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Background: Treatment of anemia in peritoneal dialysis patients often requires intravenous iron supplementation. Iron diffuses into the peritoneal cavity and is injurious to the peritoneum. We studied how intermittent exposure to iron changes the properties of the senescent peritoneal mesothelial cells (MC).Methods: Replicative senescence was induced in MC in control medium (Con) or in control medium with intermittent exposure to iron isomaltoside 15 µg/dL (Con-IIS). After 10 passages properties of MC from both groups were compared to MC not exposed to replicative senescence.Results: In senescent MC population doubling time was elongated, intracellular generation of free radicals and staining for β-galactosidase was stronger than in MC not exposed to replicative senescence. All these effects were stronger in MC intermittently exposed to IIS. In these cells intracellular iron content was also higher. Also expression of genes p21 and p53 was stronger in MC intermittently treated with IIS. In senescent cells higher release and expression of IL6 and TGFβ1 was observed and that effect was stronger in MC treated with iron. Senescent MC had reduced fibrinolytic activity, what may predispose to the peritoneal fibrosis. Synthesis of collagen was higher in senescent cells, more in MC treated with iron.Conclusion: MC aging results in change of their genotype and phenotype which lead to their profibrotic effect. Exposure to iron enhances these changes.

There have been great advances in the therapy of cancer and leukemia. However, there are still many neoplastic diseases that are difficult to treat. For example, it is often difficult to find effective therapies for aggressive cancer and leukemia. An NF-κB inhibitor named dehydroxymethylepoxyquinomicin (DHMEQ) was discovered in 2000. This compound was designed based on the structure of epoxyquinomicin isolated from a microorganism. It was shown to be a specific inhibitor that directly binds to and inactivates NF-κB components. Until now, DHMEQ has been used by many scientists in the world to suppress animal models of cancer and inflammation. Especially, it was shown to suppress difficult cancer models, such as hormone-insensitive breast cancer and prostate cancer, cholangiocarcinoma, and multiple myeloma. No toxicity has been reported so far. DHMEQ was administered via the intraperitoneal (IP) route in most of the animal experiments because of its simplicity. In the course of developmental studies, it was found that IP administration never increased the blood concentration of DHMEQ because of the instability of DHMEQ in the blood. It is suggested that inflammatory cells in the peritoneal cavity would be important for cancer progression, and that IP administration, itself, is important for the effectiveness and safety of DHMEQ. In the present review, we describe mechanism of action, its in vivo anticancer activity, and future clinical use of DHMEQ IP therapy.

To determine the mechanistic role of mobile genetic elements in causing widespread DNA damage in primary human trophoblasts.

ABSTRACTThe impaired synthesis of antigen-specific antibodies, which is indispensable for an adaptive immune response to infections, is a fundamental pathomechanism that leads to clinical manifestations in children with antibody production defects. The aim of this study was to evaluate the synthesis of antigen-specific antibodies following immunization in relation to peripheral blood B cell subsets in young children with hypogammaglobulinemia. Twenty-two children, aged from 8 to 61 months, with a deficiency in one or more major immunoglobulin classes participated in the study. Postvaccination antibodies against tetanus and diphtheria toxoids, the surface antigen of the hepatitis B virus, and the capsular Haemophilus influenzaetype b polysaccharide antigen were assessed along with an immunophenotypic evaluation of peripheral blood B lymph cell maturation. A deficiency of antibodies against the tetanus toxoid was assessed in 73% of cases and that against the diphtheria toxoid was assessed in 68% of cases, whereas a deficiency of antibodies against the surface antigen of the hepatitis B virus was revealed in 59% of the children included in the study. A defective response to immunization with a conjugate vaccine with the Haemophilus influenzaetype b polysaccharide antigen was demonstrated in 55% of hypogammaglobulinemic patients. Increased proportions of transitional B lymph cells and an accumulation of plasmablasts accompanied antibody deficiencies. The defective response to vaccine protein and polysaccharide antigens is a predominating disorder of humoral immunity in children with hypogammaglobulinemia and may result from a dysfunctional state of the cellular elements of the immune system.

The authors studied the in vitroeffect of a calcium-channel-blocker -verapamil, local anesthetic -bupivacaine and a calcium ionophore -A23187 on the permeability of the monolayer mesothelium from the rabbit's mesentery. Verapamil and bupivacaine increased transmesothelial flux of calcium while A23187 increased calcium transport only transiently. Verapamil augmented the permeability of the mesothelium to inulin and urea. However A23187 decreased the transmesothelial flux of inulin only whereas it increased the transport of urea. From this study we have concluded, that intracellular calcium may determine the permeability of the peritoneal mesothelium.Various important biological processes like muscle contraction, secretion of transmitters and hormones, and control of epithelial permability seem to be dependent on intracellular calcium-ion activity (1–4). Our previous paper suggested that a local anesthetic, bupivacaine, produces its effect on the peritoneal mesothelium through interaction with the cytoskeleton of the epithelial cells, and with the mem brane's calcium transport (5). In addition Palant et alfound that the permeability of another leaky epithelium, that from the Necturus gallbladder, depends on the extracellular calcium concentrations and may be altered by drugs, which interfere with the entrance of these ions into the epithelial cells (6). Therefore, we decided to study the role that calcium ions may play in the transport of solutes across the peritoneal mesothelium in vitro.

