1 Department of Nephrology, CharitéHumboldt University of Berlin, 2 Laboratory Medicine and Pathobiochemistry, CharitéHumboldt University of Berlin, 3 Institute for Medical Biometrics, CharitéHumboldt University of Berlin and 4 Gambro Corporate Research, Hechingen, Germany
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Abstract |
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Methods. Blood from healthy volunteers (n=15) was incubated for 4 h with 1 mg of endotoxin and then circulated through a closed extracorporeal circuit. A newly developed polyflux haemofilter (P2SX) was used. Haemofiltration, haemodialysis and albumin dialysis were tested. IL-1ra (17 kDa), interleukin-6 (IL-6) (28 kDa), tumour necrosis factor alpha (TNF-) (51 kDa), albumin (64 kDa), creatinkinase (CK) (80 kDa) and IgG (140 kDa) were measured in blood and filtrates prior to the initiation and after 5 min, 1, 2 and 4 h.
Results. Haemofiltration was superior to haemodialysis in the clearance capacity of all substances when applied in the 1 l/h ultrafiltration mode. Increasing the ultrafiltration rate/dialysate flow from 1 to 3 l/h led to a significant increase in cytokine clearances (P<0.001). At 3 l/h the differences between haemofiltration and haemodialysis vanished and both techniques achieved comparable cytokine clearances. Median clearance values ranged between 25 and 54 ml/min for interleukin-1 receptor antagonist (IL-1ra), 23 and 42 ml/min for IL-6 and 15 and 28 ml/min for TNF-. Albumin loss was highest in the haemofiltration group with albumin clearances ranging between 7 and 13 ml/min. Using diffusion instead of convection significantly reduced the loss of albumin (P<0.01 for 1 l/h, P<0.05 for 3 l/h). Albumin dialysis was able to completely inhibit albumin loss but cytokine clearance capacity was limited.
Conclusions. High cut-off haemofilters achieve high clearances for inflammatory IL-6 and TNF-. Due to the high protein loss in haemofiltration, dialysis in combination with balanced protein substitution seems to be a suitable approach for clinical trials.
Keywords: albumin dialysis; haemodialysis; haemofiltration; high cut-off haemofilter; TNF- elimination
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Introduction |
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In the course of sepsis-induced acute renal failure continuous renal replacement therapies (CRRT) are established procedures for uraemia and volume control [6,7]. Apart from representing a valuable renal replacement therapy (RRT), it has been hypothesized that CRRT may modulate the inflammatory response by non-specific extracorporeal removal of circulating cytokines while preserving their local tissue effects which are necessary to resolve infection. However, due to the structure of commercially available membranes the elimination capacity for inflammatory cytokines is limited [6,8]. We already reported about a newly developed high cut-off haemofiltration membrane which allows a good elimination of molecules up to a molecular weight of 3040 kDa such as interleukin-6 (IL-6). However, the TNF-
elimination capacity was also rather poor [9].
This in vitro study analyses a second generation high cut-off haemofilter. This haemofilter is characterized by an increased pore size which should also allow a substantial removal of TNF-. This study was designed to target two goals. First, analysing the elimination capacity for molecules of different size including interleukin-1 receptor antagonist (IL-1ra) (17 kDa), IL-6 (28 kDa), TNF-
(51 kDa), albumin (64 kDa), creatinkinase (CK) (80 kDa) and IgG (140 kDa). Secondly, to develop strategies for reducing the expected loss of albumin. Different RRT modalities were tested including haemofiltration, haemodialysis and albumin dialysis.
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Materials and methods |
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Blood preparation and incubation
300 ml of fresh whole blood was drawn from volunteers into pre-heparinized (25 000 U Heparin, Liquemin® N 25 000; Hoffmann-La Roche AG, Grenzach-Wyhlen, Germany) sterile blood collection bags. Thereafter 1 mg of endotoxin was added (lyophilized powder of lipopolysaccharide from Escherichia coli serotype 055:B5, L3012 product code; Sigma Aldrich, Munich, Germany). The bags were then placed onto a slow rotating shaker (KS 125 basic; IKA Labortechnik, Staufen, Germany) and incubated at 39°C (PCO2 5%) for 4 h. Finally, the blood was thinned with 300 ml of 0.9% saline solution (B. Braun Schiwa GmbH and Co. KG, Glandorf, Germany) to become the blood reservoir for the experiments.
