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.

Low-GDP fluid (Gambrosol trio) attenuates decline of residual renal function in PD patients: a prospective randomized study.

BACKGROUND: Residual renal function (RRF) impacts outcome of peritoneal dialysis (PD) patients. Some PD fluids contain glucose degradation products (GDPs) which have been shown to affect cell systems and tissues. They may also act as precursors of advanced glycosylation endproducts (AGEs) both locally and systemically, potentially inflicting damage to the kidney as the major organ for AGE elimination. We conducted a clinical study in PD patients to see if the content of GDP in the PD fluid has any influence on the decline of the residual renal function. METHODS: In a multicentre approach, 80 patients (GFF > or = 3 mL/min/1.732 or creatinine clearance > or =3 mL/min/1.73 m(2)) were randomized to treatment with a PD fluid containing low levels of GDP or standard PD fluid for 18 months. RRF was assessed every 4-6 weeks. Fluid balance, mesothelial cell mass marker CA125, peritoneal membrane characteristics, C-reactive protein (CRP), total protein, albumin, electrolytes and phosphate were measured repeatedly. RESULTS: Data from 69 patients revealed a significant difference in monthly RRF change: -1.5% (95% CI = -3.07% to +0.03%) with low GDP (43 patients) vs -4.3% (95% CI = -6.8% to -2.06%) with standard fluids (26 patients) (P = 0.0437), independent of angiotensin-converting enzyme (ACE) inhibitor or angiotensin receptor blocker medication. Twenty-four-hour urine volume declined more slowly with low-GDP fluid compared to standard fluids (12 vs 38 mL/month, P = 0.0241), and monthly change of phosphate level was smaller (+0.013 vs +0.061 mg/dL, P = 0.0381). CONCLUSIONS: Our prospective study demonstrates for the first time a significant benefit concerning preservation of RRF and urine volume of using a PD fluid with low GDP levels. These findings suggest that GDPs might affect patient outcome related to RRF.

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.

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.

Increased binding of beta-2-microglobulin to blood cells in dialysis patients treated with high-flux dialyzers compared with low-flux membranes contributed to reduced beta-2-microglobulin concentrations. Results of a cross-over study.

BACKGROUND: Patients on long-term dialysis eventually develop amyloid deposits with beta2-microglobulin as a predominant component. Although several studies have suggested that high-flux membranes reduce beta2-microglobulin in plasma compared with low-flux dialyzers, the mechanisms underlying this observation are still discussed. METHODS: We revisited this important subject and measured beta2-microglobulin in the plasma of healthy individuals (n = 8), and patients undergoing hemodialysis (n = 20) who for assigned periods of time were either treated with a low-flux membrane (cuprophan) or high-flux (polyamide) dialyzer with an ELISA. The number of blood cells was determined by FACS. Beta2-microglobulin was also measured on the surface of granulocytes, lymphocytes, and monocytes before, directly after, and 4 h after hemodialysis. Expression of beta2-microglobulin, c-fos, tumor necrosis factor-alpha (TNF-alpha), and interleukin-1 mRNA was determined in whole blood samples with quantitative RT-PCR using an internal standard in parallel. In the second part of the study, patients were assigned in a two-group cross-over design either to low- or high-flux dialyzers (n = 9 in each group), and dialyzer membranes were changed every 4 weeks for two consecutive periods. Serum beta2-microglobulin concentrations were measured at the end of each period. RESULTS: Healthy controls had a low plasma beta2-microglobulin level of 1.2 +/- 0.3 mg/l. Before hemodialysis, patients on low-flux dialyzers had a plasma beta2-microglobulin level of 42.0 +/- 14.0 mg/l, patients treated with high-flux dialyzers 21.5 +/- 10.8 mg/l (p < 0.05 vs. low-flux dialyzers). In contrast, there was no significant difference in plasma concentrations of active transforming growth factor-beta1 with the two different membrane types. The difference in serum beta2-microglobulin between low- and high-flux membranes was more prominent directly after hemodialysis as well as 4 h after hemodialysis compared with the values directly before the start of treatment. At all studied time-points, leukocytes and platelets were significantly higher in patients on low-flux membranes. Healthy control persons exhibited a significantly higher amount of beta2-microglobulin bound to granulocytes, lymphocytes, and monocytes compared with dialysis patients. Interestingly, beta2-microglobulin bound to granulocytes, lymphocytes, and monocytes was significantly increased in patients treated with high-flux membranes compared with low-flux filters. Quantitative RT-PCR revealed no significant difference in beta2-microglobulin expression in whole blood before hemodialysis, directly after hemodialysis, and 4 h after hemodialysis. However, TNF-alpha and c-fos transcripts were significantly higher in whole blood obtained from patients treated with low-flux membranes compared to high-flux dialyzers. The two-group cross-over study over three periods of 4 weeks revealed that switching from low-flux to high-flux dialyzers significantly reduced serum beta2-microglobulin levels. CONCLUSION: Patients treated with a polyamide high-flux membrane had lower beta2-microglobulin concentrations compared with those patients on low-flux dialyzers. This difference might not be mediated by an increase in beta2-microglobulin mRNA, but may be caused by less beta2-microglobulin released from the blood cells in patients treated with high-flux dialyzers, in addition to a better beta2-microglobulin clearance.

