Searching for mechanisms that matter in earlyseptic acute kidney injury: an experimental studyJan Benes1, Jiri Chvojka2, Roman Sykora2, Jaroslav Radej2, Ales Krouzecky2, Ivan Novak2 and Martin Matejovic2*
Abstract
Introduction: In almost half of all sepsis patients, acute kidney injury (AKI) develops. However, the pathobiologicdifferences between sepsis patients with and without AKI are only poorly understood. We used a uniqueopportunity to examine dynamic inflammatory, renal hemodynamic, and microvascular changes in two clinicallyrelevant large-animal models of sepsis. Our aim was to assess variability in renal responses to sepsis and to identifyboth hemodynamic and nonhemodynamic mechanisms discriminating individuals with AKI from those in whomAKI did not develop.
Methods: Thirty-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 livePseudomonas aeruginosa (n = 15). Eight sham operated-on animals served as time-matched controls. All animalsreceived standard intensive care unit (ICU) care, including goal-directed hemodynamic management. Before, and at12, 18, and 22 hours of sepsis, systemic and renal (ultrasound flow probe) hemodynamics, renal cortexmicrocirculation (laser Doppler), inflammation (interleukin-6 (IL-6), tumor necrosis factor-alpha (TNF-a), oxidativestress (thiobarbituric acid reactive species (TBARS), nitrite/nitrate concentrations (NOx), and renal oxygen kineticsand energy metabolism were measured.
Results: In 14 (50%) pigs, AKI developed (62% in peritonitis, 40% in bacteria infusion model). Fecal peritonitisresulted in hyperdynamic circulation, whereas continuous bacteria infusion was associated with normodynamichemodynamics. Despite insults of equal magnitude, comparable systemic hemodynamic response, and uniformsupportive treatment, only those pigs with AKI exhibited a progressive increase in renal vascular resistance. Thisintrarenal vasoconstriction occurred predominantly in the live-bacteria infusion model. In contrast to AKI-freeanimals, the development of septic AKI was preceded by early and remarkable inflammatory response (TNF-a, IL-6)and oxidative stress (TBARS).
Conclusions: The 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 vascularresistance 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 intoconsideration when evaluating hemodynamic therapeutic interventions in septic AKI.
IntroductionThe major importance of sepsis and acute kidney injury(AKI) as a clinical and public health issue has clearlybeen underscored by recent studies showing a steadilyincreasing incidence of both syndromes [1,2]. AlthoughAKI is often a complex multifactorial syndrome, sepsis
and septic shock prevail as its dominant causes [3]. Theexpanding population of patients with sepsis and AKIand the associated excess mortality provide a strongbasis for further research aimed at establishing thepathomechanisms and addressing more rigorously allpotentially modifiable factors to reduce this burden topatients and the health care system. Considering thatAKI affects 40% to 50% of all sepsis patients [4-6], oneof the fundamental, yet unresolved, questions is whyAKI develops in some patients with sepsis, whereas the
* Correspondence: [email protected], 1st Medical Dept., Charles University in Prague, Faculty of Medicine inPlzen, University Hospital Plzen, alej Svobody 80, 304 60, Czech RepublicFull list of author information is available at the end of the article
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others are spared. Because both hemodynamic andinflammatory pathogenic pathways contribute to devel-opment of septic AKI (S-AKI), it is essential to under-stand hemodynamic and nonhemodynamic differencesbetween subjects with S-AKI and those without AKI[7,8]. Although preliminary data in humans suggest thatS-AKI is associated with systemic inflammation [9,10],limited ability to analyze exactly the renal pathophysiol-ogy in humans emphasizes the need for complex,dynamic, and clinically relevant animal studies [11].Paradoxically, these animal models should replicate notonly the complexity of human sepsis, but also the varia-bility among animals closely mimicking that seen inclinical practice [11]. We took the unique opportunityto analyze dynamic inflammatory, renal hemodynamic,and microvascular changes in two clinically relevantlarge-animal models that replicate many of the biologicfeatures of human septic shock, including the integra-tion of standard day-to-day care resuscitative measures.Our aim was to assess variability in renal responses tosepsis and to identify both hemodynamic and nonhemo-dynamic mechanisms that distinguish individuals withAKI from AKI-free subjects. Because this modelingallowed us to generate septic animals both with andwithout S-AKI, the confounding effects of sepsis thatare independent of AKI could be isolated and studied[12].
