N-Acetylglucosamine modulates function of the skin fibroblasts.

BACKGROUND: Fibroblasts are an important component of the skin determining its properties. N-Acetylglucosamine (NAG) is the substrate for hyaluronan synthesis, and it also has anti-inflammatory and anti-senescent activity in mesothelial cells. METHODS: We tested in in vitro-cultured human skin fibroblasts how supplementation of culture medium with NAG 10 mmol L(-1) changes properties of these cells. RESULTS: Fibroblasts cultured in presence of NAG produced more proteins and that was mainly due to increased synthesis of collagen (+33% vs. control, P < 0.05). Hyaluronan synthesis was increased (+107% vs. control, P < 0.001), but interleukin-6 synthesis was reduced (-22% vs. control, P < 0.05). Fibroblasts cultured in medium with NAG 10 mmol L(-1) demonstrated improved ability to heal the injured layer of cells (+34% vs. control, P < 0.05). Additionally senescence of fibroblasts undergoing replicative ageing in the presence of NAG was less pronounced, as reflected by smaller increase in the population doubling time (-70% vs. control, P < 0.05). CONCLUSION: We conclude that NAG induced changes in the skin fibroblasts' properties maybe important for prevention of the age-dependent changes in its structure and function.

Sulodexide modifies intravascular homeostasis what affects function of the endothelium.

PURPOSE: Sulodexide is a mixture of heparin and dermatan sulphate which has an antithrombotic action. It was shown that it has also direct effect on the endothelial cells. We tested the effect of sulodexide on the intravascular homeostasis in patients with peripheral vascular disease. METHODS: Sulodexide was infused iv. at a dose of 1200 Lipoprotein Lipase Releasing Units (LRU) in 10 patients with peripheral vascular disease. Blood samples were collected before the infusion and 1, 6 and 24 hours after the infusion. Inflammatory and fibrinolytic parameters were studied in the collected serum samples. Additionally, ex-vivo effect of the serum samples on in vitro function of the endothelial cells was studied. RESULTS: Infusion of sulodexide caused acute and transient peak of the Hepatocyte Growth Factor (HGF) concentration in blood and decrease of the Vascular Endothelial Growth Factor (VEGF) level, what, as we found in in vitro experiments, was due to adsorption of VEGF to endothelium. We found that HGF enhanced in vitro stimulating effect of VEGF on proliferation of the endothelial cells. Serum level of interleukin-6 was gradually decreased, whereas fibrinolytic activity of serum, reflected by t-PA/PAI-1 ratio, increased. Serum samples obtained from the studied patients suppressed oxidative stress and release of interleukin-6 in endothelial cells maintained in in vitro culture. CONCLUSION: Sulodexide reduces intravascular inflammation and suppresses inflammatory reaction in the endothelial cells; both effects are desirable in patients with peripheral vascular disease.

Intravenous iron sucrose changes the intraperitoneal homeostasis.

BACKGROUND/AIMS: Intravenous iron infusion is the accepted way of supplementation of that compound in uremic patients. The aim of the study was to evaluate whether this treatment affects intraperitoneal homeostasis in patients on peritoneal dialysis. METHODS: Blood and peritoneal dialysate samples were collected from 10 patients treated with continuous ambulatory peritoneal dialysis who were given 100 mg iron sucrose (IS) intravenously. Systemic and peritoneal permeability as well as transperitoneal transport were studied. The effect of spent dialysate was tested in vitro on human peritoneal mesothelial cells (MCs). RESULTS: Dialysate total iron was increased (+19%, p < 0.01) during intravenous infusion of IS. Immediately after infusion the concentration of 8-OHdG was increased in plasma (+10%, p < 0.01) and in dialysate (+5%, p < 0.05). IS infusion caused a transient decrease in peritoneal permeability to protein (-42%, p < 0.05) and glucose (-30%, p < 0.01) and a reduction in dialysate cell count (-58%, p < 0.05). During the exchange dialysate hyaluronan was increased by 27% (p < 0.01). Spent dialysate, tested ex vivo on cultured MC, induced oxidative stress (+39%, p < 0.01), slowed their proliferation (-20%, p < 0.01), and stimulated MCP-1 synthesis (+46%, p < 0.01). Iron content in MCs exposed to dialysate obtained after IS infusion was increased by 32% (p < 0.01). CONCLUSION: Intravenous infusion of IS causes oxidative stress and inflammation within peritoneal MCs which may impair viability of the peritoneum.

