Economic evaluation of expanded hemodialysis with the Theranova 400 dialyzer: A post hoc evaluation of a randomized clinical trial in the United States.

INTRODUCTION: The Theranova 400 is a medium cut-off dialyzer that allows for superior clearance of larger middle molecules than traditional high-flux dialyzers. This study evaluates the association of expanded hemodialysis (HDx) using the Theranova dialyzer versus conventional hemodialysis (HD) with a high-flux dialyzer on hospitalization rates and healthcare costs as compared to conventional HD in a post hoc analysis of a randomized controlled trial. METHODS: In a non-concealed, 24-week clinical trial, maintenance HD patients were randomized to receive treatment with either Theranova 400 or a similar size high-flux dialyzer. Hospitalization rate and average length of stay were calculated from trial data. Use of erythropoiesis-stimulating agents and iron were assumed to be equal and therefore excluded from the model. Average cost per inpatient day was obtained from a publicly available published source. Probabilistic sensitivity analyses were conducted to account for variability in model inputs. FINDINGS: There were 86 patients (389 patient-months) in the Theranova group and 85 patients (366 patient-months) in the high-flux HD group. All-cause hospitalization rate was 45% lower with Theranova compared to high-flux HD (IRR = 0.55; p = 0.05). Average annual estimated cost of hospitalization was $6098 lower with Theranova compared to high-flux HD. Compared to high-flux HD, average annual estimated cost associated with Theranova use was $4772 lower per patient. Hospitalization rate and hospital length of stay were the main drivers of cost. CONCLUSIONS: Use of the Theranova dialyzer is associated with lower estimated costs of care among maintenance HD patients, driven by fewer hospitalization events.

Efficacy and Safety of Expanded Hemodialysis with the Theranova 400 Dialyzer: A Randomized Controlled Trial.

BACKGROUND AND OBJECTIVES: Expanded hemodialysis therapy enabled by medium cut-off membranes may promote greater clearance of larger middle molecules that comprise putative uremic solutes than conventional high-flux dialysis. This randomized trial evaluated the efficacy and safety of hemodialysis treatment with a medium cut-off dialyzer. DESIGN, SETTING, PARTICIPANTS, & MEASUREMENTS: Clinically stable patients on maintenance hemodialysis were randomized to receive dialysis with either a medium cut-off dialyzer (Theranova 400) or a high-flux dialyzer (Elisio-17H) over 24 weeks of treatment. The primary safety end point was the predialysis serum albumin level after 24 weeks of treatment. The primary efficacy end point was the reduction ratio of free λ light chains at 24 weeks of treatment. RESULTS: Among 172 patients on maintenance hemodialysis, mean age was 59±13 years, 61% were men, 40% were Black, and mean dialysis vintage was 5±4 years. Of the 86 patients randomized to each dialyzer, 65 completed the trial in each group. The reduction ratio for the removal of free λ light chains was significantly higher in the Theranova 400 group compared with the Elisio-17H group after 4 weeks (39% versus 20%) and 24 weeks (33% versus 17%; both P<0.001). Among secondary end points, the Theranova 400 group demonstrated significantly larger reduction ratios at 4 and 24 weeks for complement factor D, free κ light chains, TNFα, and ß2-microglobulin (P<0.001 for all), but not for IL-6. Predialysis serum albumin levels were similar between groups after 24 weeks (4 g/dl with the Theranova 400 and 4.1 g/dl with the Elisio-17H), consistent with noninferiority of the Theranova 400 dialyzer in maintaining predialysis serum albumin levels after 24 weeks of treatment. CONCLUSIONS: Hemodialysis therapy with the Theranova 400 dialyzer provides superior removal of larger middle molecules, as exemplified by free λ light chains, compared with a similar size high-flux dialyzer, while maintaining serum albumin level. CLINICAL TRIAL REGISTRY NAME AND REGISTRATION NUMBER: A Multi-Center, Prospective, Randomized, Controlled, Open-Label, Parallel Study to Evaluate the Safety and Efficacy of the Theranova 400 Dialyzer in End Stage Renal Disease (ESRD) Patients, NCT03257410.

Pyrogen retention: Comparison of the novel medium cut-off (MCO) membrane with other dialyser membranes.