Materials and methodsAll experiments were performed in adherence to theNational Institutes of Health Guidelines on the Use ofLaboratory Animals, and their protocols were approvedby the University Animal Care Committee. Thirty-sixdomestic pigs of either sex with a median body weightof 35 (32 to 40) kg were used. Eight time-matchedsham-operated controls and 15 animals with sepsisinduced by continuous infusion of live bacteria (Pseudo-monas aeruginosa) were studied prospectively. In addi-tion, we performed a secondary analysis of 13 septicanimals that served as control groups in two rando-mized prospective studies [13,14]. In these animals, sep-sis was induced by fecal peritonitis. These two studiesaddressed completely different questions, and the proto-cols did not interfere with each other.
Anesthesia and surgical preparationAnesthesia was induced with intravenous propofol (1 to2 mg/kg) and ketamine (2 mg/kg). Animals were intu-bated and mechanically ventilated with tidal volumes of8 to 10 ml/kg, with positive end-expiratory pressure of0.6 kPa, and FiO2 of 0.4. Respiratory rate was adjustedto maintain normocapnia (arterial carbon dioxide ten-sion, 4.0 to 5.0 kPa). During the surgical procedure, con-tinuous infusions of fentanyl (10 to 15 μg/kg/h),
thiopental (10 mg/kg/h), and pancuronium (4 to 6 mg/h) were administered. After surgical preparation, theinfusion of thiopental and fentanyl was decreased to 5mg/kg/h and 5 μg/kg/h, respectively, and maintaineduntil the end of the experiment. Continuous infusion ofPlasmalyte (Baxter Healthcare, Deerfield, IL, USA) orRingerfundin solutions (Braun Melsungen Ag, Melsun-gen, Germany) were used as a fluid replacement indoses of 15 ml/kg/h during the surgery and reduced to7 ml/kg/h thereafter. Normoglycemia (arterial blood glu-cose level, 4.5 to 7 mmol/L) was maintained throughoutthe whole experiment by using 20% glucose infusion asneeded.Before the surgical procedure, a fiberoptic arterial
catheter was inserted into the femoral artery for contin-uous blood pressure measurement, intermittent double-indicator transpulmonary dilution (COLD Z-021; Pul-sion Medical Systems GmbH, Munich, Germany), andblood sampling. Central venous and pulmonary arterycatheters were introduced via jugular veins. Afterward, amidline laparotomy was performed, and a precalibratedultrasound flowprobe (Transonic Systems, Ithaca, NY,USA) was placed around the left renal artery. LaserDoppler probe (PF 404, Suturable angled probe; Peri-med, Jarfalla, Sweden) was placed directly over the renalcortex for cortical microcirculation assessment, and adouble-lumen catheter was inserted into the left renalvein for renal venous pressure measurements and bloodsampling. Peritoneal drainage was inserted beforeabdominal wall closure, and epicystostomy was per-formed under ultrasound control. A recovery period of6 hours was provided before the baseline measurement.
Measurements and calculationsAt each time point (baseline, 12, 18, and 22 hours afterinduction of sepsis), the measurement of hemodynamicsincluded cardiac output (CO), systemic vascular resis-tance (SVR), intrathoracic blood volume (ITBV), fillingpressures of both ventricles (CVP, PAOP), renal arteryblood flow (Qren), renal venous pressure (RVP), andrenal cortical microcirculation (LDF), as reported pre-viously [13-15]. Renal vascular resistance was calculatedaccording to the formula:
RVR =[MAP
(mm Hg
) − RVP(mm Hg
)]/Qren
(l.min−1) .
Arterial, mixed venous, and renal venous blood sam-ples were analyzed for pH, pO2, pCO2, and for hemo-globin oxygen saturation. Systemic oxygen delivery,systemic oxygen uptake, and renal oxygen delivery andoxygen uptake were derived from the appropriate bloodgases and flow measurements.Arterial and renal venous lactate and pyruvate concen-
trations were measured. Arterial blood samples were
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analyzed for plasma creatinine, leukocyte and plateletcounts, tumor necrosis factor alpha (TNF-a), and inter-leukin 6 (IL-6). Oxidative and nitrosative stress wasevaluated by measuring thiobarbituric acid reactive spe-cies (TBARS) and nitrate/nitrite (NOx) concentrations.To correct for dilution effects resulting from volumeresuscitation, NOx, TBARS, ADMA, IL-6, and TNF-alevels were normalized for plasma protein content[13-15]. AKI was defined according to the AKIN criteriaas an increase of more than 26.4 μmol/L or 150% inserum creatinine from baseline [16].