Expression patterns of the lipopolysaccharide receptor CD14, and the FCgamma receptors CD16 and CD64 on polymorphonuclear neutrophils: data from patients with severe bacterial infections and lipopolysaccharide-exposed cells.

In polymorphonuclear neutrophils (PMN) CD14, one of the receptors for lipopolysaccharides (LPS) is stored intracellularly as a preformed protein, with only few receptors expressed on the surface. We now report that in patients with severe bacterial infections, CD14 expression is profoundly upregulated, as is CD64 (FcgammaRI), the high-affinity receptor for IgG, whereas CD16 (FcgammaRIII) was partly lost from the surface. To further analyze regulation of these receptors, PMN of healthy donors were exposed to low doses of LPS. By brief exposure (10-120 min) to LPS, CD14 was transferred to the surface in a cytochalasin B-sensitive manner, as were CD16 and CD64. Prolonged culture (up to 48 h) resulted in a further upregulation of CD14, sustained expression of CD64, and profound decline of CD16, yielding a similar pattern of receptor expression as seen in the patients. Subsequent studies revealed that LPS induced de novo synthesis of CD14: the increase of surface expression could be inhibited by cycloheximide and by interfering with a known LPS-induced signaling event, the translocation of NFkappaB. Moreover, an up to 10-fold increase of specific mRNA was seen, as was incorporation into CD14 of 35S-methionine. The de novo synthesis prolonged expression of CD14, whereas the CD16 expression declined, generating a PMN phenotype characteristic for severe infection and indicative of escape from apoptosis of a PMN subpopulation.

Prolonged catheter survival in intermittent hemodialysis using a less thrombogenic micropatterned polymer modification.

Adequate vascular access is a major prerequisite for hemodialysis treatment. Catheter related complications, in particular thrombus formation, are frequent, difficult to handle, and cost intensive. We investigated whether a new surface modified catheter containing a microdomain structure is beneficial for catheter survival. Surface thrombogenicity of standard double lumen catheters (STD-DC) and surface modified film-coated domain structured double lumen catheters (FCDS-DC) consisting of a novel reactive polyurethane copolymer coating was assessed by measurement of thrombinantithrombin (TAT) III complex in vitro. Furthermore, in a randomized observational study with 20 patients on hemodialysis, we analyzed catheter survival of either STD-DC (GamCath GDK 11 French, length 12.5 or 15 cm) or FCDS-DC (GamCath Dolphin GDK 11 French, length 12.5 or 15 cm). Catheter care protocol was identical, and dialysis treatment parameters were kept constant in both groups. In vitro measured surface thrombogenicity was reduced in the modified catheter compared with standard catheter. The clinical investigation revealed that both number of days before catheter removal according to clinical requirements and number of treatments per catheter were significantly higher with the modified catheter as compared with the standard catheter (14.5 vs. 10.3 days; 7 vs. 4 treatments; p < 0.03, Fisher-Yates test). Micropatterned surface coating with a polyurethane polymer significantly increased catheter survival and the number of treatments per catheter.

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.

Inconsistency of reported uremic toxin concentrations.