Vascular endothelial growth factor in dialysate in relation to intensity of peritoneal inflammation.

BACKGROUND: Peritoneal inflammation may induce changes in peritoneal microvessels, including neoangiogenesis/vasculogenesis, leading to increased peritoneal solute transport rate (PSTR) and loss of ultrafiltration capacity. We hypothesized that an inflammatory reaction in the peritoneal cavity during peritonitis induces increased synthesis of vascular endothelial growth factor (VEGF). We therefore studied the relationship between peritoneal inflammation markers, VEGF, and transport of fluid and solutes in rats during acute peritoneal inflammation induced by lipopolysaccharide (LPS) added to standard glucose-based dialysis solution. METHODS: Under ether anesthesia, male Wistar rats were injected intraperitoneally with 30 mL Dianeal 3.86% without (Control; n=6) or with LPS (microg/mL): 0.001 (LPS 0.001; n=6), 0.01 (LPS 0.01; n=7), 0.1 (LPS 0.1; n=7), 1.0 (LPS 1.0; n=8). After 8 hours, dialysate volume (IPV), peritoneal solute transport rate (PSTR) and dialysate cell count (DCC) were measured and effluent samples were collected. RESULTS: LPS i.p. resulted in increased PSTR and decreased IPV (p<0.005). DCC (cells/microL) and the neutrophil/macrophage ratio were higher for all LPS concentrations compared to the control group. After 8 hours, LPS-exposed rats had significantly higher dialysate levels of all investigated cytokines (TNF-alfa, MCP-1 and IL-10) than the control group. Addition of LPS resulted in increased dialysate VEGF concentrations (pg/mL) (LPS 0.001, 28.2+/-5.9; LPS 0.01, 38.9+/-11.6; LPS 0.1, 43.0+/-5.9; LPS 1.0, 46.6+/-11.3; Control, 14.5+/-9.8; p<0.0005 for all LPS vs. Control). CONCLUSIONS: The infusion of Dianeal 3.86% with different doses of LPS induced a strong acute intraperitoneal inflammatory reaction with increased DCC and cytokine levels, resulting in increased peritoneal solute transport and decreased IPV. LPS induced a dose-dependent parallel increase of the intraperitoneal concentrations of MCP-1, IL-10 and TNF-alfa, as well as of VEGF. These results suggest that intraperitoneal VEGF synthesis is induced in response to inflammation, and that this may be an important component in the process leading to peritoneal transport alterations.

Time to reconsider saline as the ideal rinsing solution during abdominal surgery.

BACKGROUND: Peritoneal mesothelial cells lining the peritoneal cavity play a primary role in prevention of formation of peritoneal adhesions, which depends mainly on their fibrinolytic activity. During surgical procedures, the abdominal cavity is in most cases rinsed with normal saline solution, which may modify the fibrinolytic activity of the peritoneal mesothelium and predispose to formation of adhesions. The goal of our experiments was to evaluate how normal saline and other solutions affect the fibrinolytic properties of the peritoneal mesothelial cells. MATERIAL AND METHODS: Experiments were performed on in vitro cultures of human peritoneal mesothelial cells. Mesothelial monolayers were exposed during 6 hours to the following solutions: culture medium (control), .9% NaCl, Hanks solution, Earles solution, new peritoneal dialysis fluid with low glucose degradation products (GDP) concentration (PDF), and peritoneal dialysis fluid with high concentration of GDP (PDF-GDP). Afterwards, morphology of the cells as well as leakage of lactate dehydrogenase (LDH) from their cytosol were evaluated. During the next 24 hours when the cells were cultured in standard medium synthesis of interleukin-6 (IL-6), tissue plasminogen activator (t-PA) and plasminogen activator inhibitor-1 (PAI-1) were studied. RESULTS: Mesothelial monolayers exposed to .9% NaCl or to PDF-GDP showed destruction of their morphology after 6 hours incubation and in case of PDF-GDP release of LDH from the cytosol was increased by 275% versus the control (P<.05). During subsequent culture of all cells in standard medium, the release of IL-6 was decreased in cases of cells pretreated with .9% NaCl (-58%, P<.05) or PDF-GDP (-93%, P<.001). The release of t-PA was also reduced from cells pretreated with .9% NaCl (-71%, P<.01) or with PDF-GDP (-74%, P<.01) but increased after exposure of these cells to PDF (+35%, P<.05). Statistically significant decrease of PAI-1 synthesis was observed in cells preexposed to .9% NaCl (-69%, P<.01) or to PDF-GDP (-82%, P<.05). When changes in the PAI-1/t-PA ratio were calculated, a strong tendency for increase of that value was seen in cells pretreated with .9% NaCl or Earles salts solution but not with PDF. However, in cases of Hanks solution, a significant increase in the PAI-1/t-PA ratio was observed (+104%, P<.01). CONCLUSION: Exposure of the peritoneal mesothelial cells to .9% NaCl, Hanks, Earles salts solution, or PDF-GDP results either in reduction of their viability or in loss of their fibrinolytic activity. Peritoneal dialysis fluid with a low content of glucose degradation products appears to be the optimal solution causing the least damage to mesothelial cells and therefore may be the ideal solution for rinsing the abdominal cavity with low risk of inducing deterioration of the mesothelial cells fibrinolytic activity and formation of adhesions. We postulate therefore that such hypertonic peritoneal dialysis fluids should be used not only during peritoneal dialysis but also for rinsing the abdominal cavity during any surgical procedures.