This paper describes an experimental model of the monolayer mesothelium from the ileal rabbit's mesentery. As proved by the electron micro. scopic examination, this membrane consists of the mesothelium lying on the undamaged basement membrane. To minimize the damage to the membrane's edge, we used a new type of chamber. Over a period of 90 minutes transport rate of the various solutes across the mesothelium in vitrowas constant, which proves indirectly the viability of this epithelium during this time. This experimental model seems to be suitable for the in vitrostudy of the mechanisms which regulate the permeability of the peritoneal mesothelium.Permeability of the peritoneal mesothelium to various solutes and water has been evaluated during in vitrastudies of isolated peritoneal membranes of the mesentery or on the parietal peritoneum (1,2,3). The experimental conditions avoid or significantly diminish the interference of other barriers, like endothelium or connective tissue, which play an important role in viva.Mesentery consists of two mesotheliallayers, lying symmetrically and, as a result, the permeability so measured doesn't reflect precisely the permeability of the mesothelium. On the other hand models of the parietal peritoneum include a thick layer of muscle and connective tissue which can be a source of some artifacts.To avoid these disadvantages we developed an experimental model of the monolayer mesothelium obtained from the ileal mesentery of the rabbit. We believe this model is useful for the investigation of the function of this epithelium during the peritoneal dialysis. This paper describes this in vitramodel which we have devised for the study of the peritoneal mesothelium.

A single course of antenatal corticosteroids (ACS) is associated with a reduction in respiratory distress syndrome and neonatal death. Multiple Courses of Antenatal Corticosteroids Study(MACS), a study involving 1858 women, was a multicentre randomized placebo-controlled trial of multiple courses of ACS, given every 14 days until 33+6 weeks or birth, whichever came first. The primary outcome of the study, a composite of neonatal mortality and morbidity, was similar for the multiple ACS and placebo groups (12.9% vs. 12.5%), but infants exposed to multiple courses of ACS weighed less, were shorter, and had smaller head circumferences. Thus for women who remain at increased risk of preterm birth, multiple courses of ACS (every 14 days) are not recommended. Chronic use of corticosteroids is associated with numerous side effects including weight gain and depression The aim of this postpartum assessment was to ascertain if multiple courses of ACS were associated with maternal side effects.

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 (µg/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/µL) 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-a, 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-a, 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.

AbstractBackground: To evaluate if peroxisome proliferator-activated receptor-γ (PPAR-γ) agonists have a potential protective effect on the peritoneum changes induced by bioincompatible peritoneal dialysis (PD) solution in vivo. Methods: Male Wistar rats were dialyzed three times daily for 28 days with 1.36% Dianeal® (two groups: with (D+R) or without (D) rosiglitazone) or 1.36% Physioneal® (two groups: with (P+R) or without (P) rosiglitazone). Peritoneal transport of fluid and small solutes was assessed. Nine rats that did not receive dialysis served as controls. Results: Significant morphological changes were found in the D group compared with controls. Additional use of rosiglitazone in the D+R group resulted in less morphological changes and expression of collagen I as well as an increased drainage volume. The expression of VEGF was inhibited by rosiglitazone while no apparent effect was found regarding TGF/Smad pathway. Conclusions: The addition of rosiglitazone to standard dialysis fluids can maintain the peritoneal morphology and increase ultrafiltration in a PD rat model.Copyright © 2006 S. Karger AG, Basel

♦ Background Normal saline (0.9% NaCl) is used during various abdominal surgical interventions and during peritoneal dialysis to rinse the peritoneal cavity. Although no clear clinical evidence exists for the bioincompatibility of normal saline, various experimental studies have suggested that 0.9% NaCl solution can initiate fibrosis of peritoneum.♦ Material and Methods We review the data derived from in vitroand in vivoexperimental studies demonstrating the cytotoxic effect of 0.9% NaCl and its ability to initiate peritoneal adhesions.♦ Results Normal saline reduces the viability and fibrinolytic activity of peritoneal mesothelial cells. Use of normal saline to wash the peritoneal cavity during abdominal operations or after chronic peritoneal dialysis is more likely to produce adhesions than is no irrigation at all. Chronic exposure of the peritoneum to normal saline causes overgrowth of the connective tissue and formation of new blood vessels within that tissue.Conclusion ♦ Normal saline is a bioincompatible solution that predisposes to the formation of peritoneal adhesions and fibrosis of the peritoneum. A 0.9% NaCl solution should therefore not be used to rinse the peritoneal cavity after interruption of peritoneal dialysis.

