Jaroslav Radej

Renal haemodynamic, microcirculatory, metabolic and histopathological responses to peritonitis-induced septic shock in pigs.

IntroductionOur understanding of septic acute kidney injury (AKI) remains incomplete. A fundamental step is the use of animal models designed to meet the criteria of human sepsis. Therefore, we dynamically assessed renal haemodynamic, microvascular and metabolic responses to, and ultrastructural sequelae of, sepsis in a porcine model of faecal peritonitis-induced progressive hyperdynamic sepsis.MethodsIn eight anaesthetised and mechanically ventilated pigs, faecal peritonitis was induced by inoculating autologous faeces. Six sham-operated animals served as time-matched controls. Noradrenaline was administered to maintain mean arterial pressure (MAP) greater than or equal to 65 mmHg. Before and at 12, 18 and 22 hours of peritonitis systemic haemodynamics, total renal (ultrasound Doppler) and cortex microvascular (laser Doppler) blood flow, oxygen transport and renal venous pressure, acid base balance and lactate/pyruvate ratios were measured. Postmortem histological analysis of kidney tissue was performed.ResultsAll septic pigs developed hyperdynamic shock with AKI as evidenced by a 30% increase in plasma creatinine levels. Kidney blood flow remained well-preserved and renal vascular resistance did not change either. Renal perfusion pressure significantly decreased in the AKI group as a result of gradually increased renal venous pressure. In parallel with a significant decrease in renal cortex microvascular perfusion, progressive renal venous acidosis and an increase in lactate/pyruvate ratio developed, while renal oxygen consumption remained unchanged. Renal histology revealed only subtle changes without signs of acute tubular necrosis.ConclusionThe results of this experimental study argue against the concept of renal vasoconstriction and tubular necrosis as physiological and morphological substrates of early septic AKI. Renal venous congestion might be a hidden and clinically unrecognised contributor to the development of kidney dysfunction.

Searching for mechanisms that matter in early septic acute kidney injury: an experimental study.

IntroductionIn almost half of all sepsis patients, acute kidney injury (AKI) develops. However, the pathobiologic differences between sepsis patients with and without AKI are only poorly understood. We used a unique opportunity to examine dynamic inflammatory, renal hemodynamic, and microvascular changes in two clinically relevant large-animal models of sepsis. Our aim was to assess variability in renal responses to sepsis and to identify both hemodynamic and nonhemodynamic mechanisms discriminating individuals with AKI from those in whom AKI did not develop.MethodsThirty-six pigs were anesthetized, mechanically ventilated, and instrumented. After a recovery period, progressive sepsis was induced either by peritonitis (n = 13) or by continuous intravenous infusion of live Pseudomonas aeruginosa (n = 15). Eight sham operated-on animals served as time-matched controls. All animals received standard intensive care unit (ICU) care, including goal-directed hemodynamic management. Before, and at 12, 18, and 22 hours of sepsis, systemic and renal (ultrasound flow probe) hemodynamics, renal cortex microcirculation (laser Doppler), inflammation (interleukin-6 (IL-6), tumor necrosis factor-alpha (TNF-α), oxidative stress (thiobarbituric acid reactive species (TBARS), nitrite/nitrate concentrations (NOx), and renal oxygen kinetics and energy metabolism were measured.ResultsIn 14 (50%) pigs, AKI developed (62% in peritonitis, 40% in bacteria infusion model). Fecal peritonitis resulted in hyperdynamic circulation, whereas continuous bacteria infusion was associated with normodynamic hemodynamics. Despite insults of equal magnitude, comparable systemic hemodynamic response, and uniform supportive treatment, only those pigs with AKI exhibited a progressive increase in renal vascular resistance. This intrarenal vasoconstriction occurred predominantly in the live-bacteria infusion model. In contrast to AKI-free animals, the development of septic AKI was preceded by early and remarkable inflammatory response (TNF-α, IL-6) and oxidative stress (TBARS).ConclusionsThe observed variability in susceptibility to septic AKI in our models replicates that of human disease. Early abnormal host response accompanied by subsequent uncoupling between systemic and renal vascular resistance appear to be major determinants in the early phase of porcine septic AKI. Nonuniform and model-related renal hemodynamic responses that are unpredictable from systemic changes should be taken into consideration when evaluating hemodynamic therapeutic interventions in septic AKI.