Experimental protocolsThe study consisted of three arms; intravenous live bac-teria infusion (INFUSION, n = 15), fecal peritonitis(PERITONITIS, n = 13), and sham-operated controlgroup (CONTROL, n = 8). In the infusion group, a con-tinuous central venous infusion of Pseudomonas aerugi-nosa (strain O1 isolated from a patient with suppurativeotitis, 1 × 109 colony-forming units/ml determined serialdilution and colony counts) was commenced after base-line data acquisition and maintained until the end of thestudy. The infusion rate was titrated to a clinical goal ofmoderate pulmonary hypertension (MPAP, 35 to 40mm Hg). To avoid any variations in virulence, all pigswere challenged with bacteria from the same bacterialstrain. In the peritonitis group, fecal peritonitis wasinduced by inoculating 0.5 g/kg of autologous fecesincubated in 200 ml saline for 8 hours at 37°C throughthe drains into the abdomen.In addition to crystalloid solution, 6% hydroxyethyl
starch 130 kD/0.4 (Voluven 6%; Fresenius Kabi Deutsch-land GmbH, Bad Homburg, Germany) was infused tomaintain normovolemia in a goal-directed fashion,guided by filling pressures response and ITBV measure-ment. Continuous IV noradrenaline was administered ifmean arterial pressure (MAP) fell below 65 mm Hg andtitrated to maintain MAP above 70 mm Hg. When thelast set of data had been obtained, the animals werekilled by potassium chloride injection under deepanesthesia, and section was performed.
Statistical analysisAll values shown are median and interquartile range.The calculations were done by using SigmaStat softwareversion 3.5 (Systat Software Inc., Erkrath, Germany).After exclusion of normality by using the Kolmogorov-Smirnov test, time-dependent changes within eachgroup were tested by using the Friedman ANOVA onranks and, subsequently, the Dunn test for multiplecomparisons with the Bonferroni correction. Differencesbetween the groups were analyzed by using the Kruskal-Wallis one-way analysis of variance on ranks or theMann-Whitney rank sum test when only two sets of
measurements were compared. A value of P < 0.05 wasregarded as statistically significant.
ResultsCharacterization of sepsis modelsAll animals completed the whole protocol. Hemody-namic and oxygen-exchange parameters, inflammatoryresponses, oxidative stress, and other laboratory para-meters are summarized in Table 1. Fecal peritonitisinduced a hyperdynamic circulatory state with anincreased cardiac output, whereas continuous bacteriainfusion resulted in normodynamic circulation. Sepsissignificantly decreased systemic vascular resistance inboth groups, the effect being more pronounced in thePeritonitis group. Although pigs in the Infusion groupreceived higher cumulative amount of fluids (469 (407to 516) versus 387 (344 to 425) ml/kg in the Infusionand Peritonitis groups, respectively; P = 0.007), fluidresuscitation maintained ITBV without intergroup dif-ferences, suggesting comparable cardiac preload. Twelve(85%) animals in the Infusion group received noradrena-line infusion, whereas seven (54%) pigs required vaso-pressor support in the Peritonitis group to maintainMAP ≥ 70 mm Hg. The average dose of noradrenalinwas higher in the Peritonitis group (1.47 (0.69 to 2.67)versus 0.25 (0.1 to 0.54) μg/kg/min; P = 0.012), but thetime to start the vasopressor infusion was similar inboth groups (1,277 (1,205 to 1,335) versus 1,035 (869 to1,401) minutes in the Infusion and Peritonitis groups,respectively). In the Peritonitis group, the increased car-diac output resulted in a significant increase of systemicoxygen delivery, whereas systemic oxygen consumptiondid not change in any of the three groups over time.Sepsis induced acute lung injury in both sepsis groups,as documented by significant, progressive deteriorationof the PaO2/FiO2 ratio. Both models of sepsis markedlyincreased plasma levels of TNF-a and IL-6, without sta-tistically significant intergroup differences. Thesechanges were accompanied by a remarkable increase ofthiobarbituric acid reactive species (TBARS) levels, inparticular in the peritonitis model, providing the evi-dence for oxidative stress.