N-acetylglucosamine reduces inflammatory response during acute peritonitis in uremic rats.

BACKGROUND: Peritoneal dialysis (PD) induces intraperitoneal inflammation and that process may be uremia. The goal of this study is to evaluate the effect of uremia on the kinetics of peritonitis and furthermore test the anti-inflammatory potential of N-acetylglucosamine (NAG) in a uremic environment. METHODS: Experiments were performed on healthy Wistar rats and on animals with impaired renal function. Acute PD was performed in all animals with dialysis fluid containing either glucose (GLU) or NAG as osmotic solutes. Peritonitis was induced by addition of lipopolysaccharide from Escherichia coli (LPS) to the dialysis fluid. Transperitoneal transport of water and solutes as well as intraperitoneal and systemic inflammation were evaluated. RESULTS: Uremia reduces peritoneal permeability to total protein during peritonitis (-33% vs. control, p < 0.001) and increases net ultrafiltration (+2.5 +/- 2.2 vs. -2.7 +/- 3.2 ml in control, p < 0.001). In uremic rats with peritonitis, reduced dialysate levels of the following inflammatory mediators were detected as compared to healthy animals: MCP-1 (-15%, p < 0.01); IL-1beta (-53%, p < 0.001), and elastase (-28%, p < 0.02). In the serum of uremic rats, the increase in TNFalpha and MCP-1 concentrations was smaller than in control rats: -44% (p < 0.02) and -39% (p < 0.001), respectively. NAG used as an osmotic solute in rats with preserved renal function decreases intraperitoneal and systemic inflammation during acute peritonitis. Drained dialysate volume was increased in the NAG group by 32% (p < 0.001) and transperitoneal loss of protein was reduced by 21% (p < 0.002). When NAG was used as the osmotic solute instead of GLU, intraperitoneal inflammation in uremic animals was further reduced: TNFalpha (-40%, p < 0.05); IL-1beta (-49%, p < 0.005); MCP-1 (-21%, p < 0.005). The presence of NAG also reduced the increased blood level of IL-1beta (-47%,p < 0.02) and MCP-1 (-36%, p < 0.02). CONCLUSIONS: Intensity of acute peritonitis is reduced during uremia. NAG exerts a systemic and peritoneal anti-inflammatory action under conditions of uremia that confirms the potential use of this compound as an osmotic agent in the PD fluids that also decreases inflammation.

N-Acetylglucosamine- an osmotic slute for peritoneal dialysis without inducing hyperinsulinemia.

