Effect of anticoagulation regimens on handling of interleukin-6 and -8 during continuous venovenous hemofiltration in critically ill patients with acute kidney injury

Cytokine 60 (2012) 601–607

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Effect of anticoagulation regimens on handling of interleukin-6 and -8 during continuous venovenous hemofiltration in critically ill patients with acute kidney injury Louise Schilder a,⇑, S. Azam Nurmohamed a, Pieter M. ter Wee a, Armand R.J. Girbes b, Albertus Beishuizen b, Nanne J. Paauw c, Robert H.J. Beelen c, A.B. Johan Groeneveld b a

Department of Nephrology, VU University Medical Center, Amsterdam, The Netherlands Department of Intensive Care, VU University Medical Center, Amsterdam, The Netherlands c Department of Molecular Cell Biology and Immunology, VU University Medical Center, Amsterdam, The Netherlands b

a r t i c l e

i n f o

Article history: Received 14 July 2011 Received in revised form 5 May 2012 Accepted 15 August 2012 Available online 21 September 2012 Keywords: Renal replacement therapy Critically ill patients Acute kidney injury Innate immunity Biocompatibility

a b s t r a c t Objective: During continuous venovenous hemofiltration (CVVH) to replace renal function in acute kidney injury (AKI), anticoagulation of the filter is routinely required. A survival benefit for citrate has been reported, possibly due to reduced proinflammatory effects of the filter (bioincompatibility). We hypothesized that the type of anticoagulation modulates the immune response to, and clearance by CVVH of interleukin-6 (IL-6) and -8 (IL-8). Methods: Three anticoagulation regimens were compared: trisodium citrate (n = 17), unfractionated heparin (n = 8) and no anticoagulation in case of bleeding tendency (n = 13). Immediately before initiation of CVVH (cellulose triacetate membrane) pre-filter blood was drawn. Thereafter, at 10, 60, 180 and 720 min, samples were collected from the pre- and postfilter blood and from ultrafiltrate. IL-6 and IL-8 were determined by ELISA. Results: High inlet levels of IL-6 and IL-8, particularly in the no anticoagulation group, were associated with non-survival. The inlet concentrations and mass rates of IL-6 and IL-8 decreased during CVVH. The course of fluxes across the filter were similar for the groups, however. Although increasing in time for IL-6 in the no anticoagulation group, mass removal and adsorption of IL-6 and IL-8 were low and did not differ among the anticoagulation groups. Conclusions: Blood to membrane contact, adsorption/clearance and anticoagulation do not increase nor attenuate high circulating levels of IL-6 and IL-8 during CVVH for AKI. This renders the hypothesis that the reported survival benefit for citrate anticoagulation is based on a reduction of bioincompatibility unlikely. Ó 2012 Elsevier Ltd. All rights reserved.

1. Introduction Acute kidney injury (AKI) and renal failure are common in critically ill patients, particularly in case of sepsis. In spite of modern renal replacement therapy (RRT), mortality remains as high as 50–60% [1]. Continuous venovenous hemofiltration (CVVH) is commonly used as RRT in the intensive care unit (ICU). It requires anticoagulation to prevent clot formation and hemofilter dysfunction. Unfractionated or low molecular weight heparin is often applied, but its use must be weighted against the associated bleeding risk and therefore anticoagulation is sometimes omitted. Alternatively,

⇑ Corresponding author. Address: Department of Nephrology, VU University Medical Center, De Boelelaan 1117, 1081 HV Amsterdam, The Netherlands. Tel.: +31 20 4442673. E-mail address: [email protected] (L. Schilder). 1043-4666/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.cyto.2012.08.015

regional anticoagulation by citrate, that binds calcium and reduces its ionized levels below 0.3 mmol/L in the filter only, can be used. Citrate may be associated with longer filter life and may improve patient survival [2–4]. Among other factors, reduced bioincompatibility of the CVVH filter may underlie these observations [5]. Indeed, the type of anticoagulation used may modulate biocompatibility. The filter may, depending on the membrane, activate mononuclear cell-derived innate immunity and cytokine release [5,6]. In vitro, anticoagulants differentially affect the innate immune response in whole blood upon stimuli such as endotoxin. However, the effects of the anticoagulation on the inflammatory response depend on models, stimulants, doses, contaminating endotoxins and proteins. In addition, results are variable across studies and do not necessarily apply to the in vivo situation. For unfractionated or low molecular weight heparin, pro- and anti-inflammatory effects are reported [7–13]. Calcium signalling may contribute to activation of NFjB and subsequent release of pro-inflammatory