Discrepancies in reported uremic toxin concentrations were evaluated for 78 retention solutes. For this analysis, 378 publications were screened. Up to eight publications per toxin were retained. The highest and the lowest reported concentrations, as well as the median reported concentration were registered. The ratio between the highest and the lowest (H/L) concentrations and, for some solutes, also the ratio between the highest and the median (H/M) concentrations were calculated. The compounds were arbitrarily subdivided into three groups based on their H/L ratio: group A, H/L < 3 (n = 33); group B, 3 < H/L < 8.5 (n = 20); and group C, H/L > 8.5 (n = 25). Solutes of groups A and B showed a low to intermediate scatter, suggesting a homogeneity of reported data. Group C showed a more substantial scatter. For at least 10 compounds of group C, extremely divergent concentrations were registered (H/M > 5.5) using scatter plot analysis. For all solutes of groups A and B, the highest reported concentration could be used as a reference. For some solutes of group C and for the compounds showing a divergent scatter analysis, however, more refined directives should be followed.

An oral load of the early glycation compound lactuloselysine fails to accumulate in the serum of uraemic patients.

BACKGROUND: It has been hypothesized that in renal failure, exogenous glycation compounds from food accumulate and play a major pathogenetic role when renal excretion is impaired. METHODS: To address this, a diet containing a defined amount of the lysine Amadori product (AP) lactuloselysine was used. Plasma concentrations and cumulative urinary excretion of AP were assessed in 16 healthy subjects, 12 renal failure patients and 6 continuous ambulatory peitoneal dialysis (CAPD) patients. Amadori product was measured as furosine using reverse phase high performance liquid chromatography (RP-HPLC) after acid hydrolysis. RESULTS: A diet low in glycation compounds significantly decreased excretion of APs in healthy subjects. In healthy individuals, ingestion of lactuloselysine bound to food proteins caused only a minor acute increase (8.24+/-1.11 mg/day, 2% of the administered dose) of AP excretion in the urine; in patients with renal failure not yet on dialysis, the increase in AP excretion in the urine was significantly less (4.0+/-0.51 mg/day) and the same was true in CAPD patients (0.21+/-0.09 mg/day). The plasma concentration of total APs, i.e. the sum of APs as free amino acids and residues bound to plasma proteins, did not change in any of the three groups, however. CONCLUSION: Dietary APs do not accumulate in the blood even in advanced renal failure. The amount of APs measured as furosine excreted in the urine is significantly less, however, in renal failure and CAPD patients compared with healthy subjects. Although the findings exclude accumulation of lactuloselysine in renal failure, they do not generally exclude accumulation of other food-derived advanced glycation end products (AGEs).

Damage to the peritoneal membrane by glucose degradation products is mediated by the receptor for advanced glycation end-products.

Peritoneal dialysis is limited by morphologic changes of the peritoneal membrane. Use of peritoneal dialysis fluids (PDF) that contain glucose degradation products (GDP) generates advanced glycation end-products (AGE) within the peritoneal cavity. It is unknown whether peritoneal damage is causally related to AGE-receptor for AGE (RAGE) interaction. The effects of PDF were compared with different amounts of GDP on morphologic changes of the peritoneal membrane in 48 wild-type (WT) and 48 RAGE-deficient mice. PDF (1 ml) were instilled twice daily over a period of 12 wk. Groups with eight animals each received no manipulation (sham); sham instillation (sham i.p.); or filter-sterilized, glucose-free, conventional low GDP- or high GDP PDF. In vitro (generation of AGE fluorescence in PDF) and in vivo (immunohistochemistry for carboxymethyllysine), a GDP-dependent increase of AGE formation occurred. Inflammation and neoangiogenesis were augmented in WT mice that were treated with high GDP accompanied by upregulation of CD3+ T cells, increased NF-kappaB binding activity, increased lectin, and vascular endothelial growth factor expression. Furthermore, pronounced submesothelial fibrosis was found with increased expression of TGF-beta1. Exposure to low GDP resulted in only mild inflammation and neoangiogenesis (compared with sham i.p.) and no fibrosis in WT mice. The findings in WT contrasted with those in RAGE-deficient mice, which showed no increased inflammation (CD3+ T cells and NF-kappaB binding activity), neoangiogenesis (by lectin and vascular endothelial growth factor expression), or fibrosis (expression of TGF-beta1) after long-term exposure to GDP-containing PDF. Peritoneal damage by GDP in PDF is dependent at least in part on AGE-RAGE interaction.

In vitro evidence for immune activating effect of specific AGE structures retained in uremia.