METHODS: N-Acetylglucosamine (NAG) was compared to glucose as an osmotic solute during peritoneal dialysis in rats. The effect of the tested solutes on blood glucose and insulin levels during dialysis was evaluated. RESULTS: During 6-hour exchange with NAG (220 mmol/l) solution, the dialysate volume was higher than in rats dialyzed with fluid containing glucose (220 mmol/l; GLU: 34.5 +/- 1.7 vs. 32.8 +/- 1.1 ml, respectively; p < 0.05). The peritoneal permeability to protein (D/S x 1,000) was lower in the NAG group (9.7 +/- 2.5 vs. 16.3 +/- 5.6 in GLU; p < 0.02). Dialysis with GLU-based solution resulted in hyperglycemia up to 180 +/- 39 mg/dl; in the NAG group the increase in the blood glucose level was moderate (up to 91 +/- 9 mg/dl; p < 0.001). Dialysis with GLU fluid caused an increase in blood insulin level by 53.2 +/- 62.4 pmol/l, whereas the insulin blood concentration in NAG-treated animals was increased by 5.0 +/- 5.4 pmol/l (p < 0.001). CONCLUSIONS: NAG is more effective than GLU osmotic solute during peritoneal dialysis and it reduces peritoneal permeability to protein. Dialysis with NAG results in lower hyperglycemia and hyperinsulinemia, both effects are favorable in diabetic peritoneal dialysis patients.

Effect of iron sucrose on human peritoneal mesothelial cells.

BACKGROUND: Iron supplementation is often required in uraemic patients with anaemia. Peritoneal cavity was proposed as an alternative intravenous route for iron infusion in patients treated with peritoneal dialysis. We studied the effect of iron sucrose (Venofer) on the function of human peritoneal mesothelial cells maintained in in vitro culture. MATERIALS AND METHODS: In in vitro experiments on human peritoneal, the mesothelial effect of elemental iron (in conc. 0.0001-1 mg mL-1) present in Venofer on their viability, growth and synthesis of IL-6 was studied. Additionally we evaluated with a fluorescent probe (2',7'-dichlorodihydro-fluorescein diacatate) generation of reactive oxygen species in cells exposed to iron sucrose. We also measured accumulation of iron in the cytoplasm of mesothelial cells after their in vitro exposure to Venofer. RESULTS: In in vitro conditions iron induces a dose-dependent inhibition of viability of the mesothelial cells as reflected by inhibition of the cells growth by 34% at Fe 0.1 mg mL-1 vs. control (P < 0.05) increased release of lactate dehydrogenase (LDH) from the cytosol: 67.1 +/- 30.3 mU mL-1 at Fe 1 mg mL-1 vs. 7.9 +/- 6.4 in control group (P < 0.001), and reduced synthesis of IL-6: 209 +/- 378 pg mg-1 cell protein at Fe 1 mg mL-1 vs. 38674 +/- 4146 pg mg-1 cell protein in controls (P < 0.001). Cytotoxicity of iron towards mesothelial cells was enhanced in vitro when it was tested in presence of the dialysis fluid. Iron used in vitro at concentration 0.0001 mg mL-1 and greater induces generation of oxygen-derived free radicals in mesothelial cells. Furthermore, iron is taken by these cells and stored in their cytosol, resulting in stimulation of the intracellular generation of free radicals. CONCLUSIONS: We conclude that iron used in the form of iron sucrose is cytotoxic to human peritoneal mesothelial cells. Accumulation of iron sucrose within cytoplasm of these cells may lead to induction of its chronic cytotoxic effect.

Plasma glutathione peroxidase activity in the elderly.

Schiavon et al. (1994) have reported that the measurement of plasma glutathione peroxidase activity (PGP) could provide an index of renal function. Its activity, which was depressed in patients with impaired renal function, correlated positively with creatinine clearance and negatively with serum creatinine. To evaluate the hypothesis that the plasma PGP activity may be used to assess renal function in elderly, we measured the plasma PGP and creatinine clearance (ClCr) in 65 active, community-dwelling elderly (range: 65-93 years; 47 women and 18 men). We did not include persons with advanced renal failure in our study. PGP did not correlate with PCr and it was similar among patients with normal and with increased PCr (127.0 +/- 30.7 U/l and 119.7 +/- 21.6 U/l, respectively). A positive correlation was found between PGP and ClCr (r = 0.30; p < 0.01). Plasma PGP activity was lower in patients with a ClCr lower than 70 ml/min/1.73 m2 than in those who had a higher ClCr (113.0 +/- 25.8 U/l and 131.2 +/- 26.7 U/l, p < 0.01). However, no correlation was found between ClCr and PGP in subjects with lower ClCr. PGP did not correlate with age but there was a correlation between ClCr and age (r = -0.24, p < 0.05). Our results suggest that plasma PGP activity is decreased in the patients with impaired renal function but this decrease does not correlate with age-dependent decline of kidney function.

