Cytokine production in haemodiafiltration: a multicentre study

Background: Bacterial contamination of dialysate may enhance cytokine production in haemodialysis. We tested the hypothesis that intracellular cytokines could be enhanced in a large group of patients exposed to backfiltration of dialysate over a long period of observation. Methods: The intracellular cytokine (interleukin-1 receptor antagonist and interleukin-1{beta}) concentrations in chronic uraemic patients undergoing haemodiafiltration, which is known to be associated with backfiltration (Group II, 12 patients), were compared to those found in patients treated with a modified haemodiafiltration modality without backfiltration (group I, 16 patients), in patients shifted from one modality to the other (Group III, 27 patients) and in 10 patients on haemodialysis (Group IV) in a 1-year multicentre study. Group V comprised 10 healthy volunteers. All dialysis monitors were equipped with dialysate ultrafiltration systems. Dialysate contamination was studied by the LAL and the peripheral mononuclear cell/interleukin-1{beta} assays in the presence or absence of polymyxin B. Results: Intracellular interleukin-1 receptor antagonist and interleukin-1{beta} both increased significantly (P<0.002) but slowly (after 8 months) in Groups II vs I, and during the 4-month period in haemodiafiltration with backfiltration in Group III. The incidence of post-predialysis concentration ratio (over 1.5) increased two- to threefold in patients treated with heaemodiafiltration with backfiltration with respect to haemodiafiltration without backfiltration. Results on the assays for LAL (<0.5 E/ml) and interleukin-1{beta} (range 80.1-90.2 pg/5x10⁶ cells; 70.2-81.3 pg/5x10⁶ cells with polymyxin B) showed a moderate-to-low degree of dialysate contamination. Conclusions: Backfiltration of dialysate with moderate-to-low degree of contamination may enhance cytokine synthesis in the long term. Thus, the relevance for dialytic strategies aiming at improving dialysate quality and/or at reducing backfiltration is highlighted.     

Endotoxins modulate chronically tumour necrosis factor {alpha} and interleukin 6 release by uraemic monocytes

We examined in vivo the release of tumour necrosis factor alpha(TNF) and interleukin 6 (IL-6) by uraemic monocytes upon stimulationwith endotoxin-contaminated bicarbonate concentrate. Twelveuraemic patients underwent 1-month-subsequent periods of standardhaemodialysis (SHD) with cuprophane (CU), a high-complement-activatingmembrane (6 patients), or haemodiafiltration (HDF) with polyacrylonitrile(PAN), a low-complement-activating membrane (6 patients), byusing a dialysate prepared with either non-sterile bicarbonateconcentrate tanks (phase 1) or sterile bicarbonate concentratebags (phase 2). TNF and IL-6 concentrations were determinedin monocyte supernatants by ELISA; endotoxin levels in bicarbonateconcentrates were measured by a chromogenic limulus amoebocytelysate (LAL) assay. A significant increase in LAL reactivity was found in bicarbonateconcentrate tanks compared to sterile bags (P<0.001). Non-steriledialysate caused a significant (P<0.001) predialytic increasein monocyte TNF release as compared to controls and nondialyseduraemic patients. One month treatment with sterile bicarbonatesignificantly decreased TNF predialytic activity in monocytesupernatants (P<0.001) to levels closer to those of non-dialyseduraemic patients. A similar decrease was observed for IL-6 production.Dialytic treatment induced a further increase in both TNF andIL-6 production, particularly in phase 1. When uraemic patientswere examined separately according to the different dialyticprocedures (SHD-CU or HDF-PAN), the use of sterile dialysate(phase 2) caused a significant decrease of predialysis TNF releasein both SHD CU patients (24.1±8.4 pg/ml versus 55.3±5.7pg/ml, P<0.001) and HDF PAN-treated patients (16.6±5.3pg/ml versus 29.1±5.4pg/ml, P<0.005), so that thedifferences between the dialytic procedures were completelyabolished. In conclusion, TNF and IL-6 release may be induced by endotoxin-contaminateddialysate during haemodialysis. The use of sterile bicarbonatecan ameliorate the bioincompatibility of CU membranes and probablyinfluences the biocompatibility of PAN membranes. Therefore,regardless of the type of dialyser used, all attempts to obtainan ultrapure dialysate are important to optimize dialytic treatment,in order to attenuate the chronic monocyte activation whichoccurs during haemodialysis.     

