Impairment of renal function using hyperoncotic colloids in a two hit model of shock: a prospective randomized study

Animals were premedicated via intramuscular administration of ketamin and midazolam. Thereafter, general anesthesia was induced by intravenous injection of propofol (3 to 4 mg/kg BW) and the animals were orally intubated and placed in the supine position. General anesthesia was maintained with an infusion of propofol and sufentanil. Controlled pressure mode ventilation was chosen to ventilate the animals with an inspiratory oxygen fraction of 0.4, an inspiratory/expiratory ratio of 1:2, positive end-expiratory pressure (PEEP) set to 5 and a tidal volume of 8 to 10 ml/kg BW. Respiratory rate was set with the aim to maintain a PaCO₂ of 35 to 46 mmHg. The core body temperature was kept at above 37.5°C with the use of a warming blanket. A transpulmonary thermodilution catheter, central venous line and the pulmonary artery catheter were placed as we described in detail in former papers [4]. None of the infusion systems were heparinized. In all animals, a midline laparotomy was performed and the left renal artery was exposed and a Transonic Doppler flow probe (Transonic Systems, Ithaca, NY, USA) was placed around the vessel to measure blood flow of the left renal artery (RBF).

In this model, we used an Escherichia coli laden clot to induce septic shock. We used 7 to 9 × 10⁹ colony forming units (CFU) per kg/BW. The clot was made from a sterile solution of porcine fibrinogen (Sigma-Aldrich Inc., St. Louis, MO, USA) (final volume was adjusted to the pig's weight). The clot was formed by adding thrombin from bovine plasma to the fibrinogen and E. coli solution and incubating for 30 minutes at room temperature.

All intravascular pressure measurements were referenced to mid-chest level and values were obtained at end expiration. Mean arterial pressure (MAP), central venous pressure (CVP), mean pulmonary artery pressure (MPAP) and pulmonary artery occlusion pressure (PAOP) were recorded from calibrated pressure transducers (Edwards Lifesciences, Irvine, CA, USA) and values obtained at end-expiration.

Intrathoracic blood volume (ITBV) was calculated by the mean transit time approach (transpulmonary thermo dilution technique) using a PiCCO^® System (PULSION Medical Systems SE, Munich, Germany).

Cardiac output (CO) was determined by thermodilution with a CO computer (Vigilance^®; Edwards Lifesciences Ltd, Newbury, UK).

Systemic oxygen delivery (DO₂), oxygen extraction ratio (OE) and consumption (VO₂) were calculated from hemoglobin, oxygen saturation, PaO₂ and cardiac output according to standard formulae.

Blood gas analyses were measured using a standard blood gas oximetry system (ABL 625; Radiometer, Copenhagen, Denmark) with a co-oximeter. Sodium, potassium, chloride, creatinine and hematocrit (Hct) were determined using standard laboratory techniques. We calculated the anion gap with the formula: anion gap = ((Na⁺)+(K⁺))-((Cl^-)+(HCO₃^-)).

Colloid osmotic pressure (COP) was analyzed using a membrane colloid oncometer with a 20 kD semi-permeable membrane (BMT 921, Delta-Pharma, Pfullingen, Germany). From urine specimen, N-acetyl- beta-D-glucosamidase (NAG; analyzed by a spectrophotometric method; Hoffmann-La Roche, Basel, Switzerland), an approved sensitive marker of lysosomal tubular damage [10], was measured. Creatinine clearance was measured as an estimate of glomerular filtration rate (ClCrea = Ucrea × Uvol/Pcrea × duration of urine collection period; where Ucrea = urine creatinine concentration; Uvol = urine volume during the collection period; Pcrea = serum creatinine concentration). The duration of urine collection period was 240 minutes.

Cumulative fluid balance was calculated as fluid input minus urinary output during the entire study period. Body weight of animals was measured directly before experimentation.

Left renal arteries were identified close to their origins at the aortas. A pre-calibrated ultrasonic transit time flow probe (Transonic Systems Inc., Ithaca, NY, USA) was placed around the vessels and connected to an ultrasound blood flow meter (T207; Transonic Systems Inc.).