A comparison of the biocompatibility of phosphate-buffered saline and dianeal 3.86% in the rat model of peritoneal dialysis.

Phosphate-buffered saline (PBS), an isotonic solution with a physiologic pH can be considered an example of a biocompatible dialysis fluid. This study compared the biocompatibility of PBS with that of Dianeal 3.86% (Baxter Healthcare Corporation, Deerfield, IL, U.S.A.), using a model of peritoneal dialysis in the rat. In an acute experiment, after catheter implantation, rats were infused on day 1 with PBS, on day 5 with standard dialysis solution (Dianeal 3.86%), and on day 7 again with PBS. When rats were injected with Dianeal 3.86%, the inflammatory reaction was suppressed as compared with PBS. The cell count was lower with Dianeal (-85%, p < 0.001), the neutrophil:macrophage ratio in dialysate was 80% lower (p < 0.01), total protein concentration in the Dianeal dialysate was 73% lower (p < 0.01), and the dialysate nitrite level was 45% lower (p < 0.01). In a chronic experiment, after catheter implantation, rats were dialyzed for four weeks with PBS or with Dianeal 3.86%. At the end of the study, a 1-hour peritoneal equilibration test (PET) was performed. As evaluated on a semiquantitative scale, macroscopic changes in the peritoneum were more severe in rats exposed to PBS than in those exposed to Dianeal 3.86% (8.6 +/- 3.2 vs 5.2 +/- 2.6, p < 0.05). The thickness of the visceral peritoneum was comparable in both groups; but, in PBS-treated rats, the peritoneal interstitium contained more inflammatory cells and more new vessels. During the 1-hour PET, peritoneal permeability to water and solutes was comparable in the two groups. Despite a more physiologic composition, PBS is a less biocompatible peritoneal dialysis solutions than is standard, acidic, hypertonic dialysis solution.

Intraperitoneal hyaluronan administration in conscious rats: absorption, metabolism, and effects on peritoneal fluid dynamics.

BACKGROUND: Hyaluronan (HA) is a major component of interstitial tissue that participates in fluid homeostasis, response to inflammation, and wound healing. Previous studies have shown that intraperitoneal administration of HA can affect peritoneal fluid transport during short peritoneal dialysis exchanges in anesthetized rats. We sought to investigate the effect of high molecular weight HA on peritoneal permeability in conscious rats during dialysis exchanges up to 8 hours in duration. In addition, we sought to investigate the absorption of HA from the peritoneal cavity, its accumulation in peritoneal tissues, and its metabolism in normal and uremic rats. METHODS: Experiments were performed on male Wistar rats infused with 30 mL peritoneal dialysis solution (Dianeal, Baxter Healthcare; Castelbar, Ireland) containing 10 mg/dL HA or with Dianeal alone (control). Peritoneal fluid removal (net ultrafiltration), permeability to glucose, creatinine, and total proteins, and tissue and blood levels of HA were determined in separate groups of rats at 1,2, 4, 6, and 8 hours after intraperitoneal infusion. Hyaluronan appearance and disappearance from plasma were also studied for 24 hours in separate groups of normal and uremic rats. RESULTS: Net ultrafiltration was significantly greater (27%) in rats infused with HA at 4, 6, and 8 hours (p < 0.01) compared to controls. Transperitoneal equilibration of protein was reduced by 27% (p < 0.001) at 4 hours and by 30% (p < 0.01) at 8 hours. During the 8-hour exchange, peritoneal clearance of creatinine increased by 27% (p < 0.01), whereas the clearance of total protein decreased by 27% (p < 0.005). After 8 hours, 25.7% +/- 3.1% of the administered HA was absorbed from the peritoneal cavity, peritoneal tissue HA concentration was increased by 117% (p < 0.001), and plasma HA levels increased by 435% (p < 0.001). Plasma HA levels returned to normal within 24 hours after intraperitoneal administration in both healthy and uremic rats. CONCLUSIONS: Hyaluronan added to dialysis fluid is absorbed from the peritoneal cavity and accumulates in peritoneal tissues. Hyaluronan supplementation produces changes in peritoneal permeability, leading to higher net ultrafiltration and peritoneal creatinine clearance, whereas total protein clearance decreases. The HA that is absorbed from the peritoneal cavity appears to be rapidly metabolized in both healthy and uremic rats.

