Evaluation of an ADVanced Organ Support (ADVOS) system in a two-hit porcine model of liver failure plus endotoxemia

Multiple organ failure (MOF) is a major contributor to the mortality of patients with sepsis in the intensive care unit (ICU) [1]. The majority of patients staying longer than 3 days in the ICU already have involvement of the respiratory, cardiovascular, or central nervous system upon admission [2, 3]. Moreover, the two main detoxifying organs, i.e., the liver and the kidney, are also impaired in a high number of patients, ranging from 11 to 25% and from 16 to 67%, respectively [2–5]. The diminished detoxifying function of these two organs results in an accumulation of protein-bound and water-soluble metabolic products that favors the perpetuation of organ dysfunction and contributes to the rapid dysfunction of multiple organs due to the increase of the toxic burden in the human body [6, 7].

Indeed, MOF results from an “altered organ function in an acutely ill patient such that homeostasis cannot be maintained without intervention” [8]. Bearing this in mind, interrupting this vicious cycle appears to be an essential concept in the treatment of e.g. liver and kidney dysfunction and, consequently, sepsis. As proposed by Ronco and Bellomo, single-organ support may be a simplistic view for the management of ICU patients, suggesting that multi-organ support therapy should represent the most logical future conceptual and practical evolution to achieve the goal of extracorporeal blood purification [9]. The newly introduced ADVanced Organ Support (ADVOS) system (previously known as the Hepa Wash procedure), combining liver and renal support, based on albumin dialysis, has been shown to improve dysfunction of the liver and kidney and the circulatory system and survival in an animal model of acute liver failure [10].

The major cause of MOF is sepsis, which has been recently redefined by the European Society of Intensive Care Medicine as a “life-threatening organ dysfunction due to a dysregulated host response to infection” [11]. The severity of the different organ dysfunctions in sepsis and its correlation to mortality can be estimated by the sepsis-related organ failure assessment (SOFA) score, which covers six different organ systems (and parameters) graded from 0 (no dysfunction) to 4 (severe dysfunction/failure), including the liver (bilirubin) and the kidney (creatinine) [12]. The positive correlation of mortality with the SOFA score ranges from less than 30% of deaths for patients with a SOFA score below 9 to more than 70% for a SOFA score higher than 15 [3, 13].

As already described by Meakins, MOF may occur following the two “hit” model [14], where the first hit (e.g., liver injury) would trigger an enhanced inflammatory response that might be followed by a “second hit” or insult (e.g., a nosocomial infection) [15]. In this regard, bacterial toxins (e.g., lipopolysaccharides also called endotoxins) play a major role in the cascade of events occurring in sepsis [16–18]. Animal models involving sepsis and liver injury have contributed to our understanding of many of the underlying pathophysiological pathways. These models are, however, mainly established in small animals (rats or mice) and mostly unavailable for the assessment of safety and efficacy of extracorporeal support systems [19]. To simulate multiple organ dysfunction during sepsis, we developed a two-stage pig model. In order to be validated, the model was required to have increased levels of protein-bound and water-soluble organ dysfunction markers (e.g., bilirubin, creatinine, BUN, lactate) due to a sepsis-like syndrome. To improve the feasibility of the model, death in the control group should occur within 8 h after induction of the second hit (endotoxemia) to allow for termination of the whole procedures within 16 h.

We provoked a cholestatic liver injury by ligation of the main bile ducts. In addition, we established a functional end-to-side portosystemic shunt in order to reduce liver perfusion. The severity and stability of the model and, consequently, the development of the sepsis-like syndrome were further strengthened through the administration of endotoxins.

In the present work, we evaluated the safety and efficacy of the ADVOS system in an animal model with multiple organ involvement and with a high mortality rate when treated with the standard medical treatment. Among others, blood gas, electrolytes, liver and kidney function, and hemostatic, hemodynamic, and cerebral parameters were analyzed. We paid special attention to survival rates, as well as to those parameters related to the SOFA score.

