Ales Krouzecky

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.

Selective inducible nitric oxide synthase inhibition during long-term hyperdynamic porcine bacteremia.

We have recently demonstrated that selective inducible nitric oxide (NO) synthase (iNOS) inhibition with 1400W attenuated the hemodynamic and metabolic alterations affiliated with hyperdynamic porcine endotoxemia. In contrast to endotoxemia, limited evidence is available to document a relationship between NO and organ dysfunction in large animal bacteremic models. Therefore, using the same experimental setup, we investigated the role of selective iNOS blockade in porcine bacteremia induced and maintained for 24 h with a continuous infusion of live Pseudomonas aeruginosa. After 12 h of sepsis, animals received either vehicle (Control, n = 8) or continuous infusion of selective iNOS inhibitor, L-N6-(1-iminoethyl)-lysine (L-NIL; n = 8). Measurements were performed before, and 12, 18, and 24 h after P. aeruginosa infusion. L-NIL inhibited sepsis-induced increase in plasma nitrate/nitrite concentrations and prevented hypotension without affecting cardiac output. Despite comparable hepatosplanchnic macrocirculation, L-NIL blunted the progressive deterioration in ileal mucosal microcirculation and prevented mucosal acidosis. L-NIL largely attenuated mesenteric and hepatic venous acidosis, significantly improved P. aeruginosa-induced impairment of hepatosplanchnic redox state, and mitigated the decline in liver lactate clearance. Furthermore, the administration of L-NIL reduced the hepatocellular injury and prevented the development of renal dysfunction. Finally, treatment with L-NIL significantly attenuated the formation of 8-isoprostane concentrations, a direct marker of lipid peroxidation. Thus, selective iNOS inhibition with L-NIL prevented live bacteria from causing key features of metabolic derangements in porcine hyperdynamic sepsis. Underlying mechanisms probably include reduced oxidative stress with improved microcirculatory perfusion and restoration of cellular respiration.

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.

Successful reversal of resistent hypodynamic septic shock with levosimendan.

Myocardial depression is a well-recognized feature of septic shock. End-stage hypodynamic septic shock characterized by a severely low cardiac output state invariably carries a poor prognosis. We report, for the first time, successful treatment of a fatal form of catecholamine resistent hypodynamic septic shock using a novel inotropic calcium-sensitizing drug, levosimendan. This compound was originally developed for the treatment of decompensated heart failure (1), and there are no data available regarding the effects of levosimendan in human hypodynamic septic shock. A 37-year-old woman was admitted to our intensive care unit (ICU) because of septic shock caused by methicillin-sensitive Staphylococcus aureus. The patient had multiple sclerosis lasting for 9years and her customary therapy consisted of methylprednison (16mgday ) and azathioprin (75mgday ). She was mechanically ventilated and received aggressive fluid resuscitation using both crystalloids and colloids targeting the central venous pressure (CVP) between 12 and 14mmHg. Despite volume resuscitation, noradrenaline (0.4mgkg min ) was required to maintain mean arterial pressure (MAP) above 70mmHg. Vancomycin (1.5 g), ceftazidime (2g every 8h) and fluconazol (0.2g every 12h) were infused intravenously (at that time, the results of blood cultures were not known) and stress doses of hydrocortizon were administered (50mg i.v. every 6h). During the next 12h, the patient’s condition progressively deteriorated. She required substantial, increasing doses of noradrenaline (1.3mgkg min ), her body temperature rose (39 C), and oligo-anuria developed. Repeated laboratory assessment revealed a progressive rise in arterial lactate (5.2mmol l ) and worsening of metabolic acidosis (pH7.25). A pulmonary artery catheter was inserted, showing a reduced cardiac index (CI) of 2.4 lmin m 2 despite sufficient cardiac filling pressures (CVP 19mmHg, pulmonary artery occlusion pressure 17mmHg). Mixed venous oxygen saturation was 54%. As an additional fluid challenge did not improved CI, an infusion of dobutamine was started(5mgkg min ) and the dose was increased incrementally (maximum dose 24mgkg min ). In a couple of hours the state rapidly progressed into cold shock with no capillary refill and mottled cool extremities. CI was 0.6 lmin m 2 and a bedside echocardiography showed markedly depressed cardiac contractility (left ventricular ejection fraction of approximately 15%). Despite massive doses of combined catecholamine support (noradrenaline 2.3mgkg 1 min , adrenaline 3.1mgkg min , dobutamine 24mgkg min ), MAP kept falling further (46mmHg). In a desperate attempt to reverse this fatal catecholamine-resistent hypodynamic shock, a decision was made to start an intravenous levosimendan infusion (initial loading dose 12mgkg 1 over 10min, followed by a continuous infusion of 0.2mgkg min 1 for 24h) as a last resort therapy. In approximately 30min of levosimendan infusion, we observed a sustained gradual increase in CI reaching the maximum of 3.6 lmin m . The improved CI resulted in the restoration of effective perfusion pressure (MAP 75mmHg) and recovery of urine output. This impressive effect of levosimendan allowed complete and rapid weaning from adrenaline and a significant dose reduction of noradrenaline (0.6mgkg min ) as well as dobutamine (8.1mgkg 1 min ). Repeated echocardiography, performed 5 h after the start of levosimendan, showed a dramatic improvement in myocardial contractility (left ventricular ejection fraction 60%). Subsequently, the typical multiple organ failure requiring long-term ICU care developed. She was discharged from the ICU on the 52nd day, recovering from severe critical-illness polyneuropathy and myopathy. In summary, in this immunocompromized patient, the novel calcium-sensitizing inotropic drug levosimendan was an essential, life-saving therapeutic component in an otherwise fatal course of hypodynamic septic shock. So far, only one studyhas addressed the effects of levosimedan in experimental septic shock (2), but, to our knowledge, there are no human data available. This significant observation should encourage further studies evaluating the role of levosimendan in septic shock associatedwith profoundmyocardial depression.