Haemodialysis effectively removes small solutes and smaller-sized middle molecules from the blood; however, the clearance of larger middle molecules, which have been associated with negative effects, is poor. The novel medium cut-off (MCO) dialysis membrane has larger pore sizes and a more open structure than other high-flux membranes, providing improved removal of larger middle molecules while retaining albumin. However, larger pore sizes may potentially increase permeability to pyrogens, including endotoxins and other bacterial contaminants, that could be present in the dialysis fluid. In this study, we tested the capacity of low-flux, high-flux, MCO and high cut-off dialyser membranes with different pore sizes to prevent pyrogens crossing from dialysate to the blood side in a closed-loop test system, differentiating among lipopolysaccharides, peptidoglycans and bacterial DNA using a toll-like receptor assay. Even though the bacterial contamination levels in our test system exceeded the acceptable pyrogen dose for standard dialysis fluid, levels of lipopolysaccharides, peptidoglycans and bacterial DNA in the blood-side samples were too low to identify potential differences in pyrogen permeability among the membranes. Our results suggest that MCO membranes are suitable for haemodialysis using ISO standard dialysis fluid quality, and retain endotoxins at a similar level as other membranes.

Retention of beneficial molecules and coagulation factors during haemodialysis and haemodiafiltration.

Middle molecules (MMs) are associated with the pathology of uraemia, and are not effectively removed by standard extracorporeal treatments. Increased convection used in haemodiafiltration (HDF) can enhance the removal of MMs; however, high-volume HDF is not available to all patients. The new medium cut-off (MCO) membrane has been developed to allow increased removal of MMs using standard haemodialysis (HD). Improved removal of MMs has been shown with the MCO membrane compared with standard high-flux dialysers, but it is not known whether the increased pore size affects the retention of commonly used medications or that of coagulation factors in dialysis patients. Using an in vitro model, the retention of erythropoietin, heparin, insulin, vancomycin and several coagulation factors (Factors II, VII and X, protein C and antithrombin III) was investigated with the MCO membrane dialyser, compared with high-flux dialysers with polysulfone (in HDF) or polyethersulfone membranes (in HD and HDF). The retention of all molecules investigated was comparable between the MCO membrane and the high-flux dialysers. Results from the in vitro studies suggest that switching from a high-flux dialyser to the MCO membrane should not require changes to the medication dosing or anti-coagulation protocols of dialysis patients.

Hypoalbuminemia: a price worth paying for improved dialytic removal of middle-molecular-weight uremic toxins?

Hemodiafiltration (HDF) increases the removal of middle-molecular-weight uremic toxins and may improve outcomes in patients with end-stage kidney disease (ESKD), but it requires complex equipment and comes with risks associated with infusion of large volumes of substitution solution. New high-flux hemodialysis membranes with improved diffusive permeability profiles do not have these limitations and offer an attractive alternative to HDF. However, both strategies are associated with increased albumin loss into the dialysate, raising concerns about the potential for decreased serum albumin concentrations that have been associated with poor outcomes in ESKD. Many factors can contribute to hypoalbuminemia in ESKD, including protein energy wasting, inflammation, volume expansion, renal loss and loss into the dialysate; of these factors, loss into the dialysate is not necessarily the most important. Furthermore, recent studies suggest that mild hypoalbuminemia per se is not an independent predictor of increased mortality in dialysis patients, but in combination with inflammation it is a poor prognostic sign. Thus, whether hypoalbuminemia predisposes to increased morbidity and mortality may depend on the presence or absence of inflammation. In this review we summarize recent findings on the role of dialysate losses in hypoalbuminemia and the importance of concomitant inflammation on outcomes in patients with ESKD. Based on these findings, we discuss whether hypoalbuminemia may be a price worth paying for increased dialytic removal of middle-molecular-weight uremic toxins.

Requirements and Pitfalls of Dialyzer Sieving Coefficients Comparisons.