BACKGROUND: Advanced glycation end-products (AGEs) have been identified to be accumulated in blood and tissues of patients with end-stage renal disease (ESRD). AGEs have been shown to modulate immune competent cell activities and in this way they may contribute to the progression of atherosclerosis. All studies in this context have been performed, however, with generated mix of glycation compounds, and not with structures similar to those encountered in uremia. In the present study, the immunologic effect of specific AGE compounds, known to be retained in uremia, has been evaluated. METHODS: Four albumin preparations, modified chemically at lysine or arginine residues, respectively, to contain N-epsilon-carboxymethyllysine (CML albumin), N-epsilon-carboxyethyllysine (CEL albumin), glyoxal-induced imidazolinones (Arg I albumin) or methylglyoxal-induced imidazolinones (Arg II albumin) were applied. Their effect on chemiluminescence production, CD14 expression, and the DNA synthesis of calcitriol-differentiated HL-60 (monocyte/macrophage phenotype) was studied. RESULTS: The phorbol 12-myristate 13-acetate (PMA)-stimulated chemiluminescence production of the calcitriol differentiated HL-60 cells was enhanced in the presence of CEL albumin (44.1 +/- 18.5 vs. 64.7 +/- 28.1 counts 10(3)/30 min) (P < 0.05), Arg I albumin (46.4 +/- 18.8 vs. 66.1 +/- 32.6 counts 10(3)/30 min) (P < 0.05) and CML albumin (41.9 +/- 25.5 vs. 60.9 +/- 5.5 counts 10(3)/30 min) (P= 0.0625) pointing to an increase in free radical production. The latter AGE compounds also significantly increased the calcitriol-induced CD14 expression on HL-60 cells (1675 +/- 796 vs. 2075 +/- 1044; 768 +/- 143 vs. 890 +/- 150; 647 +/- 63 vs. 716 +/- 69 mean fluorescence intensity) (P < 0.05, respectively) pointing to an increase in expression of the lipopolysaccharide (LPS) receptor. Finally, the DNA synthesis of the calcitriol-differentiated HL-60 cells was enhanced in the presence of Arg I albumin [34.5 +/- 4.6 vs. 27.7 +/- 9.7% 5-bromo-2'-deoxyuridine (BrdU)-positive cells] (P < 0.05) resulting in an increased cell proliferation. CONCLUSION: Genuine AGE compounds, as they are encountered in the uremic condition, activate leukocyte response, and hence could play a role in uremia related atherogenesis.

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.

3,4-Dideoxyglucosone-3-ene (3,4-DGE): a cytotoxic glucose degradation product in fluids for peritoneal dialysis.

BACKGROUND: Bioincompatible glucose degradation products (GDPs) in fluids for peritoneal dialysis (PD) develop during sterilization and storage. Their biological activity has successfully been monitored through the use of various in vitro methods but their molecular and chemical nature is less well understood. Many GDPs are highly reactive carbonyl compounds. Although some of the identified GDPs are extremely cytotoxic, none of them actually possess cytotoxicity at the concentrations found in PD fluids. Thus, the GDP responsible for the toxicity in PD fluids has not yet been identified. The intention of the present work was to investigate to what extent the unsaturated dicarbonyl compound, 3,4-dideoxyglucosone-3-ene (3,4-DGE) was present in PD fluids, and if it could be responsible for the in vitro effects on L-929 fibroblast cells. METHODS: A commercial preparation of 3,4-DGE and two different liquid chromatography methods were used for the chemical identification and quantification. In vitro bioincompatibility was determined as inhibition of cell growth using the L-929 fibroblast cell line. RESULTS: 3,4-DGE was present in conventionally manufactured PD fluids at a concentration of 9 to 22 micromol/L. In the newly developed PD fluid, Gambrosol trio, the concentrations were 0.3 to 0.7 micromol/L. When added as synthetic 3,4-DGE to cell growth media at the concentrations measured in conventional PD fluids, the inhibition of cell growth was significantly lower than for that seen with the conventional fluids. However, in the conventional PD fluids the total amount of 3,4-DGE available for toxic reactions most probably was higher than that measured, because 3,4-DGE was freshly recruited from a molecular pool when consumed. The speed of this recruitment was high enough to explain most of the growth inhibition seen for heat-sterilized PD fluids. CONCLUSION: 3,4-DGE is present in conventional PD fluids at a concentration between 9 and 22 micromol/L, and is the most biologically active of all GDPs identified to date. Thus, it is the main candidate to be held responsible for the clinical bioincompatibility caused by conventionally manufactured PD fluids.

