Glucose degradation products and peritoneal membrane function.

BACKGROUND: The bioincompatibility of peritoneal dialysis fluids (PDF) in current use has been partially attributed to the presence of glucose degradation products (GDPs), which are generated during heat sterilization of PDF. Several of the GDPs have been identified and we have recently demonstrated that these GDPs per se may impair the viability and function of human peritoneal mesothelial cells (HPMC) in vitro. It is also possible that GDP-related toxicity is further exacerbated by the milieu of PDF. We review the current literature on GDP and present the results of experiments comparing the impact of heat- and filter-sterilized PDF on the viability and function of HPMC. METHODS: Peritoneal dialysis fluids with low (1.5%) and high (4.25%) glucose concentrations were laboratory prepared according to the standard formula and sterilized either by heat (H-PDF; 121 degrees C, 0.2 MPa, 20 minutes) or filtration (F-PDF; 0.2 microns). The buildup of GDP was confirmed by UV absorbance at 284 nm. Confluent HPMC monolayers were exposed to these solutions mixed 1:1 with standard M199 culture medium. After 24 hours, cell viability was assessed with the MTT assay, and interleukin-1beta-stimulated monocyte chemotactic protein-1 (MCP-1) release with specific immunoassay. RESULTS: Exposure of HPMC to H-PDF resulted in a significant decrease in cell viability, with solutions containing 4.25% glucose being more toxic than 1.5% glucose-based PDF (27.4% +/- 3.4% and 53.4% +/- 11.0% of control values, respectively). In contrast, viability of HPMC exposed to F-PDF was not different from that of control cells. Moreover, treatment with H-PDF impaired the release of MCP-1 from HPMC to a significantly greater degree compared to F-PDF (17.4% and 24.9% difference for low and high glucose PDF, respectively). CONCLUSIONS: Exposure of HPMC to H-PDF significantly impairs cell viability and the capacity for generating MCP-1 compared to F-PDF. This effect is likely to be mediated by GDPs present in H-PDF but not in F-PDF.

Synthesis of C-X-C and C-C chemokines by human peritoneal fibroblasts: induction by macrophage-derived cytokines.

Leukocyte accumulation during peritonitis is believed to be controlled by chemotactic factors released by resident peritoneal macrophages or mesothelial cells. Recent data indicate, however, that in many tissues fibroblasts play a key role in mediating leukocyte recruitment. We have therefore examined human peritoneal fibroblasts (HPFBs) for the expression and regulation of C-X-C and C-C chemokines. Quiescent HPFBs secreted monocyte chemoattractant protein (MCP)-1 and interleukin (IL)-8 constitutively. This release could be dose-dependently augmented with the pro-inflammatory cytokines IL-1beta and tumor necrosis factor-alpha. Stimulated IL-8 production reached a plateau within 48 hours while MCP-1 continued to accumulate throughout 96 hours. Induction of IL-8 and MCP-1 synthesis by HPFBs was also triggered by peritoneal macrophage-conditioned medium. This effect was partly related to the presence of IL-1beta as demonstrated by IL-1 receptor antagonist inhibition. Pretreatment of HPFBs with actinomycin D or puromycin dose-dependently reduced cytokine-stimulated IL-8 and MCP-1 secretion, which suggested de novo chemokine synthesis. Indeed, exposure of HPFBs to IL-1beta and tumor necrosis factor-alpha produced a significant up-regulation of IL-8 and MCP-1 mRNA. This effect was associated with the rapid induction of nuclear factor-kappaB binding activity mediated through p65 and p50 subunits, and with a transient increase in the mRNA expression for RelB and inhibitory protein kappaB-alpha proteins. These data indicate that peritoneal fibroblasts are capable of generating large quantities of chemokines under a tight control of nuclear factor-kappaB/Rel transcription factors. Thus, peritoneal fibroblast-derived chemokines may contribute to the intraperitoneal recruitment of leukocytes during peritonitis.

IL-17 stimulates intraperitoneal neutrophil infiltration through the release of GRO alpha chemokine from mesothelial cells.

