosolic glutathione-S-transferase-A1 (GSTA1-1) and GSTP1-1, markers for proximal and distal tubule damage, respectively, was determined. In septic patients, an almost 40-fold induction of iNOS mRNA in cells that were isolated from urine was found accompanied by a significant increase in NO metabolites in blood. The mRNA expression of iNOS was induced 34-fold after endotoxin administration. LPS-treated healthy volunteers showed a higher urinary excretion of NO metabolites compared with control subjects. Urinary NO metabolite excretion correlated with urinary GSTA1-1 excretion, indicating proximal tubule damage, whereas no distal tubular damage was observed. Co-administration of aminoguanidine reduced the upregulation of iNOS mRNA, urinary NO metabolite, and GSTA1-1 excretion, indicating that upregulation of iNOS and subsequent NO production may be responsible for renal proximal tubule damage observed.

Microvascular permeability during experimental human endotoxemia: an open intervention study.

INTRODUCTION: Septic shock is associated with increased microvascular permeability. As a model for study of the pathophysiology of sepsis, endotoxin administration to humans has facilitated research into inflammation, coagulation and cardiovascular effects. The present study was undertaken to determine whether endotoxin administration to human volunteers can be used as a model to study the sepsis-associated increase in microvascular permeability. METHODS: In an open intervention study conducted in a university medical centre, 16 healthy volunteers were evaluated in the research unit of the intensive care unit. Eight were administered endotoxin intravenously (2 ng/kg Escherichia coli O113) and eight served as control individuals. Microvascular permeability was assessed before and 5 hours after the administration of endotoxin (n = 8) or placebo (n = 8) by three different methods: transcapillary escape rate of I(125)-albumin; venous occlusion strain-gauge plethysmography to determine the filtration capacity; and bioelectrical impedance analysis to determine the extracellular and total body water. RESULTS: Administration of endotoxin resulted in the expected increases in proinflammatory cytokines, temperature, flu-like symptoms and cardiovascular changes. All changes were significantly different from those in the control group. In the endotoxin group all microvascular permeability parameters remained unchanged from baseline: transcapillary escape rate of I(125)-albumin changed from 7.2 +/- 0.6 to 7.7 +/- 0.9%/hour; filtration capacity changed from 5.0 +/- 0.3 to 4.2 +/- 0.4 ml/min per 100 ml mmHg x 10(-3); and extracellular/total body water changed from 0.42 +/- 0.01 to 0.40 +/- 0.01 l/l (all differences not significant). CONCLUSION: Although experimental human endotoxaemia is frequently used as a model to study sepsis-associated pathophysiology, an endotoxin-induced increase in microvascular permeability in vivo could not be detected using three different methods. Endotoxin administration to human volunteers is not suitable as a model in which to study changes in microvascular permeability.

Severe vagal response after endotoxin administration in humans.

OBJECTIVE: Endotoxin administration to humans is a common means to study systemic inflammation. Worldwide, thousands of volunteers have received endotoxin, and adverse events are rarely reported. The aim of this report was to increase awareness of specific risks of the intravenous administration of endotoxin to human volunteers. DESIGN: Report of four cases who developed severe bradycardia or protracted asystole after administration of endotoxin. Interviews with investigators at three large centers that conduct normal volunteer endotoxin studies. SETTING: Clinical research unit. CASES: Four subjects developed severe bradycardia or protracted asystole, approximately 1 h after administration of endotoxin. Further analyses revealed that the subjects had a history of vasovagal syncope or a positive head-tilt test, indicating increased vagal sensitivity. Relative volume depletion associated with fasting overnight may have predisposed these subjects to this condition. CONCLUSIONS: These responses are very rare and are likely due to the cardioinhibitory Bezold-Jarisch reflex. A thorough screening regarding a history of vagal sensitivity and liberal oral or intravenous fluid administration prior to and during the endotoxin challenge may decrease the risk of these events.