Elimination of middle-sized uremic solutes with high-flux and high-cut-off membranes: a randomized in vivo study.

The elimination of substances between 10 and 50 kDa by conventional high-flux membranes is not satisfactory. We investigated in vivo the elimination of middle-sized uremic solutes by conventional polyflux (PF) and modified high-cut-off (HCO) membranes. All 12 patients underwent four treatments, two with the HCO dialyzer and two with the PF dialyzer, each in either a haemodialysis (HD) or haemodiafiltration (HDF) mode. The reduction ratio of urea, creatinine, ß2-microglobulin (ß2M), leptin, soluble TNF-RI, complement factor D, IL-6, sIL-6 receptor, advanced glycation end-products (AGEs) and albumin was determined. In addition, the amount removed was determined in the dialysate for ß2M, complement factor D, AGEs and albumin. Treatment with HCO removed ß2M, sTNF-RI, factor D, and high molecular AGE significantly better than conventional high-flux membranes. The albumin loss was higher when using HCO membranes. HCO membranes are a promising approach to improve removal of uremic toxins not affected by conventional high-flux membranes.

Renal toxicity mediated by glucose degradation products in a rat model of advanced renal failure.

BACKGROUND: In peritoneal dialysis (PD) residual renal function contributes to improved patient survival and quality of life. Glucose degradation products (GDP) generated by heat sterilization of PD fluids do not only impair the peritoneal membrane, but also appear in the systemic circulation with the potential for organ toxicity. Here we show that in a rat model of advanced renal failure, GDP affect the structure and function of the remnant kidney. MATERIALS AND METHODS: Sprague-Dawley rats were randomly assigned to a two stage subtotal nephrectomy (SNX) or sham operation and were left untreated for 3 weeks. The SNX + GDP group continuously received chemically defined GDP intravenously for 4 weeks; the SNX and the sham-operated rats remained without GDP. The complete follow-up for all groups was 7 weeks postoperatively. We analysed renal damage using urinary albumin excretion as well as a semiquantitative score for glomerulosclerosis and tubulointerstitial damage, as well as for immunohistochemical analyses. RESULTS: The SNX + GDP rats developed significantly more albuminuria and showed a significantly higher score of glomerulosclerosis index (GSI) and tubulointerstitial damage index (TII) as compared to SNX or control rats. In the SNX + GDP group the expression of carboxymethyllysine and methylglyoxal was significantly higher in the tubulointerstitium and the glomeruli compared to the SNX rats. Caspase 3 staining and TUNEL assay were more pronounced in the tubulointerstitium and the glomeruli of the SNX + GDP group. In SNX + GDP animals, the expression of the slit diaphragm protein nephrin, was significantly lower compared to SNX or control animals. CONCLUSION: In summary, our data suggests that GDP can significantly advance chronic kidney disease and argues that PD solutions containing high GDP might deteriorate residual renal function in PD.

Increased reduction of dimethylarginines and lowered interdialytic blood pressure by the use of biocompatible membranes.

Hypertension contributes to cardiac and cerebrovascular complications in HD patients. Endogenous inhibitors of nitric oxide synthase accumulate in renal failure and may interfere with the regulation of vascular tone. We investigated the elimination of asymmetric dimethylarginine (ADMA) by using biocompatible Polyamide Strade mark membranes in low-flux (Polyflux 6L) or high-flux (Polyflux 14S) hemodialysis or hemodiafiltration (HDF) compared with hemodialysis with cellulosic membranes. Removal rates for ADMA, symmetric dimethylarginine (SDMA), and beta2-microglobulin significantly increased in HDF. The plasma total amino acid concentration and the arginine/ADMA ratio increased, and the mean 24-hour blood pressure decreased during the study. In a second study, we investigated whether plasma amino acids and interdialytic blood pressure are influenced by the use of a biocompatible membrane and HDF. Seventeen end-stage renal disease patients were treated for six weeks with hemodialysis using cellulosic membranes, six weeks with low-flux hemodialysis using Polyflux 6L, and six weeks with HDF using Polyflux 14S. Only in the diabetic patients were the hemoglobin concentration (from 10.6 +/- 1.5 to 11.9 +/- 0.6 mg/dL) and hematocrit (from 33.6 +/- 1.9 to 36.2 +/- 1.5%) increased significantly, whereas the mean 24-hour systolic blood pressure decreased (from 154 +/- 22 to 129 +/- 18 mm Hg). No significant changes were observed in nondiabetic patients. We conclude that primarily diabetic patients seem to benefit from the use of biocompatible membranes--most in HDF--after a period of six weeks. The regulation of nitric oxide pathways by ADMA removal and changed ADMA/arginine ratio might be contributing factors. Further prospective studies are required to show whether the long-term application of HDF or other changes of dialysis treatment modalities may help to improve well-being, morbidity, and mortality in hemodialysis patients.