Tailoring high-cut-off membranes and feasible application in sepsis-associated acute renal failure: in vitro studies.

BACKGROUND: As removal of pro-inflammatory cytokines is limited in conventional diffusive or convective extracorporeal therapies, we studied in two polysulphone membranes with an industrial albumin sieving coefficient of 0.05 (Type A) and 0.13 (Type B) cytokine (IL-6, IL-8, IL-1beta, IL-1ra, TNF-alpha) and plasma protein (albumin, cystatin C, total proteins) permeability profiles. Based on the convective membrane permeability, we evaluated in vitro the dialytic modality that could provide an acceptable balance between high cytokine and low albumin clearances. METHODS: Cytokine and plasma protein sieving coefficient (SC) and clearance were studied in (i) post-dilutional haemofiltration mode at 20% fixed ultrafiltration rate; (ii) haemodialysis mode (dialysate flow rate of 3 and 5 l/h); and (iii) haemodiafiltration mode (dialysate flow rate of 3 or 5 l/h with 0.5 l/h of ultrafiltrate). RESULTS: In haemofiltration mode both Type A and Type B haemodialysers at QB 150 ml/min exhibited similar median SC nearly up to 1 for IL-1beta and IL-1ra, at about 0.6 for IL-6, 0.4 for IL-8 and 0.7 for TNF-alpha, with clearance values ranging from 15 to 30 ml/min. SC were independent of blood flow and were stable throughout the whole experiment. Albumin SC was higher in Type B than in Type A and rapidly decreased from 0.2 to 0.02 and from 0.5 to 0.04 within 3 h for haemodialyser Types A and B, respectively. Cytokine SC was lower in haemodialysis than in haemodiafiltration and haemofiltration mode, and by increasing dialysate flow from 3 up to 5 l/h in both haemodialysis and haemodiafiltration mode, SC for all tested cytokines decreased. However, at 5 l/h clearances were not different or were higher, since increased amounts of dialysate outlet compensated for the decreased SC. Albumin clearances in haemodialysis and haemodiafiltration mode after 360 min at 5 l/h were 0.81 and 0.91 ml/min, respectively. CONCLUSIONS: Our studies show that a mixed convective and diffusive technique ensures high cytokine clearances with an acceptable loss of albumin.     

Differences in the permeability of high-flux dialyzer membranes for bacterial pyrogens

AIMS: The increasing use of high-flux membranes for hemodialysis has raised concerns that patients dialyzed with these membranes may be at higher risk of being exposed to cytokine-inducing bacterial substances in the dialysate than patients dialyzed with low-flux membranes. We investigated the permeability of various high-flux membranes for both purified E. coli lipopolysaccharide (LPS) as well as for LPS derived from Stenotrophomonas (Sten.) maltophilia. MATERIALS AND METHODS: An in vitro dialysis circuit with saline in the blood compartment of 3 dialyzers containing different membranes (polysulfone, helixone and Diapes) was employed. The dialysate was challenged with increasing doses of sterile filtrates derived from Sten. maltophilia cultures or with purified LPS from E. coli. Samples from the blood compartment were tested for cytokine induction (IL-1beta, IL-6 and TNF) in mononuclear cells as well as for LPS by limulus amebocyte lysate test (LAL). RESULTS: IL-6 induction above sterile controls (< 0.02 ng/ml IL-6) was observed by samples from the blood side of DIAPES dialyzers (1.2 +/- 0.7 ng/ml IL-6) after challenging the dialysate with 4.1 +/- 3.6 U/ml E. coli LPS (9.9 +/- 4.5 ng/ml IL-6). In contrast, at the same challenge dose no significant IL-6 induction above sterile controls was observed by blood side samples of polysulfone (0.15 +/- 0.07 ng/ml) and helixone (0.09 +/- 0.05 ng/ml) dialyzers. Increasing the amount of E. coli LPS in the dialysate further augmented IL-6 induction by blood side samples of Diapes but not of polysulfone and helixone dialyzers. Similar results were obtained for IL-1beta and TNF. After challenging the dialysate with E. coli LPS as well as with cultures of Sten. maltophilia, significantly more LAL reactivity was observed in the blood compartment of Diapes compared to polysulfone and helixone. CONCLUSIONS: There are considerable differences between high-flux membranes regarding their permeability for cytokine-inducing substances from E. coli as well as for LPS derived from E. coli and Sten. maltophilia. Dialyzers that leak CIS under aqueous conditions in vivo should not be used unless the dialysate has passed through an ultrafilter.     