Glucose degradation products (GDP) are an important factor that contribute to bioincompatibility of peritoneal dialysis fluids. These substances are generated in the dialysis fluid during heat sterilization. Several approaches have been proposed to reduce the content or toxicity, or both, of GDP present in the dialysis fluid. We examined whether L-2-oxothiazolidine-4-carboxylic acid (OTZ), a precursor for glutathione synthesis, reduces the cytotoxicity of GDP in human peritoneal mesothelial cells.

In vitro experiments point to a better biocompatibility profile of new pH-neutral peritoneal dialysis fluids (PDFs) containing low levels of glucose degradation products (GDPs). The present study examines the impact on human peritoneal mesothelial cells (HPMCs) of equilibrated dialysates obtained during dialysis with either conventional or new PDFs.

Background Acidic pH and the presence of glucose degradation products (GDP) are believed to compromise the biocompatibility of peritoneal dialysis fluids (PDF). The present study examines the effects of long-term exposure to GDP and low pH by comparing conventional PDF and a new, neutral pH, low GDP solution.Methods All experiments were performed using a chronic infusion model of dialysis in nonuremic rats. The animals were treated for 6 weeks with 2 daily injections of 4.25% glucose-containing PDF. The following PDF were tested: CAPD3 (single-chamber bag, low pH, high GDP), CAPD3 pH 7.4 (single-chamber bag, neutral pH, high GDP), CAPD3-Balance (double-chamber bag, neutral pH, low GDP). All test solutions were obtained from Fresenius Medical Care, Bad Homburg, Germany.Results After 6 weeks of exposure, peritoneal permeability to water, urea, creatinine, glucose, and sodium, assessed by peritoneal equilibration test, was similar in all groups. However, compared to other PDF, dialysis with CAPD3-Balance was associated with reduced concentrations of protein and hyaluronan in the dialysate, decreased peritoneal eosinophilia, and reduced dialysate levels of chemokines CCL2/MCP-1 and CCL5/RANTES. Morphologic changes in the peritoneal membrane of CAPD3-Balance-treated animals were much less pronounced and included reduced vascular density, preservation of the mesothelial monolayer and intercellular junction, and no reduplication of the submesothelial basement membrane.Conclusions A new generation of PDF with physiologic pH and low GDP level produce less irritation to the peritoneal membrane and better preserve its structural integrity. This effect seems to be related predominantly to minimized GDP concentrations.

♦ Objectives High concentrations of glucose and/or formation of glucose degradation products (GDPs) during heat sterilization of peritoneal dialysis fluids (PDFs) are believed to be key factors in the limited biocompatibility of PDFs. We have previously shown that several identified GDPs can specifically impair human peritoneal mesothelial cell (HPMC) function. In the present study we aimed at differentiating the respective roles of glucose and GDPs in the toxicity of PDF to mesothelial cells.♦ Methods HPMCs were acutely pre-exposed to or incubated chronically in the presence of pH-neutral PDF sterilized by either heat (H-PDF) or filtration (F-PDF). In addition, HPMCs were treated with commercially available H-PDF manufactured either conventionally, that is, in single-chamber containers, or using novel dual-chamber bags that help to substantially decrease GDP formation. Functional assessment of HPMCs included viability, release of interleukin (IL)-6, and proliferation.♦ Results Viability and release of IL-6 from HPMCs pretreated with H-PDF (pH 7.3) for 1 to 4 hours were significantly reduced compared to cells exposed to corresponding F-PDF. Incubation in medium mixed (1:1) with H-PDF considerably impaired growth of HPMCs, and over a period of 10 days gradually decreased both the viability of HPMCs and their ability to generate IL-6. These effects were either absent from or significantly less in HPMCs exposed to F-PDF. Similar differences were observed when commercial GDP-containing H-PDFs were compared with newly designed H-PDFs free of GDPs.♦ Conclusions Impaired viability and function of HPMCs exposed to glucose-containing pH-neutral PDF is related predominantly to the presence of GDP and, to a significantly lesser extent, to the presence of glucose per se.Prevention of GDP formation during auto-claving markedly improves the biocompatibility of H-PDF with HPMCs.