Expression of beta2-microglobulin and c-fos mRNA: is there an influence of high- or low-flux dialyzer membranes?

BACKGROUND: Dialysis-related amyloidosis is an important complication of long-term hemodialysis (HD) therapy with several pathogenetic factors. One of them is the influence of the dialyzer membrane type on the synthesis of beta2-microglobulin (beta2m). In vitro results are controversial. Thus, the hypothesis of whether in vivo beta2m generation is induced by the HD procedure and whether this induction depends on the type of the used dialyzer membrane should be tested. The aim of the present study was to investigate the influence of "biocompatible" high-flux versus "bioincompatible" low-flux HD on in vivo beta2m generation as well as the induction of the early activation gene c-fos in peripheral blood cells. METHODS: Six nondiabetic HD patients [mean age 46 (21 to 69) years; Kt/V> 1.2] were included in a randomized crossover study using either a low-flux (cellulosic/cuprophan) or a high-flux (polyamide) dialyzer membrane. At the end of a four-week run-in period for each membrane, whole blood samples were taken before, immediately at, and four hours after the end of the dialysis session. MRNA was extracted, and after transcription to cDNA, quantitative polymerase chain reaction was performed for the beta2m gene, the early response gene c-fos, and the GAP-DH housekeeping gene. RESULTS: Based on the applied method for detection of specific mRNA, the results were given as ratio of beta2m or c-fos cDNA per GAP-DH cDNA. General cell activation during HD was indicated by increasing mRNA expression of c-fos related to the time course of the dialysis session, whereas beta2m did not change significantly. However, no difference was found when comparing the low-flux and the high-flux dialyzer membranes. Despite the evidence for activation of peripheral blood cells, as indicated by increasing c-fos message, no sign of beta2m mRNA induction during HD procedure with different dialyzer membranes was seen. CONCLUSIONS: Our results suggest that there is post-transcriptional regulation of beta2m generation and/or release as well as the influence of the dialyzer membrane type on post-translational processes, that is, advance glycation end products (AGE) or conformational modification of the beta2m protein. Furthermore, our data demonstrate that gene expression patterns during dialysis and/or uremia are not homogenous and need to be investigated further, especially with respect to the proinflammatory role of early leukocyte activation signals.

Complement components as uremic toxins and their potential role as mediators of microinflammation.

Cardiovascular disease is the major cause of death in end-stage renal disease (ESRD) patients. There is growing evidence that atherogenesis is an inflammatory rather than a purely degenerative process leading to a state of microinflammation. This raises the issue of whether treatment modalities of ESRD contribute to the microinflammatory state. One potential candidate in this context is the complement system. Here we consider three potential pathways linking complement activation to progression of atherosclerosis: (1) complement activation on artificial surfaces depends on their physicochemical characteristics, the effect of which is amplified because of the accumulation of complement factor D; (2) the exposure of ESRD patients to endotoxin creates a microinflammatory state, and this may amplify complement-induced damage; exposure to endotoxin may result from frequent infections because of the impairment of host-defense mechanisms or from transfer of bacterial contaminants across dialysis membranes into the blood stream; and (3) direct transduction of proinflammatory signals from blood-material interactions to the vascular system. We conclude that the complement system is an important candidate system in the genesis of microinflammation and accelerated atherogenesis in ESRD. We advance the hypothesis that the generation of proinflammatory signals, in which the complement system appears to be involved--both through systemic and local activation--plays a role in the development of late complications of uremia, including coronary heart disease. This hypothesis provides a rationale to maximize the biocompatibility of the dialysis procedure, that is, selection of nonactivating materials, use of ultrapure dialysis fluid, and--still theoretical--high-flux dialysis to remove factor D.

