Thymol alleviates lipopolysaccharide-stimulated inflammatory response via downregulation of RhoA-mediated NF-κB signalling pathway in human peritoneal mesothelial cells.

Thymol is one of the most important dietary constituents in the thyme species and has been shown to possess anti-inflammatory properties both in vivo and in vitro. We investigated the protective effects of thymol on the lipopolysaccharide (LPS)-induced inflammatory responses in the human peritoneal mesothelial cell line (HMrSV5) to clarify the potential mechanism. HMrSV5 cells were stimulated with LPS in the presence or absence of thymol. Our results showed that thymol markedly suppressed the production of cytokines such as tumour necrosis factor α (TNF-α), interleukin (IL)-6, monocyte chemoattractant protein 1 (MCP-1) and α-smooth muscle actin (α-SMA) in a dose-dependent manner. Western blot analysis indicated that RhoA and ROCK activation; Toll-like receptor 4 (TLR4) expression; and Nuclear factor -kappa B (NF-κB) p65, IKK and IκBα phosphorylation were also inhibited by thymol. Moreover, siRNA knockdown of RhoA suppressed the expression of pro-inflammatory cytokines and phosphorylation of NF-κB p65 and IκBα proteins in LPS-stimulated HMrSV5 cells, but did not affect TLR4 expression. In conclusion, thymol inhibits LPS-induced inflammation in HMrSV5 cells by suppressing TLR4-mediated RhoA-dependent NF-κB signalling pathway. Our study suggests that thymol may be a promising therapeutic agent against peritonitis.

Sublethal microcystin exposure and biochemical outcomes among hemodialysis patients.

Cyanobacteria are commonly-occurring contaminants of surface waters worldwide. Microcystins, potent hepatotoxins, are among the best characterized cyanotoxins. During November, 2001, a group of 44 hemodialysis patients were exposed to microcystins via contaminated dialysate. Serum microcystin concentrations were quantified with enzyme-linked immunosorbent assay which measures free serum microcystin LR equivalents (ME). We describe serum ME concentrations and biochemical outcomes among a subset of patients during 8 weeks following exposure. Thirteen patients were included; 6 were males, patients' median age was 45 years (range 16-80), one was seropositive for hepatitis B surface antigen. The median serum ME concentration was 0.33 ng/mL (range: <0.16-0.96). One hundred thirty nine blood samples were collected following exposure. Patients' biochemical outcomes varied, but overall indicated a mixed liver injury. Linear regression evaluated each patient's weekly mean biochemical outcome with their maximum serum ME concentration; a measure of the extrinsic pathway of clotting function, prothrombin time, was negatively and significantly associated with serum ME concentrations. This group of exposed patients' biochemical outcomes display evidence of a mixed liver injury temporally associated with microcystin exposure. Interpretation of biochemical outcomes are complicated by the study population's underlying chronic disease status. It is clear that dialysis patients are a distinct 'at risk' group for cyanotoxin exposures due to direct intravenous exposure to dialysate prepared from surface drinking water supplies. Careful monitoring and treatment of water supplies used to prepare dialysate is required to prevent future cyanotoxin exposure events.

Conventional but not high-purity dialysis fluid induces apoptosis and insulin resistance in adipocytes in vitro.