← Background Hyaluronan (HA) is a glycosaminoglycan found in connective tissues and tissue spaces, including the peritoneal cavity. In vivostudies in a rat model of peritoneal dialysis (PD) have shown that addition of HA to PD solution during an intraperitoneal dwell can alter peritoneal fluid transport and protect the peritoneal membrane from the effects of inflammation and repeated infusions of dialysis solution. The current study sought to evaluate the safety of intraperitoneal HA and its effect on peritoneal fluid and solute transport when administered during a dialysis dwell in humans.← Methods 13 PD patients were enrolled in a prospective, randomized crossover study involving three dialysis treatments using the following PD solutions: (1) a commercially available PD solution (Dianeal PD-4, 1.36% glucose; Baxter Healthcare Corporation, Alliston, Ontario, Canada); (2) Dianeal PD-4 containing 0.1 g/L HA, and (3) Dianeal PD-4 containing 0.5 g/LHA. Each 6-hour dialysis exchange was separated from the other exchanges by a 2-week washout period. Radioiodinated human serum albumin (RISA) was administered with the dialysis solution to evaluate intraperitoneal volume, net ultrafiltration (UF), and fluid reabsorption. Peritoneal clearances, dialysate/plasma ratios (D/P), and mass transfer area coefficients (MTACs) were determined for sodium, urea, creatinine, albumin, and glucose. Safety was evaluated by monitoring adverse events and changes in serum chemistries. Ten patients completed all three dialysis exchanges and two additional patients completed at least one treatment exchange.← Results There were no reported adverse events related to HA administration and no significant changes in serum chemistries. There were no significant differences in net UF or peritoneal volume profiles among the three treatments. Mean net UF calculated using residual volumes, estimated by RISA dilution, tended to be slightly higher during treatment with solution containing 0.1 g/L HA and 0.5 g/L HA [74 ± 86 (SE) and 41 ± 99 mL, respectively] compared to control treatment (–58 ± 129 mL). Although not statistically significant, there was a trend toward decreased fluid reabsorption during treatment with HA. Solute clearances, D/P ratios, and MTACs were similar for the three treatments. Serum levels of HA were also unaffected by the two treatment solutions.← Conclusions These data support the acute safety of HA when administered intraperitoneally with the dialysis solution to PD patients. Due to the small sample size and variability in net UF and fluid reabsorption, statistically significant differences were not demonstrated for these parameters. However, a trend toward decreased fluid reabsorption was observed, suggesting that HA may act by a mechanism similar to that observed in animal studies. Further studies are necessary to evaluate whether the beneficial effects of HA observed in animal studies can be shown in humans.

AbstractThe aim:To record blood flow velocimetry in the fetal superior mesenteric artery in normal pregnancy and to evaluate if blood flow recordings in the vessel might predict adverse outcome in high-risk pregnancy.Methods:The fetal superior mesenteric artery blood velocimetry was recorded in a cross sectional manner in 75 normal pregnancies between 27 and 41 weeks of gestation. Reference curves were performed for pulsatility and resistance indices. The superior mesenteric artery was also located in 48 singleton pregnancies complicated by pregnancy-induced hypertension and/or intra-uterine growth retardation. Middle cerebral artery, umbilical artery and vein and uterine artery velocimetry were also recorded.Results:Superior mesenteric artery PI and RI values expressed an increase in resistance to blood flow with gestational age after 32 weeks of gestation. In all except eight high-risk pregnancies the fetal mesenteric artery PI values were within normal range. Among the pregnancies with absent or reversed blood flow in the umbilical artery, all had abnormal mesenteric artery pulsatility index (PI) (>97.5thpercentiles), one fetus died intrauterine and two others died after delivery due to prematurity, growth retardation and necrotizing enterocolitis. In the remaining fetuses with increased mesenteric artery PI, necrotizing enterocolitis was diagnosed in three cases.Conclusions:Increased vascular resistance in the mesenteric artery might be a late sign of fetal circulation redistribution and frequently related to necrotizing enterocolitis in the newborn.

Objective L-2-Oxothiazolidine-4-carboxylate (OTZ), a cysteine precursor, is a substrate for intracellular glutathione synthesis. As shown previously, OTZ prevents free-radical induced cellular damage during in vitrosimulation of peritoneal dialysis. In the present study, we examined the effect of adding OTZ to peritoneal dialysis solution on peritoneal function and structure during lipopolysaccharide (LPS)-induced peritonitis in rats. In addition, we studied the effects of pretreatment with OTZ (given orally) on the effects of LPS-induced peritonitis in rats.Methods Peritonitis was induced in rats by adding LPS (5 μg/mL) to the dialysis fluid. For acute experiments, rats were exposed to a single infusion of dialysis solution containing LPS or to LPS plus 5 mmol/L OTZ; peritoneal cell counts and permeability were determined after 4 hours. Alternatively, rats were pretreated with OTZ added to the drinking water (0.1%) for 10 days prior to infusion of LPS. For chronic experiments, peritoneal dialysis was performed over a 3-week period in rats with implanted peritoneal catheters. On days 8, 9, and 10 of the experiment, the rats were infused intraperitoneally with solution containing LPS (5 μg/mL), or LPS plus 5 mmol/L OTZ, to induce acute peritonitis. At the end of dialysis (10 days after the episodes of peritonitis), peritoneal function was assessed using a peritoneal equilibration test (PET), and peritoneal biopsies were taken to assess effects on peritoneal morphology.Results In the acute experiments, exposure to LPS led to increased peritoneal cell counts (+61% vs control, p< 0.05) and enhanced permeability of the peritoneum, leading to a loss in ultrafiltration (–63%, p< 0.0005). The glutathione concentration in peritoneal leukocytes also decreased during acute peritonitis (–31%, p< 0.05). During LPS-induced peritonitis, OTZ prevented the increase in dialysate cell count and the decrease in cellular glutathione content. Simultaneous administration of OTZ did not prevent the increased peritoneal permeability induced by LPS. However, in rats pretreated with OTZ, LPS-induced permeability to protein was significantly lower than in the nontreated animals (0.049 ± 0.011 vs 0.087 ± 0.034, p< 0.05). In the chronic experiments, LPS-induced peritonitis did not lead to any functional differences in peritoneal transport at the end of 3 weeks of dialysis. However, LPS-induced peritonitis led to increased thickness of the peritoneum and neovascularization within peritoneal interstitium compared to peritonitis-free animals. In contrast to the LPS-treated animals, the peritoneum of the rats exposed to LPS in the presence of OTZ was of a thickness similar to that in the control rats.Conclusion Supplementation of dialysis fluid with OTZ prevented changes in cellular glutathione levels and dialysate cell counts during acute peritonitis in rats. During chronic dialysis in rats exposed to intermittent peritonitis episodes, OTZ prevented increased thickening and neovascularization of the peritoneum. Our results suggest this may help to protect the peritoneal membrane during episodes of peritonitis.