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mediators like interleukin-6 (IL-6) and -8 (IL-8) from mononuclear cells. Thus calcium antagonists inhibit such responses [9,14,15]. Moreover, regional citrate anticoagulation could down regulate the release of pro-inflammatory mediators by creating an almost calcium free environment in the extracorporeal circuit. In the present study, we focus on the cytokines IL-6 and IL-8. Both cytokines play a pivotal role in innate immunity and are markedly elevated in patients suffering from sepsis particularly when complicated by AKI and in case of non-survival [16,17]. It has been suggested that the production of cytokines by monocytes is impaired in AKI and this may modify effects of CVVH on innate immunity [17,18]. A potential benefit of CVVH is the removal by convection or adsorption of harmful cytokines released from the circulation during sepsis and AKI [19–21]. However, the magnitude and significance of this potential removal is hotly debated and may depend on underlying diseases, size and baseline levels of mediators, membrane properties and other factors like the anticoagulants used [18,22–32]. For the present study, we postulated that citrate favourably modulates handling of IL-6 and IL-8 in comparison to either heparin or no anticoagulation during CVVH for AKI in critically ill patients. We also compared the handling of IL-6 and IL-8 by the filter during CVVH, which may differ due to the molecular sizes of the cytokines. 2. Patients and methods The patients for the current study were recruited from two different studies performed at our hospital. The first study started one year before the availability of a custom-made citrate-based replacement fluid. From March 2004 to September 2005, patients admitted to the ICU who developed AKI necessitating CVVH but did not receive heparin due to a high bleeding tendency, either received anticoagulant-free CVVH (n = 13) or, after becoming available in 2005, regional citrate anticoagulation (n = 10) and were prospectively followed [2]. High risk for bleeding was defined as a platelet count of less than 40  109 L, an activated partial thromboplastin time of more than 60 s, a prothrombin time of more than 2.0 international normalized ratio, a recent major bleeding, or significant active bleeding. Patients with high serum lactate levels (>5 mmol/L) were routinely treated with bicarbonate-buffered rather than lactatebuffered CVVH. Because all patients were treated according to local standards at that time, the need for informed consent was waived for this study. The second study is a randomised controlled trial starting in 2006. Patients admitted to the ICU who developed AKI necessitating RRT without a high bleeding risk, were randomised between receiving heparin as anticoagulant (n = 8), which is standard at our ICU, or the citrate solution (additional n = 7 for the citrate group). For this second study, informed consent was obtained from all study participants or their next-of-kin. Study protocols were approved by the local medical committee on ethics and performed in accordance with the Declaration of Helsinki. The patients receiving no anticoagulation are defined as Group 1 (n = 13), the patients receiving heparin are defined as Group 2 (n = 8) and finally the patients receiving citrate are defined as Group 3 (n = 17). For the present study on IL-6 and IL-8, patient inclusion and blood and ultrafiltrate samples were only taken in office hours when research staff was available. We stopped this study after inclusion of at least 8 patients per group, which was considered sufficient for detecting clinically important differences. A formal power analysis was not done for this proof of principle study. 2.1. Treatment protocol CVVH was started for acute kidney injury and ongoing hypercatabolism, diuretic-resistant volume overload, respiratory