Sieving coefficients reported in dialyzer data sheets and instructions for use (IFUs) indicate the potential of different solutes to pass across a particular membrane. Despite being measured in vitro, sieving coefficient data are often used as a predictor of the clinical performance of dialyzers. Although standards for the measurement of sieving coefficients exist, the stated methodologies do not offer sufficient guidance to ensure comparability of test results between different dialyzers. The aim of this work was to investigate the relationship between sieving coefficients and published clinical performance indicators for two solutes, albumin loss and beta-2 microglobulin (ß2 M) reduction ratio (RR), and to assess the impact of different in vitro test parameters on sieving coefficient values for albumin, ß2 M, and myoglobin. Clinical albumin loss and ß2 M RR for commercially available dialyzers used in hemodialysis (HD) and post-dilution hemodiafiltration (HDF) were extracted from the literature and plotted against sieving coefficients reported in data sheets and IFUs. Albumin, ß2 M, and myoglobin sieving coefficients of a selection of dialyzers were measured per the ISO 8637 standard. The impact of in vitro testing conditions was assessed by changing blood flow rate, ultrafiltration (UF) rate, sampling time, and origin of test plasma. Results showed variation in albumin loss and ß2 M RR for the same sieving coefficient across different dialyzers in HD and HDF. Changes in blood flow rates, UF rates, sampling time, and test plasma (bovine vs. human) caused marked differences in sieving coefficient values for all investigated solutes. When identical testing conditions were used, sieving coefficient values for the same dialyzer were reproducible. Testing conditions have a marked impact on the measurement of sieving coefficients, and values should not be compared unless identical conditions are used. Further, variability in observed clinical data in part reflects the lack of definition of test conditions.

Assessment of the association between increasing membrane pore size and endotoxin permeability using a novel experimental dialysis simulation set-up.

BACKGROUND: Membranes with increasing pore size are introduced to enhance removal of large uremic toxins with regular hemodialysis. These membranes might theoretically have higher permeability for bacterial degradation products. In this paper, permeability for bacterial degradation products of membranes of comparable composition with different pore size was investigated with a new in vitro set-up that represents clinical flow and pressure conditions. METHODS: Dialysis was simulated with an AK200 machine using a low-flux, high-flux, medium cut-off (MCO) or high cut-off (HCO) device (n = 6/type). A polyvinylpyrrolidone-solution (PVP) was recirculated at blood side. At dialysate side, a challenge solution containing a filtrated lysate of two water-borne bacteria (Pseudomonas aeruginosa and Pelomononas saccharophila) was infused in the dialysate flow (endotoxin ≥ 4EU/ml). Blood and dialysate flow were set at 400 and 500 ml/min for 60 min. PVP was sampled before (PVPpre) and after (PVPpost) the experiment and dialysate after 5 and 55 min. Limulus Amebocyte Lysate (LAL) test was performed. Additionally, samples were incubated with a THP-1 cell line (24 h) and IL-1ß levels were measured evaluating biological activity. RESULTS: The LAL-assay confirmed presence of 9.5 ± 7.4 EU/ml at dialysate side. For none of the devices the LAL activity in PVPpre vs. PVPpost was significantly different. Although more blood side PVP solutions had a detectable amount of endotoxin using a highly sensitive LAL assay in the more open vs traditional membranes, the permeability for endotoxins of the 4 tested dialysis membranes was not significantly different but the number of repeats is small. None of the PVP solutions induced IL-1ß in the THP-1 assay. CONCLUSIONS: A realisitic in vitro dialysis was developed to assess membrane translocation of bacterial products. LAL activity on the blood side after endotoxin exposure did not change for all membranes. Also, none of the PVPpost solutions induced IL-1ß in the THP-1 bio-assay.

Membrane Innovation in Dialysis.

Despite advances in renal replacement therapy, the adequate removal of uremic toxins over a broad molecular weight range remains one of the unmet needs in hemodialysis. Therefore, membrane innovation is currently directed towards enhanced removal of uremic toxins and increased membrane permeability. This chapter presents a variety of opportunities where innovation is brought into dialysis membranes. It covers the membrane formation from solution, describing different approaches to control the phase inversion process through additives that either swell in the polymer solution or influence the pore shrinkage during the membrane drying process. Additionally, large-scale manufacturing is described, and the influence of raw materials, spinning, and drying processes on membrane selectivity are presented. Finally, new characterization methods developed for the latest innovations around the application of membranes in dialysis are discussed, which allow the membrane performance for removal of a broad range of uremic toxins and the expected albumin loss in clinical use.

Performance of hemodialysis with novel medium cut-off dialyzers.