Effects of tempol, a free radical scavenger, on long-term hyperdynamic porcine bacteremia*

Objectives:Pretreatment with tempol, a membrane-permeable radical scavenger, has been shown to be protective in rodent models of endotoxic and Gram-positive shock. However, neither the pretreatment design nor hypodynamic endotoxic shock in rodents mimics the clinical scenario. Therefore, we investigated the effects of tempol in a posttreatment model of long-term, volume-resuscitated, hyperdynamic porcine bacteremia. Design:Prospective, randomized, controlled experimental study. Setting:University animal laboratory. Subjects:Sixteen anesthetized, mechanically ventilated, and instrumented pigs. Interventions:Sepsis was induced and maintained for 24 hrs with continuous infusion of live Pseudomonas aeruginosa. After 12 hrs of hyperdynamic sepsis, animals were randomized to receive either vehicle (control, n = 8) or continuous infusion of tempol (n = 8, 30 mg/kg/hr). Measurements and Main Results:Systemic and hepatosplanchnic hemodynamics, oxygen exchange, metabolism, ileal mucosal microcirculation, and tonometry as well as oxidative stress and coagulation variables were assessed before and after 12, 18, and 24 hrs of P. aeruginosa infusion. Tempol significantly attenuated reduction in mean arterial pressure. Despite comparable mesenteric macrocirculation, tempol attenuated the otherwise progressive deterioration in ileal mucosal microcirculation and prevented mucosal acidosis. By contrast, treatment with tempol failed to influence the P. aeruginosa-induced derangements of hepatosplanchnic redox state, liver lactate clearance, and regional acidosis but prevented the development of renal dysfunction. In addition, tempol reduced nitrosative stress without significant effect on the gradual increase in plasma 8-isoprostanes. Finally, tempol attenuated sepsis-induced endothelial (von Willebrand factor) and hemostatic dysfunction (thrombin-antithrombin complexes, plasminogen activator inhibitor-type 1). Conclusions:The radical scavenger tempol partially prevented live bacteria from causing key features of hemodynamic and metabolic derangements in porcine hyperdynamic sepsis and beneficially affected surrogate markers of sepsis-induced endothelial and coagulation dysfunction. Incomplete reduction of oxidative stress because of dilutional effects and/or missed optimal therapeutic window for antioxidant treatment when used in posttreatment approach may account for the only partial protection by tempol in this model.

Effects of combining inducible nitric oxide synthase inhibitor and radical scavenger during porcine bacteremia.

Complex interactions of nitric oxide and other free radicals have been implicated in the pathogenesis of sepsis and organ dysfunction. We hypothesized that simultaneous inducible nitric oxide synthase inhibition (L-N6-[1-iminoethyl]-lysine [L-NIL]) and neutralization of superoxide (O2−) (4-hydroxy-2,2,6,6-tetramethylpiperidine-N-oxyl [Tempol]) would protect from detrimental consequences of long-term, volume-resuscitated, hyperdynamic porcine bacteremia. In this prospective, randomized, controlled experimental study, 16 anesthetized, mechanically ventilated and instrumented pigs were exposed to 24 h of continuous infusion of live Pseudomonas aeruginosa. After 12 h of hyperdynamic sepsis, animals were randomized to receive either vehicle (control, n = 8) or combination of L-NIL and Tempol (n = 8). Systemic and hepatosplanchnic hemodynamics, oxygen exchange, metabolism, ileal mucosal microcirculation and tonometry, oxidative stress and coagulation parameters were assessed before, 12, 18, and 24 h of P. aeruginosa infusion. Combined treatment inhibited sepsis-induced increase in plasma nitrate/nitrite, 8-isoprostane, and thiobarbituric acid reactive species concentrations, prevented hypotension, and reversed hyperdynamic circulation. Despite lower intestinal macrocirculation, combined regimen attenuated the otherwise progressive deterioration in ileal mucosal microcirculation and prevented mucosal acidosis. Treatment substantially attenuated mesenteric and hepatic venous acidosis, preserved sepsis-induced impairment of hepatosplanchnic redox state, and prevented the development of renal dysfunction. Finally, coinfusion of L-NIL and Tempol largely attenuated the sepsis-induced rise in plasma von Willebrand factor and thrombin-antithrombin complexes. Thus, hemodynamic, microcirculatory, metabolic, renal, and coagulation data indicate that combining inducible inhibition with cell permeable O2− radical scavenger afforded significant protection in porcine sepsis, thus suggesting an important interactive role of O2− and nitric oxide in mediating organ dysfunction.