Levels of interleukin 6 (IL-6), interleukin 10 (IL-10) and tumor necrosis factor alpha (TNF-α) in plasma were assessed by an enzyme-linked immunosorbent assay using commercially available kits specific for pigs (IL-6; IL-10; TNF-α: BioSource, Solingen, Germany). Measurements were made according to the manufacturer's guidelines and blood samples were collected additionally two and four hours after sepsis induction.

Biopsies of the kidney were taken directly after euthanasia of the animals, fixed in 4% formaldehyde and embedded in paraffin. Sections (4 μm) were cut and stained with hematoxylin and eosin (H&E). Renal morphology was assessed semiquantitatively using the following four criteria of tubulointerstitial renal injury: acute tubular necrosis (ATN), that is, dilatation of tubuli with flattening or loss of the tubular epithelium, interstitial bleeding (IBl), glomerulo sclerosis (GSl) and osmotic-nephrosis like lesions (OL) of the tubuli (see also Additional files 1, 2, 3). One experienced investigator blinded to the groups scored each variable using a semiquantitative scoring system (0 to 4) for each criterion in 20 randomly sampled visual fields per animal: 0 (absent), 1 (0 to 25%), 2 (25 to 50%), 3 (50 to 75%) or 4 (more than 75%) [10].

During the surgical procedure, animals received 10 ml/kg BW/h RAc (Serumwerke Bernburg AG, Bernburg, Germany). Hemorrhagic shock was induced by bleeding the animals via the carotid artery catheter. Animals were bled until half of the baseline MAP was reached, resulting in a blood loss of 32.8 ± 3.8 ml/kg BW per animal. Hemorrhagic shock was maintained for 45 minutes. Thereafter, animals were randomly allocated to receive either fluid therapy with RAc (n = 5), 4% gelatin in RAc (4% gelatin) (Gelafusal^®, Serumwerke Bernburg AG, Bernburg, Germany) (n = 5), 6% HES with a molecular weight of 130 kD and a molar substitution of 0.42 in RAc (HES 130) (Vitafusal^®, Serumwerke Bernburg AG, Bernburg, Germany) (n = 5), 10% HES with a molecular weight of 200 kD and a molar substitution of 0.5 in 0.9% sodium chloride (HES 200) (Infukoll^®, Serumwerke Bernburg AG AG, Bernburg, Germany) (n = 5), compared to a non-shock, sham-operated control group (SHAM, n = 3) receiving RAc. Fluid replacement was performed while maintaining baseline MAP for six hours. In addition, four hours after cessation of the hemorrhagic shock, the blood collected during hemorrhagic shock was retransfused.

Six hours after hemorrhagic shock, the infected clot was placed into the abdominal cavity to induce septic shock. SHAM group received a non-infected clot.

Two hours after the induction of septic shock, the hemodynamic treatment scheme aimed at maintaining a CVP of 12 mmHg was initiated. Fluid therapy during this second shock was the identical randomized solution that was administered during hemorrhagic shock; hence, each animal received only one study solution. CVP was measured continuously and a gradual continuous fluid resuscitation was carried out. After induction of sepsis, animals were treated for another 12 h. Measurements were performed at baseline, before induction of septic shock (6 h after resolution of hemorrhagic shock) and 12 h after induction of septic shock (Figure 1).

Data were analyzed using SPSS 16.0 for Windows (IBM Statistics, Ehningen, Germany) and all results are presented as mean ± standard deviation (SD). After verifying normal data distribution (skewness < 1.5) [13], effects of fluid replacement solution were statistically analyzed by analysis of variance (ANOVA) for repeated measurements. Bonferroni's correction was applied for multiple comparisons, following ANOVA for each single time point. Differences were considered significant at P < 0.05. Data are presented at baseline (BL), 6 h after hemorrhagic and before septic shock (SL) and 12 h after sepsis induction (12 h sepsis).