Background: Compared to high-flux dialysis membranes, novel medium cut-off (MCO) membranes show greater permeability for larger middle molecules. Methods: In two prospective, open-label, controlled, randomized, crossover pilot studies, 39 prevalent hemodialysis (HD) patients were studied in four dialysis treatments as follows: study 1, three MCO prototype dialyzers (AA, BB and CC with increasing permeability) and one high-flux dialyzer in HD; and study 2, two MCO prototype dialyzers (AA and BB) in HD and high-flux dialyzers in HD and hemodiafiltration (HDF). Primary outcome was lambda free light chain (λFLC) overall clearance. Secondary outcomes included overall clearances and pre-to-post-reduction ratios of middle and small molecules, and safety of MCO HD treatments. Results: MCO HD provided greater λFLC overall clearance [least square mean (standard error)] as follows: study 1: MCO AA 8.5 (0.54), MCO BB 11.3 (0.51), MCO CC 15.0 (0.53) versus high-flux HD 3.6 (0.51) mL/min; study 2: MCO AA 10.0 (0.58), MCO BB 12.5 (0.57) versus high-flux HD 4.4 (0.57) and HDF 6.2 (0.58) mL/min. Differences between MCO and high-flux dialyzers were consistently significant in mixed model analysis (each P < 0.001). Reduction ratios of λFLC were greater for MCO. Clearances of α1-microglobulin, complement factor D, kappa FLC (κFLC) and myoglobin were generally greater with MCO than with high-flux HD and similar to or greater than clearances with HDF. Albumin loss was moderate with MCO, but greater than with high-flux HD and HDF. Conclusions: MCO HD removes a wide range of middle molecules more effectively than high-flux HD and even exceeds the performance of high-volume HDF for large solutes, particularly λFLC.

Comparative Effectiveness of Dialyzers: A Longitudinal, Propensity Score-Matched Study of Incident Hemodialysis Patients.

Differences in dialyzer design may have consequences for patient outcomes. We evaluated the comparative effectiveness of commonly used dialyzers with respect to measures of dialysis treatment, anemia management, inflammation, and dialyzer clotting. Patients receiving hemodialysis between January 1, 2009, and December 31, 2013, and using polyarylethersulfone-polyvinylpyrrolidone (PAS-PVP; Polyflux Revaclear) or polysulfone (PS; Optiflux 160 or Optiflux 180) dialyzers were followed for 1 year or until end of study or censoring for dialyzer switch, modality change, or loss to follow-up. For each comparison, eligible patients were propensity score-matched 1:1 on a range of baseline characteristics. Outcomes were assessed using generalized linear mixed models. Dialysis adequacy was similar in both dialyzer groups. Erythropoiesis-stimulating agent (ESA) doses were lower for patients using PAS-PVP versus patients using PS-160 (difference range: 75-589 units/treatment; statistically significant in months 1-5 and 7) and for patients using PAS-PVP versus patients using PS-180 (difference range: 27-591 unit/treatment; statistically significant in months 1-9). Intravenous iron doses trended lower for patients using PAS-PVP versus patients using PS, but hemoglobin concentrations were equivalent. In conclusion, use of PAS-PVP versus PS dialyzers was associated with equivalent dialysis adequacy, lower ESA doses, modestly lower Intravenous iron doses, and equivalent hemoglobin concentrations.

Effect of a More Permeable Dialysis Membrane on ESA Resistance in Hemodialysis Patients--A Pilot Investigation.

BACKGROUND/AIMS: Hemodialysis (HD) patients often show impaired response to erythropoiesis-stimulating agents (ESAs). Extended HD membrane permeability may potentially improve ESA response. METHODS: Twenty-four prevalent HD patients were randomly assigned to 12 weeks use of high cut-off (HCO) membrane (in every second dialysis treatment) or continued treatment with high-flux membrane. We monitored changes in hemoglobin (Hb), ESA dose, and key biochemical markers. RESULTS: The Hb level increased in the study group (from 11.6 ± 1.0 to 12.5 ± 1.5 g/dl; p = 0.038) but was stable in the control group. Variation over time in ESA dose and ESA resistance index did not differ between groups. HCO membrane usage for 12 weeks led to decreased hepcidin level, from 303 ± 189 to 157 ± 83 ng/ml (p = 0.024); serum albumin level decreased and stabilized 15 ± 6% below baseline. CONCLUSIONS: These results indicate that use of a more permeable dialysis membrane may improve ESA responsiveness in iron-replete HD patients. Extensive albumin removal may preclude long-term use of the HCO membrane.

In Vitro Comparative Assessment of Mechanical Blood Damage Induced by Different Hemodialysis Treatments.