Characteristics of animals with and without S-AKIOverall, S-AKI developed in 14 (50%) of 28 animals;62% met criteria for AKI in the Peritonitis group, and40%, in the Infusion group. Within the period of theexperiment, 86% of these pigs reached AKIN stage I,and 14%, AKIN stage II. No statistically significant dif-ferences in any measured variables were found betweenAKI-free and AKI pigs at baseline. The gender distribu-tion was homogeneous in both groups. Similarly, thetotal amount of infused P. aeruginosa was identical (24(18 to 43) ml in the AKI-free versus 24 (16 to 35) ml in
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the AKI group). In the Peritonitis group, the exactquantification of bacterial load could not be assessed.Nevertheless, no statistically significant intergroup dif-ferences in systemic hemodynamic variables were seenduring the course of the experiment (Table 2). In addi-tion, neither total amount of fluids nor vasopressor
requirement differed between AKI and AKI-free ani-mals, suggesting comparable infectious insults. Nine of14 (64%) pigs in the AKI group and 10 (71%) of 14AKI-free animals were given noradrenaline infusion.Total dose of noradrenalin was 0.21 (0.01 to 0.42) versus0.12 (0.05 to 0.22) mg/kg; P = 0.56 in the AKI and the
Table 1 Characterization of sepsis models
Baseline 12 hours 18 hours 22 hours
Mean arterial pressure (mm Hg)
Control 95 [87-109] 86.5 [83-94] 80 [73-90] 84 [68-87]
Control, sham-operated group; DO2 systemic, systemic oxygen delivery; IL-6, interleukin 6; Infusion, sepsis induced by bacterial infusion; ITBV, intrathoracic bloodvolume; PaO2/FiO2, ratio between arterial oxygen tension and the inspired fraction; Peritonitis, peritonitis-induced group; TBARS, thiobarbituric acid-reactivespecies; TNF-a, tumour necrosis factor alpha; VO2 systemic, systemic oxygen uptake; asignificant difference within each group versus baseline (P < 0.05);bsignificant difference between the Control group and any of the septic groups (P < 0.05); csignificant difference of the Infusion versus the Peritonitis group(P < 0.05).
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AKI-free groups, respectively. Likewise, time to start thenoradrenalin infusion was identical (935 (816 to 1,368)minutes for AKI versus 1,133 (900 to 1,420) minutes forAKI-free; P = 0.87).Both groups had no changes in systemic oxygen con-
sumption (Table 2). Significantly greater metabolicacidosis and higher arterial lactate/pyruvate ratio werenoted in the animals that developed AKI over the onesthat did not (Table 2). Despite comparable systemichemodynamics and vasopressor support, only those ani-mals with AKI had very early and remarkable increase
in serum TNF-a and TBARS levels (Figure 1). Signifi-cant increase in these markers occurred later in thenon-AKI group (Figure 1). Although a clear-cut ten-dency of plasma IL-6 levels was seen to increase moremarkedly in the AKI group, the intergroup differencesdid not reach statistical significance (Figure 1, lowerpanel).
Renal effects of sepsis in pigs with and without AKISerum creatinine levels increased significantly in AKIanimals by the end of the experiment, whereas no
Table 2 Characteristics of animals with and without S-AKI
Baseline 12 hours 18 hours 22 hours
Mean arterial pressure (mm Hg)
AKI 106 (97-115) 82 (72-99)a 82 (70-96)a 76 (62-88)a
Control 1.1 (0.8-1.4) 0.6 (0.5-0.7)a 0.7 (0.6-0.9) 0.8 (0.6-1)
Control, sham-operated group; IL-6, interleukin 6; Infusion, sepsis induced by bacterial infusion; ITBV, intrathoracic blood volume; NOx, nitrite/nitrateconcentration; PaO2/FiO2, ratio between arterial oxygen tension and the inspired fraction; Peritonitis, peritonitis-induced group, TBARS, thiobarbituric acid-reactivespecies; VO2 systemic, systemic oxygen uptake; asignificant difference within each group versus baseline (P < 0.05); bsignificant difference between the Controlgroup and any of the septic groups (P < 0.05).
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Figure 1 Biomarkers of inflammatory response during the course of sepsis. Plasma levels of inflammatory and oxidative stress markers(tumor necrosis factor a, upper panel; thiobarbituric acid-reactive species, middle panel; and IL-6, interleukin 6, lower panel) in control, non-AKI,and AKI animals. AKI, septic animals with acute kidney injury; control, sham operated; non-AKI, septic animals without acute kidney injurydevelopment. *Significant difference within each group versus baseline (P < 0.05); #significant difference between the control group and any ofthe septic groups (P < 0.05); $significant difference of the AKI versus the non-AKI group (P < 0.05).
