Roman Sykora

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.

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.

Anesthesia and the microcirculation.

There is increasing evidence that the microcirculation and its regulation are severely compromised during many pathological conditions, such as hemorrhage, sepsis, or trauma. The effects of anesthetic agents on macrohemodynamics were investigated intensively in the last several decades. Research regarding modern anesthetics and anesthesia techniques has increased knowledge regarding the nonanesthetic effects of anesthetic agents, including those on organ perfusion and the microcirculation. Alterations in microvascular reactivity, nitric oxide pathways, and cytokine release are presumably the main mechanisms of anesthetic-induced tissue perfusion changes. This review summarizes current methods of microcirculatory status assessment and current knowledge regarding the microcirculatory effects of intravenous and potent volatile anesthetics and anesthesia-related techniques under both normal and pathophysiological conditions.

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

GreenLight (532 nm) laser partial nephrectomy followed by suturing of collecting system without renal hilar clamping in porcine model.

OBJECTIVES To assess the feasibility of partial nephrectomy (PN) without renal hilar clamping using the 80-W GreenLight (532 nm) laser with opening of the collecting system followed by its suture in a porcine model. METHODS We performed 12 open laser PNs in 6 farm pigs. We used the technique of incisional laser ablation of the lower pole of the right kidney. The pigs were kept alive, and 2 weeks later, underwent the same technique on the left kidney, followed by immediate killing. We recorded the acute and chronic outcomes. Renal parenchyma resection was achieved solely with the laser. The collecting system was opened with the laser in each procedure. All renal specimens underwent retrograde pyelography and histologic examination. RESULTS All procedures were completed without renal hilar clamping. The mean operative and laser time was 67.1 +/- 20.6 minutes (range 35-95) and 17.8 +/- 6.4 minutes (range 10-30), respectively. The mean resected kidney mass was 17% +/- 0.5% (range 10%-27%) of the total kidney mass. The mean blood loss was 142.5 +/- 88.9 mL (range 50-350). No evidence of urinary extravasation was seen after the acute procedures, but proven urinomas with decayed suture were found after all chronic procedures. CONCLUSIONS PN using the GreenLight laser is a feasible method for renal parenchyma incisional ablation. The laser hemostatic effect of interlobar vessel bleeding is safe and sufficient. The sutured collecting system after laser PN cannot heal in the thermically damaged tissue. The method is applicable to peripherally located lesions, without entering the collecting system. More prospective animal studies are necessary before application in humans.

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.

DIFFERENTIAL EFFECTS OF HEMOFILTRATION AND OF COUPLED PLASMA FILTRATION ADSORPTION ON CARDIAC REPOLARIZATION IN PIGS WITH HYPERDYNAMIC SEPTIC SHOCK

The aim was to investigate effects of continuous hemofiltration (CHF) and of coupled plasma filtration adsorption (CPFA) on electrophysiological properties of the septic heart. Sepsis was induced in anesthetized pigs by fecal peritonitis and continued for 22 h either without intervention (control sepsis) or with intervention (CHF or CPFA) applied for the last 10 h of this period. Electrocardiograms were recorded at baseline, before induction of peritonitis, and 22 h later, at the end of in vivo experiment. In vitro, action potentials were recorded in right ventricular trabeculae. RR, QT, and QTc (QT corrected for heart rate) intervals were shortened by sepsis. Action potential durations (APDs) were shortened by CHF, but not by CPFA, compared with control sepsis. Continuous hemofiltration prolonged APD. Coupled plasma filtration adsorption filtrate did not exert any effect on APD. Plasma separated during CPFA prolonged APD. Continuous hemofiltration shortened cardiac repolarization, and this effect was reversed by the hemofiltrate. In contrast, neither CPFA nor the CPFA filtrate influenced APD. The data indicate that some inflammatory mediators able to delay cardiac repolarization were removed from plasma to hemofiltrate by CHF but not by CPFA.ABBREVIATIONS - CHF - continuous hemofiltration; CPFA - coupled plasma filtration adsorption; APD - action potential duration

Continuous hemofiltration in pigs with hyperdynamic septic shock affects cardiac repolarization

Objective:Sepsis has been defined as the systemic host response to infection with an overwhelming systemic production of both proinflammatory and anti-inflammatory mediators. Continuous hemofiltration has been suggested as possible therapeutic option that may remove the inflammatory mediators. However, hemodialysis and hemofiltration were reported to influence cardiac electrophysiologic parameters and to increase the arrhythmogenic risk. We hypothesize that sepsis affects electrophysiologic properties of the pig heart and that the effects of sepsis are modified by hemofiltration. Design:Laboratory animal experiments. Setting:Animal research laboratory at university medical school. Subjects:Forty domestic pigs of either gender. Interventions:In anesthetized, mechanically ventilated, and instrumented pigs sepsis was induced by fecal peritonitis and continued for 22 hours. Conventional or high-volume hemofiltration was applied for the last 10 hours of this period. Measurements and Main Results:Electrocardiogram was recorded before and 22 hours after induction of peritonitis. RR, QT, and QTc intervals were significantly shortened by sepsis. The plasma levels of interleukin-6 and tumor necrosis factor-&agr; were increased in sepsis. High-volume hemofiltration blunted the sepsis-induced increase in tumor necrosis factor-&agr;. Action potentials were recorded in isolated ventricular tissues obtained at the end of in vivo experiments. Action potential durations were significantly shortened in septic preparations at all stimulation cycle lengths tested. Both conventional and high-volume hemofiltrations lead to further shortening of action potential durations measured afterward in vitro. This action potential duration shortening was reversed by septic hemofiltrates obtained previously by conventional or high-volume hemofiltration. Tumor necrosis factor-&agr; (500 ng/L) had no effect on action potential durations in vitro. Conclusions:In a clinically relevant porcine model of hyperdynamic septic shock, both sepsis and continuous hemofiltration shortened duration of cardiac repolarization. The continuous hemofiltration was not associated with an increased prevalence of ventricular arrhythmias. Tumor necrosis factor-&agr; or interleukin-6 did not contribute to the observed changes in action potential durations.

Renal Proteomic Responses to Severe Sepsis and Surgical Trauma: Dynamic Analysis of Porcine Tissue Biopsies

ABSTRACT Although the burden of septic acute kidney injury continues to increase, the molecular pathogenesis remains largely obscure. The aim of this exploratory study was a discovery-driven analysis of dynamic kidney tissue protein expression changes applied for the first time in a classic large mammal model of sepsis. To achieve this goal, analyses of protein expression alterations were performed in serial samples of kidney cortical biopsies (before, 12 and 22 h of sepsis) in mechanically ventilated pigs challenged with continuous infusion of pseudomonas aeruginosa and compared with sham-operated control data. Global protein expression was analyzed using two-dimensional gel electrophoresis and mass spectrometry-based proteomics. Normodynamic sepsis was associated with 43% reduction in glomerular filtration. The exposure to surgical stress per se altered the renal protein expression profile, while sepsis induced distinct and highly dynamic proteome evolution shifting the balance toward cellular distress phenotype. We identified 20 proteins whose expression changes discriminated effects of sepsis from those induced by surgery. The data implicate endoplasmic reticulum stress, oxidative stress, mitochondrial energy metabolism, immune/inflammatory signaling, and tubular transport as major activated pathways. Thus, by coupling the power of sequential tissue proteomics with whole-animal physiological studies, our study helped to establish a first global overview of critical renal proteomic events occurring during surgical trauma and early sepsis in a porcine model. The study supports the notion that multiple potentially subtle and even transient changes in several proteins which are members of key functional interrelated systems appear to play a role in septic acute kidney injury.