Gradual deterioration of red blood cells (RBCs) due to mechanical stress (chronic hemolysis) is unavoidable during treatments that involve extracorporeal blood circulation, such as hemodialysis (HD). This effect is generally undetectable and does not generate any acute symptoms, but it leads to an increase in plasma free hemoglobin (fHb). There are no absolute safety levels for fHb increase, indicating the need for an empirical evaluation using comparative testing. The increase in fHb levels was investigated in vitro by applying double-needle double-pump HD (HD-DNDP), a new modality in which arterial and venous pumps both run continuously. fHb was measured during typical and worst-case simulated dialysis treatments (double-needle single-pump HD [HD-DNSP], hemodiafiltration [HDF-DN], single-needle double-pump HD [HD-SNDP], and HD-DNDP) performed in vitro using bovine blood for 4 h. Hemolysis-related indices (fHb%; index of hemolysis, IH; and normalized IH) were calculated and used for comparison. The increase in fHb during either HDF-DN or HD-SNDP with Artis and AK200 dialysis machines was similar, while the fHb at the maximum real blood flow rate (Qbreal ) at the completion of the HD-DNDP treatment on Artis was higher than that for HD-DNSP using a Phoenix dialysis machine (fHb% = 1.24 ± 0.13 and 0.92 ± 0.12 for the Artis machine with HD-DNDP at Qbreal = 450 mL/min and Phoenix with HD-DNSP at Qbreal = 500 mL/min, respectively). However, the fHb levels increased linearly, and no steep changes were observed. The increases observed during HD-DNDP were the same order of magnitude as those for widely used bloodlines and treatment modes for delivering dialysis treatments. The observed results matched literature findings, and thus the measured fHb trends are not predicted to have clinical side effects. HD-DNDP treatment with Artis does not merit any additional concern regarding mechanical stress to RBCs compared with that observed for routinely used dialysis treatments, bloodlines and machines. Although the in vitro measurement of the fHb increase in bovine blood does not allow a prediction of the absolute level of blood mechanical damage or the possible effects in humans, such measurements are valuable for assessing hemolytic harm by performing tests comparing the proposed treatment with existing devices.

Treatment frequency and efficiency in hemodiafiltration.

BACKGROUND: One of the main objectives of dialysis is uremic retention product elimination. Efficiency of dialysis modalities varies both regarding the range of solutes removed and the extent of such removal. We analyzed plasma (or blood) concentrations of marker solutes in intermittent treatment schedules using hemodiafiltration (HDF). METHODS: Elimination and rebound of uremic solutes were measured in 10 patients (77 ± 12 kg, 66.5 ± 9.2 years) treated with postdilution HDF in one 4-hour treatment and in two 2-hour treatments on consecutive days (Polyflux 2.1 m(2), QB 451 ± 53 ml/min, QD 598 ± 13 ml/min). Blood urea, creatinine, phosphate, ß2-microglobulin, complement factor D and advanced glycation end products were analyzed before, during and after HDF for 24-48 h. RESULTS: Applying two 2-hour HDF treatments on consecutive days resulted in significantly lower plasma (or blood) levels of urea, creatinine, phosphate, ß2-microglobulin, and advanced glycation end products after 48 h than using one 4-hour session. CONCLUSIONS: Increased treatment frequency could further optimize blood purification in HDF therapy.

Bismuth coating of non-tunneled haemodialysis catheters reduces bacterial colonization: a randomized controlled trial.

BACKGROUND: Haemodialysis (HD) catheter-related blood stream infections are a major cause of morbidity and mortality in patients with acute and chronic renal failure. METHODS: We conducted a randomized, prospective, double-blinded trial investigating the clinical value of bismuth-coated non-tunneled HD catheters in patients in need of temporary short-term vascular access. A standard catheter (SC) was compared to a surface-modified, bismuth-film-coated catheter (FCC). After removal of the catheter for any reason, both arterial and venous lumina were rinsed and the fluid cultured for detection of bacterial colony-forming units (CFU). The catheter tip was placed in a tube containing sterile saline, sonicated and shortly centrifuged to remove debris (3 min at 1000 g). The supernatant was cultured and assayed for DNA content. RESULTS: Seventy-seven patients in three HD units were randomized. Thirteen patients suffered from acute renal failure, 60 patients from chronic renal failure, and four patients without renal insufficiency were treated with plasma exchange. The time to catheter removal was not significantly different between groups, with a mean of 18.5 +/- 2 days for SC and 15.1 +/- 2 days for FCC. In most cases, the reasons for catheter removal were related to no further need for extracorporeal therapy or establishment of a permanent vascular access. Six catheters for SC and four catheters for FCC were removed because of presumed infection. Bacterial colonization was significantly lower for coated catheters compared to standard catheters, both for cultured catheter tips as well as for CFU in rinse fluids (P < 0.05). CONCLUSIONS: Surface modification with bismuth film reduces bacterial colonization of temporary non-tunneled HD catheters in a clinical trial. Larger trials with these modified catheters are justified to further investigate the effect on catheter-related infections, complications and costs.