Extracorporeal renal replacement circuit
All experiments were performed with a roller blood pump (BM-25; Edwards Lifesciences GmbH, Unterschleissheim, Germany) using a prototype of a new polyflux high cut-off hollow fibre membrane (Polyflux P2SX; Gambro Dialysatoren, Hechingen, Germany) (Figure 1). Inside diameter of the fibre is 200 µm, wall thickness 40 µm. The effective surface area is 1.27 m2. The membrane is characterized by an increased pore diameter with an industrially estimated cut-off point of
100 kDa.
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Renal replacement procedures
Haemofiltration, haemodialysis, 4% human albumin dialysis and 2% human albumin dialysis were tested in this ex vivo study. Figure 2 shows the extracorporeal circuit for haemofiltration and haemodialysis.
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Next, the circuit was changed first to a 3 l/h dialysis mode and then switch to a 3 l/h haemofiltration mode. Blood and filtrates/dialysates were sampled as described above. Then ultrafiltration/dialysate flow was ceased. Blood flow was maintained across the circuit and the same sequences of sampling were repeated at 1, 2 and 4 h (Figure 3).
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Experiments were started with haemodialysis at a flow rate of 1 l/h. Then flow rate was increased to 3 l/h. Blood and dialysate samples were taken at 5 min, 1, 2 and 4 h as described above.
Laboratory analysis
Blood samples were collected in EDTA tubes, centrifuged at 3000 r.p.m. for 5 min and then the supernatant was removed. The supernatants and the dialysate and filtrate samples were stored in sterile silicone Eppendorf tubes at -70°C until assays were performed for IL-1ra, IL-6, TNF-, albumin, CK and IgG. IL-1ra, IL-6 and TNF-
concentrations were analysed by enzyme-linked immunosorbent assays (ELISA; BD PharMingen, Heidelberg, Germany).
The intra-assay variabilities of the assays were <5% for IL-1ra, 5% for IL-6, 3% for TNF-. The inter-assay variabilities were <10% for IL-1ra, 8% for IL-6, 6% for TNF-
.
The protein and albumin concentration as well as the biochemical data in blood and filtrate were measured by photometric pyrogallol-red reaction (Analyticon: Kit No. 911) and by an immun-turbidimetric kit (Roche-Tinaquant: Kit No. 1203622), respectively.
Calculations
Several calculations were performed for the purpose of this study.
Sieving coefficient (SC), clearances and adsorption for haemofiltration were calculated as follows:
| (001) |
| (002) |
| (003) |
For the dialysis procedures the following equations were used:
| (004) |
Statistical analyses
The data are expressed as medians with with interquartile range (25th to 75th percentile). Graphical presentation was done with box plots. Procedures of non-parametric analysis of variance for longitudinal data in factorial design [10] were used to detect differences in several RRT modalities, differences over the time and/or over flow rates and their corresponding interactions for the tested substances. A P-value <0.05 was considered significant. In case of paired analysis the significance level was adjusted by the HolmBonferroni procedure. Statistical analysis was carried out with the SAS System (Release 8.1).
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Results |
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Sieving coefficient (SC)
The sieving coefficients at 1 and 3 l/h for IL-1ra (17 kDa), IL-6 (28 kDa), TNF- (51 kDa), CK (80 kDa), albumin (64 kDa), IgG (140 kDa) are given in Table 1
. Increasing the ultrafiltrate volume from 1 to 3 l/h led to a striking decline in the SC for IL-6 of
3040% (P<0.001). Similar findings were made for TNF-
(P<0.001), albumin (P<0.001), CK (P<0.001) and IgG (P<0.001). Although for IL-1ra a decline was observed values did not reach statistical significance. SCs of all substances except for IL-1ra decreased significantly over the 4 h observation period. This was particularly pronounced in the 3 l/h mode. The decline in the 3 l/h mode (baseline versus 4 h values) was
10% for IL-1ra,
40% for IL-6,
25% for TNF-
and
60% for albumin (Table 1
).
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Clearances (Cl)
Haemofiltration achieved exceptionally high clearances for IL-1ra, IL-6 and TNF-. The clearance capacity declined for bigger molecules (albumin, CK, IgG) (Table 2
).