Permeability of cellulosic and non-cellulosic membranes to endotoxin subunits and cytokine production during in-vitro haemodialysis.

The possibility of endotoxin transfer across haemodialysis membranes remains a controversial issue. Additional concern has arisen because of the recent introduction in clinical practice of highly permeable, synthetic dialysis membranes and of bacteria-contaminated bicarbonate concentrate with potential short-term and long-term hazards for haemodialysis (HD) patients. Therefore, we performed experiments in an in-vitro dialysis recirculation system using three different types of HD membranes, namely standard regenerated cellulose (Cuprophan, CU), polyacrylonitrile AN-69 (PAN), and polysulphone F-60 (PS). When radiolabelled lipopolysaccharide (125I M-LPS) from E. coli, together with 10 micrograms/ml unlabelled LPS, was added to the recirculating solution in the dialysis compartment, radioactivity could be detected in the blood compartment after 15 min and increased progressively with time up to respectively 6.7% (CU), 10.3% (PAN), and 10.3% (PS) of initial activity on the dialysate side. The addition of albumin to the solution on the blood side led to a decreased permeability of radioactivity (7.3% vs 10.3%), compared to the absence of albumin (tested only for PS membrane). Furthermore, 73% of 125I M-LPS transferred across the PS membrane in the presence of albumin was TCA-precipitable. In contrast, free iodine (Na 125I) incubated in an albumin-containing solution did not precipitate with albumin after the addition of TCA (precipitation of only 0.6%). Moreover, kinetics of transmembranous transfer of Na-125I were strikingly different from that of 125I M-LPS. Analysis by the method of sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) of the blood side solution, after LPS addition in the dialysis solution and 30 min of back-filtration, revealed the presence of several silver-stainable and autoradiographic bands of low-molecular-weight range, probably LPS fragments. Finally, the presence of LPS in the dialysate compartment led to a moderate increase in interleukin 1 (IL-1) and tumour necrosis factor alpha (TNF) concentrations in plasma as well as in monocyte culture supernatants after isolation from recirculating normal human whole blood exposed to CU, PAN, or PS membrane. In conclusion, our study provides evidence for the permeation of low-molecular-weight LPS subunits across cellulosic and non-cellulosic HD membranes. The clinical significance, if any, of such a transfer has, however, still to be demonstrated.     

A study on limulus amebocyte lysate (LAL) reactive material derived from dialyzers

The amebocytes of horseshoe crab (Limulus) hemolymph contain a coagulation system highly sensitive to bacterial endotoxins. Limulus amebocyte lysate (LAL) reactive material derived from cuproammonium rayon membranes, however, is not an endotoxin and acts upon the alternative pathway in the coagulation cascade found in Limulus amebocyte lysate. This study confirmed these facts by using the coagulation system of Limulus without factor G, which is a substrate of the alternative pathway. LAL reactive material lingered in the circulation for a relatively long time. In acute hemodialysis, its plasma concentration increased by an average of 100 pg/ml with each dialysis and eventually reached a plateau of approximately 300 pg/ml. In patients with chronic renal failure under regular hemodialysis, the mean level of LAL reactive material was 330.0 +/- 8.0 pg/ml before hemodialysis which increased by 70.6 +/- 20.7 pg/ml after four hours of hemodialysis.     

