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Critical Care Medicine  |   April 2010
Renal Effects of Saline-based 10% Pentastarch versus  6% Tetrastarch Infusion in Ovine Endotoxemic Shock
Author Affiliations & Notes
  • Christian Ertmer, M.D.
    *
  • Gabriele Köhler, M.D., Ph.D.
  • Sebastian Rehberg, M.D.
    *
  • Andrea Morelli, M.D.
  • Matthias Lange, M.D., Ph.D.
    §
  • Björn Ellger, M.D., Ph.D.
    §
  • Bernardo Bollen Pinto, M.D.
  • Eva Rübig
    #
  • Michael Erren, M.D.
    **
  • Lars G. Fischer, M.D., Ph.D.
    ††
  • Hugo Van Aken, M.D., Ph.D., F.R.C.A., F.A.N.Z.C.A.
    ‡‡
  • Martin Westphal, M.D., Ph.D.
    ††
  • * Resident and Research Fellow, § Associate Professor, ∥ Resident, # Medical Student, †† Professor, ‡‡ Chair and Professor, Department of Anesthesiology and Intensive Care, † Professor, Department of Pathology, ** Consultant, Department of Laboratory Medicine, University of Muenster, Muenster, Germany. ‡ Consultant Anesthesiologist and Intensive Care Physician, Department of Anesthesiology and Intensive Care, University of Rome “La Sapienza,” Rome, Italy.
Article Information
Critical Care Medicine / Coagulation and Transfusion / Critical Care / Renal and Urinary Systems / Electrolyte Balance
Critical Care Medicine   |   April 2010
Renal Effects of Saline-based 10% Pentastarch versus  6% Tetrastarch Infusion in Ovine Endotoxemic Shock
Anesthesiology 4 2010, Vol.112, 936-947. doi:10.1097/ALN.0b013e3181d3d493
Anesthesiology 4 2010, Vol.112, 936-947. doi:10.1097/ALN.0b013e3181d3d493
What We Already Know about This Topic
  • ❖ Starch-based colloid solution use during septic shock may be associated with renal dysfunction
  • ❖ It is unknown whether newer solutions of low substituted starch colloids (6% HES 130/0.4) also produce renal dysfunction
What This Article Tells Us That Is New
  • ❖ In ovine endotoxemic shock, 6% HES 130/0.4 produces less anatomic and functional renal injury than an older starch colloid (10% HES 200/0.5)
ADEQUATE fluid resuscitation represents one of the key features of early hemodynamic optimization in patients with severe sepsis and septic shock.1 Although colloids reduce the total amount of fluids needed to achieve hemodynamic stabilization when compared with sole crystalloid infusion,2 it is still unclear whether crystalloids or colloids should be preferred in this indication. Previous clinical studies report negative renal effects associated with the infusion of “classic” hydroxyethyl starches (HES; 10% HES 200/0.5 = pentastarch or 6% HES 200/0.60–0.66 = hexastarch) in patients with sepsis. These adverse events include higher rates of acute renal failure and requirements for renal replacement therapy.3,4 Notably, these studies exclusively used HES preparations typically accumulating in the plasma after repetitive use and/or markedly exceeded the clinically recommended maximum doses.3,4 In addition, the exact pathomechanism of starch-induced renal injury is still unknown.5 
The renal effects of modern, low-substituted, and medium– molecular weight preparations, that is, 6% HES 130/0.4 (tetrastarch), in the presence of severe sepsis have not yet been investigated. Likewise, few studies have directly compared the effects of HES 200/0.5, HES 130/0.4, and balanced crystalloids (acetate/malate based) on renal function,6,7 and none of these was conducted in the setting of sepsis or systemic inflammation.
Although 6% HES 130/0.4 has been suggested to be safe in patients who underwent nonseptic cardiac surgery, even in the presence of preexisting renal dysfunction,8 the recently published VISEP trial demonstrated that 10% HES 200/0.5 dose-dependently impaired renal function.4 In addition, a recent prospective observational study suggested that hyperoncotic colloids may increase the risk of renal failure in patients with shock.9 
We hypothesized that 10% HES 200/0.5 impairs renal function and tubular integrity when compared with 6% HES 130/0.4 or balanced crystalloids. The objective of the current study was, therefore, to compare the effects of saline-based 10% HES 200/0.5 (the study drug of the VISEP trial),4 saline-based 6% HES 130/0.4 (a modern third-generation tetrastarch), and a commonly used balanced, isotonic crystalloid on hemodynamics, colloid-osmotic pressure, surrogate variables of renal function, and light and transmission electron microscopic renal tubular injury, using an established and clinically relevant ovine model of endotoxemic shock.10–12 
Materials and Methods
Animals
After approval by the Local Animal Care Committee (State Office for Nature, Environment, and Consumer Protection, Recklinghausen, Germany), 30 healthy adult ewes were chronically instrumented with strict adherence to the National Institutes of Health's Guide and the American Physiologic Society's Guide for the Care and Use of Laboratory Animals using an established protocol.11–13 
Anesthesia and Instrumentation
Induction of anesthesia was performed by intramuscular injection of S-ketamine (Ketanest® S, 10 mg/kg; Parke-Davis, Berlin, Freiburg, Germany) and midazolam (Dormicum®, 0.3 mg/kg; Hoffmann-La Roche AG, Grenzach-Wyhlen, Germany). After catheterization of a peripheral vein, ceftriaxone (1 g ceftriaxone; Rocephin®; Hoffmann-La Roche AG) was administered intravenously as perioperative infection prophylaxis. Anesthesia was maintained using a continuous intravenous propofol infusion (Disoprivan®, 4–8 mg · kg−1· h−1; AstraZeneca, Schwetzigen, Germany). The ewes remained unconscious but were spontaneously breathing during the entire instrumentation period. All punctures were performed under sterile conditions after local anesthesia with 2% mepivacain (Scandicain® 2%; AstraZeneca GmbH, Wedel, Germany). An indwelling pulmonary artery catheter was inserted via  the right jugular vein through an introducer sheath (7.5 French, Edwards Swan Ganz, Edwards Critical Care Division, Irvine, CA; 8.5 French, Catheter Introducer Set, pvb Medizintechnik GmbH, Kirchseeon, Germany). In addition, sheep were instrumented with a left femoral arterial catheter (18-gauge Leader Cath; Vygon, Aachen, Germany) and a Foley catheter (12 French, urinary catheter, Porgès S.A., Le Plessis Robinson-Cedex, France) to monitor arterial blood pressure and urinary output, respectively.