distress, multiorgan failure, or any combination of these features, at the discretion of the treating physician and consulting nephrologist. Continuous venovenous hemofiltration was performed using a Braun Diapact hemofiltration machine (DIAPACT, B. Braun Medical, Melsungen, Germany). Vascular access was obtained by the insertion of an 11F double-lumen catheter (GAMCATH, Gambro, Hechingen, Germany) into the femoral, subclavian, or internal jugular vein. A 1.9 m2 highly permeable cellulose triacetate filter (NIPRO UF-205, Nissho Corp., Osaka, Japan; cutoff approximately 40 kDa) was used in all treatments. For bicarbonate-based CVVH, a commercially prepared buffer solution was available (HF 32 bic, Dirinco, Rosmalen, the Netherlands). For the use of citrate, the replacement fluid was custom-made, according to the protocol described before [2]. Blood flow rate was set at 180 mL/min in all groups. Replacement fluid was administered at a standard rate of 2000 mL/h in the patients receiving no anticoagulation and bicarbonate or lactate replacement fluid. Replacement rates in the citrate groups were set at 2400 mL/h. In the heparin group, replacement rates were set at 2000–2400 mL/h. Replacement fluids were delivered in predilutional mode. Patients receiving citrate-based therapy had a separate intravenous infusion with calciumglubionate (Calcium SandozÒ containing 0.225 mmol/mL calcium, Novartis Consumer Health, Breda, The Netherlands). Calcium administration was adapted to concentrations of ionized calcium by a specially designed algorithm, as described before [2]. 2.2. Study protocol At inclusion, demographic variables were recorded and reason of ICU admission. Assessment of disease severity on ICU admission was done according to the simplified acute physiology score II (SAPS II) and the sequential organ failure assessment score (SOFA). We collected systemic inflammatory response syndrome (SIRS) criteria: body temperature >38 °C; a heart rate of >90 beats/min; a respiratory rate of >20 breaths/min or mechanically ventilation or white blood cells of <4.0  109/L or >12.0  109/L. When SIRS (at least 2 of the criteria mentioned) and an infection were present (either clinically suspected or microbiologically confirmed), patients were classified as having sepsis. Routine laboratory measurements including coagulation variables and renal data were collected. Mortality is mortality in the ICU. Venous blood samples were collected from each patient before the initiation of CVVH from the afferent port of the CVVH. After initiation of CVVH (first filter), samples were collected at 10, 60, 180 and 720–1440 min, or until filter termination. Prefilter blood was drawn before addition of replacement fluids. Postfilter blood and ultrafiltrate were drawn from the appropriate ports. Samples were collected in standard Vacutainer tubes (Becton, Dickinson and Company, Erembodegem, Belgium) containing ethylenediaminetetraacetic acid (EDTA), benzamidine and soybean trypsin inhibitor. In all patients a zero fluid balance was achieved during the time points at which blood samples were drawn. Samples were centrifuged at 1300g for 10 min at 4 °C and stored at 80 °C until assayed. 2.3. Measurements and calculations IL-6 and IL-8 were measured by enzyme-linked immunosorbent assay employing a monoclonal capture antibody to polyclonal detecting antibody with a peroxidase-detecting system. For IL-6 (Biosource International, Nivelles, Belgium, Human IL-6 CytoSet™, CHC1263), the lower detection limit was 4 pg/mL. Plasma samples and ultrafiltrate were measured in separate assays with IL-6 standards prepared in a 0.5% bovine albumin serum phosphate buffered saline Tween solution or fresh ultrafiltrate as appropriate. For IL-8, the detection limit was 3 pg/mL (Invitrogen Corporation, Nivelles, Belgium, Human IL-8 CytoSet™ CHC1303) and standards

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were prepared as described above. All measurements were done according to the protocols provided by the manufacturer. Each sample was run in duplicate and the mean cytokine concentration was calculated. Formulas used to calculate the study variables of interest are explained in Table 1.

Table 1 Formulas used. Qi = Qb  (1 Ht), Qo = Qi Mi = Qi  C Mo = Qo  Co Muf = Quf  Cuf Mtr = Mi – Mo Mad = Mtr Muf Ci = C  Qi/(Qi + RF) SC = 2  Cuf/(Ci + Co)

2.4. Statistical analysis

Abbreviations: C = concentration in inlet plasma before addition of replacement fluid (pg/mL), Co = concentration in outlet plasma (pg/mL), Cuf = concentration in ultrafiltrate (pg/mL), Qb = inlet blood flow rate (mL/min), Qi = inlet plasma flow rate (mL/ min), Qo = outlet plasma flow rate (mL/min), Quf = ultrafiltration flow rate (mL/min), Mi = mass inlet rate (pg/min), Mo = mass outlet rate (pg/min), Muf = mass ultrafiltration rate (pg/min), Mtr = mass removal rate (in ng/min), Mad = mass adsorption rate (in ng/min), Ci = concentration in inlet plasma after addition of replacement fluid (pg/mL), RF = replacement flow, SC = Sieving coefficient.

Due to the non-normal distributions, data are summarised as median and range. Where appropriate for analysis, they were log-transformed to achieve normal distributions. The values for total mass removal and adsorption rates were ranked, since some of the values were negative and could not be log-transformed. Since there are no baseline differences between the two different citrate groups (citrate vs. no-anticoagulation and citrate vs. heparin), the citrate data were pooled (Group 3 n = 17). Group differences were

Table 2 Baseline characteristics no anticoagulation vs. heparin vs. citrate.