A study on limulus amebocyte lysate (LAL) reactive material derived from dialyzers

The amebocytes of horseshoe crab (Limulus) hemolymph contain a coagulation system highly sensitive to bacterial endotoxins. Limulus amebocyte lysate (LAL) reactive material derived from cuproammonium rayon membranes, however, is not an endotoxin and acts upon the alternative pathway in the coagulation cascade found in Limulus amebocyte lysate. This study confirmed these facts by using the coagulation system of Limulus without factor G, which is a substrate of the alternative pathway. LAL reactive material lingered in the circulation for a relatively long time. In acute hemodialysis, its plasma concentration increased by an average of 100 pg/ml with each dialysis and eventually reached a plateau of approximately 300 pg/ml. In patients with chronic renal failure under regular hemodialysis, the mean level of LAL reactive material was 330.0±8.0 pg/ml before hemodialysis which increased by 70.6±20.7 pg/ml after four hours of hemodialysis.     

Removal of morphine with the new high-efficiency and high-flux membranes during haemofiltration and haemodiafiltration

We present three critically ill patients with severe renal failure who required haemofiltration or haemodiafiltration, with high-efficiency or high-flux membranes, while receiving an intravenous infusion of morphine. We show that despite the very high ultrafiltrability/diffusability of free morphine, only 1-3% of the total amount of infused morphine is removed in 24 h. This is in marked contrast to haemodialysis where, owing to much higher dialysate flow rate, a significant quantity of free morphine is removed.     

The Effects of Endotoxin–Contaminated Dialysate and Polysulfone or Cellulosic Membranes on the Release of TNFα during Simulated Dialysis

Abstract: Simulated dialysis of whole blood was used to determine whether membrane factors (biocompatibility), endotoxin (ET) membrane diffusion, or transmembrane monocyte–ET interactions would stimulate tumor necrosis factor (TNFα) release. Whole blood containing EDTA and aprotinin was recirculated in the blood compartment of hollow fiber dialyzers containing either regenerated cellulose or polysulfone membranes. ET–free and ET–spiked dialysate were recirculated consecutively in the dialysate compartment for 30 min each. Blood and dialysate samples were collected at t _o and after each 30 min of simulated dialysis for determination of TNFa and ET concentrations. TNFa was not detected in any blood samples collected after simulated dialysis with regenerated cellulose (RC) membranes and ET–free or ET–spiked dialysate. However, blood ET concentrations, as determined by the Limulus amebocyte lysate (LAL) assay, increased in RC dialyzers after each 30 min of simulated dialysis even with ET–free dialysate. Since TNFa was not detected in these blood samples, the material detected by the LAL assay probably was not ET but an LAL–reactive material. After simulated dialysis with polysulfone dialyzers and ET–free dialysate, TNFa and ET were not detected in blood samples. ET also was not detected in blood samples after dialysis with ET–spiked dialysate. However, TNFa was detected in 7 of 13 (54%) of the blood samples following the 500 ng/ml of ET dialysate spike. TNFα release during simulated dialysis with polysulfone membranes and ET–contaminated dialysate may be due to transmembrane stimulation of circulating mononuclear cells and not diffusion of ET across the membrane.     

The role of plasma coating on the permeation of cytokine-inducing substances through dialyser membranes

We studied the effects of coating of dialyser membranes withplasma proteins on the permeation of bacteria-derived cytokine-inducingsubstances (CIS). An in vitro dialysis circuit using polysulphone(PS) or modified cellulose triacetate (mCT) dialysers was used.Precoating of the dialysers was performed by recirculation of10% normal human plasma for 30 min in the blood compartmentand subsequent rinse with pyrogen-free saline. Samples fromthe blood compartment were tested for induction of interleukin-1(IL-1), interleukin-1ß (IL-1ß) and tumournecrosis factor (TNF) at various time points after challengingthe dialysate with sterile culture supernatants from Pseudomonasaeruginosa. Contamination of the dialysate resulted in the appearanceof CIS in the blood compartment of both polysuphone modifiedcellulose triacetate (IL-1: PS, time 0: 81±11 pg/ml,time 60min: 4747±1822 pg/ml, P<0.05; mCT, time 0:235±141 pg/ml, time 60 min: 1632±531 pg/ml, P<0.05).The plasma protein layer reduced the penetration of CIS significantlyonly for polysulphone (IL-1: PS, time 60: 4747±1822 versus880±525 pg/ml, P<0.05; modified cellulose triacetate,time 60 min: 1632±531 pg/ml versus 930±326 pg/ml).Samples from the blood compartment contained <6 pg/ml LAL-reactivematerial at all time points. We conclude that plasma coatingof polysulphone dialysers reduces the permeability for CIS derivedfrom Pseudomonas, either by reducing the effective pore sizeor by adsorption of proteins that bind CIS. When precoated dialyserswere used, transfer of CIS through both dialysers was comparable,suggesting that under the conditions of in vivo haemodialysisthere may be no difference between the dialysers regarding thepenetration of CIS from the dialysate.     