After the instrumentation, intravascular catheters were connected to a physiologic recorder (Hellige Servomed, Hellige, Freiburg, Germany) via  pressure transducers (DTX pressure transducer, Ohmeda, Erlangen, Germany). The instrumentation was followed by a 24-h period of recovery. During this time, the sheep that were awake were housed in metabolic cages with free access to water and food until baseline measurements were performed.
Hemodynamic and Oxygen Transport Variables
Hemodynamic monitoring included mean arterial pressure (MAP), mean pulmonary arterial pressure, central venous pressure (CVP), and pulmonary arterial occlusion pressure. Heart rate was determined by calculating the mean frequency of arterial pressure curve peaks. The thermodilution technique (9520A cardiac output computer; Edward Lifescience, Irvine, CA) was applied to measure cardiac output by three-fold central venous injection of 10 ml of isotonic saline solution at a temperature of 2°–5°C. Cardiac index (CI), systemic vascular resistance index, pulmonary vascular resistance index, stroke volume index, and left and right ventricular stroke work indices were determined using standard equations.14 Core body temperature was continuously measured by the thermistor positioned at the tip of the pulmonary artery catheter.
Arterial and mixed venous blood samples (0.5 ml each) were collected in heparinized tubes designed to determine blood gases (Sarstedt; Nümbrecht, Germany). Potentia hydrogenii (pH), and partial pressures of oxygen and carbon dioxide (Po2and Pco2, respectively) were determined using an ABL 725 blood gas analyzer with SAT 100 calibration (Radiometer Copenhagen; Copenhagen, Denmark). In addition, hemoglobin concentration, hematocrit, arterial and mixed-venous oxygen saturation (Sao2and Svo2, respectively), and arterial lactate concentrations were assessed. Oximetry-corrected base excess (BEox) was calculated from hemoglobin concentration, Pco2, pH, and Sao2. Systemic oxygen delivery index (DO2I), oxygen consumption index, and oxygen extraction rate were determined by using standard formulae.14 
Laboratory Analyses
At specific time points (see Experimental Protocol), arterial blood (7.5 ml of lithium heparinate blood) was withdrawn and immediately centrifuged at 3,000 rpm for 10 min. The isolated plasma and urine samples (3 ml) were then immediately stored at −70°C for determination of creatinine, urea, electrolyte, and total protein concentrations (Hitachi 747 automatic analyzer, Roche Diagnostics GmbH, Mannheim, Germany) at a later time point (see Experimental Protocol). Plasma colloid osmotic pressure (COP) was measured using a colloid osmometer (Colloid Osmometer; Knauer, Berlin, Germany).
Experimental Protocol
Inclusion criteria for the current study were an initial heart rate less than 100 beats/min, core body temperature less than or equal to 39.8°C (normal range for sheep 38.5°–39.8°C), mean pulmonary arterial pressure less than 20 mmHg, and arterial lactate less than or equal to 1 mm. During the experimental protocol, all ewes were spontaneously breathing room air and were studied in a conscious state.
After a baseline measurement in the healthy state (BL1), 4 ml · kg−1· h−1of a balanced, isotonic crystalloid solution containing acetate/malate buffer (Sterofundin® ISO, B. Braun Melsungen, Germany) were continuously infused to compensate for basal fluid requirements, because balanced electrolyte solutions (rather than isotonic saline) represent the standard of care for basal fluid substitution in Germany and other parts of Europe. In addition, all animals received a continuous infusion of 100 mg of Salmonella typhosa  endotoxin (Sigma Chemicals, Deisenhofen, Germany, Catalogue number L6386) starting at a rate of 5 ng · kg−1· min−1. The endotoxin infusion rate was doubled every hour until MAP decreased less than 65 mmHg (shock time). At this time, the baseline measurement in endotoxemia (BL2) was performed, and endotoxin infusion was maintained at the dosage determined at shock time. Sheep were then randomly allocated to one of the three study groups, that is, 6% HES 130/0.4, 10% HES 200/0.5, and crystalloid (each n = 10). The 6% HES 130/0.4 and 10% HES 200/0.5 groups received volume resuscitation with repeated bolus infusions of 5 ml/kg saline-based 6% HES 130/0.4 (Voluven® 6%; Fresenius Kabi, Bad Homburg, Germany) or saline-based 10% HES 200/0.5 (Hemohes® 10%; B. Braun Melsungen), respectively. The crystalloid group received repeated bolus infusions of 10 ml/kg of Sterofundin® ISO. Fluid resuscitation was performed according to the recommendations of the current sepsis guidelines, aiming to maintain CVP at 8–12 mmHg and pulmonary arterial occlusion pressure at 12–15 mmHg.15 After infusion of the maximum colloid dose of 20 ml/kg (i.e.  , recommended maximum dose for Hemohes® 10%), only crystalloids were infused in all study groups. If MAP was less than predefined threshold values of 70 ± 5 mmHg despite fluid therapy, norepinephrine (Arterenol®; Sanofi-Aventis Deutschland GmbH, Frankfurt, Germany) was continuously administered as a titrated infusion to achieve threshold values. Hemodynamic measurements and blood gas analyses were performed hourly. In addition, blood and urine samples were withdrawn for laboratory analyses at BL1 and BL2 and at 2, 4, 8, and 12 h after randomization.