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changes were observed in AKI-free and Control groups(Table 3). Urine output decreased markedly in AKI ani-mals, whereas it transiently and significantly increasedin AKI-free animals (Table 2). As opposed to subjectsthat remained AKI free, those in which AKI developed
exhibited an increase in renal vascular resistance at theend of the experiment (Figure 2, upper panel). As com-pared with the Control group, the renal-to-systemic vas-cular resistances index more than doubled in the AKIgroups (Figure 2, middle panel). This renal vasoconstric-tion, also apparent from the relative changes from base-line (RVR change, Table 3), was accompanied by areduction in renal blood flow. No changes in renalblood flow occurred in AKI-free animals (Figure 2,lower panel). Despite maintained mean arterial pressurewith noradrenaline above 70 mm Hg, renal perfusionpressure decreased significantly in both groups, partly asa result of gradually increased renal venous pressure(Table 3). Nevertheless, no significant intergroup differ-ences occurred. Reduced renal blood flow observed inAKI animals was accompanied by decreased renal oxy-gen delivery and oxygen consumption at the end of theexperiment, whereas no changes were detected in AKI-free pigs over time (Table 3). As compared with baselinevalues, the renal cortical microcirculatory blood flowdecreased both in AKI-free and in the AKI group, withearlier deterioration observed in pigs with AKI (Table3). Kidneys in AKI animals showed more marked meta-bolic stress, as evidenced by a significant developmentof renal venous metabolic acidosis and a tendencytoward increased renal venous lactate/pyruvate ratio(Table 3).
Renal effects of sepsis in pigs with AKI: comparison ofsepsis modelsData comparing renal responses to different septic mod-els are shown in Table 4 in the following order: bacteriainfusion with AKI (AKI-INF; n = 6) and without AKI(NONAKI-INF; n = 9), and peritonitis with AKI (AKI-PERIT; n = 8) and without AKI (NONAKI-PERIT; n =5). Individual responses are depicted in Figures 3 and 4.Increase in serum creatinine levels reached similarvalues in both groups at the end of the experiment(Table 4). No significant differences were noted in theamount of hydroxyethylstarch between any of the sub-groups analyzed. Although renal vascular resistanceincreased gradually in the AKI group induced by livebacteria infusion, it remained unchanged in AKI animalschallenged with peritonitis (Table 4 Figure 3). However,wide interindividual responses were observed even inthe Peritonitis group (Figure 3). In addition, when com-pared with baseline values, the renal-to-systemic vascu-lar resistances ratio increased in both groups of animalsdeveloping AKI, whereas it remained unchanged inAKI-free pigs in the Peritonitis group (Table 4). Indivi-dual relations between renal and systemic vascular resis-tances in the particular groups are shown in Figure 4.Renal blood flow and cortical microvascular perfusiondecreased in both AKI models, without any intergroup
Table 3 Renal effects of sepsis in pigs with and withoutacute kidney injury
Baseline 12 hours 18 hours 22 hours
Creatinine (μmol/L)
AKI 98 (88-105) 105 (91-134) 124 (89-164) 137 (130-176)abc
systemic, systemic oxygen uptake; asignificant difference within each groupversus baseline (P < 0.05); bsignificant difference between the Control groupand any of the septic groups (P < 0.05); csignificant difference of the AKIversus the Non-AKI group (P < 0.05).
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Figure 2 Changes in renal hemodynamics during the course of sepsis. Changes in renal vascular resistance (upper panel), renal-to-systemicvascular resistance ratio (middle panel), and renal blood flow (lower panel) in control, non-AKI, and AKI animals. AKI, septic animals with acutekidney injury; control, sham operated; non-AKI, septic animals without acute kidney injury developement; *significant difference within eachgroup versus baseline (P < 0.05); #significant difference between the control group and any of the septic groups (P < 0.05); $significantdifference of the AKI versus the non-AKI group (P < 0.05).
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Table 4 Renal effects of sepsis in pigs with and without acute kidney injury: comparison of sepsis models
AKI, septic animals with acute kidney injury; Control, sham-operated group; DO2 systemic, systemic oxygen delivery; IL-6, IL-6, interleukin 6; interleukin 6; INF,sepsis induced by bacterial infusion; ITBV, intrathoracic blood volume; NONAKI, septic animals without acute kidney injury development; Nox, nitrite/nitrateconcentrations;PaO2/FiO2, ratio between arterial oxygen tension and the inspired fraction; PERIT, peritonitis-induced group; TBARS, thiobarbituric acid-reactivespecies; TNF-a, tumor necrosis factor alpha; VO2 systemic, systemic oxygen uptake; asignificant difference within each group versus baseline (P < 0.05);bsignificant difference of the AKI-INF versus the NONAKI-INF group (P < 0.05); csignificant difference of the AKI-PERIT versus the NONAKI-PERIT group (P < 0.05);dsignificant difference of the AKI-PERIT versus the AKI-INF group (P < 0.05).