♦ 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 vitrocultured rat mesothelial cells (ex vivostudy). 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.

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 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-γ (INF-γ) 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-γ 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.

Objective To evaluate changes in volume of mesothelial cells exposed to hypertonic medium and the role of volume regulatory mechanisms in adaptation to hyperosmolality.Design Experiments were performed on primary cultures of human peritoneal mesothelial cells. Cell volume was estimated by measuring equilibrated (intracellular/ extracellular space) 14C-urea in cellular water. Cells in monolayers were exposed to hyperosmotic media and changes in cellular water or intracellular uptake of 3H-proline were measured.Results Exposure of mesothelial cell monolayers to hyperosmotic media reduced the cell volume; the effect was proportional to the osmolality of the medium. Volume of cells exposed to medium supplemented with glucose (180 mmol/L) decreased by 26%, p< 0.001, after 30 minutes’ incubation. Prolonged exposure of mesothelial cells to hyperosmotic medium resulted in gradual recovery, after initial decline, of their volume. Intracellular uptake of amino acid 3H-proline increased after 240 minutes’ exposure of the mesothelial cells to medium supplemented with glucose (90 mmol/L) (+40%, p< 0.05). When cells cultured for 7 days in medium supplemented with glucose (45 mmol/L) were exposed to medium with low glucose content (5 mmol/L) their volume increased by 17%, p< 0.05.Conclusion Mesothelial cells shrink after exposure to hypertonic medium. Increased intracellular uptake of amino acids may be one of the regulatory mechanisms that ensure subsequent volume increase in these cells. Mesothelial cells chronically exposed to hypertonic medium swell after transfer to a medium with physiologic osmolality.

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 vivoisolated rat peritonealleukocytes.Materials Peritoneal leukocytes were isolated from rats on chronic peritoneal dialysis. On alternate days, fluid exchanges were performed with NAG-based or glucosebased dialysis solutions. After a 4-hour dwell, dialysate was drained and peritoneal leukocytes were incubated in vitro:I= lipopolysaccharide (LPS). Production of nitrites (index of NO synthesis), tumor necrosis factor α (TNFα), interleukin-1 β (IL -1 β), and interferon gamma (IFN-y) 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 :I= 615 cells/μL) as compared to glucose-based fluid (3643 :I= 1108 cells/μL; p < 0.01). Differential cell count was similar in both studied groups. Unstimulated peritoneal leukocytes from NAGbased dialysate produced more NO (nitrites) (0.65 ± 0.07 μmol per 106 cells) than did cells from glucose-based dialysate (0.26 :I= 0.09 μmol per 106 cells, p < 0.01). Stimulated peritoneal leukocytes from NAG-based dialysate produced more cytokines than did cells from glucose-based dialysate: TNFα, 135.2 ± 37.0 pg versus 70.2 :I= 21.8 pg per 106 cells respectively, p < 0.01; IL -1 β, 143.2 :I= 60.9 pg versus 99.1 :I= 22.4 pg per 106 cells respectively, p < 0.05; IFN-y, 16.2:1= 12.5 pg versus 6.0:1= 1.8 pg per 106 cells respectively, p <0.01.Conclusions We demonstrated that rat peritonealleukocytes exposed in vivotoNAG-based dialysis fluid have better ability to produce inflammatory mediators than do peritoneal leukocytes from the same donor, but exposed in vivoto glucose-based dialysis solution.

