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

Background The EMiC2 membrane is a medium cut-off haemofilter (45 kiloDalton). Little is known regarding its efficacy in eliminating medium-sized cytokines in sepsis. This study aimed to explore the effects of continuous veno-venous haemodialysis (CVVHD) using the EMiC2 filter on cytokine clearance. Methods This was a prospective observational study conducted in critically ill patients with sepsis and acute kidney injury requiring kidney replacement therapy. We measured concentrations of 12 cytokines [Interleukin (IL) IL-1β, IL-1α, IL-2, IL-4, IL-6, IL-8, IL-10, interferon (IFN)-γ, tumour necrosis factor (TNF)-α, vascular endothelial growth factor, monocyte chemoattractant protein (MCP)-1, epidermal growth factor (EGF)] in plasma at baseline (T0) and pre- and post-dialyzer at 1, 6, 24, and 48 h after CVVHD initiation and in the effluent fluid at corresponding time points. Outcomes were the effluent and adsorptive clearance rates, mass balances, and changes in serial serum concentrations. Results Twelve patients were included in the final analysis. All cytokines except EGF concentrations declined over 48 h (p < 0.001). The effluent clearance rates were variable and ranged from negligible values for IL-2, IFN-γ, IL-1α, IL-1β, and EGF, to 19.0 ml/min for TNF-α. Negative or minimal adsorption was observed. The effluent and adsorptive clearance rates remained steady over time. The percentage of cytokine removal was low for most cytokines throughout the 48-h period. Conclusion EMiC2-CVVHD achieved modest removal of most cytokines and demonstrated small to no adsorptive capacity despite a decline in plasma cytokine concentrations. This suggests that changes in plasma cytokine concentrations may not be solely influenced by extracorporeal removal. Trial registration: NCT03231748, registered on 27th July 2017.


Introduction
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].
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.

Material and methods
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.

Subjects
Eligible patients were critically ill adult patients with acute kidney injury and sepsis in whom a decision had been made by the clinical team to start citrate-based CVVHD. Exclusion criteria were lack of consent, preexisting dialysis-dependent kidney failure, life expectancy < 24 h, haemoglobin < 7 g/dL, and need for extracorporeal membrane oxygenation (ECMO).

Kidney replacement therapy (KRT) setting
CVVHD was performed with the dialysis machine multifiltrate using the medium cut-off dialyzer EMiC2 and a bicarbonate-buffered dialysate (Fresenius Medical Care, Bad Homburg, Germany) at 25-30 ml/kg/h [30]. Regional citrate anticoagulation was used in all patients.

Laboratory analyses
Blood and effluent samples were centrifuged at 3000 rpm for 15 min and stored in a − 80 °C freezer until batch analysis at the end of the study. The cytokine concentrations were determined by electro-chemiluminescent immunoassay (ECLIA) method using an Evidence Investigator Bioship system (Randox Laboratories Limited, the United Kingdom).

Outcomes
The primary outcome of interest was cytokine clearance during EMiC2-based CVVHD. The secondary endpoints were adsorption by the EMiC2 filter, changes in cytokine concentrations in plasma, and reduction ratios of all cytokines over 48 h.

Clearances
Effluent clearance (Cl eff ) at each sampling time point was estimated using the following equation [31]: where Q df represents dialysate flow rate (ml/h).
Adsorptive clearance (K ad ) was calculated as: M ad represents mass removal rate by membrane adsorption (pg/min) (see below): The average hourly effluent clearance (Cl total ) during the study period was calculated using the following formula: The period between starting CVVHD and the 1-h time point was not included due to the necessary equilibration process. Average clearance per minute (Cl mean ) was determined from Cl total as follows:

Mass balance
Mass balance equations describe the transport of molecules and account for material entering and leaving a system. They allow the estimation of contribution from adsorption and removal into the effluent. Mass balance of the cytokines at each time point was calculated as follows: (1) where Q i is inlet plasma flow rate (ml/min); Q b is blood flow rate (ml/min); Hct is haematocrit at sampling time; Q o is outlet plasma flow rate (ml/min); Q uf is ultrafiltration rate (ml/min); Q df is dialysate flow rate (ml/min); M predialyzer is inlet mass rate (pg/min); M postdialyzer is outlet mass rate (pg/min); M total is total mass removal rate (pg/ min); M df is mass removal rate by CVVHD (pg/min); M ad is mass removal rate by membrane adsorption (pg/min). We only included subjects with detectable pre-dialyzer concentrations when analysing the effluent and adsorptive clearance rates and mass balances.

Reduction ratios
The reduction ratio (RR) of plasma cytokine concentrations at each time point was calculated as follows [24]: where Ctime0 = plasma concentrations of cytokines at baseline before CVVHD initiation.

Statistical analyses
The Kolmogorov-Smirnov test was performed to test for normal distribution of continuous variables. Normally distributed data were summarised as mean ± standard deviation. Missing data were not imputed. Nonparametric variables were summarised as median with interquartile range. Changes in median levels over time were compared using generalised estimating equations (GEE). Spearman's correlation was performed to assess the correlation between plasma cytokine concentrations and clearance rates. Linear regression was performed to investigate the link between molecular weight and clearance. A p value < 0.05 was considered statistically significant. Data were analysed using Stata 16 (StataCorp, College Station, Texas).

Patient characteristics
Thirteen patients were recruited to the study, but one patient was excluded because KRT was not started for clinical reasons. Baseline characteristics, severity scores, clinical and laboratory data at KRT initiation of the remaining 12 patients are presented in Table 1. The median dialysate volume was 2400 mL (IQR 2300-3000), and the median ultrafiltration rate was 40 mL/h (IQR 0-190). Eight patients were discharged alive from the ICU.

Cytokine plasma concentrations
Median and interquartile range of pre-dialyzer plasma cytokine concentrations at baseline and pre-determined time points are displayed in Table 2. IL-2, EGF, IFN-γ, IL-1β were undetectable in 3, 3, 2, and 1 patient, respectively, throughout the whole study period. The plasma concentrations of all cytokines except EGF significantly decreased over time (p < 0.001). Figure 1 demonstrates the pre-filter cytokine concentrations at each time point relative to baseline levels (T0, 100%). At 48 h, the pre-filter cytokine concentrations decreased to 38.98 ± 18.89% for IFN-γ and to 90.57 ± 52.21% for IL-2, corresponding to reduction ratios of -61.02% and -9.43%, respectively. (Additional file 2) In contrast, for EGF, the pre-filter cytokine concentrations at 48 h were 161.74 ± 97.84% higher compared with baseline.

Cytokine removal Clearance rates
The adsorption and effluent clearance rates at each time point are shown in Table 3

Mass balance
The total mass transfer (M total ) and mass balance via adsorption (M ad ) and CVVHD (M df ) are demonstrated in Additional file 5. Total mass transfer and the contributions from M df and M ad were calculated in percentage of M predialyzer and shown in Fig. 2

Discussion
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][33][34][35][36][37][38][39][40][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 proinflammatory 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][26][27][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  Cytokines  T1  T6  T24  T48  Time-weighted  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.

Table 3 Serial cytokine clearance rates by adsorption and diffusion at 1, 6, 24, and 48 h after continuous veno-venous haemodialysis initiation and time-weighted mean clearance rates
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 predialyzer 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].

Conclusion
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.