Clearance of inflammatory cytokines in patients with septic acute kidney injury during renal replacement therapy using the EMiC2 filter (Clic-AKI study)

Sepsis is a life-threatening condition in which a dysregulated release of pro- and anti-inflammatory cytokines can lead to multiple organ failure and increased mortality [1]. Management of sepsis is supportive [2].

Critically ill patients with sepsis-associated acute kidney injury (AKI) requiring kidney replacement therapy (KRT) are at particularly increased risk of death [3]. However, KRT may offer the opportunity to provide extracorporeal blood purification therapy (BPT). With regards to the potential benefit of BPT, there are several hypotheses. The ‘peak concentration hypothesis’ proposes that during BPT excessive pro- or anti-inflammatory mediators are removed and plasma concentrations decrease to levels below a toxic threshold [4, 4]. The ‘cytokinetic theory’ proposes that cytokine removal creates a decreased cytokine gradient between tissues and the bloodstream and promotes leukocyte chemotaxis towards the infected tissues [6]. The ‘cytotoxic threshold immune modulation theory’ relates to the removal of cytokines from the cytokine-concentrated interstitium and tissues into the circulation [7]. Finally, a ‘cellular theory’ refers to removing leucocytes directly or through immune cell reprogramming [8, 9]. The proposed techniques include high-volume haemofiltration (HVHF), use of high cut-off (HCO) membrane and adsorption techniques, or plasmapheresis [10]. HCO haemofiltration with a cut-off up to 60 kDa has been shown to achieve higher cytokine clearance than conventional membranes (15–30 kDa), but the effects on clinical outcomes such as haemodynamic improvement, severity scores, and survival are inconclusive [11‐17]. Besides, the concomitant loss of albumin, proteins, micronutrients, and antibiotics is a concern [18, 19].

The EMiC2 filter (Fresenius, Bad Homburg, Germany) is a polysulfone (PS)-based membrane with a cut-off of 45 kDa. Case reports showed reduction in serum k-free light chains and myoglobin levels with the EMiC2 filter, but actual removal by the filter was not investigated [20‐23]. Other studies reported higher removal of kappa light chains (23 kDa), β2-microglobulin (17 kDa), myoglobin (17 kDa), IL-6, and IL-8 in patients receiving treatment with EMiC2 filters compared with standard high-flux membranes [24‐27]. EMiC2-continuous veno-venous haemodialysis (CVVHD) was well-tolerated, and albumin loss was limited [28]. However, in-vitro data showed no adsorptive capacity of the EMiC2 filter compared with other membranes [29].

The exact role of the EMiC2 filter in the treatment of sepsis-associated AKI is unknown. Furthermore, the causal link between reduction in plasma cytokine concentration and extracorporeal cytokine removal has not been demonstrated [26, 28]. Before proceeding to a trial comparing the EMiC2 filter with other commercially available filters to manage sepsis-associated AKI, it is important to determine the characteristics and the actual magnitude of cytokine removal in vivo. In this pilot study, we aimed to measure the clearance of middle molecular weight cytokines using the EMiC2 filter in patients receiving CVVHD.

This study was a prospective observational study in a 62-bed mixed tertiary-care intensive care unit (ICU) between June and September 2017. The study was approved by the Research Ethics Committee (16/LO/0313), registered on clinicaltrials.gov (NCT03231748) and conducted in accordance with the Declaration of Helsinki 2013. Written informed consent was obtained from all patients or their legal representatives.

This is the first study which investigated the transport characteristics of 12 molecules across the EMiC2 membrane. The key findings were: first that the plasma concentrations of all molecules declined over 48 h, except for EGF. Second, the effluent clearance rates were low for most cytokines, except for IL-4, IL-8, and MCP-1. Third, minimal or negative adsorption was observed for all cytokines. Finally, the total removal rates and contributions from CVVHD and adsorption were heterogeneous but were mostly low to moderate.

Extracorporeal blood purification to attenuate the effects of pro-inflammatory and anti-inflammatory mediators in sepsis remains a controversial issue. Although some studies showed potentially promising results and a reduction in cytokine concentrations in plasma with particular extracorporeal techniques, it is not always clear whether these declines in cytokine levels are related to the filter, or simply a reflection of the dynamic nature of sepsis [10]. Baseline cytokine concentrations of our patient cohort were similar or lower than previous reports in the literature [32‐41]. This possibly reflects the heterogeneity in phenotypes and severity of sepsis and also underlines the complexity of cytokine profile interpretation in this setting. Whilst some cytokines are pro-inflammatory and associated with poor outcomes [42], others have a potentially beneficial role. For instance, EGF represents tissue recovery or regeneration after injury and is associated with cellular proliferation and survival in sepsis [43].

Previous studies explored cytokine changes and cytokine removal by the EMiC2 filter, but none have fully investigated the various mechanisms of cytokine clearance in critically ill patients with sepsis [25‐28]. Despite reported decent removal of IL-6, IL-8, IL-1β, and TNF-α (MW 8.4–25 kDa) in-vitro, the effluent clearance rates varied markedly in humans [26, 27, 29]. Several factors might affect in vivo clearance, e.g. duration of blood contact, binding to protein or plasma components, dialysate rate or ultrafiltration rate, molecular weight, serum concentration, or sampling time after filter installation. In a previous study using a HCO membrane, a decline in plasma IL-1ra and IL-6 was observed in patients with high baseline concentrations [11]. This corresponds with our results showing that serum IL-2, IL-6, and IL-1β concentrations were positively correlated with effluent clearance rates. We also noted that the effluent clearance rates remained constant over time which may be explained by the use of citrate-based anticoagulation [44]. Citrate can prolong filter life by reducing both filter clotting (i.e. thrombosis within the lumen of the filter) and clogging (i.e. formation of protein layer which progressively obliterates the membrane pores) [45, 46].