Age (years) Sex (male) Weight (kg) Admission category: Respiratory failure Circulatory failure Trauma Post-resuscitation Surgical Acute renal failure Sepsis SAPS II SOFA Mechanical ventilation Norepinephrine (lg/kg/min) ICU stay (days) Mortality in ICU

No anticoagulation N = 13

Heparin N = 8

Citrate N = 17

P

70 (34–84) 7 (54) 70 (50–100)

57 (23–81) 6 (75) 73.5 (55–135)

61 (32–79) 11 (65) 75 (60–110)

0.41 0.61 0.50 0.27

2 (15) 3 (23) 2 (15) 2 (15) 4 (31) 0 5 (39) 75 (43–112) 14 (7–21) 12 (93) 0.72 (0–2.8) 5 (2–82) 9 (69)

5 (63) 1 (13) 0 0 1 (13) 1 (13) 5 (63) 47 (37–77) 11 (8–15) 5 (63) 0.15 (0–1.2) 19.5 (4–48) 1 (13)

5 (29) 2 (12) 1 (6) 2 (12) 7 (41) 0 7 (41) 52 (32–86) 13 (8–18) 7 (41) 0.12 (0–1.1) 20 (3–81) 5 (29)

0.52 <0.001 0.02 0.74 0.20 0.11 0.02

Median (range) or number (percentage). Abbreviations: SAPS II = simplified acute physiology score II, SOFA = sequential organ failure assessment score, ICU = intensive care unit.

Table 3 Biochemical data. A

No anticoagulation n = 13

Heparin n = 8

Citrate n = 17

P

Leukocytes (109/L) 0h 12 h 24 h

7.8 (1–17.2) 8.1 (1.2–24.8) 10.1 (1.10–26.2)

11.6 (6.5–19.7) 12.4 (5.2–24.2) 12.8 (5.6–25.9)

11.4 (1.3–25.7) 11.6 (0.8–116) 12.5 (2.3–114)

0.33 0.39 0.83

Thrombocytes (109/L) 0h 12 h 24 h

65 (22–777) 72 (8–131) 59 (13–148)

167 (44–332) 140 (45–203) 117 (27–239)

117 (35–332) 100 (18–335) 103 (5–349)

0.03 0.04 0.02

aPTT (s) 0h 12 h 24 h

48 (39–72) 46 (41–118) 49 (37–152)

40 (36–81) 62 (40–125) 61 (46–150)

48 (34–94) 46 (33–68) 42 (32–47)

0.56 0.03 0.04

Creatinine (lmol/L) 0h 24 h

249 (100–410) 206 (140–250)

420 (156–626) 284 (125–463)

311 (47–626) 236 (46–441)

0.02 0.04

Urea (mmol/L) 0h 24 h

21 (6–48) 17 (6–34)

32 (12–97) 17 (11–102)

20 (6–41) 13 (6–26)

0.18 0.15

Diuresis during First filter (mL/h) Filter survival (h)

11.5 (0–83) 10 (3–66)

27 (0–45) 16.5 (1–70)

16 (0–113) 30 (5–74)

0.46 0.07

Median (range). P-value by Krukall–Wallis test. aPTT = activated partial prothrombin time.

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Fig. 1. Mass inlet rate (Mi), mass outlet rate (Mo) and mass ultrafiltration rate (Muf) of IL-6 and IL-8 over time during CVVH (median and interquartile range). Symbols: d no anticoagulation j heparin N citrate. A IL-6. Mi: decreases over time irrespective of anticoagulation (P = 0.049), Muf: no anticoagulation > other groups (P = 0.027), Muf: decreases over time irrespective of anticoagulation (P < 0.001). B IL-8. Mi: no anticoagulation > other groups (P = 0.036), Mi: decreases over time (P = 0.004) particularly for no anticoagulation (P = 0.029), Mo: no anticoagulation > other groups (P = 0.023), Mo: decreases over time irrespective of anticoagulation (P = 0.001), Muf: no anticoagulation > other groups (P = 0.031).

calculated by use of the Kruskal–Wallis test or X2 test. To compare the differences according to anticoagulation regimens we used generalized estimating equations (GEEs). The focus of GEE is on estimating differences between groups and times, and their first order interaction, i.e. differences between groups over time. A Spearman correlation coefficient was used to express relations. A forward multiple logistic regression was used to evaluate differences among groups for mortality when adjusted for SAPS II scores. A p-value <0.05 was considered statistically significant. 3. Results Patient characteristics are presented in Table 2. Thirteen patients were treated without anticoagulation (Group 1), 8 patients received unfractionated heparin (Group 2) and 17 patients received citrate for anticoagulation (Group 3). Group 1 patients had a higher SAPS II score than the other groups. In Group 2, the activated partial thromboplastin times were higher than in the other groups. The creatinine levels at baseline were lower in Group 1 than in the other groups. Filter life times did not differ among groups (see Table 3).