Hepatotoxic substance(s) removed by high-flux membranes enhances the positive acute phase response.

BACKGROUND: Acute phase proteins (APPs) are enhanced in end-stage renal disease patients (ESRD) requiring dialysis treatment. They are involved in a variety of pathologic processes like muscle proteolysis, cachexia, regulation of appetite, and atherosclerosis. They are predictive for mortality. APPs are not only makers but also active substances. They are mainly produced in liver cells and are primarily, but not exclusively, regulated by proinflammatory cytokines. To what extent hepatic APPs are influenced by uremic toxins is still unclear. Therefore, we investigated the effects of different ultrafiltrates (UFs) on the synthesis of alpha1-acid glycoprotein (AGP) in HepG2 cells. METHODS: A cross-sectional as well as a crossover study with high-/low-flux membranes was conducted to investigate the impact of UFs on bioactivity of liver cell cultures. Metabolic activity (MTT test), cytotoxicity (lactate dehydrogenase release), and the positive APP AGP were measured in HepG2 cells. RESULTS: Cultured hepatocytes treated with UFs from high-flux membranes exhibited a higher cytotoxicity (18.6 +/- 0.3% high-flux vs. 13.9 +/- 0.2% low-flux, P < 0.001) and a lower metabolic activity (29.3% high-flux vs. 50.3% low-flux, P < 0.001) in comparison with low-flux UFs. In addition, enhanced APP secretion could be observed under costimulatory conditions (high-flux 5.0 +/- 0.7 vs. low-flux 3.1 +/- 0.6 ng/microg protein, P < 0.05). The effects of high- and low-flux UFs were strongly expressed at the beginning and were still significantly different after 120 minutes of hemodialysis (HD) treatment. The crossover experiments confirmed that UFs collected during high-flux HD had a higher capacity to stimulate AGP synthesis in liver cells. CONCLUSION: The effects of UFs from dialysis patients demonstrate that hepatotoxic substances can be removed by dialysis. Stimulating the acute phase response UF collected during high-flux HD had a higher impact on liver cells in comparison with low-flux UF. These substances are putative cofactors involved in cytokine regulation.

Uremic toxicity: present state of the art.

The uremic syndrome is a complex mixture of organ dysfunctions, which is attributed to the retention of a myriad of compounds that under normal condition are excreted by the healthy kidneys (uremic toxins). In the area of identification and characterization of uremic toxins and in the knowledge of their pathophysiologic importance, major steps forward have been made during recent years. The present article is a review of several of these steps, especially in the area of information about the compounds that could play a role in the development of cardiovascular complications. It is written by those members of the Uremic Toxins Group, which has been created by the European Society for Artificial Organs (ESAO). Each of the 16 authors has written a state of the art in his/her major area of interest.

Glucose degradation products in peritoneal dialysis fluids may have both local and systemic effects: a study of residual fluid and mesothelial cells.

OBJECTIVE: When peritoneal dialysis (PD) fluids are heat sterilized, glucose is degraded to carbonyl compounds. These compounds are known to interfere with many cellular functions and to promote the formation of advanced glycation end-products. However, little is known about what actually happens with glucose degradation products (GDPs) after infusion into the peritoneal cavity. The aim of the present study was to investigate possible targets for GDPs in the peritoneal cavity. DESIGN: In vitro reactions between residual fluid and GDPs were studied by incubating unused PD fluid with overnight dialysate. Confluent monolayer cultures of human mesothelial cells were used as a model to study the reactions of GDPs with the cells lining the peritoneal cavity. METHODS: Samples were analyzed, using high pressure liquid chromatography, for the presence of formaldehyde, acetaldehyde, 5-hydroxymethyl-2-furaldehyde (5-HMF), methylglyoxal, and 3-deoxyglucosone (3-DG). Cytotoxicity was determined as inhibition of proliferation of cultured fibroblasts. RESULTS: None of the analyzed GDPs reacted with overnight dialysate. Formaldehyde and methylglyoxal, in contrast to 3-DG and 5-HMF, reacted with the cultured mesothelial cells. CONCLUSIONS: Low molecular weight carbonyls such as formaldehyde and methylglyoxal most probably react with the mesothelial cells lining the peritoneal cavity, and could be responsible for the disappearance of these cells during long-term treatment. 3-Deoxyglucosone showed remarkably low reactivity and was most probably transported within the patient.

