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J Witowski

Publications and source records attributed to J Witowski.

30 records · Page 2Linked to original sources
PubMed1994

In vitro culture of human peritoneal mesothelium for investigation of mesothelial dysfunction during peritoneal dialysis.

There is a growing need for more biocompatible peritoneal dialysis fluid formulations. Using a method of in vitro cell culture we have examined the effect of osmotic agents, antiseptics and drug additives on various functions of human peritoneal mesothelial cells (HPMC). We have demonstrated that glucose and amino acids reduce the HPMC proliferation rate in a dose-dependent manner. 3H-methyl-thymidine incorporation into HPMC exposed to either 1.1% amino acid mixture or 2.0% glucose was decreased to (mean +/- SD) 32.5 +/- 6.1% and 16.8 +/- 1.2% of the control level respectively. Cultured HPMC have been shown to produce a fibrinolytic activity which may be estimated by the rate of plasma clot lysis. This activity may be diminished following the pre-incubation with 2.0% glucose and 1.1% amino acid mixture. We have found that povidone-iodine exerts a dose-dependent cytotoxic effect towards HPMC as measured by the increased 86Rb or 3H-inulin release from radiolabelled cells. We have also demonstrated that insulin enhances the 86Rb uptake and stimulates the Na, K-ATPase activity in HPMC but on the other hand is capable of reducing the phospholipids secretion from HPMC. Glucose, hydrocortisone or verapamil have also been shown to inhibit the release of mesothelial phospholipids. These data indicate that the mesothelial cell culture provides a convenient model for testing the biocompatibility of peritoneal dialysis solutions and assessing the dysfunction of mesothelial cells during peritoneal dialysis.

Cell Division↗
PubMed1994

Glycerol toxicity for human peritoneal mesothelial cells in culture: comparison with glucose.

Glycerol has been proposed as a substitute osmotic agent for glucose in peritoneal dialysis fluids. We have compared the effect of glycerol and glucose on the function of human peritoneal mesothelial cells (HPMC) in vitro. The viability of HPMC was not affected by glycerol (up to 250 mM), whereas it was reduced by glucose in a time- and dose-dependent manner, as assessed by the LDH release. Although the incubation of HPMC with glycerol induced a dose-dependent decrease in HPMC proliferation, the effect was significantly less inhibitory than that produced by glucose. In HPMC treated with 90 mM of glycerol or glucose the incorporation of [3H]-thymidine had reached 79.0 +/- 19.3% and 55.3 +/- 4.0% of the control (p < 0.05 and p < 0.01), respectively. As measured by the [methyl-14C]-choline incorporation, the intracellular amount of newly synthesized phospholipids was reduced from (cpm/microgram cellular protein) 147 +/- 58 in control HPMC to 59 +/- 15 in cells exposed to 90 mM of glucose (p < 0.01), but not affected by glycerol (163 +/- 65). On the other hand, both glycerol and glucose (90 mM) decreased the synthesis of proteins (as assessed by the [3H]-proline incorporation) and interfered with potassium (86Rb) transport mechanisms in HPMC. Our data suggest that there exist some possibly advantageous aspects of glycerol as far as mesothelial cell biocompatibility profile is concerned.

Cell Division↗
PubMed1993

Effect of phosphatidylcholine on the function of human mesothelial cells in vitro.

We tested the hypothesis that phosphatidylcholine (PC) molecules present in the dialysis solution may interact with the mesothelial cell membrane and modify its function. In vitro experiments were performed on human mesothelial cells (HMC) in culture. PC decreased proliferation of HMC when used at concentrations of 200 mg/l and higher. PC was also cytotoxic to HMC as measured by the release of lactate dehydrogenase from their cytosol. Cells exposed to PC had a diminished capacity for taking up 86Rb from medium. PC decreased the fibrinolytic properties of HMC and increased their procoagulant activity. Our results suggest that the positive short-term effect of the addition of PC to the dialysis solution (i.e., an increase in ultrafiltration) may be over-shadowed by its deleterious action on HMC membrane.

Cell Division↗
PubMed1993

Toxicity of free radicals to mesothelial cells and peritoneal membrane.

We studied the toxicity of free radicals to human mesothelial cells in vitro and to the peritoneal membrane of rats during peritoneal dialysis. Free radicals cause damage to mesothelial cells as measured by release of cytosolic markers such as 86Rb and lactate dehydrogenase. Vitamin E neutralized the toxic effect of free radicals in vitro. Human mesothelial cells exposed over 6 h to a mixture of essential and nonessential amino acids in medium are more vulnerable to the cytotoxic effect of free radicals than control cells exposed to medium alone. Cells exposed previously to glucose or glycerol are less vulnerable than controls. In rats free radicals generated intraperitoneally by a xanthine-xanthine oxidase system induce changes in peritoneal permeability similar to those observed during peritonitis: loss of ultrafiltration, increased glucose absorption from the dialysate and augmented transperitoneal loss of albumin. In addition lipids in the peritoneum became peroxidated. The addition of vitamin E to the peritoneal fluid with xanthine-xanthine oxidase prevents peroxidation of lipids and the subsequent loss of ultrafiltration. Our results show that free radicals may exert a potentially toxic effect on the peritoneal membrane during peritonitis. In such circumstances the addition of free radical scavenger to the dialysis fluid may preserve intact structure and function of peritoneum.