A three-fold increase in beta-NAG was regarded as clinically relevant. A total sample size of at least 23 animals was necessary in order to achieve a power of at least 80%.

Hemodynamic parameters are summarized in Table 1. Baseline values were comparable in all groups. Twelve hours after sepsis induction CO, as well as MAP, CVP and PAOP, did not differ between groups. MPAP increased significantly in the HES200 group 12 h after sepsis induction compared to the RAc, 6% HES130 and SHAM group (Table 1).

Treatment with RAc led to a significantly higher fluid input and fluid balance in the RAc group as compared to all other groups after 12 h of sepsis. The degree of Hct was similar in all groups at baseline and 12 h after induction of sepsis (Table 1). COP was significantly higher in 10% HES200 and 4% gelatin group after 12 h of sepsis compared to 6% HES130 and RAc (Table 1).

The inflammatory response was enhanced in all septic groups two hours after sepsis induction. IL-6 (Figure 2) was significantly higher in the 10% HES200 group compared to all other groups two hours after sepsis induction and TNF-α increased significantly in the 10% HES200 group (4,122 ± 1,417 pg/ml; P < 0.05) compared to all other groups (6% HES130 1,199 ± 1,237 pg/ml; 4% gelatin 1,939 ± 1,691 pg/ml; RAc 1,101 ± 498 pg/ml; SHAM 285 ± 385 pg/ml) two hours after sepsis induction (Table 2).

IL-10 was significantly elevated after 12 h of sepsis in the 10% HES200 group compared to 6% HES130, RAc and SHAM (Table 3).

Results of systemic oxygenation are summarized in Table 3. SvO₂ was comparable at baseline in all groups. Twelve hours after sepsis induction a significantly lower SvO₂ was observed in the 4% gelatin and 10% HES200 group compared to SHAM (Table 3). Oxygen extraction ratio (OE) increased significantly in the 4% gelatin and 10% HES200 group compared to SHAM.

Electrolyte levels at baseline were comparable in all groups (Table 4). There were no differences in sodium levels between groups. Chloride levels were significantly lower in the 4% gelatin group compared to 10% HES200 and RAc after 12 h of sepsis.

Serum creatinine (as percent of baseline, a negative value means creatinine is decreasing compared to baseline) was significantly higher in the 10% HES200 group compared to RAc and 6% HES130 12 h after sepsis, and creatinine clearance was significantly lower in the 10% HES200 and the 4% gelatin group compared to 6% HES130 and SHAM 12 h after sepsis. Urine output at study end was lower in the 10% HES200 group compared to all other groups (Table 4).

The renal artery flow of the left kidney as percentage of CO 12 h after sepsis was lower, but not significant, in the 10% HES200 group compared to all other septic groups, and significantly lower compared to the SHAM group (Figure 3).

Baseline values of NAG in urine (in units per gram creatinine (U/g creatinine)) were comparable in all groups. Fluid replacement with 10% HES200 led to a significantly higher NAG at study end compared to 6% HES130, RAc and SHAM (Figure 4).

The histological analyses are presented in Table 5. They showed a significant increase in OL for 4% gelatin compared to RAc, 6% HES130 and SHAM. ATN was significantly higher in 10% HES200 compared to SHAM. Also IBl was significantly higher in 10% HES200 compared to RAc, 6% HES130 and SHAM. There was a non- significant increase in GSl in the 10% HES200 group compared to all other groups (Table 5).

In macro-circulation there were no differences between the fluid replacement solutions tested. There were significant differences in oxygen extraction and SvO2 between 10% HES200 and 4% gelatin compared to SHAM and no differences between septic groups.

Sepsis is often associated with impaired renal function. The effect of HES infusion on renal function in sepsis patients has spurred ongoing research on the underlying pathology.