The ultrastructure of the peritoneal membrane in chronically dialysed rats.

The model to estimate peritoneal function in chronically dialysed rats was previously presented by us. The aim of the paper is to report the findings obtained in electron microscopy of peritoneal biopsies from Wistar rats dialysed for 1 month with high glucose dialysis solution. In control animals, thin mesothelial cells were covered with microvilli. The submesothelial tissue was composed of sparse bundles of parallelly oriented collagen fibers with a few resting cells. In chronically dialysed rats, mesothelial cell layer was thicker and cells were fully packed with intracellular structures, mainly secretory granules with a homogeneous content. The submesothelial tissue was expanded due to the increased amount of collagen fibers, oedema and increased amount of submesothelial cells which were activated. The use of electron microscopy to study the peritoneum in dialysed rats is an excellent supplement to the chronic functional model of peritoneal dialysis in rats.

Hyaluronan modifies inflammatory response and peritoneal permeability during peritonitis in rats.

The effect of high-molecular-weight hyaluronan (HA) on peritoneal and systemic inflammation and peritoneal permeability to water and solutes was studied during endotoxin-induced peritonitis in rats. Acute peritonitis was induced by adding lipopolysaccharide (LPS) to the dialysis fluid (Dianeal 3.86; Baxter Healthcare, Ireland, Castlebar). HA was added to the dialysis solution in a concentration of 10 mg/dL. During 4- and 8-hour dwells of the dialysis fluid, we studied the intensity of peritoneal (dialysate) and systemic (blood) inflammation (dialysate cell count and differential, cytokine and HA levels), as well as the transperitoneal transport of solutes and water. In rats, the addition of LPS to the dialysis fluid induced changes in inflammatory reaction and transperitoneal transport similar to those seen in continuous ambulatory peritoneal dialysis patients with peritonitis. During peritonitis, the addition of HA to the dialysis fluid reduced the loss of ultrafiltration, which resulted in a greater peritoneal creatinine clearance during the 8 hours of dwell (29.9 +/- 6.7 mL/8 h in the HA-LPS group versus 19.7 +/- 7.8 mL/8 h in the LPS group; P < 0.05). Dialysate interferon-gamma (INF-gamma) levels during peritonitis were greater in HA-treated animals (536.8 +/- 296.6 pg/mL in the HA-LPS group versus 169.8 +/- 137.8 pg/mL in the LPS group; P < 0.05). Dialysate elastase activity increased during peritonitis (44.4 +/- 9.3 versus 14.2 +/- 4.1 U/mL in peritonitis-free rats); during peritonitis, the increase in dialysate elastase activity was less pronounced in the rats that had HA in the dialysate (27.3 +/- 4.1 U/mL versus the LPS group; P: < 0.01). We conclude that HA added to the dialysis fluid reduces loss of ultrafiltration during peritonitis in rats. In the presence of HA dialysate, INF-gamma levels during peritonitis increased, whereas elastase activity decreased; these changes might improve the peritoneal immune reaction during peritonitis and at the same time prevent peritoneal membrane injury.

Multidirectional approach to study peritoneal dialysis fluid biocompatibility in a chronic peritoneal dialysis model in the rat.

BACKGROUND: Peritoneal dialysis causes the functional and morphological changes in the peritoneum that result from the bioincompatibility of dialysis solutions. We present a model of chronic peritoneal dialysis in the rat that can be used for testing the biocompatibility of dialysis fluids. Methods and Results. Long-term exposure of the peritoneum to dialysis solutions can be performed in rats with implanted peritoneal catheters. Sampling of the dialysate allows the evaluation of intraperitoneal inflammation by examining cell differential and dialysate cytokine levels. Peritoneal permeability can be evaluated at designed time intervals with the peritoneal equilibration test (PET). At the end of dialysis, peritoneal histology is studied with light and electron microscopy. CONCLUSIONS: Such a multidirectional approach is an effective way to test biocompatibility of dialysis solutions.

Influence of neutral-pH dialysis solutions on the peritoneal membrane: a long-term investigation in rats.