IL-17 is a newly discovered cytokine implicated in the regulation of hemopoiesis and inflammation. Because IL-17 production is restricted to activated T lymphocytes, the effects exerted by IL-17 may help one to understand the contribution of T cells to the inflammatory response. We investigated the role of IL-17 in leukocyte recruitment into the peritoneal cavity. Leukocyte infiltration in vivo was assessed in BALB/Cj mice. Effects of IL-17 on chemokine generation in vitro were examined in human peritoneal mesothelial cells (HPMC). Administration of IL-17 i.p. resulted in a selective recruitment of neutrophils into the peritoneum and increased levels of KC chemokine (murine homologue of human growth-related oncogene alpha (GROalpha). Pretreatment with anti-KC Ab significantly reduced the IL-17-driven neutrophil accumulation. Primary cultures of HPMC expressed IL-17 receptor mRNA. Exposure of HPMC to IL-17 led to a dose- and time-dependent induction of GROalpha mRNA and protein. Combination of IL-17 together with TNF-alpha resulted in an increased stability of GROalpha mRNA and synergistic release of GROalpha protein. Anti-IL-17 Ab blocked the effects of IL-17 in vitro and in vivo. IL-17 is capable of selectively recruiting neutrophils into the peritoneal cavity via the release of neutrophil-specific chemokines from the peritoneal mesothelium.

Nightly intermittent peritoneal dialysis: targets and prescriptions.

Adequate peritoneal dialysis can be achieved in most ESRD patients, provided that the prescription is individualized according to body surface area, body weight, residual renal function, and peritoneal transport characteristics. NIPD is particularly indicated in patients with significant residual renal function or those with a high-transport peritoneal membrane (accounting for about 10% of the PD patient population). "Dry day" NIPD reduces small-solute clearance by at least 10%-15% and middle-molecule clearance by almost 50%. Thus, most NIPD patients without residual renal function are at risk of inadequate treatment. The target dose of NIPD should be a weekly Kt/V of at least 2.2 and a weekly total creatinine clearance of 66 L/1.73 m2. However, the periodic clinical evaluation of patients should have priority over the mere achievement of a numerical clearance target.

Haemodialysis-membrane biocompatibility and mortality of patients with dialysis-dependent acute renal failure: a prospective randomised multicentre trial. International Multicentre Study Group.

BACKGROUND: There is controversy as to whether haemodialysis-membrane biocompatibility (ie, the potential to activate complement and neutrophils) influences mortality of patients with acute renal failure. We did a prospective randomised multicentre trial in patients with dialysis-dependent acute renal failure treated with two different types of low-flux membrane. METHODS: 180 patients with acute renal failure were randomly assigned bioincompatible Cuprophan (n=90) or polymethyl-methacrylate (n=90) membranes. The main outcome was survival 14 days after the end of therapy (treatment success). Odds ratios for survival were calculated and the two groups were compared by Fisher's exact test. Analyses were based on patients treated according to protocol (76 Cuprophan, 84 polymethyl methacrylate). FINDINGS: At the start of dialysis, the groups did not differ significantly in age, sex, severity of illness (as calculated by APACHE II scores), prevalence of oliguria, or biochemical measures of acute renal failure. 44 patients (58% [95% CI 46-69]) assigned Cuprophan membranes and 50 patients (60% [48-70]) assigned polymethyl-methacrylate membranes survived. The odds ratio for treatment failure on Cuprophan compared with polymethyl-methacrylate membranes was 1.07 (0.54-2.11; p=0.87). No difference between Cuprophan and polymethyl-methacrylate membranes was detected when the analysis was adjusted for age and APACHE II score. 18 patients in the Cuprophan group and 20 in the polymethyl-methacrylate group had clinical complications of therapy (mainly hypotension). INTERPRETATION: There were no differences in outcome for patients with dialysis-dependent acute renal failure between those treated with Cuprophan membranes and those treated with polymethyl-methacrylate membranes.

Biocompatibility and buffers: effect of bicarbonate-buffered peritoneal dialysis fluids on peritoneal cell function.

BACKGROUND: Conventional peritoneal dialysis fluids (PDF) have been shown to compromise the function of both leukocytes and human peritoneal mesothelial cells (HPMC). Various in vitro studies have identified the low initial pH in combination with high lactate content, as well as the hyperosmolality and high glucose concentration present in currently used solutions as the primary determinants of their bioincompatibility. Bicarbonate buffered PDF (at neutral pH) display improved in vitro biocompatibility as compared to conventional, lactate buffered PDF. However, little information is currently available regarding the potential impact of PDF on the function of human peritoneal fibroblasts (HPFB), the major cell population present in peritoneal interstitium. METHODS: The current study compares the effect of bicarbonate and lactate buffered PDF in a model system of resting peritoneal mesothelial cells and fibroblasts cultured from human omentum. Interleukin-1 beta-stimulated IL-6 release from HPMC and HPFB was used as the cell functional parameter. RESULTS: While short (30 min) pre-exposure to lactate buffered PDF significantly reduced the IL-1 beta-stimulated IL-6 release from HPMC during a subsequent recovery period (24 hr), a significant decrease in HPMC IL-6 secretion with bicarbonate buffered PDF was only observed after prolonged (> or = 60 min) exposure. In contrast, no significant IL-6 inhibition was detected with HPFB pre-exposed to PDF for up to 90 minutes. A significant suppression of HPFB IL-6 secretion was only observed in coincubation experiments (24 hr) with dilutions of both types of PDF. CONCLUSIONS: These results indicate that (i) bicarbonate buffered PDF are less inhibitory to peritoneal cell function as compared to conventional, lactate buffered PDF; and (ii) HPFB may be more resistant than HPMC to bioincompatible PDF.