Pharmacokinetics of vancomycin in patients undergoing haemodialysis and haemofiltration

Vancomycin is widely used for the treatment of infections with Gram-positive bacteria in patients with end-stage renal disease. The concentration of vancomycin in serum, in ultrafiltrate, and in dialysate was measured during nine haemofiltration and seven haemodialysis procedures with high-permeability membranes. The t1/2 of vancomycin was 101 +/- 19 h in the interdialytic and interhaemofiltration period. There was no significant difference between the haemodialysis clearance (55.2 +/- 18.5 ml/min) and the haemofiltration clearance (66.8 +/- 13.6 ml/min). The redistribution phenomenon was about 25% in the post haemofiltration period and only 10% in the post haemodialysis period. Approximately 270 mg of vancomycin was recovered in dialysate or ultrafiltrate. With high-permeability membranes more commonly used in patients with end-stage renal disease, continuous monitoring of vancomycin therapy is recommended.     

No evidence for endotoxin transfer across high flux polysulfone membranes

Recently, there has been some concern that high-flux membranes may expose dialysis patients to the risk of endotoxin transfer secondary to backfiltration within the dialyzer. To evaluate the safety of high-flux polysulfone dialyzers, we examined in an in vitro recirculation system whether lipopolysaccharides (LPS) and lipid A respectively penetrate from the dialysate to the blood compartment and vice versa using a F-60 hemofilter (Fresenius AG). For the detection of endotoxin, a sensitive, kinetic limulus amebocyte lysate (LAL) microtiter test was used. It can be concluded that LPS and lipid A do not pass from either side through the filter system used when saline was recirculated for more than 10 h on both sides of the membrane.     

Measurement of operative plasma endotoxin levels in jaundiced and non-jaundiced patients

A study of portal plasma endotoxin levels was performed using a chromogenic limulus amoebocyte lysate (LAL) assay. The assay proved sensitive and reproducible. In only 1 of 25 healthy subjects was the systemic plasma endotoxin level above 100 pg/ml (equivalent Escherichia coli 0111B4). In 30 non-jaundiced patients undergoing surgery the mean (+SEM) portal plasma endotoxin level (60 + 9 pg/ml) was significantly higher (p less than 0.05) than the mean level in the systemic blood (46 + 6 pg/ml), supporting the concept of endotoxin absorption from the intestine into the portal blood. In 20 patients with obstructive jaundice undergoing surgery 42% of portal, 45% of inferior mesenteric and 35% of systemic venous plasma endotoxin levels were above 100 pg/ml. There were significantly higher levels in the portal (p less than 0.05) and inferior mesenteric (p less than 0.05) compared with the systemic blood. Neither the presence of malignancy nor the duration of surgery appeared to influence endotoxin absorption. The significance of raised plasma endotoxin levels in obstructive jaundice is discussed.     

Effect of dialysis on serum/plasma levels of free immunoglobulin light chains in end-stage renal disease patients.