Protein loss in on-line hemofiltration.

BACKGROUND: The albumin serum level is one of the most important nutritional indices and is directly correlated to the uremic patient's hemodialysis outcome. One of the factors that can interfere with protein metabolism is the possible loss of albumin through the dialysis membrane that can contribute to keeping levels chronically low, especially for high-flux convective treatments requiring high permeability membranes and the removal of high volumes of plasma water. METHODS: Twenty stable patients undergoing chronic renal replacement therapy for at least 3 months were included. Each patient performed four hemofiltration treatments, 2 in post-dilution and 2 in pre-dilution (post-D, pre-D) with a polyamide membrane (Poliflux, 2.1 m2). RESULTS: The amount of albumin found in the ultrafiltrate was 2.9 +/- 1.5 g in post-D and 1.7 +/- 0.8 g in pre-D (p < 0.01). Albumin loss during online HF was lower than 3 g per treatment, and significantly lower in pre-D than in post-D. Furthermore, we observed a correlation between the transmembrane pressure and the albumin loss in both techniques, but with different slopes (y = 0.351x - 10.014 in post-dilution and y = 0.0639x + 8.2403 in pre-dilution; p = 0.01): the same transmembrane pressure determines larger albumin losses in post-dilution than in pre-dilution. CONCLUSIONS: Convective treatments that utilize high exchange volume can be performed with no risk of a significant albumin loss, particularly in pre-D where the proteic component's contact with the dialysis membrane is lower. In post-dilution the transmembrane pressure is a relevant factor in determining the protein loss.

Advanced glycation end products in hemodialyzed patients with diabetes mellitus correlate with leptin and leptin/body fat ratio.

Advanced glycation end products (AGEs) and other carbonyl and oxidative stress compounds are supposed to play a critical role in the pathogenesis of several diseases and their complications, i.e., diabetes mellitus, diabetic retinopathy, atherosclerosis, and chronic renal failure. In the present investigation, we were interested in the relationship of AGEs in plasma to other prominent factors in the patients on chronic hemodialysis treatment-27 patients with diabetes mellitus, 35 patients without diabetes mellitus. AGE-group reactivity was estimated using a spectrofluorometric method (excitation 350 nm, emission 430 nm) and is expressed in arbitrary units (AU). We found significantly higher AGEs levels in diabetics than in non-diabetics on regular hemodialysis treatment both before (2.7 +/- 0.7 x 10(4) AU vs. 2.2 +/- 0.6 x 10(4) AU, p < 0.001) and after the dialysis session (2.3 +/- 0.5 x 10(4) AU vs. 1.8 +/- 0.7 x 10(4) AU, p < 0.005). AGEs were significantly reduced during hemodialysis in both groups of patients--by 15.4% in the diabetic go (p < 0.001) and by 17.3% in non-diabetics (p < 0.005). In the patients with diabetes mellitus, AGEs did not correlate with parameters of the glucose metabolism correction (blood glucose, HbA1c). We observed a significant correlation between AGEs and leptin (r = 0.48, p < 0.05) as well as the leptin/body fat ratio (r = 0.56, p < 0.05) only in hemodialyzed patients with diabetes mellitus. These findings suggest more detailed studies to identify the molecular links between carbonyl stress, i.e., advanced glycation end products, and leptin metabolism, sign of microinflammation and hypertension.

Glucose degradation products in PD fluids: do they disappear from the peritoneal cavity and enter the systemic circulation?