Histologic Analyses
Animals surviving the 12-h intervention period were deeply anesthetized with propofol (4 mg/kg) and killed with a lethal dose of 100 ml of potassium chloride solution (7.45%). Subsequently, an autopsy was performed, and tissue samples from the left kidney were taken and fixated in 3.7% formaldehyde solution. At a later time point, kidney samples were stained with hematoxylin-eosin and analyzed by a pathologist, being unaware of the study protocol and grouping. Tissue injury was quantified using a modified score originally described by Cox et al.  ,16 where 1 = normal tissue and 10 = necrosis.
Transmission Electron Microscopy
Kidney tissue samples were fixed for 12 h at 4°C in 3% cacodylate-buffered glutaraldehyde (pH 7.35) and then transferred into 5% sucrose for electron microscopy. Postfixation was performed with 1% osmium tetroxide and 50 mm potassium ferricyanide. Specimens were then washed with distilled water, dehydrated in graded alcohols, and embedded in araldite resin. The ultrathin sections (50 nm) were cut and placed on copper grids. The sections were stained with 5% uranyl acetate and 0.2% lead citrate. Transmission electron microscopic examination was performed using a Philips CMlO electron microscope (Philips, Eindhoven, The Netherlands) operating at 80 kV.17 Because there is no established quantitative scoring system for tubular injury in acute renal failure, kidney samples were analyzed as follows: in each of the 30 study animals, tubules of 10 fields of view were scored according to the following criteria by a pathologist unaware of the study protocol and group assignment: (1) vacuolar degeneration and swelling of organella (0 = none; 1 = occasional; 2 = moderate; and 3 = ubiquituous), (2) dissociation of epithelium and basal membrane (0 = none; 1 = single cells; 2 = more than half of tubule; and 3 = whole tubule), (3) epithelial cell injury (0 = none; 1 = moderate; 2 = severe; and 3 = complete destruction), and (4) intratubular precipitation (0 = none; 1 = moderate protein precipitation; 2 = lumen obstructed by protein; and 3. intratubular cell organella). The sum of the four criteria was quantified as renal tubular injury score.
Study Endpoints
The study hypothesis and statistical analysis were two tailed. Primary study endpoints were differences in urinary output, plasma creatinine, and urea concentrations among study groups. Secondary endpoints included total volume requirements and electron microscopic tubular injury.
Statistical Analysis
Data are expressed as means ± SEM. Sigma Stat 3.10 software (Systat Software Inc., Chicago, IL) was used for statistical analysis. After confirming normal distribution of all variables (Kolmogorov-Smirnov test), overall differences between groups over the whole study period were analyzed using one-way ANOVA. Only if significant overall differences were detected, a two-way ANOVA for repeated measurements with group and time as factors was performed to analyze the different time points within and between groups using appropriate post hoc  comparisons (Student-Newman-Keuls test). Time-independent variables and microscopic scores were analyzed by one-way ANOVA or one-way ANOVA on ranks, as appropriate, and post hoc  comparisons (Student-Newman-Keuls test) were performed in case of significant overall differences. Correlation between electron microscopic scores and diuresis was tested using the Pearson Product Moment Correlation formula. For all statistical tests, an α-error probability of P  < 0.05 was considered as statistically significant.
Results
There were no significant differences among groups in any of the investigated variables at BL1 and BL2.
Body Weight, Endotoxin Dose, and Shock Time
Body weight, lipopolysaccharide dose, and time to the onset of shock were comparable between study groups (each P  > 0.05; table 1).
Table 1.  Body Weight, LPS Dose, and Time to Shock Onset in Endotoxemic Sheep
Image not available
Table 1.  Body Weight, LPS Dose, and Time to Shock Onset in Endotoxemic Sheep
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Hemodynamic and Oxygen Transport Variables
The effects of endotoxin on cardiopulmonary and global oxygen transport variables are depicted in figure 1and table 2, respectively. Endotoxin infusion was associated with increases in heart rate, mean pulmonary arterial pressure, and pulmonary vascular resistance index and reductions in CVP, left ventricular stroke work index, MAP, stroke volume index, and systemic vascular resistance index (each P  < 0.001 BL2 vs.  BL1). Volume resuscitation was associated with an increase in CVP, pulmonary arterial occlusion pressure, MAP, CI, and DO2I in all three study groups (each P  < 0.05 at 8 h vs.  BL2). DO2I was lowest in the HES 200/0.5 group when compared with the other two groups (P  = 0.02 vs.  HES 130/0.4; P  = 0.04 vs.  crystalloid). In addition, there was a strong trend toward a higher oxygen extraction rate in the HES 200/0.5 group when compared with the crystalloid group (P  = 0.05). Sao2and Svo2were similar among groups (table 3).
Fig. 1. Hemodynamic effects of volume therapy in endotoxemic sheep. Data are presented as means ± SEM. BL = baseline; DO2I = systemic oxygen delivery index; HES = hydroxyethyl starch; HR = heart rate; MAP = mean arterial pressure; SVI = stroke volume index. *P  < 0.05 HES 130/0.4 versus  HES 200. †P  < 0.05 HES 200/0.5 versus  crystalloid. ‡P  < 0.05 HES 130/0.4 versus  crystalloid.
Fig. 1. Hemodynamic effects of volume therapy in endotoxemic sheep. Data are presented as means ± SEM. BL = baseline; DO2I = systemic oxygen delivery index; HES = hydroxyethyl starch; HR = heart rate; MAP = mean arterial pressure; SVI = stroke volume index. *P 
	< 0.05 HES 130/0.4 versus 
	HES 200. †P 
	< 0.05 HES 200/0.5 versus 
	crystalloid. ‡P 
	< 0.05 HES 130/0.4 versus 
	crystalloid.