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differences. Despite apparent intrarenal vasoconstriction,only two (33%) animals in the AKI group induced bylive bacteria infusion developed oliguria. By contrast,seven (88%) pigs became oliguric in the Peritonitis-induced AKI. No statistically significant differences in
systemic inflammatory responses were found betweenpigs in both sepsis models with AKI, although those ani-mals that developed AKI in the Peritonitis group hadearlier and apparently greater increases of IL-6 levels. Inaddition, animals with AKI in the Peritonitis group had
Figure 3 Individual changes in renal vascular resistance in the AKI, non-AKI, and control groups. Dashed line, infusion groups; solid line,Peritonitis and Control; bold line, median; TP 1 to 4, time points 1 to 4; AKI, septic animals with acute kidney injury; Control, sham-operated group;Infusion, sepsis induced by bacterial infusion; non-AKI, septic animals without acute kidney injury developement; Peritonitis, peritonitis-inducedgroup; *significant difference within each group versus baseline (P < 0.05); $significant difference of the AKI versus the non-AKI group (P < 0.05).
Figure 4 Individual changes in renal and systemic vascular resistances in the AKI and non-AKI groups. Lines indicate the individualconnection between the baseline values of renal (x-axis) and systemic (y-axis) vascular resistances and the values measured after 22 hours ofsespis. Arrow, the direction of change. Only AKI and non-AKI groups are displayed. Dashed line, infusion groups; solid line, peritonitis; bold line,median; AKI, septic animals with acute kidney injury; infusion, sepsis induced by bacterial infusion; non-AKI, septic animals without acute kidneyinjury developement; peritonitis, peritonitis-induced group.
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larger production of nitric oxide and reactive nitrogenspecies, as documented by significantly increased NOxand TBARS levels at 12 and 18 hours of sepsis (Table4).
DiscussionWe have developed two clinically relevant large-animalmodels allowing us to study the renal responses to sep-sis. A unique feature of these models is variability insusceptibility to the development of S-AKI that closelyreplicates that of human disease. This heterogeneityallowed us to isolate and study factors discriminatingAKI from non-AKI. Abnormal host response to sepsis,characterized by early systemic inflammation and oxida-tive stress coupled with subsequent renal vasoconstric-tion, was found to be a major discriminating factorassociated with the initiation phase of S-AKI. Further-more, our data suggest that in pigs developing S-AKI,renal circulation might not participate in sepsis-inducedsystemic vasodilatation. However, the intensity of renalvasoconstriction varies widely and appears to be modelspecific.Ideally, animal models of sepsis-induced AKI should
reproduce the mechanisms and consequences of humansepsis, including the complex supportive treatment. Toincrease the relevance of these models, they also shouldexhibit a heterogeneous response to sepsis that is com-parable to that in humans [11]. In our study, 50% ofanimals developed S-AKI, a finding consistent withrecent multicenter epidemiologic studies showing thatin 40% to 50% of sepsis patients, concomitant AKIdevelops [4-6]. In addition, an early (that is, within 22hours) development of S-AKI observed in our modelsalso closely mimics a pattern seen typically in clinicalstudies [17]. The separation of animals into those withand without S-AKI according to the AKIN definitionwas associated with a number of significant physiologi-cal differences, adding validity and usefulness to thisapproach.The most salient feature of the present study is the
considerable variability in renal hemodynamic responsesto sepsis despite a homogeneous and reproducible pat-tern of systemic hemodynamics and a uniform level ofsupportive treatment. This finding is somewhat contra-dictory to the results of a recent comprehensive reviewof the available experimental evidence, demonstratingthat in the presence of preserved or increased cardiacoutput, renal blood flow is typically maintained orincreased [18]. Similar predictive value was shown forcalculated renal vascular resistance, although extraordin-ary heterogeneity exists [19]. We could consistentlyobserve the link between well-maintained cardiac outputand preserved renal blood flow only in those septic ani-mals that remained AKI free. By contrast, in animals
that developed AKI, sepsis is often accompanied by anuncoupling between systemic (reduced) and renal vascu-lar resistance (increased), resulting in reduced renalblood flow despite maintained or even increased cardiacoutput. In contrast to all previous experiments, ourstudy is the first to determine renal vascular resistanceby using the directly measured renal venous pressureand to compare the differences between dynamicchanges in renal vascular resistance in AKI-free andAKI-positive subjects. In this context, our results sug-gest that renal circulation might behave differently inpigs with S-AKI as opposed to S-AKI-free animals andthat the renal circulatory response to sepsis cannot reli-ably be predicted from changes in systemic hemody-namics. Moreover, the divergent renal circulatoryresponse observed in a majority of S-AKI animals sup-ports the existence of the phenomenon of selective renalvasoconstriction, even in large-animal models withapparent sepsis-induced systemic vasodilatation [20].Our observation is congruent with the clinical data ofLerolle et al. [21], who recently demonstrated that theincreased Doppler resistive index, assessed at ICUadmission as a surrogate measure of intrarenal vascularresistance, was significantly higher in septic shockpatients in whom S-AKI subsequently developed. Con-versely, our results markedly differ from a series of stu-dies done by the Australian research group in a sheepsepsis model induced by continuous Escherichia coliinfusion, in which S-AKI was uniformly associated withsignificant renal vasodilatation and increased renal arteryblood flow [22-25]. Although the reasons for this discre-pancy are not readily apparent, differences between spe-cies (sheep are ruminants) in regard to renal responsesto sepsis, different bacterial insults (E. coli versus Pseu-domonas vs. polymicrobial peritonitis) and the use ofconscious versus anesthetized animals can be taken intoconsideration [18,26].Based on the data presented here, we cannot unam-