BACKGROUND: Free immunoglobulin light chains (FLCs) have previously been shown to be uraemic toxins. In this work we investigated the effect of haemodialysis and haemodiafiltration on the level of FLCs in serum/plasma of uraemic patients. METHODS: Serum/plasma proteins were separated by non-reducing SDS-PAGE and transferred to a nitro-cellulose membrane. FLCs were detected by specific antibodies and an enhanced chemiluminescence detection system. The FLC concentrations were calculated. We studied 15 healthy subjects, 10 patients with chronic renal failure, 71 patients undergoing haemodialysis treatment and 33 patients treated with haemodiafiltration. Different membranes were compared: low- and high-flux polysulfone membranes, low- and high-flux cellulose triacetate membranes, high-flux polymethylmethacrylate and polyacrylonitrile membranes. RESULTS: Chronic renal failure patients showed elevated FLC concentrations as compared with controls. In haemodialysis or haemodiafiltration patients these values were even higher. This was mainly due to an increased concentration of FLC of the lambda-type. The treatment modality per se did not influence the FLC concentrations. Only haemodialysis or haemodiafiltration with the polymethylmethacrylate membrane lead to a significant reduction in FLC concentrations; however, these did not reach control levels. We did not observe differences in FLC levels between patients with different underlying diseases, nor did we find a correlation between age or the duration of the dialysis treatment and FLC concentrations. We found a positive correlation between FLC concentrations at the beginning of dialysis treatment and the amount of IgLCs removed during treatment. However, the average FLC level after treatment did not reach control values. CONCLUSIONS: Currently available haemodialysis or haemodiafiltration treatments are unable to normalize the elevated serum/plasma levels of FLCs in end-stage renal disease patients.     

Bacteria and endotoxin removal from bicarbonate dialysis fluids for use in conventional, high-efficiency, and high-flux hemodialysis

The use of bicarbonate-based dialysis fluids in hemodialysis centers in the United States has increased with the advent of high-efficiency and high-flux hemodialysis. However, bicarbonate dialysis fluids can support rapid bacterial growth and high endotoxin concentrations. This study determined the efficacy of an ultrafiltration device in reducing the bacterial and endotoxin concentrations in bicarbonate dialysis fluids. A polysulfone hollow fiber dialyzer was used to ultrafilter bicarbonate concentrate before entering the central proportioner and bicarbonate dialysate after exiting the proportioner in single patient dialysis machines. Pre- and post-ultrafilter samples were collected for bacterial and endotoxin assays over 10 months. Ultrafiltration of bicarbonate concentrate reduced bacterial and endotoxin concentrations from 288,330 colony forming units (CFU)/ml and 42,804 pg/ml to 0.47 CFU/ml and 109 pg/ml, respectively. Ultrafiltration of the dialysate in single patient systems decreased bacterial and endotoxin concentrations from 15,889 CFU/ml and 1,746 pg/ml to 0.003 CFU/ml and 0.109 pg/ml, respectively. These results demonstrate that ultrafiltration of bicarbonate dialysis fluids is effective in reducing bacterial and endotoxin contamination inherently associated with the use of bicarbonate-based dialysates.     

Removal of uraemic plasma factor(s) using different dialysis modalities reduces phosphatidylserine exposure in red blood cells.

BACKGROUND: Solute(s) retained during uraemia cause increased exposure of aminophospholipid phosphatidylserine (PS) on the outer surface of erythrocyte membranes, and this phenomenon may be involved in the pathophysiology of uraemia by promoting abnormal erythrocyte interactions. METHODS: We examined in a prospective randomized cross-over fashion the ability of various dialysis modalities to remove the circulating uraemic factor(s) causing increased PS externalization in red cells. Each patient was treated with haemodialysis (HD) and with on-line haemodiafiltration (HDF) using standard high-flux polysulphone membranes or with the new polisulphone-based Helixone membrane to compare the effects of dialysis technique and membrane type on PS exposure. Removal of PS was assessed indirectly by measuring PS-expressing normal erythrocytes exposed to uraemic plasma or to ultrafiltrate obtained at various time points during the extracorporeal session. RESULTS: Removal of the uraemic plasma factor(s) causing PS exposure was demonstrated by the reduced ability of uraemic plasma at the end of dialysis to induce PS exposure in normal erythrocytes, and by the capacity of ultrafiltrate from the dialysate side of the dialyzer membrane to markedly increase PS-positive red cells. However, the degree of removal varied according to the dialyzer type and to dialysis technique. Removal was greater for on-line HDF using the Helixone membrane, intermediate and comparable with HD with Helixone and with on-line HDF using standard polysulphone, and lower for HD using polysulphone membrane. The putative uraemic compound causing PS exposure seems to be highly lipophilic, somehow associated with plasma proteins, and apparently having a molecular weight between 10 and 10.8 kDa. CONCLUSIONS: Uraemia is associated with retention of compound(s) that are lipophilic, possibly protein-bound and which cause an abnormal exposure of PS in erythrocytes. Our findings, that such compound(s) can be removed during dialysis and at higher rates with convection techniques, indicate a potential benefit for uraemic patients. The present results also seem to confirm the marked ability of high-flux Helixone membranes to eliminate high molecular weight solutes.     