Fig. 1. Hemodynamic effects of volume therapy in endotoxemic sheep. Data are presented as means ± SEM. BL = baseline; DO2I = systemic oxygen delivery index; HES = hydroxyethyl starch; HR = heart rate; MAP = mean arterial pressure; SVI = stroke volume index. *P  < 0.05 HES 130/0.4 versus  HES 200. †P  < 0.05 HES 200/0.5 versus  crystalloid. ‡P  < 0.05 HES 130/0.4 versus  crystalloid.
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Table 2.  Hemodynamic Effects of Volume Therapy in Endotoxemic Sheep
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Table 2.  Hemodynamic Effects of Volume Therapy in Endotoxemic Sheep
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Table 3.  Changes in Oxygen Transport, Arterial Lactate, Electrolyte Concentrations, and Body Temperature in Endotoxemic Sheep
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Table 3.  Changes in Oxygen Transport, Arterial Lactate, Electrolyte Concentrations, and Body Temperature in Endotoxemic Sheep
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Volume Requirements
Volume requirements of the three study groups are displayed in figure 2. The maximum dose of colloids was infused after 4 h in both HES-treated groups. Thus, from 4–12 h, only crystalloids were infused in all study groups. HES-treated animals required significantly less total fluids to achieve the predefined cardiac filling pressures when compared with the crystalloid group (P  = 0.03, HES 200/0.5 vs.  crystalloid, P  = 0.04, HES 130/0.4 vs.  crystalloid). In addition, crystalloid requirements were lower in the two saline-based HES groups when compared with the balanced crystalloid group (P  = 0.002, HES 200/0.5 vs.  crystalloid, P  = 0.02, HES 130/0.4 vs.  crystalloid).
Fig. 2. Cumulative colloid, crystalloid, and total volume requirements over 12 h in endotoxemic sheep. Data are presented as means ± SEM. HES = hydroxyethyl starch. †P  < 0.05 HES 200/0.5 versus  crystalloid; ‡P  < 0.05 HES 130/0.4 versus  crystalloid.
Fig. 2. Cumulative colloid, crystalloid, and total volume requirements over 12 h in endotoxemic sheep. Data are presented as means ± SEM. HES = hydroxyethyl starch. †P 
	< 0.05 HES 200/0.5 versus 
	crystalloid; ‡P 
	< 0.05 HES 130/0.4 versus 
	crystalloid.
Fig. 2. Cumulative colloid, crystalloid, and total volume requirements over 12 h in endotoxemic sheep. Data are presented as means ± SEM. HES = hydroxyethyl starch. †P  < 0.05 HES 200/0.5 versus  crystalloid; ‡P  < 0.05 HES 130/0.4 versus  crystalloid.
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Norepinephrine Requirements
There were no overall differences among groups in norepinephrine requirements over the whole study period (0.32 ± 0.07, 0.34 ± 0.08, and 0.37 ± 0.07 μg · kg−1· min−1in the HES 130/0.4, HES 200/0.5, and crystalloid groups, respectively; P  = 0.52, HES 130/0.4 vs.  HES 200/0.5; P  = 0.27, HES 130/0.4 vs.  crystalloid; P  = 0.28, HES 200/0.5 vs.  crystalloid).
COP and Plasma Protein Concentration
Endotoxin infusion was linked to a significant decrease in total plasma protein concentration and COP in all three groups (each P  < 0.001 BL2 vs.  BL1; fig. 3). There was no overall difference between groups in total plasma protein concentrations. Within the initial 4 h of volume resuscitation, however, COP was significantly higher in both colloid-treated groups when compared with the crystalloid group (P  < 0.001 HES 200/0.5 vs.  crystalloids; P  = 0.02, HES 130/0.4 vs.  crystalloids at 4 h).
Fig. 3. Changes in total plasma protein concentration and colloid-osmotic pressure in endotoxemic sheep. Data are presented as means ± SEM. BL = baseline; HES = hydroxyethyl starch. †P  < 0.05 HES 200/0.5 versus  crystalloid. ‡P  < 0.05 HES 130/0.4 versus  crystalloid.
Fig. 3. Changes in total plasma protein concentration and colloid-osmotic pressure in endotoxemic sheep. Data are presented as means ± SEM. BL = baseline; HES = hydroxyethyl starch. †P 
	< 0.05 HES 200/0.5 versus 
	crystalloid. ‡P 
	< 0.05 HES 130/0.4 versus 
	crystalloid.
Fig. 3. Changes in total plasma protein concentration and colloid-osmotic pressure in endotoxemic sheep. Data are presented as means ± SEM. BL = baseline; HES = hydroxyethyl starch. †P  < 0.05 HES 200/0.5 versus  crystalloid. ‡P  < 0.05 HES 130/0.4 versus  crystalloid.
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Core Body Temperature, Electrolytes, and Acid–Base Balance
Core body temperature and arterial lactate increased (each P  < 0.001 BL2 vs.  BL1), whereas BEoxdecreased (P  = 0.04, BL2 vs.  BL1) in response to endotoxin infusion. The decrease in BEoxwas more pronounced in saline-based HES 200/0.5—than in saline-based HES 130/0.4—(P  = 0.04, HES 130/0.4 vs.  HES 200/0.5) and balanced crystalloid-treated sheep (P  = 0.08, crystalloid vs.  HES 200/0.5). Although plasma sodium concentrations did not significantly change, chloride concentrations increased with time in all groups (BL2 vs.  12 h: P  < 0.001 in the HES 130/0.4 and crystalloid group; P  = 0.03 in HES 200/0.5; table 3). However, there were no differences between groups at any time point.