biguously determine the cause of elevated renal vascularresistance. Recently, we observed gradually increasedrenal venous pressure with an early reduction in renalcortex microvascular perfusion during progressive por-cine sepsis, each potentially contributing to the increasein renal resistance [15]. Although both renal venouscongestion and deterioration in cortex microcirculationdeveloped in this study, none of these variables differedfrom those in AKI-free animals. Renal interstitial tissueedema, more pronounced microvascular blood-flowderangement in deeper cortex layers and medulla notdetected by our approach, and altered renal vasculaturemight have been factors implicated in the increased flowresistance in the S-AKI group. Whatever the mechan-isms accounting for the increased renal resistance, thedesign of the present study does not allow one to
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determine a causative link between intrarenal vasocon-striction and kidney injury nor to clarify its relative con-tribution to S-AKI. However, reduced renal oxygenconsumption, combined with signs of metabolic stress,as evidenced by increased renal venous base deficitimplies an impairment of normal aerobic respiration.Because we did not calculate renal oxygen consumptionper sodium reabsorbed, we can only speculate to whatextent the oxygen consumption was affected by per-turbed mitochondrial function or altered efficiency fortubular sodium transport. Nevertheless, considering theunchanged renal lactate clearance and oxygen extractionratio (data not shown), it is plausible to speculate thatthe reduction in renal oxygen consumption representsdecreasing oxygen demand rather than exhaustion ofrenal oxidative metabolism.The onset of AKI was preceded by early and remark-
able inflammatory response and oxidative stress. Despitecomparable septic insult and indistinguishable systemichemodynamic response, only those pigs that developedAKI had a very early increase in the plasma levels ofTNF-a and TBARS. A similar pattern, albeit not statisti-cally significant, was observed in the time-dependentchanges of plasma IL-6 levels. These findings corrobo-rate emerging evidence from laboratory and clinical stu-dies suggesting that AKI in sepsis has a prominentinflammatory component in both the initiation and theextension phases of the kidney injury [12,27,28]. Severallarge cohorts of critically ill patients demonstrated thatIL-6 could be a robust predictor of AKI [9]. In keepingwith our findings, Murugan et al. [10] recently demon-strated different immune response to pulmonary infec-tion in patients with AKI as opposed to patients withoutAKI. Patients admitted with pneumonia and who pre-sented with or developed concomitant S-AKI had higherplasma levels of IL-6 and TNF-a than did non-AKIpatients. Although our study is not the first to assessthe association between inflammation and the suscept-ibility to S-AKI, it extends the clinical observations bydemonstrating the temporal relation between early,exaggerated inflammatory and oxidative stress responseand AKI, thus supporting the cause-effect interplaybetween AKI and abnormal immune response.Another major feature of this study is the comparison
of renal responses in two different sepsis models. Toour knowledge, we are the first to demonstrate renalphysiology in two different large-animal models of sep-sis. First, they displayed differences with respect to therenal vascular response to sepsis. Although the ratio ofrenal to systemic vascular resistance increased in bothgroups when compared with baseline values, theincrease in renal vascular resistance was more remark-able in pigs challenged with intravenous bacteria infu-sion. However, as shown in Figures 3 and 4, the
individual responses were heterogeneous. The exactmechanism(s) responsible for different renal vascularresponses cannot be inferred from our data. The preser-vation of urine output in the Infusion group was likelycaused by an increase in the hydrostatic pressure of theglomerular capillaries, indicating that infusion of livebacteria elicited more vasoconstriction in the efferentthan in the afferent arteriole of the glomerulus, which isin sharp contrast to results observed in a sheep modelby Langenberg et al. [23]. Different renal vascularresponses might be attributable to different immuneresponses. Only in those pigs in the Peritonitis groupthat developed S-AKI, was a significant, albeit transient,increase present in the production of nitric oxide andoxidative stress, as revealed by changes in plasma levelsof NOx and TBARS, respectively. These results couldexplain the relatively preserved renal vascular resistancein the Peritonitis-induced AKI model. In addition, theinflammatory response evaluated by plasma IL-6 levelsappeared to be more pronounced in this model thanthat elicited by bacteria infusion. Of note, S-AKI devel-oped more frequently in the Peritonitis group (62%) ascompared with the Infusion group (40%). Collectively,these data support a prominent role of abnormal hostresponse to the infection in the development of S-AKIand suggest that renal vasoconstriction might not neces-sarily be a prerequisite for AKI to develop, at least inthe Peritonitis model [7]. Although inconclusive dataexist regarding the association of AKI and primaryinfectious source [5,17,29], our data admit the depen-dence of physiological renal responses on the causativeagent(s) and infectious source. Certainly, knowing histo-pathologic findings in all particular subgroups wouldallow us to put the data into a more comprehensive pic-ture. Because renal tissue samples taken from our ani-mals are scheduled for detailed genomic and proteomicanalyses, we were unable to provide morphologic data atthis stage.