Inhibition of cytokine synthesis by peritoneal dialysate persists throughout the CAPD cycle.

The current study focused on the effect of continuous ambulatory peritoneal dialysis (CAPD) dialysate obtained following different intraperitoneal dwell periods on the release of interleukin-6 (IL-6) and tumor necrosis factor-alpha (TNF alpha) from mononuclear leukocytes (PBMC). Aliquots of 5 x 10(6)/ml healthy peripheral PBMC were exposed to fresh or spent CAPD dialysate (10-240 min of intra-peritoneal dwell) and stimulated with Escherichia coli endotoxin (10 micrograms/ml, 2h). IL-6 and TNF alpha in cell supernatants were determined by specific enzyme immunoassays. Control PBMC in physiological buffer released 361 +/- 70 pg/ml IL-6 and 717 +/- 147 pg/ml TNF alpha (mean +/- SEM, n = 8), whereas exposure to fresh dialysis fluids severely suppressed cytokine release from PBMC (less than 30 pg/ml IL-6 and less than 15 pg/ml TNF alpha). A significant inhibition of IL-6 and TNF alpha release was also observed in PBMC exposed to spent dialysate. The inhibitory capacity of the spent fluids was pronounced with increasing intra-peritoneal dwell time (10 min: 183 +/- 45 pg/ml IL-6 and 538 +/- 109 pg/ml TNF alpha; 240 min: 26 +/- 5 pg/ml IL-6 and 105 +/- 30 pg/ml TNF alpha; mean +/- SEM, n = 16). These data indicate that the impairment of cell responsiveness following exposure of PBMC to peritoneal dialysate is not restricted to the unused fluids, but is also observed following intra-peritoneal equilibration. Moreover, our findings suggest the presence of cytokine inhibitory factors in the peritoneal dialysate of CAPD patients which appear to accumulate in the peritoneal effluent during the CAPD cycle.     

Pyrogen retention by highly permeable synthetic membranes during in vitro dialysis

Pyrogen permeability of the new highly permeable synthetic membrane polyethersulfone (DIAPES) was compared to polysulfone in vitro dialysis experiments with heparinized human donor blood in the blood compartment. After sterile dialysis for 5 min, dialysate was contaminated with a culture filtrate of Pseudomonas aeruginosa using high and moderate challenge doses (Limulus assay reactivity 20,000 EU/ml and 50 EU/ml, respectively). Whole blood samples were separated from the blood compartment during the sterile (5 min) and contaminated (60 min) phases of dialysis and incubated for 6 h at 37 degrees C. Blood samples were lysed, and interleukin-1beta and tumor necrosis factor alpha were measured by specific ELISAs. Moderate dialysate contamination (50 EU/ml) did not induce detectable cytokine production in whole blood. High challenge dose (20,000 EU/ml) induced whole blood interleukin-1beta and tumor necrosis factor alpha production in the blood compartment, which was higher with DIAPES than with polysulfone after 30 min. After 60 min, membrane-dependent differences were no longer detectable. Pyrogen concentrations in the dialysate decreased with time indicating adsorption of cytokine-inducing substances to the dialyzer membrane. Pyrogens adsorbed to dialyzer membranes were resuspended during recirculation of sterile phosphate-buffered saline in the dialysate compartment and retained cytokine-inducing activity as seen from whole blood incubation experiments. DIAPES and polysulfone adsorbed pyrogens in the presence of whole blood. Pyrogen adsorption to the membrane polymer and/or to the protein coat on the membrane prevented the passage of pyrogens in the presence of moderately contaminated dialysate. High grade dialysate contamination caused breakthrough of pyrogens into the blood with DIAPES and polysulfone. In order to reduce the risk of a dialysis-dependent inflammatory response, dialysate of high bacteriological quality (ultrapure dialysate) should be mandatory.     