Renal Function
Surrogate variables of renal function are depicted in figure 4. Plasma creatinine concentrations significantly increased, and creatinine clearance decreased after endotoxin infusion in all groups (each P  < 0.001, BL2 vs.  BL1). However, urinary output was lower during the initial 7 h of volume resuscitation in the saline-based HES 200/0.5 group when compared with the saline-based HES 130/0.4 (P  = 0.04 at 7 h) and the balanced crystalloid group (P  = 0.03 at 7 h). Plasma creatinine concentrations were higher in HES 200/0.5-treated sheep than in HES 130/0.4 over the entire intervention period (P  = 0.02). In addition, plasma creatinine concentrations were higher in the saline-based HES 200/0.5 than in the balanced crystalloid group from 4–8 h (P  = 0.002 at 8 h). Plasma urea concentrations significantly increased in all study groups (each P  < 0.001 vs.  BL2) and were significantly higher in the HES 200/0.5 group when compared with the HES 130/0.4 (P  = 0.03) and the crystalloid group (P  = 0.001) after 8 h. Creatinine clearance was 62% higher in the saline-based HES 130/0.4 group and 58% higher in the balanced crystalloid group than in the saline-based HES 200/0.5 group after 4 h of treatment. However, this trend was not statistically significant.
Fig. 4. Changes in renal function parameters in endotoxemic sheep. Data are presented as means ± SEM. BL = baseline; HES = hydroxyethyl starch. *P  < 0.05 6% HES 130/0.4 versus  10% HES 200/0.5. †P  < 0.05 10% HES 200/0.5 versus  crystalloid.
Fig. 4. Changes in renal function parameters in endotoxemic sheep. Data are presented as means ± SEM. BL = baseline; HES = hydroxyethyl starch. *P 
	< 0.05 6% HES 130/0.4 versus 
	10% HES 200/0.5. †P 
	< 0.05 10% HES 200/0.5 versus 
	crystalloid.
Fig. 4. Changes in renal function parameters in endotoxemic sheep. Data are presented as means ± SEM. BL = baseline; HES = hydroxyethyl starch. *P  < 0.05 6% HES 130/0.4 versus  10% HES 200/0.5. †P  < 0.05 10% HES 200/0.5 versus  crystalloid.
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Survival
Six of 10 animals in the saline-based HES 130/0.4 and the balanced crystalloid group survived the 12-h intervention period when compared with 3 of 10 sheep in the saline-based HES 200/0.5 group (n.s., P  = 0.34).
Histologic Examination
Light microscopy of the kidney revealed acute tubular cell injury with intraluminal protein precipitation. These alterations were equally distributed among groups without significant differences (see fig. 5for illustrative examples).
Fig. 5. Histologic images of renal tubular injury in endotoxemic sheep. This figure represents illustrative examples of histologic findings in endotoxemic sheep. (A  ) 100-fold magnification; (B  ) 400-fold magnification.
Fig. 5. Histologic images of renal tubular injury in endotoxemic sheep. This figure represents illustrative examples of histologic findings in endotoxemic sheep. (A 
	) 100-fold magnification; (B 
	) 400-fold magnification.
Fig. 5. Histologic images of renal tubular injury in endotoxemic sheep. This figure represents illustrative examples of histologic findings in endotoxemic sheep. (A  ) 100-fold magnification; (B  ) 400-fold magnification.
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Transmission Electron Microscopy
Renal tubular injury score was inversely correlated with the diuresis rate determined during the last hour of the protocol (r  =−0.403; P  = 0.03). Renal tubular injury score was higher in sheep treated with saline-based 10% HES 200/0.5 or sole balanced crystalloids when compared with saline-based 6% HES 130/0.4. In addition, intratubular precipitations and cellular injury were most pronounced after treatment with saline-based 10% HES 200/0.5 (table 4; fig. 6).
Table 4.  Electron Microscopic Evaluation of Renal Tubular Injury in Endotoxemic Sheep
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Table 4.  Electron Microscopic Evaluation of Renal Tubular Injury in Endotoxemic Sheep
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Fig. 6. Transmission electron microscopic images of renal tubular injury in endotoxemic sheep. Exemplary presentation of renal tubules of endotoxemic sheep treated with 6% hydroxyethyl starch (HES) 130/0.4 (A  ), 10% HES 200/0.5 (B  ), or sole crystalloids (C  ) (1,000-fold magnification each). Dissociation of epithelium from basal lamina (closed arrow  ), vacuolar cell degeneration (open arrow  ), edematous tubular epithelial cell organella (open arrowheads  ), and intraluminal precipitation of proteins (dashed closed arrows  ).
Fig. 6. Transmission electron microscopic images of renal tubular injury in endotoxemic sheep. Exemplary presentation of renal tubules of endotoxemic sheep treated with 6% hydroxyethyl starch (HES) 130/0.4 (A 
	), 10% HES 200/0.5 (B 
	), or sole crystalloids (C 
	) (1,000-fold magnification each). Dissociation of epithelium from basal lamina (closed arrow 
	), vacuolar cell degeneration (open arrow 
	), edematous tubular epithelial cell organella (open arrowheads 
	), and intraluminal precipitation of proteins (dashed closed arrows 
	).
Fig. 6. Transmission electron microscopic images of renal tubular injury in endotoxemic sheep. Exemplary presentation of renal tubules of endotoxemic sheep treated with 6% hydroxyethyl starch (HES) 130/0.4 (A  ), 10% HES 200/0.5 (B  ), or sole crystalloids (C  ) (1,000-fold magnification each). Dissociation of epithelium from basal lamina (closed arrow  ), vacuolar cell degeneration (open arrow  ), edematous tubular epithelial cell organella (open arrowheads  ), and intraluminal precipitation of proteins (dashed closed arrows  ).
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Discussion
The key finding of the current study is that in the setting of ovine endotoxemic shock, infusion of saline-based 10% HES 200/0.5, when given in pharmaceutically recommended doses, was associated with significantly impaired renal function and tubular integrity, whereas infusion of saline-based 6% HES 130/0.4 and a balanced crystalloids was not linked to such alterations.