The role of polymer surface degradation and barium sulphate release in the pathogenesis of catheter-related infection.

BACKGROUND: Susceptibility to infection and thrombosis of intravascular catheters is increased by surface irregularities, which might be prevented by coating. METHODS: BaSO4 release from conventional haemodialysis catheters (CC) and modified catheters (MC) which had been coated with a surface-modifying additive (SMA) was assessed in vivo and in vitro. For the in vivo part, patients were randomized to receive a temporary CC or MC, with crossover after 1 week. After retrieval, catheters were examined using scanning electron microscopy to assess surface integrity, and an in vitro model of catheter exposure to the bloodstream was used to evaluate surface morphology and susceptibility to bacterial adhesion and proliferation. RESULTS: BaSO(4) moieties covered 14.7 +/- 3.7% of the surface of unused CC. After in vivo use in 16 patients, 62.7 +/- 32.9 x 10(3) holes/mm(2) were detected, indicating BaSO(4) detachment from 3.3 +/- 1.7% of the catheter surface. No defects were observed in unused CC and in MC, whether used or unused. After incubation of four catheters (two of each type) with Staphylococcus epidermidis, the two degraded CC showed an immediate and strong bacterial growth as indicated by an increase in medium impedance of 0.512%/10 min compared to -0.021%/10 min in MC (P < 0.001). CONCLUSIONS: Short-term exposure of CC to the bloodstream causes BaSO(4) particle release, resulting in surface irregularities predisposing to bacterial proliferation. BaSO(4) release can be prevented by SMA coating.

Differential effects of dialysis and ultrafiltrate from individuals with CKD, with or without diabetes, on platelet phosphatidylserine externalization.

Individuals with chronic kidney disease (CKD) and/or diabetes mellitus (DM) are at increased risk of cardiovascular events and have elevated externalization of phosphatidylserine (PS; which propagates thrombus formation) in a small subpopulation of platelets. The purpose of this study was to examine the effect of 1) removing uremic toxins by hemodialysis on PS externalization in patients with either CKD or CKD and DM and 2) ultrafiltrate (UF) from these individuals on PS externalization in healthy platelets. PS externalization was quantified by a fluorescence-activated cell sorter using annexin V in platelet-rich plasma. PS externalization was elevated threefold in CKD patients and returned to basal values during 3-h hemodialysis. In contrast, it was elevated fivefold in individuals with CKD and DM and was still threefold above control after 3-h treatment. UF significantly increased PS externalization in a small subpopulation of platelets from healthy controls. The effect of UF from individuals with CKD and DM was significantly greater than that from patients with CKD alone, and the responses were partially inhibited by the protein kinase Cdelta (PKCdelta) inhibitor rottlerin and the 5-hydroxytryptamine (5-HT)(2A/2C) receptor antagonist ritanserin. The data suggest that uremic toxins present in UF mediate PS externalization in a small subpopulation of platelets, at least in part, via the 5-HT(2A/2C) receptor and PKCdelta and demonstrate that DM further enhances platelet PS externalization in CKD patients undergoing hemodialysis. This may explain, at least in part, the additional increase in vascular damage observed in CKD patients when DM is present.

Advanced glycation end products elicit externalization of phosphatidylserine in a subpopulation of platelets via 5-HT2A/2C receptors.