Objective To assess the in vivoperitoneal inflammatory reaction in rats dialyzed with neutral, bicarbonatelactate-buffered dialysis fluid.Methods Chronic peritoneal dialysis was performed for 4 weeks in Wistar rats with two solutions: (1) 40 mmol/l lactate-buffered fluid, pH 5.2, with a glucose concentration of 2.27 gldl (lac); and, (2) 15 mmolll lactate and 25 mmolll bicarbonate-buffered fluid, pH 7.0 -7.5, with a glucose concentration of 2.27gldl (Bic-lac). After 4 weeks, two peritoneal equilibration tests (PET 1 and PET 2) were performed in all animals with each respective solution. PET 1 was done with test solutions alone, whereas, on a subsequent day, PET 2 was performed with test solutions supplemented with endotoxin [lipopolysaccharide (IPS)] to induce peritonitis.Results During PET 1 no consistent differences were detected in peritoneal permeability between the lac and Bic-lac groups. Total dialysate cell count in the Bic-lac animals was lower than in rats treated with lac fluid: that is, at 8 hours, the respective counts were 1858 ± 524 cellslμl versus 2785 ± 1162 cellslμl (p < 0.01). Dialysate from animals dialyzed with Bic-lac contained more macrophages (at 4 hours: 53.6% ± 35.8% versus 35.8% ± 8.8%, p < 0.001) and fewer neutrophils (at 4 hours: 3.6% ± 1.8% versus 15.4%± 6.1%, p < 0.001) as compared to those dialyzed with the lac solution. Concentration of nitrites in 8-hour dwell dialysate samples from Bic-lac rats was lower than that in the lac group (0.98 ± 0.28 μmollml versus 2.32 ± 0.87 μmollml, p < 0.002), but cytokine levels in the dialysates were comparable. During PET 2, the in -crease in peritoneal permeability resulting from the lPS induced inflammatory response was similar for both test solutions. Dialysate cell count was higher in the lac group versus the Bic-lac group (at 8 hours: 8789 ± 4862 cellslμl versus 3961 ± 581 cellslμl, p < 0.001), contained more neutrophils (at 8 hours: 80.0% ± 11.3% versus 54.8% ± 4.4%, p < 0.001) and fewer macrophages (at 8 hours: 6.8% ± 5.6% versus 21.2% ± 3.3%, p < 0.05). During peritonitis, we found a higher overall dialysate concentration of both tumor necrosis factor (TNFα: +53%, p < 0.05) and of interferon gamma (lFN-y: +303%, p < 0.02), in the Bic-lac group than in the lac group.Conclusions A lower dialysate cell count, higher percentage of macrophages, and lower percentage of neutrophils in dialysate suggest that Bic-lac fluid induces a diminished nonspecific inflammatory response of the peritoneal cavity during dialysis. However, after in vivostimulation, peritoneal cells from animals dialyzed with Bic-lac solution possess an augmented ability to produce inflammatory cytokines.

The influence of the glutathione precursor, l-2-oxothiazolidine-4-carboxylic acid (OTZ), on the function of human peritoneal mesothelial cells in vitro, in conditions that mimic the in vivo effect of peritoneal dialysis solutions on mesothelium, was studied. Mesothelial monolayers were exposed to dialysis fluids (Dianeal 1.36 or Dianeal 3.86; Baxter Healthcare Corp, Round Lake, IL) that were diluted gradually with pooled-effluent dialysate obtained from patients undergoing continuous ambulatory peritoneal dialysis. In vitro exposure of mesothelium to standard dialysis fluid enhances their susceptibility to injury by hydrogen peroxide. OTZ added to dialysis solution in concentrations of 1 mmol/L prevented the toxic effect of hydrogen peroxide, probably by increasing intracellular glutathione. Mesothelial cells exposed to dialysis fluid become activated, evidenced by increased release of interleukin-6 and hyaluronan. OTZ used in concentrations of 1 mmol/L reduced that effect. Furthermore, the addition of glucose to the culture medium in a concentration of 45 mmol/L inhibits the proliferation of mesothelial cells; the presence of OTZ, 1 mmol/L, partially prevents the inhibitory effect of glucose. The results presented in this report show that by augmenting the intracellular concentration of glutathione in mesothelial cells by the addition of OTZ to the dialysis fluid, we can increase their resistance to the acute toxicity of free radicals and long-term toxicity of glucose. In addition, mesothelial cells with an increased glutathione level are less activated after their exposure to dialysis fluid.