One-year immunological evaluation of chronic hemodialysis in end-stage renal disease patients.

BACKGROUND: Much research has been devoted to the determination of acute leukocyte activation as well as acute cytokines production during and after blood hemodialysis membrane interaction. In contrast, few studies deal with chronic immunological evaluation of T-cell activation markers in hemodialysis. METHODS: We evaluated different immune parameters using a modified cellulose low-flux hemophan vs. synthetic high-flux polyamide membrane during 1 year in 35 stable chronic hemodialysis patients. Leukocyte counts, lymphocyte subpopulations, T-cell activation markers (CD69, CD25, HLA-DR, CD54, CD62L, CD45RO, CD11a, CD28), complement-activation products (C3a) and serum elastase were measured at 0, 3, 6 and 12 months in the two patient groups and compared to 13 healthy control subjects. RESULTS: Over dialysis time, all patients showed a significant level elevation of CD69/CD3 (p < 0.005) and CD25/ CD3 (p < 0.005) phenotypes. In contrast, HLA-DR and CD45RO remained unchanged suggesting a truncated pattern of activation. T lymphocyte subset analysis showed in both hemodialyzed groups a significant decrease in the expression of CD54 (ICAM-1) when compared to controls (p < 0.005). C3a and elastase measurements showed a significant upward trend with dialysis time in both hemodialyzed groups. CONCLUSION: Although the immunological changes seen in chronic hemodialyzed patients must be interpreted in conjunction with their basal uremic states and the membrane permeability properties, our study suggests that 1-year immunological evaluation of hemodialysis membranes biocompatibility is associated with changes in the pattern of chronic T-cell activation, which is in part related to the use of a particular membrane type. Moreover, some key molecules (CD54) are affected in patients with end-stage renal disease undergoing hemodialysis.

Biological significance of reducing glucose degradation products in peritoneal dialysis fluids.

UNLABELLED: Carbohydrates are not stable when exposed to energy; they degrade into new molecules. In peritoneal dialysis (PD) fluids, degradation of glucose occurs during the heat sterilization procedure. The biological consequences of this degradation are side effects such as impaired proliferation and impaired host defense mechanisms, demonstrated in vitro for a great variety of cells. Several highly toxic compounds--such as formaldehyde and 3-deoxyglucosone--have been identified in PD fluids. Carbonyl compounds, apart from being cytotoxic, are also well-known promoters of irreversible advanced glycation end-products (AGEs), which might participate in the long-term remodeling of the peritoneal membrane. Various approaches can be used to reduce the formation of glucose degradation products (GDPs) during heat sterilization. Some examples are shortening the sterilization time, lowering the pH, removing catalyzing substances, and increasing glucose concentration. The latter three factors are employed in the multi-compartment bag with a separate chamber containing pure glucose at high concentration and low pH. Gambrosol trio, a PD fluid produced in this way, shows reduced cytotoxicity, normalized host defense reactions, less AGE formation, and reduced concentrations of formaldehyde and 3-deoxyglucosone. Moreover, in the clinical situation, the fluid turns out to be more biocompatible for the patient, causing less mesothelial cell damage, which in the long term could lead to a more intact peritoneal membrane. CONCLUSION: Glucose degradation products in heat-sterilized fluids for peritoneal dialysis are cytotoxic, promote AGE formation, and cause negative side effects for the patient. Using improved and well-controlled manufacturing processes, it is possible to produce sterile PD fluids with glucose as the osmotic agent but without the negative side effects related to GDPs.

In vitro cytotoxicity of four different buffers for use in peritoneal dialysis.