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Implying dialysis and using diffusion instead of convection as the elimination technique still revealed high cytokine clearances. Again, increasing the volume from 1 to 3 l/h led to a significant increase in the clearances of IL-1ra, IL-6 and TNF- (P<0.001). Significant differences were also found for albumin (P<0.01), CK and IgG (P<0.05) (Table 2
).
Comparing haemofiltration with haemodialysis in the 1 l/h mode revealed a clear superiority for the haemofiltration mode in the clearance capacity for TNF- (P<0.005), albumin (P<0.01), CK (P<0.05) and IgG (P<0.05). No difference between haemodialysis and haemofiltration was found for IL-1ra and IL-6. These advantages in favour for haemofiltration vanished almost completely when the procedures were switched to the 3 l/h mode. Although haemofiltration still achieved the highest peak clearances for IL-1ra, IL-6 and TNF-
, it did not reach statistical significance. Only albumin clearance still remained statistically higher in the haemofiltration group (P<0.05) (Table 2
). Figure 4
shows box plot presentations of pooled data obtained from the 1 and 3 l/h haemofiltration and haemodialysis procedure.
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Albumin dialysis led to a back-filtration of albumin from the dialysate into the blood reservoir. While the blood albumin concentration decreased steadily during the haemofiltration and dialysis procedure, during albumin dialysis the blood albumin concentration reached a steady state at 2 or 4 g/dl depending on the albumin concentration in the dialysate.
Adsorption
We found no adsorption for IL-1ra, IL-6 or TNF-. This was true for all tested RRT modalities. Albumin adsorption was observed during albumin dialysis but not during haemofiltration or haemodialysis. Absorption during 4% albumin dialysis was 0.53±0.07 g at 5 min after initiation of therapy and declined rapidly to 0.07±0.05 g at 4 h (P<0.01). No significant differences were observed between the 1 and the 3 l/h mode. In the 2% albumin dialysis group, corresponding values were 0.31±0.07 g at 5 min and 0.02±0.02 g at 4 h (P<0.01), respectively.
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Discussion |
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We recently studied the impact of such a new high cut-off haemofilter in septic patients with multiorgan failure and demonstrated that it is not only effective for uraemia and volume control but that it allows also a substantial filtration of inflammatory IL-6 [9]. Unfortunately, the filtration capacity for TNF- was limited and did not differ from that of commercially available membranes. These results were reproduced by Uchino et al. [12] in an in vitro approach. For IL-6, they found an in vitro clearance of
15 ml/min at 1 l/h and 37 ml/min at 6 l/h of ultrafiltration. Similar to our results, they observed no clearance for TNF-
.
Based on our clinical data we developed a second-generation membrane with a superior cut-off point that should enable us to better eliminate molecules in the molecular weight range of TNF-. Apart from possibly beneficial effects, high cut-off haemofiltration endures substantial risks such as the loss of proteins like albumin or coagulation factors. Severe albumin depletion can promote circulatory dysfunction by reducing the effective arterial blood volume and by activating the vasoconstrictor systems. Depletion of coagulation factors may enhance haemostasiological disorders which are already present in sepsis. In this in vitro approach we tried to detect a feasible RRT modality, which combines high cytokine clearances while keeping protein loss the minimal possible.
Haemofiltration achieved the highest peak clearances for all tested substances. Increasing the ultrafiltration volume led to an increase in the clearance capacity. The extent of the increase depended on the molecular size of the substance and was highest in smaller molecules. We found a 100% increase for IL-1ra (17 kDa), a 60% increase for IL-6 (28 kDa) and a 50% increase for TNF- (51 kDa). No significant increase was observed for albumin (64 kDa), CK (80 kDa) or IgG (140 kDa). Median TNF-
clearance at 3 l/h of ultrafiltration ranged between 21 and 29 ml/min with the highest values achieved in the initial phase of haemofiltration. Clearances for IL-6 were even higher and ranged between 24 and 42 ml/min in the 3 l/h mode. However, these encouraging results were narrowed by a substantial loss of proteins through the haemofilter. The sieving coefficient for albumin (at 4 h after initiation of therapy) was 10% in the 3 l/h mode associated with an albumin clearance of 7 ml/min. In a clinical setting this would be associated with an albumin loss of
9 g/h. Although protein loss declines over time as a consequence of filter clotting the expected protein loss makes large pore haemofiltrationin its present formnot suitable for clinical use.