The contribution of adsorption to total cytokine removal with EMiC2 filters was minimal. We noted a small degree of adsorption of IL-2 and VEGF which contributed to total mass removal. However, “negative adsorption” was also observed for all cytokines consistent with similar reports in the literature [15, 28, 31]. The mechanisms for this “de-sorption” phenomenon are unclear and might be explained by effects of haemoconcentration on the outflow side from fluid removal albeit in a significantly smaller degree than in CVVH mode, release of previously bound cytokines from cells [47], activation of the inflammatory system through reverse diffusion [28], the ‘back-filtration/back-diffusion’ phenomenon including the movement of molecules from a higher concentration in the dialysate to a lower concentration in the blood at the distal end of the hollow fibres in high-flux membranes [48], cytokine induction from dialyzer bio-incompatibility [49], activation or deactivation by enzymes after sampling, or sampling errors.

We found that the changes in cytokine concentrations seen in plasma were discordant with the extent of removal by clearance and adsorption. Despite a decline in serum concentrations, we found low total mass removal rates across the filter for most cytokines. The clearance rates were highest for IL-4, IL-8, and MCP-1, but their plasma reduction ratios varied significantly from − 13.46 to − 59.94%. Although VEGF showed the highest total mass removal at 48 h, the reduction ratio was − 34.88% which was lower than others.

Some of our findings are compatible with data in the literature but not all. For instance, a previous study demonstrated higher IL-6 and IL-8 clearance by the EMiC2 filter than the standard membrane, but showed no significant impact on plasma cytokine concentrations [26]. Another study showed comparable IL-6 clearance between the EMiC2 and high-flux membrane [27]. Similarly, studies using the EMiC2 or HCO membranes reported no changes in plasma concentrations despite detectable clearance in the ultrafiltrate [11, 15, 28, 50]. These results in the literature, together with our findings highlight that a rise or fall in serum concentrations during KRT might be related to factors other than extracorporeal removal, e.g. changes in cytokine production, endogenous clearance, intradialytic cytokine release, general improvement of the underlying disease, or response to treatment [51]. It should also be noted that cytokine half-lives are extremely short. (Additional file 6) Thus, their endogenous metabolism might be more rapid than clearance during extracorporeal therapy. In general, the kidneys contribute to 15–20% of cytokine metabolism [52]. Our study showed that CVVHD using the EMiC2 filter contributed little to total clearance of most cytokines. This suggests that extracorporeal removal does not substitute renal clearance, similar to lactate removal [53]. In addition, the interaction of soluble forms and their endogenous modulators (receptors or antagonists) indicates different states of immune activation. Thus, the measurement of plasma cytokine concentrations in isolation might not reflect the true immune status or indicate the impact of extracorporeal removal on the dynamics of that particular cytokine system [54].

This is the first study to investigate a comprehensive panel of molecules which are representative of pro- and anti-inflammatory cytokines in real clinical settings. Adsorption and diffusive clearances were evaluated extensively by determination of their clearance rates, and mass balances across the membrane over a 48-h period. However, some limitations need to be acknowledged. First, the objective of this pilot study was to investigate the mechanistic impact of using the EMiC2 filter. It was not powered to assess an association with clinical outcomes. Second, we did not intend to compare the EMiC2 filter with other filters. Therefore, there was no control group. However, this is an exploratory study to characterize the transport characteristics of cytokines when using the EMiC2 filter. This investigation is essential before proceeding to larger clinical studies investigating the role of blood purification with the EMiC2 filter as an adjunctive therapy in sepsis. Third, we selected 12 different molecules but did not measure all potential pro- and anti-inflammatory cytokines. We acknowledge that our conclusions only apply to the cytokines measured and that it is possible that other cytokines or medium-sized molecules are removed at higher quantities when using the EMiC2 filter. Fourth, the cohort of included patients was heterogenous and there were some patients with undetectable cytokine concentrations throughout the whole study period. Given the mechanistic nature of this project, we only included subjects with detectable pre-dialyzer concentrations when analysing the effluent and adsorptive clearance rates and mass balances. Although there was minimal correlation between initial cytokine concentrations and clearance rates, we are unable to comment on whether cytokine removal in patients with different cytokine concentrations, including cytokine storm/septic shock would have different clinical effects. We acknowledge that cytokines may have different effects independent of the concentration, including paracrine and endocrine actions [55]. Finally, our aim was to describe clearance and adsorption of cytokines during CVVHD with an EMiC2 filter. Although we showed that only small amounts of cytokines were actually removed, we acknowledge that we cannot exclude any immunomodulatory effects [56, 57].

Our study has shown that in patients with sepsis and acute kidney injury requiring KRT with the EMiC2 filter, clearance of cytokines by CVVHD was modest and adsorption was minor. We observed a decline in serum concentrations of most cytokines during the study period but were unable to detect an obvious correlation between serum concentration and cytokine clearance. The results suggest that mechanisms other than extracorporeal removal contribute to changes in plasma cytokine concentrations. Further work to determine the role of the EMiC2 filter in clinical practice is required.