Various buffers can be used in fluids for peritoneal dialysis (PD). Lactate is the most frequently used buffer, but bicarbonate and pyruvate have been suggested as more biocompatible alternatives. In the past, acetate was used as a buffer in PD fluids, but was abandoned after being linked with sclerosing peritonitis and loss of ultrafiltration. When a new buffer for PD fluids is introduced, one of its most important characteristics is that it must be non-toxic, i.e. that it does not influence fundamental cellular functions. The aim of this study was to investigate the basal cytotoxicity of bicarbonate, acetate, lactate and pyruvate at neutral pH. As target cells, we used cultured mouse fibroblast-like L-929 cells, a well-known cell line approved by the authorities for regulatory use, and primary human mesothelial cells, which are the cells that line the peritoneal cavity and are exposed to the PD fluid in vivo. Pyruvate was more cytotoxic than lactate and bicarbonate, and no significant difference in cytotoxicity was found between lactate and bicarbonate. The human mesothelial cells were more sensitive to exposure to pyruvate than the L-929 fibroblast-like cells, but less sensitive to exposure to pure PD fluids. Thus, we recommend that both types of cell are used for the evaluation of the biocompatibility of PD fluids.

Advanced glycated end-products (AGE) during haemodialysis treatment: discrepant results with different methodologies reflecting the heterogeneity of AGE compounds.

BACKGROUND: There has been much recent interest in accumulation of advanced glycation end-products (AGE) in uraemic patients. Analysis of AGE has been difficult, because commonly used methodologies, i.e. immunodetection assays or fluorescence measurements, reflect group reactivity and are not specific for chemically defined substances. Some investigators measured individual AGE compounds, e.g. pentosidine, carboxymethyllysine, pyrraline or imidazolone, but a systematic assessment of known compounds using specific HPLC methods in diabetic and non-diabetic end-stage renal disease (ESRD) patients during treatment has not been performed. METHODS: For the present study, the concentrations of early and late products of the Maillard reaction in plasma and ultrafiltrate were monitored during high-flux dialysis sessions in diabetic and non-diabetic patients. AGE were analysed by fluorescence spectroscopy and size exclusion chromatography with fluorescence detection. Specific HPLC methods were used to quantify the Amadori product fructoselysine and the AGE compounds pentosidine and pyrraline in acid or enzymatic hydrolysates. RESULTS: Using size exclusion chromatography, we confirmed a similar fluorescent peak distribution for diabetic and non-diabetic ESRD patients. Main fractions were found at approximately 70, approximately 14 and <2 kDa, confirming results obtained by other authors. In diabetic patients, the fluorescence intensity of the low molecular weight fraction was higher. Uraemic patients differed from controls mainly by the fluorescence of the low molecular weight fraction. The peak spectrum in ultrafiltrates was similar to that in plasma regarding low molecular weight fractions and the 14 kDa peak, but no protein-bound fluorescence was found at 70 kDa. HPLC analysis revealed a significant reduction of plasma pentosidine during high-flux dialysis in non-diabetic (from 9.1+/-5.1 to 8.5+/-4.7 pmol/mg protein; P<0.05) and diabetic patients (from 10.0+/-9.1 to 6.8+/-4.0 pmol/mg protein; P<0.05). In contrast, plasma fructoselysine showed only a non-significant trend to decrease in diabetic (from 3.24+/-0.88 to 3.05+/-0.77 nmol/mg protein) and non-diabetic patients (from 2.69+/-0.52 to 2.56+/-0.50 nmol/mg protein). Pyrraline, a nonfluorescent late AGE product derived from reaction of 3-deoxyglucosone with lysine, could not be detected (detection limit approximately 40 pmol/mg protein). Comparing HPLC and size exclusion analysis, it was found that pentosidine accumulated in the range of low molecular weight substances and was removed by high-flux dialysis. CONCLUSIONS: High-flux dialysis reduces the plasma concentration of fluorescent AGE compounds, i.e. pentosidine, but the Amadori product fructoselysine is not removed, indicating that this compound is protein associated.

3-Deoxyglucosone, a promoter of advanced glycation end products in fluids for peritoneal dialysis.