In vitro biocompatibility evaluation of a novel bicarbonate-buffered amino-acid solution for peritoneal dialysis.

BACKGROUND: Conventional lactate-buffered peritoneal dialysis fluids containing glucose as the osmotic agent have been shown to compromise important peritoneal host defence functions. The current study employed an in vitro model using activated peripheral blood mononuclear leukocytes (PBMC) for the preclinical biocompatibility assessment of a novel bicarbonate-buffered peritoneal dialysis fluid containing 1.0% amino acids as the osmotic agent. METHODS: PBMC (5 x 10(6)/ml) were pre-exposed (10-30 mm, 37 degrees C) to bicarbonate-buffered 1% amino-acid solution, bicarbonate- or lactate-buffered 1.5% glucose fluid, or control medium (RPMI). The cells were then washed and stimulated for 2 h at 37 degrees C in RPMI containing 100 ng/ml E.coli endotoxin from strain O55:B5. The cytokines IL-6 and TNF alpha in cell supernatants were assessed using specific enzyme immunoassays, cytokine mRNA expression by reverse transcription polymerase chain reaction. RESULTS: Short, i.e. 10 min, exposure to conventional, lactate-buffered glucose fluid resulted in a significant and time-dependent inhibition of cytokine release and mRNA expression by activated PBMC, whereas the cytokine response was improved even following prolonged (up to 2 h) exposure to bicarbonate-buffered 1% amino-acid solution or bicarbonate-buffered 1.5% glucose fluid. CONCLUSIONS: Our results suggest that very short, i.e. potentially clinically relevant, exposure to conventional dialysis fluid impairs the cytokine response by activated leukocytes. In this respect, the use of bicarbonate-buffered solutions containing 1.0% amino acids or 1.5% glucose may result in improved biocompatibility properties.

Inhibition of tumour necrosis factor production in endotoxin-stimulated human mononuclear leukocytes by the prostacyclin analogue iloprost: cellular mechanisms.

The prostacyclin analogue iloprost has been shown to inhibit effectively TNF-alpha production in human peripheral blood mononuclear leukocytes (PBMC) stimulated with bacterial lipopolysaccharide (LPS). The current paper set out to analyse further the possible mechanisms involved in the regulation of TNF-alpha synthesis by iloprost. Healthy human PBMC were challenged with Escherichia coli LPS and assessed for TNF-alpha gene transcription, mRNA stability and protein secretion. Iloprost reduced both steady-state TNF-alpha mRNA expression and protein release as assessed by Northern blot analysis, polymerase chain reaction and enzyme immunoassay. This effect was related both to a reduction of TNF-alpha transcriptional activity (as evaluated by nuclear run-on transcription analysis) and a decrease in TNF-alpha mRNA stability (as assessed by serial Northern blot analysis of TNF-alpha mRNA content in PBMC blocked with actinomycin D). When collectively assessed, these data demonstrate that iloprost regulates TNF-alpha synthesis at both transcriptional and post-transcriptional level.

Establishment and functional characterization of human peritoneal fibroblasts in culture: regulation of interleukin-6 production by proinflammatory cytokines.

The functional and morphologic integrity of the peritoneal membrane is of major importance for the successful treatment of patients with chronic peritoneal dialysis. This study aimed at the establishment and functional characterization of human peritoneal fibroblasts (HPFB) in cell culture. HPFB were isolated from human omentum by enzymatic digestion and cultured. Confluent HPFB could be identified as spindle-shaped cells, growing in parallel arrays and whorls which stained positive for vimentin and negative for factor VIII, cytokeratin 18, and desmin. Maximum cell growth was observed after 24 h in medium supplemented with 10% fetal calf serum. HPFB could be growth arrested and maintained in fetal calf serum-depleted medium (0.1%) for > 48 h without loss of cell viability as evaluated by intracellular ATP determination. Stimulation of resting HPFB for 0.5 to 48 h with increasing doses of interleukin (IL)-1 beta and/or tumor necrosis factor-alpha (1 to 10,000 pg/mL) resulted in a dose- and time-dependent induction of IL-6 messenger RNA and an increase in immunoreactive IL-6 protein secreted into HPFB supernatants, which was significant with IL-1 beta or tumor necrosis factor-alpha doses as low as 1 pg/mL. HPFB IL-6 production could be inhibited by both actinomycin D or cycloheximide, which suggests that the induction of IL-6 occurs both on a transcriptional and a post-transcriptional level. In summary, this cell culture model is expected to facilitate further investigation of the potential role of the HPFB in the peritoneal cytokine network of patients treated with chronic peritoneal dialysis.