提升透析液水质改善维持性血液透析患者微炎性反应状态

目的 探讨改善透析液水质对长期血液透析患者微炎性反应状态的影响.方法 以53例维持性血液透析(MHD)终末期肾病患者为对象,前瞻性观察透析液水处理系统升级前后患者病情变化.以脱离血液透析(死亡、转为腹膜透析或肾移植)和(或)生存至入组后8年为观察终点.比较水处理系统升级前后透析液内毒素含量及患者血清白细胞介素6(IL-6)、肿瘤坏死因子α(TNF-α)、C反应蛋白(CRP)、白蛋白水平等的变化.结果 水处理系统和中央供液系统经高频热消毒结合内毒素过滤器升级改造后,与升级前比较,透析液内毒素年平均值显著下降[(0.046±0.012) EU/ml比(0.454±0.002) EU/ml,P＜0.01],并在观察期间内维持稳定水平;患者的血清IL-6显著下降[(3.947±3.624) ng/L比(13.779±7.106) ng/L,P=0.036];血清TNF-α显著下降[(7.935±3.864) ng/L比(12.804±8.017) ng/L,P=0.012];血清CRP年平均值显著下降[(0.194±0.149) mg/L比(0.561±0.309) mg/L,P＜0.01],并在观察期间内维持稳定水平;血清白蛋白年平均水平显著增加[(41.900±6.803) g/L比(38.140±7.083) g/L,P=0.042];患者的年平均血红蛋白水平无显著改变,但红细胞生成素应用剂量显著下降[(93.0±12.7)U·kg-1·周-1比(131.0±10.1)U·kg-1·周-1,P=0.015].结论 采用含双级反渗、高频热消毒以及内毒素过滤器在内的水处理和中央供液系统,能明显提升透析液水质.透析液水质提升显著改善了MHD患者的微炎性反应状态及减少相关并发症的发生.     

Kinin kinetics during different dialysis protocols with AN69 dialyser in ACEI-treated patients

The effect of different dialysis modes on kinin kinetics wasstudied in seven stable haemodialysis patients treated withAN69 dialysers and ACE inhibitors (ACEI). AN69 haemodiafiltrationwith calcium-enriched substitution (HDF), AN69 haemodialysiswith 1.75 (HD 1.75) and 1.50 (HD 1.50)mmol/l dialysate calcium,AN69 haemodialysis with 1.25mmol/l dialysate calcium and substitutionof 2.25 mmol/h calcium (HD +Ca), and cellulose acetate haemodiafiltration(CA HDF) were compared. Dialysis was uneventful in all patients.During dialysis, serum calcium, sodium, pH, albumin, and bradykininwere measured at the start and after 5 min at arterial, venous,and postinfusion side of the extracorporeal circuit. Serum predialysisbradykinin was 107±18fmol/ml (mean±SEM) in patientson HDF, 61±9 fmol/ml in patients on HD 1.50, 49±13fmol/ml in patients on HD 1.75, 35±3 fmol/1 in patientson HD±Ca, and 75±27 fmol/ml in CA HDF. No significantchange of mean bradykinin levels occurred after 5 min at thearterial and venous side of the dialyser or postinfusion. Individualhigh bradykinin levels, up to 2672 fmol/ml, were observed butwithout clinical consequences, suggesting that the thresholdvalue is difficult to determine. No significant correlationswere evidenced between bradykinin levels and any of the biochemicalmeasurements. The present data show an intraindividual variabilityof the bradykinin levels with variation coefficients rangingfrom 0.386 to 2.783. The present study illustrates that haemodialysisand haemodiafiltration with AN69 in ACEI-treated patients, underthe present conditions, does not result in anaphy-lactoid reactionsor in a clinically significant release of bradykinin. The occurrenceof anaphylactoid reactions with high bradykinin levels is probablythe result of several concurrent bioincompatible factors insensitized patients.