Data were collected in an established large animal model of endotoxemic shock that closely reflects hemodynamic and metabolic derangements typically seen in the early stage of human septic shock.11,12,18 The observed alterations in response to endotoxin challenge are in accordance with previous data of our study group and others.11,19 
In the current study, all sheep suffered from vasodilatory shock (as indicated by reduced systemic vascular resistance index and MAP, and increased CI) and absolute hypovolemia (as reflected by increases in hematocrit before volume resuscitation). The reductions in COP and total plasma protein concentrations were indicative of endothelial barrier dysfunction with extravasation of plasma proteins.20,21 The increases in arterial lactate concentrations and decreases in BEoxcan best be explained by impaired tissue perfusion in response to arterial hypotension.22 These alterations were associated with increased surrogate markers of renal injury, suggesting the presence of organ dysfunction.23,24 
Volume resuscitation was initiated as soon as MAP decreased less than the threshold values of 65 mmHg. Fluid therapy was guided to optimize CVP and pulmonary arterial occlusion pressure.15 Although the latter approach is recommended by the current sepsis guidelines,15 dynamic markers of volume responsiveness (i.e.  , variations in stroke volume or pulse pressure) may be more accurate variables to guide volume therapy. However, the latter dynamic markers are only reliable in mechanically ventilated subjects without spontaneous breathing activity,25 unlike in the current study. Passive leg lifting seems to be the most suitable method in awake, spontaneously breathing subjects,26 but it could not be applied in the current study because of the anatomy of sheep. Thus, filling pressures were chosen to guide resuscitation. Volume resuscitation established hemodynamic goal values in all groups within 1 h and contributed to a hyperdynamic circulation characterized by a low systemic vascular resistance index and an increase in CI,27 which resulted in higher DO2I in the HES 130/0.4 when compared with the HES 200/0.5 group. Reduced BEoxvalues in the saline-based HES 200/0.5 group suggest that the reduction in DO2I may have negatively impacted on tissue oxygenation. This assumption is supported by a study of Chiara et al.  ,22 showing that base excess is a suitable marker of tissue hypoperfusion after hemorrhage in pigs. Marx et al.  20 also conducted experiments in this area and reported similar hemodynamic differences after infusion of tetrastarch and pentastarch solutions in porcine septic shock. In addition, the authors of the latter study noticed that pentastarch (6% HES 200/0.5) markedly increases the albumin escape rate (a marker of vascular leakage) when compared with 6% HES 130/0.42.
In the current study, administration of both saline-based 6% HES 130/0.4 and 10% HES 200/0.5 resulted in a significantly higher COP when compared with balanced crystalloid resuscitation. This may be explained by the different in vitro  COP of the compounds. Although 6% HES 130/0.4 is isooncotic (COP = 34–36 mmHg) and 10% HES 200/0.5 is hyperoncotic (COP = 60–80 mmHg), crystalloids are hypooncotic (COP = 0 mmHg). However, in vivo  COP values were relatively low in both colloid groups and decreased over time (fig. 3). This finding suggests the presence of pronounced vascular leakage with a subsequent transition of plasma proteins from the intravascular to the extravascular compartment.20 In this context, it is important to note that infusion of equivalent volumes of two colloids with markedly different in vitro  COP resulted in comparable in vivo  COP values. This finding may be explained (1) by a shift of extracellular protein-free water into the intravascular space after hyperoncotic pentastarch infusion and (2) by marked differences in pharmacokinetics between HES 200/0.5 and HES 130/0.4. Although the latter compound is rapidly degraded into many small fragments and thus exerts a high in vivo  COP, pentastarch is degraded slowly and, therefore, exerts an in vivo  COP that is lower than would be expected from the in vitro  molecular weight.5 
In the current study, administration of saline-based 10% HES 200/0.5 was associated with a significantly reduced urinary output when compared with the other two groups during the initial 7 h of volume resuscitation. This difference was accompanied by increases in plasma creatinine and urea concentrations (fig. 4). Thus, a reduced glomerular filtration rate contributed to the renal impairment noticed in the HES 200/0.5 group. It can be excluded that differences in sodium or chloride load between groups have contributed to the differences in renal function, because both HES groups received similar amounts of saline-based starch solutions, and plasma electrolyte concentrations were similar among groups (table 3). Because MAP and COP were also comparable between the two colloid groups, it is unlikely that these variables impacted on the group differences in the current study. However, it cannot be excluded that differences in CI may have resulted in reduced glomerular filtration.
The phenomenon that differences in renal function diminished during the late phase of the intervention period (fig. 4) represents another interesting finding of the current study and may be explained by the fact that the maximum dose of colloids was already infused after 4 h. Thereafter (from hours 5 to 12, when the maximum HES dose had been reached), only crystalloids were infused in all study groups. Thus, it seems that impaired renal function associated with saline-based 10% HES 200/0.5 has partially been offset by prolonged administration of balanced crystalloids.
Tubular injury, as assessed by standard histologic techniques, was comparable between groups. However, cellular and subcellular tubular epithelial injury was more pronounced in sheep treated with saline-based 10% HES 200/0.5 when compared with 6% HES 130/0.4. In particular, infusion of 10% HES 200/0.5 was associated with vacuolar degeneration of tubular cells. These pathologic alterations were not seen in sheep treated with 6% HES 130/0.4. Interestingly, intracellular edema and cellular injury accounted for marked tubular injury in sheep treated only with balanced crystalloids. Extracellular overhydration caused by maximum amounts of crystalloids may have resulted in tubular cell edema and subsequent cellular injury. However, these hypotheses need to be tested in future (experimental) studies.