Fluid challenge in patients at risk for fluid loading‐induced pulmonary edema

Background:  This study evaluated the effects of protocol‐guided fluid loading on extravascular lung water (EVLW) and hemodynamics in a group of patients at high risk for volume expansion‐induced pulmonary and systemic edema.

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.

Reduced L-type calcium current in ventricular myocytes from pigs with hyperdynamic septic shock.

Objective: To hypothesize that reduced L-type calcium current with consequent shortening of cardiac repolarization is present in a clinically relevant porcine model of hyperdynamic septic shock. Myocardial depression is a well-recognized manifestation of sepsis and septic shock. Reduction of L-type calcium current was demonstrated to contribute to the myocardial depression in endotoxemic rodents. Design: Laboratory animal experiments. Setting: Animal research laboratory at a university. Subjects: Twenty-two domestic pigs of either gender. Interventions: In anesthetized, mechanically ventilated, and instrumented pigs, sepsis was induced by bacteremia (central venous infusion of live Pseudomonas aeruginosa) and continued for 22 hrs. Measurements and Main Results: Electrocardiogram was recorded before and 22 hrs after induction of bacteremia. RR, QT, and QTc intervals were significantly shortened by sepsis. In vitro, action potentials were recorded in right ventricular trabeculae. Action potential durations were shortened in septic preparations. Tumor necrosis factor-&agr; did not influence action potential durations. L-type calcium current was measured in isolated ventricular myocytes. Peak L-type calcium current density was reduced in myocytes from septic animals (8.3 ± 0.4 pA/pF vs. 11.2 ± 0.6 pA/pF in control). The voltage dependence of both L-type calcium current activation and inactivation was shifted to more negative potentials in myocytes from septic animals. Action potential-clamp experiments revealed that the contribution of L-type calcium current to the septic action potential was significantly diminished. In cardiac myocytes incubated with tumor necrosis factor-&agr;, L-type calcium current was not further affected. Conclusions: In a clinically relevant porcine model, hyperdynamic septic shock induced shortening of ventricular repolarization and reduction of L-type calcium current. The contribution of L-type calcium current to the action potential in septic ventricular myocytes was significantly diminished. Tumor necrosis factor-&agr; probably did not contribute to this effect.

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.

Hypothermic anticoagulation: testing individual responses to graded severe hypothermia with thromboelastography.

Selective incircuit blood cooling could be an effective anticoagulation strategy during hemodialysis. However, it is currently unknown what blood temperature would ensure sufficient anticoagulation. Similarly, no information exists about potential interindividual variability in response to graded hypothermia. Therefore, the aim of this study was to analyze effects of profound hypothermia on human coagulation. Furthermore, a mathematical relationship between blood temperatures and coagulation was sought to predict individual responses to blood cooling. It was designed as a laboratory study. Thromboelastography (TEG) measurements were taken at a temperature range of 38–12°C. To enable measurements below 20°C, the TEG device was placed into an air conditioned chamber allowing for setting of the temperatures over a wide range. The data were analyzed by regression analysis for pooled and individual measurements. Decreasing temperatures always led to a progressive reduction in blood coagulation by delaying the initiation of thrombus formation, as well as by decreasing the speed of its creation and growth. However, the response to cooling was not uniform and the interindividual variability exists. The relationship between blood temperature and coagulation is not linear but exponential (parameters R and K) and sigmoid (parameter &agr;-angle). The lower the blood temperature, the more significant effect on blood coagulation decline. To predict an individual response of the coagulation system over a wide range of temperatures, a mathematical modeling can be used.

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.