Sepsis and Acute Kidney Injury Are Bidirectional

Sepsis is the most common cause of acute kidney injury (AKI). There has been a growing body of evidence demonstrating the association between worsening of kidney function during sepsis and the risk of short- and long-term mortality. AKI in sepsis is associated with poor outcome and independently predicts increased mortality. Sepsis-associated AKI may therefore serve as a biomarker of adverse physiological events that portends worse outcome. Conversely, the important role of sepsis among intensive care unit patients with nonseptic AKI is increasingly being recognized. Indeed, sepsis represents a significant contributing factor to the overall mortality and incomplete recovery of kidney function in subjects who developed nonseptic AKI. Because AKI portends such an ominous prognosis in sepsis and vice versa, there has been a surge of interest in elucidating mechanisms underlying the complex and bidirectional nature of the interconnections between AKI, sepsis and multiorgan dysfunction. Accumulating data indicate that AKI can trigger several immune, metabolic and humoral pathways, thus potentially contributing to distant organ dysfunction and overall morbidity and mortality. The expanding population of patients with sepsis and AKI, and the associated excess mortality provide a strong basis for further research aimed at addressing more rigorously all potentially modifiable factors to reduce this burden to patients and health care systems. Better insights into bidirectional and synergistic pathways linking sepsis and AKI might open the window for new therapeutic approaches that interrupt this vicious circle. Here, we discuss the rationale for and the current understanding of the bidirectional relationship between AKI and sepsis.

Coagulation and endothelial dysfunction during longterm hyperdynamic porcine bacteremia--effects of selective inducible nitric oxide synthase inhibition.

Coagulation abnormalities have been implicated in the pathogenesis of sepsis and organ dysfunction. Nitric oxide (NO) is regarded as a critical mediator of many vascular pathologies, including sepsis. However, limited evidence is available to document a relationship between NO generated by inducible NO synthase (iNOS) and hemostatic abnormalities in sepsis. Therefore, we evaluated the effects of selective iNOS inhibition on markers of endothelial and coagulation homeostasis in a clinically relevant model of porcine bacteremia induced and maintained for 24 hours (h) with a continuous infusion of live P. aeruginosa. After 12 h of sepsis, animals received either vehicle (Control, n=7) or continuous infusion of selective iNOS inhibitor L-NIL (n=7). Before as well as 12, 18 and 24 h after starting P. aeruginosa following variables related to i) endothelial dysfunction (von Willebrand factor [vWf]; tissue plasminogen activator activity [t-PA]; ii) coagulation (thrombin-antithrombin complexes [TAT]; platelet count); iii) fibrinolysis (t-PA activity, activity of plasminogen activator inhibitor type 1 (PAI-1 act); and iv) oxidative/nitrosative stress (isoprostanes, nitrate/nitrite levels) were measured. L-NIL inhibited sepsis-induced increase in plasma nitrate/nitrite and isoprostanes concentrations, prevented hypotension and acidosis. L-NIL significantly attenuated sepsis-induced rise in plasma vWF and TAT. P. aeruginosa-induced drop in t-PA activity was blunted by iNOS inhibition, while increased PAI-1 and reduced platelet count were not reversed by the treatment. In conclusion, selective iNOS inhibition was associated with attenuation of sepsis-induced coagulation and endothelial dysfunction suggesting the interplay between mediators of vascular system and hemostatic balance. Reduction of oxidative stress probably contributes to the beneficial effects afforded by iNOS blockade.

Coupled plasma filtration adsorption in experimental peritonitis-induced septic shock.