PURPOSE: Previous study has shown an independent association of low body fat percentage and fat loss over time with higher mortality in hemodialysis (HD) patients. High-purity dialysis fluid (HPDF) used in place of conventional dialysis fluid (CDF) may decrease inflammation and improve nutritional status. Its effect on adipocytes and therefore fat storage is unclear. The purpose of this study was to assess the effects of these dialysates on apoptosis and insulin resistance in cultured adipocytes to determine a basis for the superiority of HPDF. METHODS: Flow cytometry and fluorescence staining were used to evaluate apoptosis in adipocytes. Insulin receptor substrate-1 (IRS-1) and -2 and serine phosphorylation of IRS-1 levels were assayed by Western blot. Tumor necrosis factor a (TNFa) levels were measured by enzyme-linked immunosorbent assay (ELISA). RESULTS: Although CDF contained more bacteria (66 ± 6 CFU/mL) than HPDF (7 ± 3 CFU/mL) and higher levels of bacterial DNA, endotoxin levels were similar. Combined with uremic serum (containing high TNFa levels), CDF (vs. HPDF) induced more apoptosis in adipocytes. Exposure to CDF also decreased expression of IRS-1 protein, increased expression of IRS-2 protein and insulin-stimulated serine phosphorylation of IRS-1, and decreased glucose consumption. Additionally, the culture supernatant from adipocytes exposed to CDF plus uremic serum for 48 hours contained significantly higher levels of TNFa (15.17 ± 2.89 vs. 8.12 ± 0.54 pg/mL [HPD plus uremic serum], p<0.0001). CONCLUSION: The advantage of HPDF over CDF may be due to lower levels of dialysis fluid impurities.

Impact of 3,4-dideoxyglucosone-3-ene (3,4-DGE) on cytotoxicity of acidic heat-sterilized peritoneal dialysis fluid.

Of the glucose degradation products (GDPs) in glucose-rich peritoneal dialysate, we investigated the influence of 3,4-dideoxyglucosone-3-ene (3,4-DGE) on the cytotoxicity of acidic heat-sterilized peritoneal dialysis fluid (L-H PDF) using human peritoneal mesothelial cells (HPMC). We prepared acidified filtration-sterilized PDF (glucose concentration 3.86%) containing eight types of added GDP [3,4-DGE, glyoxal (GO), methylglyoxal (MGO), 3-deoxyglucosone (3-DG), formaldehyde (FA), acetaldehyde (AA), 5-hydroxymethyl-2-furaldehyde (5-HMF), and furfural (FF)] or seven types of GDP (GO, MGO, 3-DG, FA, AA, 5-HMF, and FF). HPMC were exposed to these two types of solution and acidic heat-sterilized PDF (glucose concentration 3.86%, L-H 3.86) for 4 h. Cell viability was determined by 3,(4,5-dimethythiazol-2-yl)2,5-diphenyl-terazolium bromide (MTT) assay. MTT viability was decreased significantly compared with the control when treated with L-H 3.86 or acidified neutral filtration-sterilized PDF (glucose concentration 3.86%) containing eight GDPs. However, no significant decrease in MTT viability was observed when HPMC were treated with acidified neutral filtration-sterilized PDF (glucose concentration 3.86%) containing seven GDPs. Thus, 3,4-DGE strongly affects the cytotoxicity of L-H PDF. It is suggested that the cytotoxicity of L-H PDF is based on the presence of 3,4-DGE.

Characterization of the DialGuard device for endotoxin removal in hemodialysis.

Bacterial pyrogens, capable of penetrating dialyzer membranes, are responsible for a systemic inflammatory reaction in hemodialysis patients. Dialyzer reuse, involving rinsing of the dialyzer with pyrogen-containing water, may exacerbate this situation. Studies of the mechanism of action of endotoxin suggest that it irreversibly damages the vascular endothelium. The novel endotoxin removal method described here, is based on affinity-binding of endotoxin by the adsorbent ClarEtox, a USP Class VI-certified resin that is the active component of the medical device DialGuard. Under standard hemodialysis operating conditions, challenge of DialGuard with Pseudomonas maltophilia supernatant-spiked dialysate, containing 35-193 EU/ml endotoxin, resulted in endotoxin levels below 0.05 EU/ml in the treated dialysate. DialGuard was able to decrease endotoxin concentrations in the dialysate from a range of 2.39-8.49 to <0.005 EU/ml. DialGuard supports high fluid velocities at low back pressures and can be sanitized using the heat sanitization cycle of hemodialysis machines. DialGuard offers a simple, user-friendly way to reduce the concentration of endotoxin in dialysate and water for dialysis at a low cost.