In the present study, an animal model was developed following the two-hit etiology for MOF proposed by Meakins [14]. The model encompassed two steps, starting with the induction of a cholestatic liver dysfunction (1st hit) resulting in cholangitis, which was followed by a superimposed endotoxemia (2nd hit). The rationale behind this is that cholestasis has been described to be present in 20% of the patients during their stay in the ICU, being the most common feature of liver dysfunction and being associated with increased morbidity and mortality in this hospital ward [5, 26, 27]. Indeed, pre-existing liver dysfunction plays a pivotal role as a risk factor for the progress of infection into sepsis [7].

Three days post-surgery, comparable groups of animals were obtained with elevated mean levels of bilirubin, ammonia, fibrinogen, leukocytes, and aminotransferases, confirming a liver dysfunction and an inflammatory process. In addition, the renal system was also affected, revealed by an alteration in creatinine. This renal impairment at this point, adds a grade of severity to the model and highlights the multiple organ implication. Kidneys play a major role in ammonia clearance [28], and renal involvement worsens liver failure in 30–50% of the patients [29], which is associated with a poor prognosis once renal failure develops [30].

With further administration of endotoxins in an hourly twofold stepwise increments protocol, we were able to induce a septic shock in the pigs within 2 h after endotoxemia. The animals developed a capillary leak syndrome, thrombocytopenia, leukopenia, and deterioration of respiratory function. The administration of endotoxins at relatively low doses with gradual increments results in deterioration of respiratory function as part of the multiple organ failure, while the injection of a single high dose bolus is more likely to cause early death from pulmonary artery hypertension [31].

During the whole study and, particularly, during the treatment phase (T0 to T10), special attention was put to avoid biases between groups and more than 25 standard operating procedures were followed. In this regard, invasive hemodynamic monitoring based on transpulmonary thermodilution and pulse contour analysis has been shown to be useful to guide and monitor extracorporeal organ support [32, 33]. Therefore, we adjusted fluid therapy in both groups according to PiCCO parameters [10]. Similarly, acid-base and electrolyte levels, especially hyper- and hypokalemia, were also controlled by additional infusions [10]. Moreover, by definition, septic shock involves the administration of vasopressors in order to control the low MAP [34]. As done in our previous trial [10], this was avoided to prevent the addition of a confounding factor that could affect the proper interpretation of the results. Despite the lack of use of vasopressors, we provided standard critical care to the animals in many other aspects, i.e., in an ICU-like environment.

As previously shown for an acute liver failure (ALF) model [10], also in this different animal model including endotoxemia and further dysfunction of multiple organs, the ADVOS procedure prevented death in treated animals. In the previous model, we induced ischemic liver injury (by ligating hepatic artery and diverting portal venous blood through functional end-to-side portosystemic shunt). In contrast, in the present model, we performed portal vein diversion and ligation of major biliary ducts, without interrupting arterial supply, and then followed by endotoxemia. The latter step constitutes a major difference between the two models and, in our opinion, accelerated the inflammatory process. Some of the lab values were also different (e.g., fibrinogen). Our results on survival improvement are supported by the efficacy of the ADVOS procedure in eliminating protein-bound and water-soluble organ dysfunction markers [10] like ammonia. In the liver and the kidney, the ADVOS procedure replaces only the detoxification function and thereby lacks other functions, e.g., synthesis or hormonal regulation. We speculate that efficient detoxification lead to the overall improvement of organ function. In this line, results may be interpreted as progress for SOFA-related systems (cardiovascular, cerebral, renal, respiratory, and hepatic systems). In comparison to the control animals, the ADVOS group was able to maintain surrogate markers such as MAP, creatinine, PaO₂/FiO₂, and bilirubin at significantly better values. However, the reduction of serum creatinine and BUN is rather a function of dialysis and may not reflect improvement in renal function. In addition, taking into account that Glasgow Coma Scale (GCS) was not measurable in the animals, CPP levels were significantly higher in pigs treated with ADVOS, which has been shown to positively correlate with GCS [35].