The coupled plasma filtration adsorption (CPFA) was developed as an adsorptive hemopurification method aimed at nonselective removal of circulating soluble mediators potentially involved in the pathogenesis of sepsis. We hypothesized that this nonselective hemopurification could protect from detrimental consequences of long-term, volume-resuscitated porcine septic shock. In 16 anesthetized, mechanically ventilated, and instrumented pigs, the hyperdynamic septic shock secondary to peritonitis was induced by intraperitoneally inoculating feces and maintained for 22 h with fluid resuscitation and norepinephrine infusion as needed to maintain MAP above 65 mmHg. After 12 h of peritonitis, animals were randomized to receive either supportive treatment (control, n = 8) or CPFA treatment (CPFA, n = 8). Systemic, hepatosplanchnic, and renal hemodynamics; oxygen exchange; energy metabolism (lactate/pyruvate and ketone body ratios); ileal mucosal and renal cortex microcirculation; systemic inflammation (TNF-&agr;, IL-6); nitrosative/oxidative stress (thiobarbituric acid reactive species, nitrates + nitrites); and endothelial/coagulation dysfunction (asymmetric dimethylarginine, von Willebrand factor, thrombin-antithrombin complexes, platelet count) were assessed before and 12, 18, and 22 h of peritonitis. Coupled plasma filtration adsorption neither delayed the development of hypotension nor reduced the dose of norepinephrine. The treatment failed to attenuate sepsis-induced alterations in microcirculation, surrogate markers of cellular energetics, endothelial injury, and systemic inflammation. Similarly, CPFA did not protect from lung and liver dysfunction and even aggravated sepsis-induced disturbances in coagulation and oxidative/nitrosative stress. In this porcine model of septic shock, the early treatment with CPFA was not capable of reversing the sepsis-induced disturbances in various biological pathways and organ systems. Both the efficacy and safety of this method require further rigorous experimental validation in clinically relevant models.ABBREVIATIONS-CPFA-coupled plasma filtration adsorption; SDF-side-stream dark field; MFI-microvascular flow index; FHI-flow heterogeneity index; L-Lactate; P-Pyruvate; ALT-alanine aminotransferase; KBR-ketone body ratio; TBARS-thiobarbituric acid reactive species; NOx-arterial nitrate + nitrite concentrations; vWF-von Willebrand factor; TAT-thrombin-antithrombin complexes; ADMA-asymmetric dimethylarginine; TNF-&agr;-tumor necrosis factor alfa; IL-6-Interleukin 6; NPY-neuropeptide Y; VIP-vasoactive intestinal peptide; CVP-central nervous pressure; PAOP-pulmonary artery occlusion pressure

Identification of a New Delhi metallo-β-lactamase-4 (NDM-4)-producing Enterobacter cloacae from a Czech patient previously hospitalized in Sri Lanka.

In recent two years, a global spread of NDM-type metallobeta-lactamase-producing bacteria has been observed. NDM enzymes are mainly produced by Enterobacteriaceae, but have also been detected in Acinetobacter baumannii and Vibrio spp. as well (Tzouvelekis et al. 2012; Walsh et al. 2011). Two cases of the new variant, NDM-4, have been recently described in isolates recovered from patients previously hospitalized in India and in Cameroon (Nordmann et al. 2012; Dortet et al. 2012). In Czech Republic, only a case of an NDM-1-producing A. baumannii isolated from a Czech citizen repatriated from Egypt has been observed (Hrabak et al. 2012a). Here, we describe an NDM-4producing Enterobacter cloacae strain recovered from a patient previously hospitalized in Sri Lanka. We also present data on the localization and the genetic environment of the blaNDM-4 gene. The patient, a 62-year-old female, who traveled to Sri Lanka in August 2012, was hospitalized for 28 days in three different hospitals in Sri Lanka for sepsis of a putative wound origin. The patient was treated with amoxicillin/clavulanic acid, followed by a combination therapy with meropenem, clindamycin, and doxycycline as an empiric therapy because no infectious agent was identified. After cultivation of Streptococcus pyogenes from wound swab, clindamycin was changed to penicillin. The patient was mechanically ventilated for 11 days. In September, the patient was repatriated to the Czech Republic. Immediately after repatriation to Czech Republic, screening for carbapenemase-producing Enterobacteriaceae was performed according to the national guidelines. Rectal swab and another clinical material standardly collected from patients hospitalized in ICUs (e.g., sputum/bronchoalveolar lavage, nose swabs, throat swabs) were inoculated on the selective plates for the detection of cephalosporin-resistant isolates (ChromIDTM, ESBL Agar, bioMérieux, Paris, France). Colonies grown on the media after 24 h cultivation were identified to the species level. Susceptibility to imipenem and meropenem was determined according to the EUCAST recommendation (EUCAST 2003). Matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry (MALDI Biotyper; Bruker DaltoniK, GmbH, Bremen, Germany) identified an E. cloacae clinical isolate (Encl-922), which exhibited elevated minimum inhibitory concentrations to carbapenems (Table 1). Encl-922 was resistant to all β-lactams, including meropenem, as determined by broth dilution method (EUCAST 2003). The isolate was also resistant to various non-β-lactam antibiotics (Table 1). Carbapenemase production was verified byMALDITOF MS hydrolysis assay (Hrabák et al. 2011, 2012b). Additionally, ethylenediaminetetraacetic acid–meropenem combined disk test confirmed metallo-β-lactamase production, while boronic acid-based tests using meropenem appeared negative. PCR and sequencing showed that Encl-922 carried C. C. Papagiannitsis :V. Studentova : E. Chudackova : T. Bergerova : J. Hrabak Department of Microbiology, Faculty of Medicine and University Hospital, Charles University, Plzen, Czech Republic