Schortgen and colleagues showed HES to be an independent risk factor for acute renal failure in severe sepsis [6]. The methodology of this study has been questioned, although it was randomized and controlled [9, 14]. Recently, in a German multicenter randomized controlled trial (efficacy of fluid substitution and insulin therapy in severe sepsis (VISEP) study), it was shown that the use of 10% HES 200/0.5 compared with lactated Ringer's solution (RL) in patients with severe sepsis or septic shock is associated with an increased need for renal replacement therapy. Furthermore, the cumulative dosage of 10% HES200/0.5 was significantly correlated with the need for renal replacement therapy [7]. In contrast, a recent large prospective observational study of over 3,000 critically ill patients, showed that in those patients with an ICU stay of more than 24 hours, sepsis, heart failure and hematological cancer were all significantly associated with the need for dialysis or hemofiltration therapy, but fluid replacement with HES was not [14]. Comparing this result with the data of the VISEP study, one important difference is the total amount of administered HES. In the VISEP study, patients received HES for up to 21 days with a median cumulative dose of 70.4 ml/kg (interquartile range: 33.4 to 144.2 ml/kg), whereas in the observational study, the median total amount of HES per patient was lower (1,000 ml, interquartile range: 500 to 2,250 ml corresponding to a cumulative dose of less than 15 ml/kg).

These data as well as the results of our study on HES and renal function suggest that the degree of functional impairment might depend on the HES formulation used. In our study, in comparison to 6% HES130 and RAc, the use of 10% HES200 was associated with a significant lower creatinine clearance and higher creatinine levels. Furthermore, significantly higher NAG levels in the urine indicated a higher degree of tubular damage in this group.

Our results are in line with a recent pharmacokinetic study of healthy volunteers demonstrating that repeated administration of 6% HES 130/0.42 shows no accumulation compared to 10% HES 200/0.5 [15].

However, injury mechanisms remain unclear but the oncotic force of the fluid replacement solutions seems also to be important. When the oncotic pressure exceeds the hydraulic pressure of glomerular filtration it may suppress urine output [16]. Furthermore, glomerular filtration of hyperoncotic molecules from colloids causes hyper viscose urine and a stasis of tubular flow, resulting in obstruction of tubular lumen [17]. Recently, the occurrence of renal adverse events after resuscitation of shock in 822 ICU patients using crystalloids, hypooncotic (gelatin and 4% albumin) and hyperoncotic colloids (HES and dextran), and hyperoncotic 20% albumin was investigated in an observational multicenter cohort study [18]. The occurrence of renal adverse events was more frequent in both hyperoncotic groups compared to crystalloids and isooncotic colloids, suggesting that the hyperoncocity was more relevant than the nature of the product itself. Also in our study, the COP was significantly higher in the 10% HES200 and 4% gelatin groups after 12 h of sepsis compared to 6% HES130 and RAc. This might be one of the potential reasons for the pronounced kidney injury in the 10% HES200 group. However, as isooncotic 6% HES 130/0.42 (in vitro COP 37.8 mmHg) and hyperoncotic 10% HES 200/0.5 (in vitro COP 80 to 85 mmHg) were compared, it cannot be differentiated whether the oncotic force, the molecular weight, the degree of molar substitution, molecular size, the solvent for colloid solution, or a combination of these were determining factors of the HES-induced adverse effects on renal function and structure.

HES is partly eliminated by the reticuloendothelial system, and a dose dependent uptake in macrophages, endothelial and epithelial cells was detected [19]. Additionally, tissue deposition of HES was found in the kidneys. The HES containing vacuoles in the kidneys are called osmotic-like lesions (OL). This tissue deposition of HES seems to be transitory and dose-dependent [19, 20]. In our investigation we found OL elevated in all colloid groups achieving a significant level in the 4% gelatin group.

In the renal histological analysis, we found in the 10% HES200 group significantly elevated acute tubular necrosis and interstitial bleeding compared to the other groups, indicating tubular injury, thereby explaining the increased NAG and reduced creatinine clearance.