Advanced glycation end products (AGE) are substantially elevated in individuals with diabetes and/or chronic kidney disease (CKD). These patients are at greatly increased risk of cardiovascular events. The purpose of this study was to investigate the novel hypothesis that AGE elicit externalization of the platelet membrane phospholipid phosphatidylserine (PS). This contributes to hemostasis through propagation of the coagulation cascade leading to thrombus formation. Platelet-rich plasma (PRP) was prepared by differential centrifugation, and PS externalization was quantified by a fluorescence-activated cell sorter using annexin V-FITC. Human serum albumin (HSA)-AGE was generated by incubating HSA with glucose for 2, 4, or 6 wk, and total HSA-AGE was assessed by fluorescence intensity. The 2-wk HSA-AGE preparation (0-2 mg/ml) stimulated a concentration-dependent increase in PS externalization in a subpopulation of platelets that was threefold at 2 mg/ml. In contrast, the 4- and 6-wk preparations were maximal at 0.5 mg/ml and fivefold in magnitude. These effects mirrored the change in total HSA-AGE content of the preparations. The PS response was maximal at 10 min and inhibited by the PKC-delta inhibitor rottlerin and the serotonin [5-hydroxytryptamine (5-HT)](2A/2C) receptor antagonist ritanserin in a dose-dependent manner. Moreover, the 5-HT(2A/2C) receptor agonist 1,2,5-dimethoxy-4-iodophenyl-2-aminopropane mimicked the effect of HSA-AGE on PS externalization. These data demonstrate, for the first time, that HSA-AGE stimulates PS externalization in a subpopulation of platelets via the 5-HT(2A/2C) receptor. This may have important consequences for platelet involvement in inflammatory responses and the increased cardiovascular risk observed in individuals with diabetes and/or CKD.

Reduced cytokine induction and removal of complement products with synthetic hemodialysis membranes.

The increasing use of high-flux membranes for hemodialysis (HD) has raised concerns that these membranes may confer a higher risk of exposure to cytokine-inducing, bacterial substances (CIS) in the dialysate. Several studies, however, reported higher transfer of CIS through low-flux cellulosic than high-flux synthetic membranes. This surprising paradox was explained by adsorption of CIS to certain high-flux membranes. In order to investigate flux and membrane type independently, we studied two synthetic Polyflux (PF) membranes of the same type but with different flux properties and compared them to a cellulosic membrane (Cuprophan). Three different approaches were employed: (1) cytokine induction in whole blood during in vitro HD contaminated with bacterial filtrates, (2) removal of recombinant C5a, and (3) transfer of purified lipopolysaccharide (LPS). After 90 min recirculation of whole blood, the appearance of IL-6-inducing substances on the blood side was lowest with high-flux PF (1.1 +/- 0.2 ng/ml), slightly higher with low-flux PF (1.9 +/- 0.7 ng/ml) and highest with Cuprophan (4.1 +/- 1 ng/ml). Recombinant C5a added to plasma on the blood side was markedly removed by high-flux PF (by 83%), to a lesser degree and only in the presence of ultrafiltration with low-flux PF (by 54%) and not significantly with Cuprophan (by 11%). Significant transfer of purified LPS from the dialysate onto the blood side was only observed with the cellulosic membrane. We conclude that in contrast to cellulosic membranes, certain synthetic membranes do not permit transfer of LPS. Cytokine induction on the blood side is further reduced by the use of high-flux membranes due to removal of activated complement factors.

Short bacterial DNA fragments: detection in dialysate and induction of cytokines.

A number of bacterial cytokine-inducing substances (CIS) such as lipopolysaccharides (LPS) and exotoxins have been detected in dialysate and may contribute to inflammation in hemodialysis patients. Short DNA fragments, oligodeoxynucleotides (ODN) of 6 to 20 nucleotides, are able to bind to Toll-like receptors and are stimulatory on immune cells. ODN induce natural killer cell activity and induce IFN-gamma, TNF-alpha, and IL-6 from mononuclear cells. The presence of ODN in dialysate samples and bacterial cultures was investigated. ODN were extracted from fluids by adsorption to reverse-phase columns. ODN were detected in 18 of 20 investigated dialysate samples, in eight of 10 reverse-osmosis water samples, and in all cultures from various bacterial strains. The presence of bacterial DNA in dialysate was confirmed by PCR specific for bacterial tRNA gene sequences. Saline for intravenous use contained 0.02 +/- 0.01 microg/ml DNA, dialysate samples contained 0.28 +/- 0.02 microg/ml, and Pseudomonas cultures contained 1.0 +/- 0.03 microg/ml DNA. ODN from bacterial cultures were only partially removed by ultrafiltration and were able to diffuse through regular high-flux dialyzer membranes. Synthetic cytosine-guanosine dinucleotide-containing ODN were able to induce IL-6 in human mononuclear cells. It is concluded that short bacterial-derived DNA fragments are present in clinically used fluids, e.g., dialysate. These fragments are of sufficient small size to pass through dialyzer membranes. Bacterial DNA fragments may be an overlooked factor contributing to inflammation in hemodialysis patients.