Objective To assess the effect of insulin on the Na+/ K+-ATPase expression and activity in human peritoneal mesothelial cells (HPMC).Methods HPMC were isolated from the omental tissue of non-uremic patients, grown to confluence and rendered quiescent by serum deprivation for 24 hours. The activity of Na+/K+-ATPase was determined by measuring the ouabain-sensitive86Rb uptake. To assess whether the effect of insulin was related to changes in [Na+]i the sodium influx was measured with 22Na and the activity of Na+/K+ -A TPase was assessed in the presence of amiloride. Expression of Na+/K+ -A TPaseα1’ α2 and β1-subunit mRNAs was determined by RT/PCR.Results Exposure of HPMC to insulin resulted in a time and dose-dependent increase in the Na+/K+-ATPase activity. After 60 minutes the ouabain-sensitive 86Rb up take (cpm/104 cells) was increased from 6650±796 in control cells to 9763±1212 in HPMC exposed to 100 mU/ mL insulin (1.5-fold increase; n=4, P<0.05). In addition, incubation of HPMC with 100 mU/mL insulin resulted in a time-dependent increase in the 22Na influx. Pre-exposure of HPMC to 1 mM amiloride reduced the activity of Na+/K+-A TPase but did not block the stimulatory effect of insulin. RT/PCR analysis revealed that HPMC constitutively expressed α1 and β1-subunit mRNAs while the α2-subunit mRNA was barely detectable. Exposure of HPMC to insulin for up to 24 hours was not associated with any changes in the expression of either α1’ α2 or B1-subunit.Conclusion Insulin stimulates the Na+/K+-ATPase activity in HPMC in a time and dose-dependent manner. This effect appears to mediated by an increase in [Na+]i and is not related to alterations in Na+/K+-ATPase subunit mRNAs expression.

The aim of this study was semiquantitative evaluation of tissue blood flow in the fetal lung before and after administration of betamethasone. This was carried out by means of computer analysis of ultrasound Doppler signals obtained by the color Doppler energy (CDE) technique. CDE signals were recorded in 20 singleton pregnancies with appropriate growth and imminent preterm delivery between 26 and 33 weeks of gestation. The CDE signal recordings were made before and after intramuscular administration of betamethasone 8 mg/day for 3 days. Fixed preset CDE system control settings for the fetal right lung were used during the examinations. Images from CDE scans were recorded on S‐VHS videotape and transmitted for computer analysis of 8‐bit images at 2.56 gray‐scale levels. The mean flow signal intensity was recorded for the fatal lung before and after betamethasone administration. Additionally, blood velocity waveforms were measured in the intrapulmonary arteries and veins in the peripheral part of the lung.

Objective Evaluation of peritoneal surface area and its permeability during dialysis in rats of various ages.Design Study I: planimetry of peritoneum and its topographic areas was performed in 47 rats of various ages (8 -30 weeks). Study II: net ultrafiltration (UF), dialysate-to-serum ratios for urea, creatinine, albumin, and total protein as well as their peritoneal permeability coefficients, were measured during a 1-hour peritoneal exchange with Dianeal 2.5%, in 21 rats of different ages (9 30 weeks) and with various peritoneal surface areas. Animals: Male Wistar rats.Results The peritoneal surface area in rats increases during aging, but young animals with lower body weight have a relatively larger peritoneal surface area than older, larger animals. The area of the topographic fragments of peritoneum expressed as a percentage of the total peritoneal surface is steady during aging. Efficiency of transperitoneal water removal expressed as net UF per amount of absorbed glucose declines in older animals, with larger peritoneal surface areas. Dialysate/serum ratio of solutes transported from blood to dialysate is proportional to peritoneal surface area. Permeability coefficient (K) of peritoneum to urea and creatinine is unchanged during the aging of animals. However peritoneal permeability (K) to albumin increases during aging, with the opposite tendency for total proteins.Conclusions Kinetics of peritoneal dialysis in rats of different ages is determined by peritoneal surface area and permeability of peritoneum to individual solutes.

Previous protocols of administration of recombinant human erythropoietin to patients with end-stage renal disease have been by the i.v. route. Because this method would be impractical for the continuous ambulatory peritoneal dialysis patient, we designed an i.p. dosing protocol in uremic rabbits to examine whether significant amounts of this hormone could be absorbed from the peritoneal cavity. Our results demonstrate that almost all of the erythropoietin is absorbed (or adsorbed) during a prolonged dwell when administered undiluted by dialysate.

We have attempted to determine whether human mesothelial cells (MC) have the power to influence their own proliferation. A serum -free medium was conditioned with the mesothelial monolayer for 24 hours and then applied to proliferating MC. Conditioned medium increased proliferation rate of MC. When the medium was heated at 60°C for 60 minutes, the growth-promoting activity of the conditioned medium decreased by 50%, suggesting that MC produce at least 2 growth factors, 1 heat-Iabile and the other heat-stable. When MC were exposed continuously to a medium containing 90 mM glucose growth factor, production was decreased by 35%. However, when the cells were exposed to glucose only on alternate days, growth-factor production was similar to that in the control medium. On the other hand, MC exposed continuously for 10 days to 90 mM of glucose exhibited a weaker response to endogenous growth factor, even in a normotonic medium with low glucose concentration. Our results suggest that MC syn thesize factor(s), which stimulate their own proliferation, and that high glucose concentrations interfere with this production and the subsequent action of growth factor.