BACKGROUND: Glucose degradation products (GDP) are generated in peritoneal dialysis (PD) fluid during heat sterilization and storage. They are thought to adversely affect the peritoneal membrane. The fate of GDP within the peritoneal cavity has not been well characterized. METHODS: A clinical study was designed to determine (1). whether during the dwell in the peritoneal cavity GDP concentration decreases in the PD fluid as assessed by ex vivo formation of AGE; (2). whether exposure to GDP-containing PD fluids increases plasma fluorescence (as an index of plasma AGE concentration) as well as plasma carboxymethyllysine (CML) concentration; and (3). whether exposure to GDP-containing PD fluids adversely affects glycoprotein CA 125 concentration. A two-group crossover design was adopted comprising two consecutive observation periods of eight weeks each. Stable PD patients were exposed in random order either to conventional PD fluid (heat sterilized at pH 5.5) and subsequently to PD test fluid (or the 2 fluids in reverse order). The PD test fluid was sterilized using a multicompartment bag system separating highly concentrated glucose at pH 3 from the buffer solution. Conventional and test fluids differed with respect to concentrations of GDP, that is, 3-deoxyglucosone (118 vs. 12.3 micromol/L), methylglyoxal (5.3 micromol/L vs. below detection threshold), 3, 4-dideoxyglucosone-3-ene (10 micromol/L vs. below detection threshold) and acetaldehyde (226 vs. <1 micromol/L). RESULTS: The following results were obtained. First, methylglyoxal disappeared completely as early as two hours after intraperitoneal instillation of conventional PD fluid. Second, when spent conventional dialysate was recovered after a two hour and particularly an eight hour dwell and subsequently incubated ex vivo with 40 mg of human serum albumin, there was a continuous decrease of AGE-forming capacity, that is, less generation of fluorescence (AGE) and pyrraline (non-fluorescent Amadori product), and an increase of advanced oxidation protein products (AOPP) in the spent dialysate. Third, plasma fluorescence (exc. 350/em. 430 nm) as an index of circulating AGE compounds as well as plasma CML concentrations were significantly higher in the conventional PD fluid period versus low GDP PD fluid period. Fourth, CA 125 concentrations in spent dialysate were higher during the low GDP PD fluid period compared to the conventional PD fluid period. CONCLUSION: Conventional PD fluid undergoes modifications during intraperitoneal dwell with a loss of AGE forming capacity, suggesting breakdown, precipitation or resorption of GDP in vivo. This is accompanied by an increase in plasma AGE compounds.

Review on uremic toxins: classification, concentration, and interindividual variability.

BACKGROUND: The choice of the correct concentration of potential uremic toxins for in vitro, ex vivo, and in vivo experiments remains a major area of concern; errors at this level might result in incorrect decisions regarding therpeutic correction of uremia and related clinical complications. METHODS: An encyclopedic list of uremic retention solutes was composed, containing their mean normal concentration (CN), their highest mean/median uremic concentration (CU), their highest concentration ever reported in uremia (CMAX), and their molecular weight. A literature search of 857 publications on uremic toxicity resulted in the selection of data reported in 55 publications on 90 compounds, published between 1968 and 2002. RESULTS: For all compounds, CU and/or CMAX exceeded CN. Molecular weight was lower than 500 D for 68 compounds; of the remaining 22 middle molecules, 12 exceeded 12,000 D. CU ranged from 32.0 ng/L (methionine-enkephalin) up to 2.3 g/L (urea). CU in the ng/L range was found especially for the middle molecules (10/22; 45.5%), compared with 2/68 (2.9%) for a molecular weight <500 D (P < 0.002). Twenty-five solutes (27.8%) were protein bound. Most of them had a molecular weight <500 D except for leptin and retinol-binding protein. The ratio CU/CN, an index of the concentration range over which toxicity is exerted, exceeded 15 in the case of 20 compounds. The highest values were registered for several guanidines, protein-bound compounds, and middle molecules, to a large extent compounds with known toxicity. A ratio of CMAX/CU <4, pointing to a Gaussian distribution, was found for the majority of the compounds (74/90; 82%). For some compounds, however, this ratio largely exceeded 4 [e.g., for leptin (6.81) or indole-3-acetic acid (10.37)], pointing to other influencing factors than renal function, such as gender, genetic predisposition, proteolytic breakdown, posttranslation modification, general condition, or nutritional status. CONCLUSION: Concentrations of retention solutes in uremia vary over a broad range, from nanograms per liter to grams per liter. Low concentrations are found especially for the middle molecules. A substantial number of molecules are protein bound and/or middle molecules, and many of these exert toxicity and are characterized by a high range of toxic over normal concentration (CU/CN ratio). Hence, uremic retention is a complex problem that concerns many more solutes than the current markers of urea and creatinine alone. This list provides a basis for systematic analytic approaches to map the relative importance of the enlisted families of toxins.