Moreover, the improvement of tissue perfusion and tissue oxygenation through adequate cardiovascular function helped in the stabilization of blood pH and allowed more protective airway pressures. During the observation period, both groups of animals developed high-output heart failure as shown by an increased cardiac index and a reduced vascular resistance [36]. Animals in the ADVOS group were able to longer survive due to the improvement of cardiac performance, highlighted by more than double cardiac power index at T6 (0.41 vs. 0.83 W/m²). Even if an improvement in cardiac performance was observed, the direct pathogenic link between the removal of disease markers by the ADVOS procedure and the increase in the cardiac index still needs to be investigated. The improvement of the cardiac index could be a result of several mechanisms: (i) The significantly higher PEEP values due to decreased oxygenation in the control group could have lowered the cardiac output. (ii) As indicated by a higher capillary leak, a lower preload could have contributed to a decreased cardiac output. The infusion rate in the control group was limited by a higher ELWI but might have also been too low for an optimal cardiac output. (iii) A possible higher pulmonary pressure due to the endotoxin-induced arterial vasoconstriction in the control group could have resulted in an increased afterload [37], even worsening the effect of the increased PEEP. However, animals in the ADVOS group received three times more endotoxin amount during the whole observation period, indicating that the effect of either endotoxins or other factors contributing to the vasoconstriction might have been positively influenced by the ADVOS treatment. (iv) Additionally, the higher CVP observed due to one of the above cited mechanisms could have also reduced the venous return and contributed to a lower preload, resulting in decreased blood pressure in the control group.

If any of the two well-known liver and kidney dysfunction markers (i.e., bilirubin and creatinine, respectively) are also markers for this detoxification function needs further investigation.

Furthermore, no differences were shown in coagulation parameters between both groups. The number of platelets remained above 100,000/μl during the whole study, which reflects the lack of influence of the ADVOS system on platelet count.

This study might be limited by a small sample size and the restrictions of the animal model in terms of life expectancy. The early start of the ADVOS procedure (which was necessary due to the short lifetime of the animals and the expedited nature of events in the model) is indeed a disadvantage that may restrict extrapolation to critically ill patients. Nonetheless, our results refer to a promising system that may improve survival in different groups of patients requiring intensive care. So far, the main target groups for the application of liver support devices (e.g., MARS and Prometheus™) were patients with acute or with acute-on-chronic liver failure [38–40].

Even if good evidence has been provided, one should be cautious in view of the fact that preclinical data for many other sepsis models does not always correlate with similar outcomes in clinical trials [41]. In fact, different extracorporeal procedures and endotoxin adsorbers have been shown to improve endotoxin-induced organ failure and hypoxemia in different animal models [42–44], but no breakthrough treatment was observed in the last years. In contrast, compared to other devices, the ADVOS procedure provides several additional advantages such as a stable blood purification during the whole treatment and not only during the first 2 h [45, 46], a lower use of albumin (2 vs. 20% with MARS) with the corresponding decrease of the costs, and higher flow rates of dialysate (up to 60 l/h) in comparison to those routinely used by MARS (200 ml/min) or single pass albumin dialysis (1 l/h) [47, 48].

Considering these advantages, it would be of great help to directly compare the ADVOS system with other devices in animal studies. However, the lack of a standardized animal model and guidelines for its handling continues hampering the development of effective treatments.

In the present work, we have developed a swine model with a sepsis-like syndrome with dysfunction of multiple organs consisting of two phases: induction of cholestatic liver injury and endotoxin administration. This model allowed us to analyze the safety and efficacy of the ADVOS procedure, resulting in an improvement of the survival rates; a decrease of bilirubin and creatinine levels; an improvement of the cardiovascular, respiratory, and central nervous system parameters; and a safe profile demonstrated by the absence of any treatment-related coagulation problems.