Longitudinal evaluation of peritoneal macrophage function and activation during CAPD: maturity, cytokine synthesis and arachidonic acid metabolism.

The release of cytokines and prostaglandins (PG) by peritoneal macrophages (PM luminal diameter of) may influence the cytokine network controlling peritoneal inflammation and in the long-term the function of the peritoneum as a dialysis membrane. In the present study, an evaluation of the long-term effects of peritoneal dialysis on the release of cytokines and prostaglandins, and the expression of surface markers of cellular maturation on blood and mononuclear cells has been performed in patients during their first year on CAPD. Spontaneous release of tumour necrosis factor alpha (TNF alpha) and interleukins 6 (IL-6) by PM luminal diameter of, after 4 or 24 hours in culture, increased significantly with time on CAPD, while there was a small but significant decrease in release of prostaglandin E2 (PGE2). Production of TNF alpha and IL-6 was enhanced following incubation of the cells with lipopolysaccharide (LPS), but the effect of LPS was proportionally greater on blood monocytes than on PM luminal diameter of. There was a significant increase in the concentrations of PGE2 and 6-keto-prostaglandin F1 alpha in overnight dwell peritoneal dialysis effluent with time on CAPD. The levels of TNF alpha and IL-6 in uninfected PDE were below the detection limit of the immunoassay over the whole time period studied. Expression of CD15, which correlates with immaturity, by PM luminal diameter of and blood monocytes increased with time on CAPD, while expression of CD11c, a marker of maturation, decreased on blood monocytes, but did not change significantly on PM luminal diameter of. There was also a slight increase in expression of transferrin receptor in both PM luminal diameter of and monocytes, but this did not reach statistical significance. These findings suggest that peritoneal macrophages and blood monocytes isolated from CAPD patients over a one year period become increasingly immature with time, and this is accompanied by a significant modulation of their ability to secrete inflammatory cytokines. Dysregulation of macrophage function may have important consequences with respect to inflammatory processes and the long-term function of the peritoneal membrane in CAPD patients.

In vitro studies on the effect of dialysis solutions on peritoneal leukocytes.

Within the limitations of the various experimental protocols there appears to be agreement in the literature that unused dialysis fluids, at least when studied in vitro, adversely affect multiple leukocyte functions. The effects of dialysis fluids on leukocytes that have been reported to date include: 1. Decreased cell viability of PMNs, PM phis, PBMCs, and lymphocytes; 2. Inhibited phagocytosis and bacterial killing of various microorganisms by PM phis, PMNs, and peripheral blood leukocytes; 3. Reduced secretion of leukotrienes (LTB4, LTC4) from peritoneal and peripheral blood PMNs and PBMCs; 4. Reduced secretion of prostaglandins (PGE2, TXB2 and 6-keto-PGF1 alpha) from PM phi; 5. Decreased production of many cytokines including TNF alpha, IL-8, and IL-6 in PM phis and PBMCs. In addition, several studies targeting the potential mechanisms by which dialysis solutions inhibit leukocyte function identified the initial low pH of the fluids in combination with their lactate content as being of primary relevance, since they may lead to a rapid intracellular acidification of leukocytes. Moreover, some studies indicated the importance of fluid hyperosmolarity and excessive glucose concentrations. These results are indirectly supported by recent in vitro investigations of alternative fluids, which showed improved leukocyte function following exposure to solutions with neutral pH, bicarbonate buffer instead of lactate, or normal osmolarity due to use of an alternative osmotic agent (e.g., glucose polymer). In conclusion, the evidence obtained during in vitro experimentation suggests that current dialysis fluids are, indeed, not biocompatible. However, whether this also bears physiological relevance in vivo remains to be established in controlled clinical trials comparing conventional fluids to alternative solutions with improved biocompatibility. With regard to the future development of in vitro models for biocompatibility assessment, the following guidelines are suggested: 1. Cell functional parameters should be studied in more than one cell population; 2. Depending on which fluid aspect is under investigation, short or even very short exposure times should be used (e.g., < 30 min for pH/buffer studies; < 4 hours for osmolality/osmotic agent studies); 3. In case the parameter/readout of interest requires longer study periods than indicated above (e.g., studies of cytokine induction or surface receptor expression), preincubation/recovery models should be preferred over coincubation experiments.