ConclusionsIn conclusion, we aimed to assess variability in renalphysiology and to study both hemodynamic and nonhe-modynamic factors discriminating AKI from non-AKI intwo different large-animal models that closely resemblehuman sepsis. The lessons that can be drawn from thesemodels are that (a) they exhibited marked heterogeneityin susceptibility to the development of septic AKI that iscomparable to that in humans; and (b) the differencesbetween those animals that developed AKI and thosethat did not are noteworthy from both the clinical andresearch perspectives. First, although sepsis appears tobe a uniform clinical entity, much variation can be seenin the renal responses. This variability has to be takeninto account when effects of therapeutic interventions
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are to be assessed. Second, renal circulatory response tosepsis cannot reliably be predicted simply from changesin systemic hemodynamics. Renal blood flow can exhibitsignificant variability, even though cardiac output ismaintained or increased. Third, the development of S-AKI is often accompanied by an uncoupling betweensystemic and renal vascular resistance, suggesting thatrenal circulation does not participate in sepsis-inducedsystemic vasodilatation. Fourth, early abnormal hostresponse seems to be a major pathobiologic factor asso-ciated with the development of S-AKI. Fifth, the inten-sity of immune and renal hemodynamic responsesappears to be model specific. The different renalresponses of these models to sepsis could allow futurestudies to tease apart the underlying pathophysiology ofS-AKI.
Key messages• Although sepsis appears to be a uniform clinicalentity, much variation can occur in the renal hemo-dynamic and functional responses. This variabilityhas to be taken into account when effects of thera-peutic interventions are to be assessed.• Early abnormal host immune response appears tobe the major pathobiologic factor predicting thedevelopment of porcine septic AKI.• Renal circulatory response to sepsis in animalsdeveloping AKI cannot reliably be predicted fromchanges in systemic hemodynamics.• Development of septic AKI is often accompaniedby an uncoupling between systemic and renal vascu-lar resistance, suggesting that renal circulation doesnot participate in sepsis-induced systemicvasodilatation.
AcknowledgementsThe study was supported by a research project MSM 0021620819(Replacement of and support to some vital organs).
Author details1Department Of Anesthesia and Intensive Care, Charles University in Prague,Faculty of Medicine in Plzen, University Hospital Plzen, alej Svobody 80, 30460, Czech Republic. 2ICU, 1st Medical Dept., Charles University in Prague,Faculty of Medicine in Plzen, University Hospital Plzen, alej Svobody 80, 30460, Czech Republic.
Authors’ contributionsMM obtained research funding, conceived the study, designed the studyprotocol, supervised and coordinated the study, and finalized themanuscript. JB carried out the experiments, statistical analysis, and draftedthe article. JC and RS performed the experiments and data acquisistion. AK,JR, and IN participated in the design of the study, contributed to theinterpretation of the results, and helped to draft the manuscript. All authorsread and approved the final manuscript for publication.
Authors’ informationThe study was performed in the animal research laboratory of the 1st
Medical Department at Charles University Medical School.The study was presented in part at the 31st International Symposium onIntensive Care and Emergency Medicine, Brussels, March 2011.
Competing interestsThe authors declare that they have no competing interests.
Received: 28 July 2011 Revised: 30 August 2011Accepted: 26 October 2011 Published: 26 October 2011
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doi:10.1186/cc10517Cite this article as: Benes et al.: Searching for mechanisms that matterin early septic acute kidney injury: an experimental study. Critical Care2011 15:R256.
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