Kidney injury in response to HES 200/0.5 infusion has been noticed recently in a randomized controlled clinical trial comparing fluid therapy with 10% HES 200/0.5 and a modified lactated Ringer's solution (45 mm lactate) in 537 patients with severe sepsis.4 Notably, patients allocated to the HES 200/0.5 group had a higher incidence of acute renal failure and were more likely to require renal replacement therapy. Future studies are now needed to evaluate the hypothesis that modern, third-generation tetrastarch solutions do not negatively impact on renal function in critically ill patients. In this context, Boldt et al.  8,28 demonstrated the safety of 6% HES 130/0.4 in several small-scale studies, even in elderly patients with preexisting renal dysfunction undergoing coronary artery bypass grafting. To date, however, only one clinical comparison (n = 20) of 6% HES 130/0.4 and 20% human albumin in patients with septic shock has been performed. In the latter study, patients treated with HES 130/0.4 had a significant reduction in Acute Physiology and Chronic Health Evaluation II Score and an improvement in gas exchange when compared with patients treated with 20% human albumin.29 However, these results may have been influenced by the fact that 20% human albumin is hyperoncotic and has been reported to contribute to organ dysfunction.9 
The mechanisms by which saline-based 10% HES 200/0.5 may have impaired renal function in the current study cannot be entirely described by the data but seem to include increased tubular epithelial injury.20 The large spectrum of HES molecules with diverse molecular weights in 10% HES 200/0.5 may have been associated with excessive glomerular filtration and reabsorption of starch molecules by tubular epithelial cells, which might partly explain vacuolar degeneration. In this context, it has been reported that renal impairment after colloid infusion is dependent on the concentration (with 10% dextran 40 kD posing the highest risk among all synthetic colloids).30 In the latter experimental study, it has also been shown that infusion of 10% HES 200/0.5 increases urine viscosity by a factor of 2.5. Using a porcine model of hemodilution, Eisenbach et al.  31 noticed that tissue storage of hexastarch is more pronounced when compared with pentastarch. However, considerable amounts of all three colloids (6% solutions of HES 200/0.62, HES 200/0.5, and HES 100/0.5) were detected in kidney tissue.
The current study has some limitations. (1) Because we did not analyze HES plasma concentrations, the exact amount of oncotically active HES molecules remains unknown. However, it has to be considered that COP more accurately reflects the amount of oncotically active molecules than HES plasma concentrations per se  .20 (2) Cardiac filling pressures were used to guide volume therapy. Although the latter approach is recommended by the current sepsis guidelines,15 filling pressures are associated with considerable weaknesses as detailed earlier. (3) Because of the high mortality among the study subjects, the sample size decreased over time. This may have contributed to the absence of significant differences in some variables at the end of the experiment. (4) Finally, the electron microscopic tubular injury score used in the current study has not yet been validated in healthy subjects or subjects with standardized acute kidney injury, respectively. Therefore, the current electron microscopic data should be interpreted with caution.
Conclusions
In summary, the current study directly compared the effects of saline-based 6% HES 130/0.4, saline-based 10% HES 200/0.5, and a balanced crystalloid on renal function and tubular injury in fulminant ovine endotoxemia. The data provide evidence that 10% HES 200/0.5 is associated with negative effects on renal function and tubular epithelial integrity in contrast to 6% HES 130/0.4. Because of significant pharmacologic and physicochemical differences among second- and third-generation HES preparations,5 the negative effects of 10% HES 200/0.5 noticed in clinical studies4 should not be extrapolated to modern tetrastarch preparations. Randomized clinical trials elucidating the effects of third-generation tetrastarch solutions versus  crystalloids in patients with severe sepsis and septic shock (e.g.  , CRYSTMAS and CHEST) are currently ongoing and will hopefully shed more light on this important issue.
The authors thank Frederike von Roth, Medical Student, University of Muenster, Muenster, Germany, for expert technical assistance during the study.
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Fig. 1. Hemodynamic effects of volume therapy in endotoxemic sheep. Data are presented as means ± SEM. BL = baseline; DO2I = systemic oxygen delivery index; HES = hydroxyethyl starch; HR = heart rate; MAP = mean arterial pressure; SVI = stroke volume index. *P  < 0.05 HES 130/0.4 versus  HES 200. †P  < 0.05 HES 200/0.5 versus  crystalloid. ‡P  < 0.05 HES 130/0.4 versus  crystalloid.
Fig. 1. Hemodynamic effects of volume therapy in endotoxemic sheep. Data are presented as means ± SEM. BL = baseline; DO2I = systemic oxygen delivery index; HES = hydroxyethyl starch; HR = heart rate; MAP = mean arterial pressure; SVI = stroke volume index. *P 
	< 0.05 HES 130/0.4 versus 
	HES 200. †P 
	< 0.05 HES 200/0.5 versus 
	crystalloid. ‡P 
	< 0.05 HES 130/0.4 versus 
	crystalloid.
Fig. 1. Hemodynamic effects of volume therapy in endotoxemic sheep. Data are presented as means ± SEM. BL = baseline; DO2I = systemic oxygen delivery index; HES = hydroxyethyl starch; HR = heart rate; MAP = mean arterial pressure; SVI = stroke volume index. *P  < 0.05 HES 130/0.4 versus  HES 200. †P  < 0.05 HES 200/0.5 versus  crystalloid. ‡P  < 0.05 HES 130/0.4 versus  crystalloid.
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Fig. 2. Cumulative colloid, crystalloid, and total volume requirements over 12 h in endotoxemic sheep. Data are presented as means ± SEM. HES = hydroxyethyl starch. †P  < 0.05 HES 200/0.5 versus  crystalloid; ‡P  < 0.05 HES 130/0.4 versus  crystalloid.
Fig. 2. Cumulative colloid, crystalloid, and total volume requirements over 12 h in endotoxemic sheep. Data are presented as means ± SEM. HES = hydroxyethyl starch. †P 
	< 0.05 HES 200/0.5 versus 
	crystalloid; ‡P 
	< 0.05 HES 130/0.4 versus 
	crystalloid.