OBJECTIVE: The accumulation of irreversible formed advanced glycosylation end products (AGE) in the peritoneal cavity might play an important role in the development of ultrafiltration failure and peritoneal membrane destruction. 3-Deoxyglucosone (3-DG), more formally named 3-deoxy-D-erythro-hexos-2-ulose or 3-deoxy-D-erythro-hexosulos is known to be a potent cross-linker responsible for the polymerization of proteins and a precursor of AGE. The purpose of the present study was to determine if the dicarbonyl compound 3-DG, is formed as a glucose degradation product during heat sterilization of fluids for peritoneal dialysis (PD). DESIGN: Four fluids were examined: a commercially available PD fluid Gambrosol (Gambro, Lund, Sweden); Gambrosol-Bio (Gambro), a new PD-fluid produced under conditions that minimize the generation of toxic glucose degradation products; a fluid prepared in the laboratory by sterile-filtration; and a fluid prepared in the laboratory by heat sterilization. METHODS: The concentration of 3-DG was analyzed by measuring the concentration of its diaminonaphthalene derivative by HPLC using a Waters Symmetry C18 column. RESULTS: The 3-DG concentrations in the commercially- and laboratory-prepared heat-sterilized fluids were 118 and 154 micromol/L, respectively. Gambrosol-Bio and the sterile-filtered fluid produced in the laboratory contained 3-DG in concentrations of 12.3 and less than 1.2 micromol/L, respectively. CONCLUSION: Our results demonstrate that during the heat sterilization of conventional PD-fluids, 3-DG is produced as a degradation product of glucose. It was also demonstrated that, through an alteration of the manufacturing condition, the production of 3-DG could be considerably reduced. We speculate that the presence of 3-DG in unused conventional PD-fluid could act as a local promoter, and increase local AGE formation within the peritoneal cavity.

Impact of membrane choice and blood flow pattern on coagulation and heparin requirement--potential consequences on lipid concentrations.

BACKGROUND: We reasoned that procoagulant activity, and by implication heparin requirement, during haemodialysis are influenced, amongst other factors, by the type of membranes and the geometry of the blood line system. In addition, there are indications that heparin has dose-dependent effects on the lipid status of chronic haemodialysis patients. METHODS: In a parallel group design we compared patients treated with cuprophane (CU) and polycarbonate-polyether (PC-PE) plate dialysers. In both groups, blood line geometry was varied by including in a first phase and omitting in a second phase drip chambers in the arterial blood line. End-points were changes in coagulation parameters, i.e. thrombin-antithrombin III complex (TAT), plasmin-anti-plasmin complex (PAP), and prothrombin fragment (F1 + 2) concentrations measured by sandwich ELISA. Subsequently all patients were switched to PC-PE dialysers for 6 months and the heparin dose was reduced in a stepwise fashion. Lipid levels and coagulation parameters were monitored. Finally, in an ancillary study, the correlation between heparin dose and LDL/HDL ratio was assessed in patients chronically exposed to PC-PE membranes and low doses of heparin. RESULTS: Post-dialytic concentrations of coagulation and fibrinolysis parameters were significantly lower in the PC-PE group (TAT 31.0 +/- 4.4 micrograms/l; PAP 1180 +/- 148 micrograms/l; F1 + 2 4.2 +/- 0.4 nmol/l) compared to the CU group (TAT 57.3 +/- 10.8 micrograms/l; PAP 1789 +/- 185 micrograms/l; F1 + 2 8.8 +/- 1.0 nmol/l), independently of the use of an arterial drip chamber. Omission of the arterial drip chamber led to lower TAT in the CU group (42.2 +/- 5.8 micrograms/l, P < 0.05), but not in the PC-PE group. In contrast, PAP and F1 + 2 concentrations did not change significantly in either group. Down-titration of heparin dose (from 20.4 +/- 1.1 to 9.4 +/- 0.9 IU/kg/h) was associated with a significant decrease in serum triglycerides (from 2.9 +/- 0.9 to 2.0 +/- 0.6 mmol/l, P < 0.05), LDL-cholesterol (from 3.4 +/- 0.2 to 2.7 +/- 0.4 mmol/l, P < 0.05) and LDL/HDL-ratio (from 3.2 +/- 0.3 to 2.0 +/- 0.3, P < 0.05) with no significant change of total or HDL-cholesterol after 6 months. In an ancilliary analysis, a correlation between lipid parameters (LDL/HDL ratio) and heparin dose was confirmed in 24 patients chronically exposed to PC-PE membranes (r = 0.473, P < 0.05). CONCLUSIONS: In a prospective exploratory study (i) heparin requirement is lower with the use of a polycarbonate-polyether membrane compared to a cuprophane membrane, (ii) heparin requirement is influenced by blood line geometry (decreased with omission of an arterial drip chamber), and (iii) in patients on polycarbonate-polyether membranes down-titration of heparin is associated with a reduction of serum triglycerides, LDL cholesterol, and LDL/HDL ratio. Our data suggest that reduction of heparin dose improves lipid profile. These preliminary observations require confirmation by parallel group controlled studies with controlled dietary intake.