3.1. Interleukins Prior to CVVH, circulating IL-6 was detectable in all patients and the inlet concentration decreased over time during CVVH (P = 0.048, data not shown), as did the mass inlet rate (P = 0.049, Fig. 1), without group differences. Outlet concentrations and mass rates did not differ among groups or times. Ultrafiltrate concentrations and mass rates were higher in Group 1 (P = 0.027) and decreased over time (P < 0.001) without group differences. The total mass removal rate of IL-6 was somewhat higher for Group 1 than the other groups (P = 0.041) and this difference increased over time (P = 0.015, Fig. 2), whereas mass adsorption rate increased in Group 1 over time, as compared to the other groups (P = 0.011). Prior to CVVH, circulating IL-8 was detectable in all patients and the inlet concentration was higher in Group 1 (P = 0.038) and decreased in time (P = 0.005), more than in the other groups (P = 0.032, data not shown). The mass inlet rate was highest in Group 1 (P = 0.036) and decreased over time (P = 0.004), more than in the other groups (P = 0.029, Fig. 2). The outlet concentration and mass rate was highest in Group 1 (P = 0.023) and decreased over time (P = 0.001) but groups did not differ in this respect. The ultra-

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Fig. 2. Total mass removal rate (Mtr), mass adsorption rate (Mad) and sieving coefficient (SC) of IL-6 and IL-8 over time during CVVH (median and interquartile range). Symbols: d no anticoagulation j heparin N citrate (A) IL-6. Mtr: no anticoagulation > other groups (P = 0.041), particularly in time (P = 0.015), Mad: no anticoagulation > other groups in time (P = 0.011), SC: decreases over time irrespective of anticoagulation (P < 0.001). (B) IL-8. Mad: increases over time irrespective of anticoagulation (P = 0.014), SC: decreases more over time for IL-6 than IL-8 (P < 0.001).

filtration concentrations and rates were also highest (P = 0.031 or lower, Fig. 1) in Group 1 but did not decrease over time in any group. The mass adsorption rate increased over time (P = 0.014), without differences between groups (P = 0.058), and total mass removal rates of IL-8 did not differ among groups, nor changed over time. Hence, there was no statistically significant net (>0) mass adsorption or removal of either IL-6 or IL-8 in any group (Fig. 2).

3.2. Interleukin 6 vs interleukin 8 Inlet concentrations of IL-8 related to those of IL-6 (rs = 0.61, P < 0.001), for all time points together (n = 172 observations). However, the sieving coefficient of IL-6 was lower than that of IL-8 (P = 0.004) and only the former decreased over time (P < 0.001). Calculated from sieving coefficient  Quf, the clearance of IL-6 decreased from about 11–5 mL/min from 10 to 720 min, irrespective of anticoagulation regimens. In contrast, the clearance of IL-8 remained at about 15 mL/min in the groups. The sieving coefficient

of IL-6 was >1 in 14 of 132 observations and of IL8 in 18 of 136 observations. 3.3. Mortality Of the 38 patients in this study, 15 died in the ICU. The crude mortality rate in Group 1 was highest. Logistic regression on the mortality adjusted for SAPS II scores at admission showed no differences between the Groups (P = 0.24). Inlet concentrations of IL-6 and IL-8 over time were higher in non-survivors than survivors (P = 0.001), irrespective of anticoagulant regimens. Sepsis (n = 17) and non-sepsis (n = 21) patients did not differ in this respect. 4. Discussion This proof of principle study suggests that prognostically important IL-6 and IL-8 concentrations and fluxes across CVVH