OBJECTIVE: Glucose degradation products (GDPs) and low pH are potential causes of bioincompatibility of peritoneal dialysis fluids (PDFs). The aim of the present study was to compare the effect of 6 weeks' exposure of the peritoneum in rats to two different PDFs: a standard PDF with a low pH and high level of GDPs (CAPD 3: Fresenius Medical Care, Bad Homburg, Germany), and a modified PDF with a low level of GDPs and a physiologic pH (CAPD 3 Balance: Fresenius Medical Care). METHODS: After catheter implantation, rats were exposed twice daily for 6 weeks to CAPD 3 fluid or to CAPD 3 Balance. At the beginning and at the end of the study, a 4-hour dwell was performed in every rat to evaluate intraperitoneal inflammation and its effect on total collagen synthesis in the in vitro cultured rat mesothelial cells (ex vivo study). Additionally, after 6 weeks' exposure, the peritoneal cavity was opened, and macroscopic changes were evaluated according to a semiquantitative scale. Peritoneal samples were also taken for morphology study. RESULTS: In rats treated with CAPD 3 fluid, intraperitoneal inflammation was comparable at the beginning and at the end of the experiment. In animals exposed to CAPD 3 Balance, the intensity of the intraperitoneal inflammation decreased during the study (cell count, p = 0.0781; neutrophil:macrophage ratio, p < 0.01; nitrite concentration, p < 0.05; hyaluronan level, p < 0.05). The capacity of effluent dialysate from CAPD 3 rats to activate collagen synthesis in in vitro-cultured mesothelial cells was the same at the beginning and at the end of the study. In the CAPD 3 Balance group, this capacity was statistically significantly lower at the end of the study than at the beginning (p < 0.05). The mean thickness of the visceral peritoneum was comparable in both groups of animals, but, macroscopically, more severe fibrosis was found in the peritoneum of rats exposed to CAPD 3 as compared with animals treated with CAPD 3 Balance (p < 0.05). CONCLUSION: We showed that, in the rat model of peritoneal dialysis, chronic exposure of the peritoneum to PDFs with low GDPs and a physiologic pH diminished the intraperitoneal inflammatory reaction induced by dialysis, and reduced peritoneal fibrosis.

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.

Hypertonicity of dialysis fluid suppresses intraperitoneal inflammation.

We studied acute and chronic intraperitoneal inflammation during dialysis performed in rats injected with phosphate-buffered saline alone (PBS) or PBS supplemented with glucose (Glu) or with mannitol (Man). In acute experiments, the result of a first dialysis with PBS in every rat (dialysis I) was compared with a second dialysis performed 24 hours later (dialysis II) using a different dialysis fluid: either PBS with 3.86 g/dL Man or PBS with 3.86 g/dL Glu. In rats exposed to hypertonic dialysis solutions (both Glu and Man), inflammatory reaction was decreased (cell count: p < 0.05; nitric oxide secretion: p < 0.05; protein in dialysate: p < 0.05). In control animals treated only with PBS (dialysis I and dialysis II), the inflammatory reactions during dialysis I and dialysis II were comparable. In chronic experiments, rats were dialyzed with the tested fluids for four weeks. Weekly, dialysate samples were taken and analyzed. At the end of the study, cell counts and protein losses were higher in the PBS-treated rats than in the other groups (cell count: p < 0.05, Glu vs PBS, and p < 0.05, Man vs PBS; protein in dialysate: p < 0.001, Glu vs PBS, and p < 0.01, Man vs PBS). We conclude that hypertonicity of the dialysis fluid inhibits the inflammatory reaction in the peritoneal cavity during peritoneal dialysis.

Dialysis solution containing hyaluronan: effect on peritoneal permeability and inflammation in rats.