Effect of hemofiltration on hemodynamics and systemic concentrations of anaphylatoxins and cytokines in human sepsis.

OBJECTIVE: To determine whether hemofiltration (HF) can eliminate cytokines and complement components and alter systemic hemodynamics in patients with severe sepsis. DESIGN: Prospective observation study. SETTING: Surgical intensive care unit of a university hospital. PATIENTS: 16 patients with severe sepsis. INTERVENTIONS: Continuous zero-balanced HF without dialysis (ultrafiltrate rate 2 l/h) was performed in addition to pulmonary artery catheterization, arterial cannulation, and standard intensive care treatment. MEASUREMENTS AND MAIN RESULTS: Plasma and ultrafiltrate concentrations of cytokines (the interleukins IL-1 beta, IL-6, IL-8, and tumor necrosis factor alpha) and of complement components (C3adesArg, C5adesArg) were measured after starting HF (t0) and 4 h (t4) and 12 h later (t12). Hemodynamic variables including mean arterial pressure (MAP), mean central venous pressure, mean pulmonary artery pressure, pulmonary capillary wedge pressure, and cardiac output were serially determined. During HF, cytokine plasma concentrations remained constant. However, C3adesArg and C5adesArg plasma concentrations showed a significant decline during 12-h HF (C3adesArg: t0 = 676.9 +/- 99.7 ng/ml vs t12 = 467.8 +/- 71, p < 0.01; C5adesArg: 26.6 +/- 4.7 ng/ml vs 17.6 +/- 6.2, p < 0.01). HF resulted in a significant increase over time in systemic vascular resistance (SVR) and MAP (SVR at t0: 669 +/- 85 dyne.s/cm5 vs SVR at t12: 864 +/- 75, p < 0.01; MAP at t0: 69.9 +/- 3.5 mmHg vs MAP at t12: 82.2 +/- 3.7, p < 0.01). CONCLUSIONS: HF effectively eliminated the anaphylatoxins C3adesArg and C5adesArg during sepsis. There was also a significant rise in SVR and MAP during high volume HF. Therefore, HF may represent a new modality for removal of anaphylatoxins and may, thereby, deserve clinical testing in patients with severe sepsis.

Activation of human neutrophils after contact with cellulose-based haemodialysis membranes: intracellular calcium signalling in single cells.

PURPOSE OF STUDY: In vitro contact of human leukocytes with cellulose-based dialysis membranes under complement-independent conditions results in activation of various leukocyte functions. To analyse signals involved in the mechanism of cell activation, we measured changes in cytosolic free calcium ([Ca2+]i) in individual human blood neutrophils (PMN) upon contact with flat sheet haemodialysis membranes. RESULTS: By confocal laser-scanning microscopy (CLSM), changes in [Ca2+]i were monitored in Fluo-3-labelled cells up to 10 min after contact with a regenerated cellulose (RC) membrane. Multiple [Ca2+]i transients were observed for cells in contact with RC; biostochastic analysis showed that up to 67% of the PMN responded with a high increase in [Ca2+]i, the rest were low- or non-responding cells. After contact with the new synthetic polycarbonate-polyether (PC-PE) membrane only non-responding cells were seen, indicating reduced cellular contact activation. The increase in [Ca2+]i of cells on RC could be inhibited by 5mM L-fucose. This monosaccharide was recently found to be present in cellulose-based polymers in picomolar concentrations. CONCLUSIONS: The data supports the hypothesis that dialysis-membrane-associated L-fucose residues participate in complement-independent leukocyte activation during haemodialysis therapy.