Epidemic parenteral exposure to volatile sulfur-containing compounds at a hemodialysis center.

OBJECTIVE: To determine the cause of acute illness on August 30, 2000, among patients at an outpatient dialysis center (center A). DESIGN: We performed a cohort study of all patients receiving dialysis on August 30, 2000; reviewed dialysis procedures; and analyzed dialysis water samples using microbiologic and chemical assays. SETTING: Dialysis center (center A). PATIENTS: A case-patient was defined as a patient who developed chills within 5 hours after starting hemodialysis at center A on August 30, 2000. RESULTS: Sixteen (36%) of 44 patients at center A met the case definition. All case-patients were hospitalized; 2 died. Besides chills, 15 (94%) of the case-patients experienced nausea; 12 (75%), vomiting; and 4 (25%), fever. Illness was more frequent on the second than the first dialysis shift (16 of 20 vs 0 of 24, P < .001); no other risk factors were identified. The center's water treatment system had received inadequate maintenance and disinfection and a sulfurous odor was noted during sampling of the water from the reverse osmosis (RO) unit. The water had elevated bacterial counts. Volatile sulfur-containing compounds (ie, methanethiol, carbon disulfide, dimethyldisulfide, and sulfur dioxide) were detected by gas chromatography and mass spectrometry in 8 of 12 water samples from the RO unit and in 0 of 28 samples from other areas (P < .001). Results of tests for heavy metals and chloramines were within normal limits. CONCLUSIONS: Parenteral exposure to volatile sulfur-containing compounds, produced under anaerobic conditions in the RO unit, could have caused the outbreak. This investigation demonstrates the importance of appropriate disinfection and maintenance of water treatment systems in hemodialysis centers.

What can we do to preserve the peritoneum?

BACKGROUND: Long-term peritoneal dialysis may lead to peritoneal membrane failure. Loss of ultrafiltration is the most important clinical abnormality. Loss of ultrafiltration is associated with an increased number of peritoneal blood vessels, with fibrotic alterations, and with loss of mesothelium. Continuous exposure to bioincompatible dialysis solutions is likely to be important in the pathogenesis of these alterations. METHODS: This article reviews the toxicity of various constituents of dialysate, current assessments of interventions, and the results of interventions aimed at preserving the peritoneum. RESULTS: Glucose, possibly in combination with lactate, and glucose degradation products (GDPs) are likely to be the most toxic constituents of dialysate. Diabetiform peritoneal neoangiogenesis is likely to be mediated by vascular endothelial growth factor (VEGF). Release of VEGF might be influenced by glucose-induced cellular pseudohypoxia, which is likely to be increased by exposure to lactate. Glucose and GDPs are both toxic to peritoneal cells. Glucose degradation products induce the formation of advanced glycosylation end-products at a much faster rate than does glucose itself, but the relative importance of GDPs and glucose in clinical PD has not been clarified. The effects of interventions should first be assessed in long-term animal models, followed by clinical studies on peritoneal transport and on effluent markers that may reflect the status of the peritoneum. Possible interventions aim at reducing peritoneal exposure to glucose, GDPs, and lactate. Techniques include peritoneal resting, replacing some glucose-based exchanges with amino acid-based and icodextrin-based dialysate, using bicarbonate as a buffer, and administering solutions that have a low GDP content. Exposure to various dialysis solutions with a reduced GDP content has resulted in an increase in the effluent concentration of the mesothelial cell marker CA125, irrespective of the buffer used. Experimental studies in a long-term peritoneal exposure model in rats showed that the combination of a reduction in the concentration of lactate and replacement of lactate with pyruvate resulted in a reduction of the number of peritoneal blood vessels. Results of drug therapy have been studied in various animal models. Their use in patients is still experimental. CONCLUSIONS: Strategies to preserve the peritoneum aim at reducing membrane exposure to bioincompatible solutions. Currently available dialysis fluids that are more biocompatible are likely to have some beneficial effects. Further research on the development of dialysis solutions that use combinations of osmotic agents and alternative buffers is necessary.