Fig. 2. Cumulative colloid, crystalloid, and total volume requirements over 12 h in endotoxemic sheep. Data are presented as means ± SEM. HES = hydroxyethyl starch. †P  < 0.05 HES 200/0.5 versus  crystalloid; ‡P  < 0.05 HES 130/0.4 versus  crystalloid.
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Fig. 3. Changes in total plasma protein concentration and colloid-osmotic pressure in endotoxemic sheep. Data are presented as means ± SEM. BL = baseline; HES = hydroxyethyl starch. †P  < 0.05 HES 200/0.5 versus  crystalloid. ‡P  < 0.05 HES 130/0.4 versus  crystalloid.
Fig. 3. Changes in total plasma protein concentration and colloid-osmotic pressure in endotoxemic sheep. Data are presented as means ± SEM. BL = baseline; HES = hydroxyethyl starch. †P 
	< 0.05 HES 200/0.5 versus 
	crystalloid. ‡P 
	< 0.05 HES 130/0.4 versus 
	crystalloid.
Fig. 3. Changes in total plasma protein concentration and colloid-osmotic pressure in endotoxemic sheep. Data are presented as means ± SEM. BL = baseline; HES = hydroxyethyl starch. †P  < 0.05 HES 200/0.5 versus  crystalloid. ‡P  < 0.05 HES 130/0.4 versus  crystalloid.
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Fig. 4. Changes in renal function parameters in endotoxemic sheep. Data are presented as means ± SEM. BL = baseline; HES = hydroxyethyl starch. *P  < 0.05 6% HES 130/0.4 versus  10% HES 200/0.5. †P  < 0.05 10% HES 200/0.5 versus  crystalloid.
Fig. 4. Changes in renal function parameters in endotoxemic sheep. Data are presented as means ± SEM. BL = baseline; HES = hydroxyethyl starch. *P 
	< 0.05 6% HES 130/0.4 versus 
	10% HES 200/0.5. †P 
	< 0.05 10% HES 200/0.5 versus 
	crystalloid.
Fig. 4. Changes in renal function parameters in endotoxemic sheep. Data are presented as means ± SEM. BL = baseline; HES = hydroxyethyl starch. *P  < 0.05 6% HES 130/0.4 versus  10% HES 200/0.5. †P  < 0.05 10% HES 200/0.5 versus  crystalloid.
×
Fig. 5. Histologic images of renal tubular injury in endotoxemic sheep. This figure represents illustrative examples of histologic findings in endotoxemic sheep. (A  ) 100-fold magnification; (B  ) 400-fold magnification.
Fig. 5. Histologic images of renal tubular injury in endotoxemic sheep. This figure represents illustrative examples of histologic findings in endotoxemic sheep. (A 
	) 100-fold magnification; (B 
	) 400-fold magnification.
Fig. 5. Histologic images of renal tubular injury in endotoxemic sheep. This figure represents illustrative examples of histologic findings in endotoxemic sheep. (A  ) 100-fold magnification; (B  ) 400-fold magnification.
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Fig. 6. Transmission electron microscopic images of renal tubular injury in endotoxemic sheep. Exemplary presentation of renal tubules of endotoxemic sheep treated with 6% hydroxyethyl starch (HES) 130/0.4 (A  ), 10% HES 200/0.5 (B  ), or sole crystalloids (C  ) (1,000-fold magnification each). Dissociation of epithelium from basal lamina (closed arrow  ), vacuolar cell degeneration (open arrow  ), edematous tubular epithelial cell organella (open arrowheads  ), and intraluminal precipitation of proteins (dashed closed arrows  ).
Fig. 6. Transmission electron microscopic images of renal tubular injury in endotoxemic sheep. Exemplary presentation of renal tubules of endotoxemic sheep treated with 6% hydroxyethyl starch (HES) 130/0.4 (A 
	), 10% HES 200/0.5 (B 
	), or sole crystalloids (C 
	) (1,000-fold magnification each). Dissociation of epithelium from basal lamina (closed arrow 
	), vacuolar cell degeneration (open arrow 
	), edematous tubular epithelial cell organella (open arrowheads 
	), and intraluminal precipitation of proteins (dashed closed arrows 
	).
Fig. 6. Transmission electron microscopic images of renal tubular injury in endotoxemic sheep. Exemplary presentation of renal tubules of endotoxemic sheep treated with 6% hydroxyethyl starch (HES) 130/0.4 (A  ), 10% HES 200/0.5 (B  ), or sole crystalloids (C  ) (1,000-fold magnification each). Dissociation of epithelium from basal lamina (closed arrow  ), vacuolar cell degeneration (open arrow  ), edematous tubular epithelial cell organella (open arrowheads  ), and intraluminal precipitation of proteins (dashed closed arrows  ).
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Table 1.  Body Weight, LPS Dose, and Time to Shock Onset in Endotoxemic Sheep
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Table 1.  Body Weight, LPS Dose, and Time to Shock Onset in Endotoxemic Sheep
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Table 2.  Hemodynamic Effects of Volume Therapy in Endotoxemic Sheep
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Table 2.  Hemodynamic Effects of Volume Therapy in Endotoxemic Sheep
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Table 3.  Changes in Oxygen Transport, Arterial Lactate, Electrolyte Concentrations, and Body Temperature in Endotoxemic Sheep
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Table 3.  Changes in Oxygen Transport, Arterial Lactate, Electrolyte Concentrations, and Body Temperature in Endotoxemic Sheep
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Table 4.  Electron Microscopic Evaluation of Renal Tubular Injury in Endotoxemic Sheep
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Table 4.  Electron Microscopic Evaluation of Renal Tubular Injury in Endotoxemic Sheep
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