Pharmacokinetic evaluation of voriconazole treatment in critically ill patients undergoing continuous venovenous hemofiltration.

Introduction: Voriconazole represents an essential part of antimicrobial therapy in critically ill patients. The aim of this study was to exclude a significant alteration in voriconazole pharmacokinetics in critically ill patients undergoing continuous venovenous hemofiltration (CVVH). Methods: Six patients dependent on CVVH with evidence of an invasive mycotic infection treated with intravenous voriconazole at the standard dosing regimen were investigated. The total serum concentration of voriconazole in arterial blood and the concentration in ultrafiltrate were measured by reverse-phase high-performance liquid chromatography with ultraviolet detection. The authors profiled a 5-point pharmacokinetic concentration-time curve during the 12-hour standard maintenance dosing interval and derived the basic pharmacokinetic parameters. Results: The serum voriconazole concentration did not decrease <1.0 mg/L at any time point, and the mean was 4.3 ± 2.6 mg/L and the median (range) 3.6 (9.0) mg/L. The sieving coefficient of the drug did not exceed 0.30 in any patient (0.22 ± 0.08). The mean serum AUC0-12, the mean total clearance, and the mean clearance via CVVH were 53.52 ± 29.97 mg·h/L [the median (range) of 57.74 (62.34) mg·h/L], 0.11 ± 0.07 L·h−1·kg−1, and 0.007 ± 0.003 L·h−1·kg−1, respectively. The clearance by the CVVH method ranged from 4% to 20% of the total drug clearance. The disposition of voriconazole was not compromised. The mean elimination half-life was 27.58 ± 35.82 hours [the median of 13.10 (92.21) hours], and the mean distribution volume value was 3.28 ± 3.10 L/kg [the median of 2.01 (8.10) L/kg]. Marked variability in serum concentrations, elimination half-life, distribution volume, and total clearance was seen. Half of the patients showed some drug accumulation. Conclusions: The clearance of voriconazole by CVVH is not clinically significant. In view of this finding, voriconazole dose adjustment in patients undergoing the standard method of CVVH is not required. However, the observed potential for an unpredictable voriconazole accumulation suggests the usefulness for monitoring its levels in critically ill patients.

A 24-h Work Shift in Intensive Care Personnel: Biological Pathways between Work Stress and Ill Health

This study evaluated inflammatory, coagulation and microvascular responses to a continuous 24-h work day in 13 healthy intensive care physicians. Inflammatory markers (interleukin [IL]-2, IL-6, IL-10, tumour necrosis factor-α, matrix metalloproteinase [MMP]-9 and adiponectin), adhesion molecules (vascular cellular adhesion molecule-1 and intercellular adhesion molecule-1 [ICAM-1]), coagulation parameters (thrombin—anti thrombin, von Willebrand factor and tissue factor) and sublingual micro circulation were assessed before and after a 24-h work shift. The 24-h work shift had no effect on inflammatory markers and ICAM-1. Direct visualization of microcirculation did not reveal stress-related perfusion abnormalities. A 24-h work shift in the intensive care unit was associated with significantly increased plasma levels of tissue factor — a potentially important mechanism linking acute job strain, haemostasis and atherosclerosis. The long-term consequences warrant further evaluation.