A review of drinking-water-associated endotoxin, including potential routes of human exposure.

In the past decade efforts have been made to reduce the formation of harmful disinfection byproducts during the treatment and distribution of drinking water. This has been accomplished in part by the introduction of processes that involve the deliberate encouragement of indigenous biofilm growth in filters. In a controlled environment, such as a filter, these biofilms remove compounds that would otherwise be available as disinfection byproduct precursors or support uncontrolled biological activity in distribution systems. In the absence of exposure to chlorinated water, most biofilm bacteria are gram negative and have an outer layer that contains endotoxin. To date, outbreaks of waterborne endotoxin-related illness attributable to contamination of water used in hemodialysis procedures have been only infrequently documented, and occurrences linked to ingestion or through dermal abrasions could not be located. However, a less obvious conduit, that of inhalation, has been described in association with aerosolized water droplets. This review summarizes documented drinking-water-associated incidents of endotoxin exposure attributable to hemodialysis and inhalation. Typical endotoxin levels in water and conditions under which substantial quantities can enter drinking water distribution systems are identified. It would appear that endotoxin originating in tap water can be inhaled but at present there is insufficient information available to quantify potential health risks.

Silicon and aluminium interactions in haemodialysis patients.

BACKGROUND: Aluminium toxicity in dialysis patients is well described. Aluminium has a close chemical affinity with silicon. Silicon may have a role in protection against aluminium toxicity. METHODS: We measured serum aluminium and silicon levels from haemodialysis patients from four different centres. RESULTS: Though no relationship was seen across all centres combined, in one centre there was a reciprocal relationship in patients on home haemodialysis (who did not require reverse osmosis). Median (range) aluminium levels were higher, 2.2 (0.4-9.6) micromol/l when serum silicon was less than 150 micromol/l, and lower, 1.1 (0.2-2.8) micromol/l when serum silicon levels were greater than 150 micromol/l (P = 0.03). CONCLUSIONS: In patients treated by haemodialysis without reverse osmosis high serum silicon concentrations were associated with lower serum aluminium concentrations than those with low serum silicon. Further work needs to confirm a preventative role for silicon in the accumulation and subsequent toxicity of aluminium in dialysis patients.

[Effect of re-utilization of cuprophan capillary dialysers with different liquids on their biocompatibility and effectiveness of elimination]. / Wplyw reutylizacji róznymi plynami na biozgodnosc kuprofanowych dializatorów kapilarnych i efektywnosc eliminacji.

In 22 patients cuprophane capillary dialyzers reutilized in turn with four sets of liquids were used four times (Andante type in 13 and TAF-12 in 9 patients). The degree of biocompatibility and efficiency of elimination of small molecules was evaluated. During four-time reuse of dialyzers reutilized with sodium hypochlorite and with formaldehyde a reduction of intra-dialysis leukopenia, granulocytopenia and thrombocytopenia was not stated in blood of the patients. Activation of the complement system measured with the quantity of decrease of C3c fraction of the complement in the patients blood after 20 minutes of dialysis reduced essentially only at the fourth reuse of dialyzers (p < 0.01). Creatinine clearance measured always one hour after starting of the dialysis, did not change in succeeding reuse of dialyzers. Reutilization of dialyzers with hydrogen peroxide solution and formaldehyde caused essential reduction of ++intra-dialysis leukopenia and neutropenia (p < 0.001). There was lack of changes in ++intra-dialysis thrombocytopenia. Activation of the complement system was reduced essentially only after the fourth reuse of dialyzers (p < 0.001), but was also essentially lower (p < 0.05) than with dialyzers reutilized with sodium hypochlorite and with formaldehyde. Creatinine clearance practically did not change and at the fourth reuse of dialyzers it decreased on the average by 1.8%. Reutilization with acetic acid already at the second reuse of dialyzers essential (p < 0.001) and deepened decrease of intradialytic leukopenia and neutropenia and the activation of the complement system in course of succeeding reuses. Intradialytic thrombocytopenia was subjected to vestigal, not essential decrease. Creatinine clearance lowered a little but not essentially. At the fourth reuse of dialyzers it was lower on the average by 3.6% than the initial one. Reutilization with Dialina (stabilized blend of peracetic, acetic acid and hydrogen peroxide solution) caused essential (p < 0.001) and, in course of further reuses, deepening of lowering of intradialytic leukopenia and neutropenia as well as the activation of the complement system already at the second reuse. At the same time at the second and fourth reuse of dialyzers reutilized with Dialina the activation of the complement system was essentially lower than reutilized with the other liquids (p < 0.02). At the fourth reuse intradialytic thrombocytopenia also lowered essentially (p < 0.01). Creatinine clearance lowered a little more than with other liquids and at the second reuse of dialyzers was lower on the average by 5.6%, and at the fourth reuse--by 6.7% in relation to the new dialyzers.(ABSTRACT TRUNCATED AT 400 WORDS)