In an ex vivo renal perfusion model, the administration of HES 130 and HES 200 solutions induced a significant decrease in diuresis and creatinine clearance compared with Ringer's lactate (RL). Interestingly, 10% HES 200/0.5 was associated in this experimental setting with significant elevated inflammatory response in tubular monocytes. This pronounced pro-inflammatory effect of 10% HES 200/0.5 compared with 6% HES 130/0.42 caused more tubular damage than 6% HES 130/0.42 and RL [12]. In our present in vivo study, 10% HES200 in comparison to 6% HES130, 4% gelatin and RAc induced higher inflammatory response as suggested by higher Il-6, IL-10 and TNF-α levels, which is in accordance with our previous data. This increased inflammatory response in the 10% HES200 group may be another explanation for the pronounced kidney injury. In line with these findings, Murugan et al. demonstrated a context between elevated immune response and kidney injury [21].

Lv et al. showed in endotoxaemic rats that 6% HES200 in comparison to 0.9% saline reduced the intestinal production of proinflammatory cytokines, including TNF and IL-6 [22].

The acetate seems not to play a role in the inflammatory modulation. Davies et al. found in a study of 30 adult patients undergoing cardiopulmonary bypass for elective cardiac surgery that the use of acetate buffered prime solutions for cardiopulmonary bypass result in supra-physiological concentrations of acetate. Nonetheless, there was no difference in interleukin-6 release compared to bicarbonate-balanced fluid [23].

Hoffmann et al. demonstrated that HES 130 is effective to prevent LPS-induced leukocyte adherence, to attenuate LPS-induced capillary perfusion failure, and to reduce LPS-induced macromolecular leakage indicating even an anti-inflammatory potential of HES 130 [24].

The concept of balanced fluid replacement and its relevance with respect to mortality remains unclear. So far, only limited data are available and to the best of our knowledge, the present study is the first one to investigate balanced fluid replacement solutions in a two-hit model of hemorrhagic and septic shock. Resuscitation using RAc in experimental hemorrhagic shock could attenuate acidosis better than 0.9% saline solution, resulting in an improved survival rate [25]. Kellum et al. demonstrated in septic rats that the use of a balanced Hextend solution in comparison with 0.9% saline solution was associated with improved acid balance and even survival [10]. In line with Kellums data, we found that using 6% HES 130/0.42 in RAc and RAc resulted in significantly less negative base excess than resuscitation using 10% HES 200/0.5 in 0.9% saline. The administered amount of the latter was less compared to RAc. This result indicates that solvent for colloid solutions may be of relevance in resuscitation of shock in order to avoid further acid-based derangements. Interestingly, we could not find any differences in the concentration of chloride and sodium in the blood, except for a significantly lower chloride level in the 4% gelatin group at study end, most likely due to the fact that 4% gelatin contains less chloride than RAc (4% gelatin 85 mmol/l; RAc 112 mmol/l). This is also reflected in a significantly higher calculated anion gap in the 4% gelatin group; the anion gap did not change in all other groups compared to baseline. Thus, the use of 0.9% saline as a solvent was not associated with unwanted electrolyte disturbances in our model.

Furthermore, it was not possible to prevent metabolic acidosis in the 10% HES 200 group with no change in the anion gap compared to baseline. The metabolic acidosis in the 10% HES 200/0.5 group may be in part explained by the elevated chloride levels (111 ± 2 mmol/l) due to the carrier solution. In addition, acetate is metabolized to HCO₃^- in the other solutions and is acting as a buffer in situ.

A limitation of our model may be the experimental duration of 19 hours. On the other hand, Rivers et al. have shown that early goal-directed fluid resuscitation in patients with severe sepsis and septic shock provides significant benefits related to outcome in the first six hours after admission to an accident and emergency hospital (A&E) [2].

We chose CVP as our target for the infusion of the fluid replacement solutions according to the Surviving Sepsis Campaign: International guidelines for management of severe sepsis and septic shock [3]. Nevertheless there are several studies in critically ill patients [26, 27] suggesting that ITBV is a valuable preload indicator. In our study, the data of ITBV suggest sufficient fluid status until the end of the experiment.

Another potential confounding factor may be gender differences in the inflammatory response and survival in sepsis [28]. Ovariectomized mice had a higher death rate than proestrus mice after cecal ligation and puncture [29]. This potential difference could not be addressed in our study because we used female pigs only.