Urinary excretion of thromboxane and markers for renal injury in patients undergoing cardiopulmonary bypass.

Urinary excretion of selected markers for renal injury, as well as urinary excretion rates of the thromboxane metabolite, 11-keto-thromboxane B2 (11k-TXB2), was studied in 36 male patients undergoing coronary bypass surgery using cardiopulmonary bypass (CPB). In all patients, excretion of both tubular (N-acetyl-beta-D-glucosaminidase [beta NAG]; alpha 1-microglobulin [alpha 1-MG]) and glomerular markers (albumin [Alb]; transferrin [Trf]; immunoglobulin G [IgG]) sharply increased on Day 1 after CPB, and they remained elevated throughout the observation period of 5 days. Urinary excretion rates of 11k-TXB2 markedly increased on Day 1 after surgery, and they rapidly decreased thereafter. In 12 of the 36 patients, a temporary increase of serum creatinine levels (> 1.30 mg/dl) was noted following surgery. A positive correlation was found between serum creatinine levels and excretion of the tubular enzyme beta NAG (r = 0.36; p < 0.05), but not between creatinine levels and alpha 1-MG or the glomerular markers. Furthermore, no correlation between urinary excretion of 11k-TXB2 and any of the urinary markers for renal injury could be detected. Our data do not strengthen the hypothesis that acute renal injury observed during CPB is related to exaggerated thromboxane biosynthesis in these patients. Monitoring of urinary markers for incipient renal damage, particularly excretion of beta NAG, might be of additional diagnostic value for detection of otherwise subclinical renal injury in patients undergoing CPB.

[Initial experiences with combined balloon angioplasty and rtPA thrombolysis in dialysis shunt failure]. / Erste Erfahrungen mit der kombinierten Ballonangioplastie und rtPA-Thrombolyse bei Dialyseshuntversagen.

The 23 interventions performed by us on 18 patients had a very high success rate both in intraarterial lysis therapy of obstructed haemodialysis shunts and in balloon angioplasty (19/23). Shunt failure had to be primarily treated by surgery in 3 cases only. The complications were confined to a rupture of a vessel that required surgical care. 14 shunts were found to be patent in a total of 18 patients (18 being of course as small patient group). Our experience leads us to believe that patients suffering from shunt insufficiency should always be primarily treated endoluminally if angiography confirms that intervention is possible. Surgical intervention is always advisable even if the result may occasionally be negative in a few rare cases.

In-vitro biocompatibility of alternative CAPD fluids; comparison of bicarbonate-buffered and glucose-polymer-based solutions.

Evidence is accumulating that conventional dialysis fluids for CAPD are incompatible with peritoneal host defence. We therefore investigated the effect of alternative CAPD fluids on mononuclear leukocyte (PBMC) viability and cytokine production in vitro. Fluids tested were bicarbonate-buffered solutions containing 1.5% or 4.25% glucose, 7.5% glucose polymer dialysis fluid (GPDF), and conventional 1.5% glucose fluid (G1.5%). PBMC were stimulated (2 h, 37 degrees C) in the different test fluids with a clinical isolate of Staphylococcus epidermidis or Escherichia coli lipopolysaccharide. The cytokines TNF alpha and IL-6 in PBMC supernatants were measured by specific enzyme immunoassays. Induction of cytokine messenger RNA was evaluated by reverse transcription-polymerase chain reaction. Conventional G1.5% (pH 5.5) inhibited cytokine release from activated PBMC by > 95%, whereas cell responses in low-glucose bicarbonate fluid were not significantly reduced. In contrast, high-glucose bicarbonate fluid exerted > 80% inhibition despite its neutral pH. GPDF was inhibitory at its initial low pH, whereas cytokine release was restored following pH neutralization. Cytokine mRNA expression was suppressed by conventional G1.5% fluid and by high-glucose bicarbonate fluid. These data indicate that pH neutralization leads to a substantial improvement of dialysis fluid biocompatibility; however, hyperosmolality and/or high glucose content inhibit cell responsiveness even at normal pH. Replacement of glucose by glucose polymer might prove beneficial provided that the initial low pH is neutralized.