[Effect of hemodialysate and its peptide fractions on stromal cells and heme synthesis in bone marrow culture and the activity of selected enzymes and GSH level in human erythrocytes. I. Effect on human bone marrow stromal cells in vitro]. / Wplyw hemodializatu i jego frakcji peptydowych na komórki podscieliska i synteze hemu w hodowli szpiku oraz na aktywnosc wybranych enzymów i stezenie GSH w krwinkach czerwonych czlowieka. Czesc I: Wplyw na komórki podscieliska szpiku ludzkiego in vitro.

Hemodialysate or its three peptide fractions added to the culture of human bone stromal cells in vitro in concentration equal to 5 micrograms/ml of medium calculated against the protein content showed varying toxicity extent as expressed by shortening of cell survival time. Fraction III and hemodialysate showed the most toxic effect and shortened the cell survival time by three 24-hour periods in comparison to the control culture. Fraction III contained peptides having molecular weights in the range 1 to 5 kda that is was rich in compounds of the so-called middle molecular weights. Both hemodialysate and peptide fractions altered in the culture the participation of stromal cells reflected by diminishing the number of fibroblasts or adipocytes and an increase in the macrophage count. Above alterations speak in favor of concept that both hemodialysate and peptide fractions contain the macrophage proliferation stimulatory factor(s).

Importancia del aluminio en la hemodiálisis crónica / Improtance of aluminum in chronic hemodialysis

A study is carried out of aluminum concentration in the water supply of the city of Montevideo and in the plasma of 86 patients with chroni renal failure, under periodic hemodialytic treatment with non-demineralized water. The determinations were performed by atomic absoption spectrophotometry with graphite furnace. The aluminum rates of the water supply were always high, with a 286 ug/l mean. All of the patients exhibited high aluminemia (a 102 ug/l mean), in relation with a control group of healthy persons unexposed to the hazard. The study of 18 patients following a period of 3 to 9 months of hemodialysis with demineralized water showed a statistically significant decrease of aluminemia. Stress is laid on the importance of using demineralized water in hemodialysis in order to prevent aluminum poisoning and its serious complications.

Importancia del aluminio en la hemodiálisis crónica / Improtance of aluminum in chronic hemodialysis

A study is carried out of aluminum concentration in the water supply of the city of Montevideo and in the plasma of 86 patients with chroni renal failure, under periodic hemodialytic treatment with non-demineralized water. The determinations were performed by atomic absoption spectrophotometry with graphite furnace. The aluminum rates of the water supply were always high, with a 286 ug/l mean. All of the patients exhibited high aluminemia (a 102 ug/l mean), in relation with a control group of healthy persons unexposed to the hazard. The study of 18 patients following a period of 3 to 9 months of hemodialysis with demineralized water showed a statistically significant decrease of aluminemia. Stress is laid on the importance of using demineralized water in hemodialysis in order to prevent aluminum poisoning and its serious complications. (AU)