The effect of chondroitin sulphate (CS) on peritoneal fluid and solute transport was studied in rats undergoing peritoneal dialysis. In the presence of CS, net ultrafiltration increased, while absorption of glucose and horseradish peroxidase from the peritoneal cavity decreased. Albumin, used instead of CS, did not modify either fluid or solute transport.In in vitroexperiments on isolated rabbit mesentery, CS decreased transmembrane water flow induced by hydrostatic pressure, and its effect was not fully reversed 60 minutes after “wash-out” of this glycosaminoglycan.We postulate that the polyanionic CS molecules are trapped in the peritoneal interstitium, thus decreasing its hydraulic conductivity and permeability, which in turn increases net fluid removal during peritoneal dialy sis because of its slower absorption from the peritoneal cavity.

Objective To assess the effect of an inhibitor of nitric oxide synthesis [NG-nitro-L-arginine methyl ester (L-NAME)] on peritoneal transport during peritoneal dialysis (PD) and peritonitis in rats.Methods The authors studied peritoneal transport of small and large solutes, and net ultrafiltration (UF) in rats during PD with Dianeal 3.86 (Baxter, McGaw Park, IL, U.S.A.). They evaluated the effect of L-NAME used as an additive to dialysis fluid in concentrations 0.5 -5 mg/m L on peritoneal transport of small and large molecules and on transperitoneal UF. In addition, they studied the effect of L-NAME (5 mg/mL) during acute peritonitis induced by lipopolysaccharides (5 μg/mL) given intraperitoneally.Results The addition of L-NAME to dialysis fluid increased the selectivity of the peritoneum and net UF during dialysis. Lipopolysaccharides used as an additive to the dialysis fluid, together with L-NAME, did not induce changes in transperitoneal transport of small and large solutes and did not cause a significant decline in net UF. L-NAME given intraperitoneally reduced both local and systemic production of nitric oxide, which might explain its effects on peritoneal transport.Conclusions Nitric oxide is an important mediator of changes in peritoneal transport and its effect is especially significant during peritonitis.

Objective To evaluate the effect of supplementation of dialysis fluid with N-acetylglucosamine (NAG) on the permeability of peritoneum during chronic peritoneal dialysis in rats.Design Experiments were performed on rats with surgically implanted peritoneal catheters. Dialysis solution [DianeaI1.5% (Baxter, Deerfield, IL, U.S.A.) supplemented with either NAG 50 mmol/L or glucose 50 mmol/L (control)] was infused intraperitoneally twice, every day, for 8 weeks. Peritoneal equilibration tests (PET) were performed in all animals at the beginning of the study and after 8 weeks of dialysis. Additionally, at the end of each week, dialysis solution infused in the morning was drained after 4 hours of intraperitoneal dwell. White blood cell count, creatinine, and total protein concentrations were measured in the effluent dialysate. After 8 weeks of dialysis, the morphology of the peritoneum was studied.Results In rats exposed to dialysis fluid supplemented with NAG, peritoneal permeability to creatinine and proteins was reduced when compared to animals dialyzed with glucose solution. In NAG treated animals, staining with alcian blue for polyanions in the peritoneal interstitium was significantly stronger than in rats dialyzed with glucose solution.Conclusions Chronic peritoneal dialysis with dialysis solution supplemented with N-acetylglucosamine causes accumulation of glycosaminoglycans in the peritoneal interstitium, which results in a change of peritoneal permeability.

The carcinoembryonic antigen (CEA) was produced and released by human colorectal carcinomata and also the normal attached mucosa when maintained in both monolayer and organ culture. Immunoperoxidase cytochemical methods were employed for the cellular localization of CEA which was demonstrable only on the neoplastic cells. Gel filtration and immunological methods confirmed that CEA, produced by normal and neoplastic cells, had properties similar to "authentic" CEA derived from metastatic colorectal carcinomata. In addition, two other CEA cross-reacting macromolecules, neither of which was CCEA-2, were produced by these tumours in culture.

The authors created transmesothelial ultrafiltration in vitroby inducing an osmotic gradient (vasc-300 mOsm: meso-400 mOsm) across the membrane. Calculated value of hydraulic conductivity of the mesothelium is comparable with analogous value for endothelium of the microvessels.They propose that transperitoneal ultrafiltration develops in two phases: Transmesothelial ultrafiltration, which augments tonicity of the interstitial tissue which is followed by transendothelial ultrafiltration due to the osmotic gradient between the dehydrated interstitium and the vascular space. Hypertonic medium on the mesothelial side of the membrane modified its permeability to various solutes: transport rate of urea and inulin was increased whereas transport rate of albumin decreased.Transperitoneal ultrafiltration of fluid during peritoneal dialysis conditions the removal of water from the organism. According to Nolph et al, the endothelium of the peritoneal microvessels plays a crucial role in this process (I). Little is known about the role of the mesothelium in transperitoneal ultrafiltration but patients with damaged mesothelium show decreased removal of water across the peritoneal membrane (2).This paper describes the results of an in vitrostudy on the transmesothelial, osmotically induced ultrafiltration.