Applying dialysis and using diffusion instead of convection as elimination modality revealed comparable cytokine clearances while albumin loss could be reduced. Albumin clearances at 4 h were 1.4 ml/min in the 1 l/h and 4.5 ml/min in the 3 l/h mode corresponding to a calculated protein loss of 2 and 5 g/h, respectively.
Albumin dialysis, either applied in a 2% or 4% albumin solution, was able to reduce albumin loss by albumin back-filtration from the dialysate into the blood circuit. Unfortunately albumin dialysis reduced significantly the clearance capacity of IL-6 and TNF- when applied in the 3 l/h mode. One reason for this phenomenon could be a reduction in the effective pore size of the membrane by albumin deposition on both sides of the haemofilter. Albumin dialysis has proven to exert beneficial effects in various types of liver failure. To our knowledge, albumin dialysis has never been applied in sepsis, although liver failure often occurs in the course of a septic multiorgan failure syndrome. High levels of bilirubin, bile salts and bile acids have been shown to exert toxic effects not only on liver [16] but also on kidney function [17,18]. In addition, albumin dialysis has been proven to increase systemic vascular resistance and mean arterial pressure [19]. These beneficial effects on the cardiovascular system are thought to be related to a removal of albumin-bound vasoactive substances, primarily nitric oxide [20,21]. Although albumin dialysis did not achieve high cytokine clearances in our setting, it might exert beneficial effects by eliminating mediators other than cytokines. A limitation of this procedure could be the high costs. Albumin dialysis is an expensive procedure. A 2% albumin solution applied in a 3 l/h dialysate mode would cost 75 Euro per hour (assuming a price of 25 Euro per 100 ml of 20% human albumin). In addition, prices for haemofilter, haemofiltration replacement fluid and circuit system have also to be calculated. A different approach to compensate for the protein loss in high cut-off RRT is balanced albumin or fresh frozen plasma substitution. This would allow effective cytokine clearances while preventing the undesired side effects of protein depletion. Calculating a protein loss of
5 g/h in a 3 l/h dialysis mode, makes a substitution of 200 ml of 20% human albumin or 300 ml of FFP (assuming a minimum protein content of 60 g/l) every 4 h necessary. Although supplementation with albumin has not yet been demonstrated to have measurable therapeutic effects in intensive care patients [22], it has a completely different value in this setting where albumin loss is home made. The implementation of FFP should be favoured as this may compensate for the loss of coagulation factors. Again costs are high. Fresh frozen plasma in Germany costs
40 Euro (200 ml). A 24 h high cut-off RRT would therefore cost
360 Euro.
Continuous RRT is an unspecific elimination modality which eliminates both pro-inflammatory cytokines but also anti-inflammatory cytokines as shown with IL-1ra. We cannot comment on the clinical consequences of an elimination of anti-inflammatory mediators in sepsis. Theoretically, it might be possible, that systemic inflammation gets triggered, further aggravating the clinical condition of the patient. We do not believe that this scenario will really take place. In sepsis the negative effects of the pro-inflammatory mediators clearly dominate over the beneficial effects of anti-inflammatory mediators. Being able to reduce the burden of these inflammatory cytokines should hopefully translate in an amelioration of systemic inflammation. However, final evidence is lacking and further studies are necessary to evaluate this question. Our results are based on in vitro findings and have thus be interpreted with caution. We cannot comment on how high cut-off RRT will be tolerated haemodynamically and whether our in vitro cytokine clearances can be achieved in vivo. Another serious problem that should not be underestimated is drug interference. Many drugs are protein bound and thus can be eliminated to some extent. Drug dosage adjustment will be necessary for protein-bound drugs such as fentanyl (84% albumin binding), midazolam (>90%) and many other drugs including antibiotics and antimycotics. In conclusion, our results demonstrate that the P2SX high cut-off haemofilter achieves high clearances for pro-inflammatory TNF- and IL-6. However, anti-inflammatory mediators (IL-1ra) are also eliminated and a substantial loss of albumin occurs. Using diffusion instead of convection significantly reduces the loss of protein while maintaining high cytokine clearances. Albumin dialysis prevents albumin loss but cytokine clearance capacity is limited. In our view, high cut-off haemodialysis applied for a short time period in a 3 l/h mode in combination with a balanced substitution of FFP might be a suitable clinical approach in the treatment of septic patients, when TNF-
clearances is desired.
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Acknowledgments |
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Conflict of interest statement. H.G. is an employee of Gambro.
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Notes |
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References |
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