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filters in critically ill patients with AKI are not affected by production or removal by adsorption or clearance of cytokines by the filters or by the anticoagulation regimens applied. Hence, citrate may not be superior to heparin anticoagulation in this respect. The patients in Group 1 did not receive anticoagulation because of a bleeding tendency and, indeed, were more severely ill than those in the other groups. This explains the higher cytokine levels, although less hemodilution by relatively low replacement fluid rates may have contributed. For IL-6, the ultrafiltrate concentration and mass ultrafiltration, adsorption and removal rates were highest in the course of CVVH in the absence of anticoagulation, probably because of a tendency for high inlet concentration and mass rate, so that outlet concentrations and mass rates did not differ among groups. Also, the higher mass inlet was probably responsible for higher IL-8 mass ultrafiltrate and outlet levels in the no anticoagulation group, so that the mass adsorption and removal rates did not differ from those in the anticoagulated groups. However, ultrafiltrate clearance, mass adsorption and removal rates were low and unlikely to have affected the patients plasma (inlet) levels of both cytokines (Figs. 1 and 2). The sieving coefficient was higher for IL-8 than IL-6, in agreement with a smaller size of IL-8 (molecular weight 8 kDa vs 21– 24 kDa for IL-6 [33]). The sieving coefficient for both interleukins exceeded unity in some cases, indicating production of the interleukins in the extracorporeal circuit. For IL-8, red blood cells are known to bind IL-8 and this binding is rapidly reversible [34]. Contact of red blood cells with the filter may release IL-8 thereby contributing to a high sieving coefficient of IL-8. In spite of greater sieving of IL-8, total mass removal rates of in the filter did not differ between the cytokines, even though they were ultrafiltrated to some extent. The reason for a decrease in IL-6 sieving and clearance over time is possibly concentration polarization and secondary layer formation by proteins. The decrease was not associated with any change in mass adsorption and removal rates, and thereby unlikely to account for the decrease in IL-6 patient’s plasma (inlet) concentrations. Hence, the decrease in IL-6 with time was probably caused by less production in the patients. The decrease of IL-8 levels over time was highest for the no anticoagulation group, probably due to higher plasma levels at baseline. However, the clearance of IL-8 was low (15 mL/min) and remained relatively unchanged during the study period. We cannot rule out, however, that the (low) clearance of IL-8 by CVVH somewhat contributed to the decreasing levels of IL-8 at inlet over time, particularly in the no anticoagulation group. Our data disagree with some studies, suggesting significant adsorption/removal of IL-6/IL-8 by CVVH filters (at comparable membranes, anticoagulation and ultrafiltration rates) resulting in a fall in patient’s blood levels, whereas our study agrees with other studies denying this effect [18,22–29,30–32]. Moreover, the type of anticoagulation did not appear to affect these study variables. There was no indication of a pro-inflammatory effect of heparin during blood membrane contact in the filter, as compared to no anticoagulation when taking the higher baseline levels in the latter into account. The apparent lack of pro-inflammatory properties of heparin are in agreement with in vitro studies [8,10,11,13]. Conversely, in the citrate group, there was no sign of any modulation of adsorption, removal and thus of inlet cytokine levels, and this does therefore not explain the survival benefit of citrate anticoagulation reported by others [3,4]. There was no significant difference in the survival time of the first filter between our groups, but the citrate group tended to have better first filter survival times. Inlet levels in non-survivors were higher than in survivors, irrespective of sepsis, CVVH and anticoagulants. This is in concordance with previous studies on AKI [16,17] and underscores the rationale of our study to evaluate the effects of anticoagulation during CVVH on the handling of IL-6 and IL-8. The mortality rates when adjusted

for disease severity, however, were similar for the groups, thereby arguing against influence of less clot formation in the filter on mortality. Limitations of the present study include the relatively small size of groups, rendering differences in mortality rates less meaningful, and absence of randomisation in some of the groups. At baseline there were differences between the groups, by virtue of the study design. However, we corrected for baseline levels by analyzing groups differences in the course of study variables over time. Apart from IL-6 and IL-8, other markers of biocompatibility such as other cytokines, complement activation and degranulation of neutrophils must be studied to evaluate the influence of anticoagulation on biocompatibility of membranes in filters used for CVVH [5]. The mass removal and adsorption rates represent net rates since any production in the filter could be offset by adsorption. Finally, we cannot exclude that synthetic membranes are more biocompatible and adsorb more cytokines than membranes composed of cellulose [5,6,21,23,25]. We do not have a group on low molecular weight heparin, which is also used for filter anticoagulation [4]. We cannot exclude earlier start of CVVH in Group 1 than in the other groups, since initial creatinine was lower, but this does not invalidate our conclusions. We have evaluated the course of the study variables for a period up to 720 min only and do not exclude differences beyond that time interval. In conclusion, blood to membrane contact, adsorption/clearance and anticoagulation do not increase nor attenuate high circulating IL-6 and IL-8 levels associated with non-survival during CVVH for AKI. This renders the hypothesis that the reported survival benefit for citrate anticoagulation is based on a reduction of bioincompatibility concerning IL-6 and IL-8 unlikely. Conversely, the prognostic value of the cytokines is not abolished by filter handling and (type of) anticoagulation during CVVH.

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