BACKGROUND: Hyaluronan (HA), a high molecular weight mucopolysaccharide found in interstitial tissues and fluid, is lost from the peritoneal cavity during peritoneal dialysis. In order to determine the role of HA in peritoneal function, we investigated the effects of exogenous HA on peritoneal permeability, markers of intraperitoneal inflammation, and peritoneal morphology in rats exposed to peritoneal dialysis solution for four weeks. METHODS: Wistar rats were infused intraperitoneally, twice daily, with conventional, hypertonic dialysis solution (Dianeal 3.86%; control) or Dianeal solution containing 10 mg/dL of high molecular weight HA. Peritoneal permeabilities and clearances of solutes and protein were determined using a modified peritoneal permeability test (peritoneal equilibration test) at the beginning and the end of the treatment. Peritoneal volume and ultrafiltration were determined using a macromolecular marker and by gravimetric methods. Peritoneal inflammation was determined by cell counts and differential and by the measurement of cytokine concentrations in the dialysate effluent. Peritoneal thickness and HA content were determined in liver and mesentery biopsies taken at the end of the experiment. RESULTS: After four weeks of exposure to the dialysis solution, transperitoneal protein equilibration was significantly lower in HA-treated rats compared with rats treated with Dianeal alone (46% lower for albumin, P < 0.003; 33% lower for total protein, P < 0.001). The total drained volume after a four hour dwell was 29% higher in the HA group compared with the control (P < 0.001), yielding a positive net ultrafiltration in the HA group versus a negative net ultrafiltration in controls. Peritoneal clearances of urea and creatinine tended to be elevated in HA-treated rats, while clearances of total protein and albumin tended to be lower. Dialysate effluent from rats exposed to HA contained a lower percentage of neutrophils (8.8 +/- 22.8 +/- 9.5%, P < 0.01) and lower levels of the cytokines, tumor necrosis factor-alpha (11.2 +/- 14.7 vs. 42.3 +/- 35.3 pg/mL, P < 0.05) and monocyte chemoattractant protein-1 MCP-1 (72.0 +/- 86.5 vs. 402.4 +/- 258.3 pg/mL, P < 0.02), compared with rats treated with Dianeal alone. The thickness of the peritoneal interstitium showed a similar increase in both groups, but mesenteric tissue from the HA group contained more HA (48%, P < 0.01) than tissue from control animals. CONCLUSIONS: The addition of HA to peritoneal dialysis solution decreases protein permeability, increases ultrafiltration, and decreases cytokine levels and the proportion of peritoneal neutrophils in dialysate from rats exposed to hypertonic dialysis solution. These results suggest that exogenous HA may help to protect the peritoneal membrane during exposure to dialysis solutions. These benefits, if sustained in the clinical setting, could lead to improvements in the therapy of peritoneal dialysis.

Effect of N-acetylglucosamine on function of peritoneal leukocytes.

OBJECTIVE: To compare effects of N-acetylglucosamine (NAG)-based and glucose-based dialysis fluids on the function of peritoneal leukocytes in conditions of peritoneal dialysis. DESIGN: In vitro experiments on ex vivo isolated rat peritoneal leukocytes. MATERIALS: Peritoneal leukocytes were isolated from rats on chronic peritoneal dialysis. On alternate days, fluid exchanges were performed with NAG-based or glucose-based dialysis solutions. After a 4-hour dwell, dialysate was drained and peritoneal leukocytes were incubated in vitro +/- lipopolysaccharide (LPS). Production of nitrites (index of NO synthesis), tumor necrosis factor alpha (TNFalpha), interleukin-1beta (IL-1beta), and interferon gamma (IFN-gamma) by unstimulated or stimulated peritoneal leukocytes originating from NAG-based or glucose-based fluid was measured. RESULTS: Dialysate cell count was lower during exchanges with NAG-based fluid (2113+/-615 cells/microL) as compared to glucose-based fluid (3643+/-1108 cells/microL; p < 0.01). Differential cell count was similar in both studied groups. Unstimulated peritoneal leukocytes from NAG-based dialysate produced more NO (nitrites) (0.65+/-0.07 micromol per 10(6) cells) than did cells from glucose-based dialysate (0.26+/-0.09 micromol per 106 cells, p < 0.01). Stimulated peritoneal leukocytes from NAG-based dialysate produced more cytokines than did cells from glucose-based dialysate: TNFalpha, 135.2+/-37.0 pg versus 70.2+/-21.8 pg per 10(6) cells respectively, p < 0.01; IL-1beta, 143.2+/-60.9 pg versus 99.1+/-22.4 pg per 10(6) cells respectively, p < 0.05; IFN-gamma, 16.2+/-12.5 pg versus 6.0+/-1.8 pg per 10(6) cells respectively, p < 0.01. CONCLUSIONS: We demonstrated that rat peritoneal leukocytes exposed in vivo to NAG-based dialysis fluid have better ability to produce inflammatory mediators than do peritoneal leukocytes from the same donor, but exposed in vivo to glucose-based dialysis solution.