Animals↗
PubMed1993

Enhancement of viability of human peritoneal mesothelial cells with glutathione precursor: L-2-oxothiazolidine-4-carboxylate.

This study tested the effect of L-2-oxothiazolidine-4-carboxylate (procysteine), a precursor of the intracellular cysteine, on the function of human mesothelial cells (HMCs) in in vitro culture. Procysteine stimulated the proliferation of HMCs and decreased the spontaneous death rate of these cells. HMCs pretreated with procysteine were resistant to injury by free radicals. Procysteine also reversed the cytotoxic effects of a mixture of essential and nonessential amino acids on HMCs, such as increased susceptibility to injury by free radicals and inhibition of proliferation.

Amino Acids↗
PubMed1993

In vitro study of the mechanism of potassium transport into human mesothelial cells. I: Effect of hyperosmolality.

OBJECTIVE: To study the mechanism(s) of potassium transport into human mesothelial cells (HMC) exposed to osmotic solutes. DESIGN: Using potassium analog 86Rb, we evaluated its intracellular transport through three pathways: 1. blocked by ouabain; 2. blocked by furosemide but not by ouabain; 3. blocked by neither furosemide nor ouabain. Experiments were performed in a normotonic medium (control) or in a medium supplemented with osmotic solutes (glucose, glycerol, mannitol). Both the acute and chronic effects of osmotic solutes on potassium transport were studied. RESULTS: The acute exposure of mesothelial cells to osmotic solutes modifies the intracellular transport of potassium through all studied channels, and the effect is specific for every solute. In mesothelial cells exposed over 7 days to glucose (90 mM), the intracellular transport via ouabain- and furosemide-blocked channels is decreased, whereas it is increased through the third pathway. Total intracellular accumulation of 86Rb (potassium) ions in mesothelial cells cultured in a medium supplemented with various concentrations of glucose is decreased, and this effect is proportional to the concentration of glucose in the medium. CONCLUSIONS: The intracellular transport of potassium in mesothelial cells is regulated through at least three independent mechanisms. Acute or chronic exposure of mesothelial cells to a hypertonic medium affects the intracellular accumulation of potassium, and this effect is specific for the various osmotic solutes.

Biological Transport↗
PubMed1993

Impact of peritoneal dialysis solutions on peritoneal immune defense.

In summary, conventional CAPD fluids may interfere with most pathways of the intraperitoneal network of cytokines (Figure 4), potentially resulting in an impairment of important host defense functions. Thus alternative approaches are needed in order to improve dialysis fluid biocompatibility. Initial results indicate that pH neutralization and a restriction to moderate osmolality might constitute important determinants of modern dialysis fluid technology; however, clinical studies will have to prove that these fluids indeed improve the clinical outcome of the patients on long-term CAPD therapy.

Cytokines↗
PubMedDate not supplied

Effect of methotrexate, doxorubicin and mitoxantrone on human peritoneal mesothelial cell function in vitro.

Recent studies have demonstrated that intraperitoneal instillation of dialysis solutions and drug additives may adversely affect the function of peritoneal cell populations. Therefore the aim of the present investigation was to examine the short-term effects of antineoplastic agents on human peritoneal mesothelial cells (HPMC). We have assessed the integrity of HPMC membrane and mechanisms of intracellular potassium transport. There was no evidence of significant cytotoxicity (as measured by 86Rb release) during a 60-min exposure of HPMC to either methotrexate (10(-6)-10(-4) M), doxorubicin (10(-7)-10(-5) M) or mitoxantrone (10(-7)-10(-5) M). In HPMC exposed to doxorubicin (10(-6) M) the intracellular transport of potassium, as assessed with 86Rb as its analogue, was not affected. Methotrexate (10(-5) M) diminished Na,K-ATPase activity and simultaneously enhanced the 86Rb transport via furosemide-sensitive pathway. Mitoxantrone reduced the furosemide-sensitive 86Rb influx in a dose-dependent manner and at a concentration of 10(-4) M it also impaired the ouabain-dependent 86Rb influx. These data demonstrate that antineoplastic agents interfere with HPMC function which might contribute to the oncostatic-induced peritoneal toxicity.

Cells, Cultured↗
PubMedDate not supplied

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.

Animals↗
PubMedDate not supplied

Insulin stimulates the activity of Na+/K(+)-ATPase in human peritoneal mesothelial cells.

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-sensitive 86Rb 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(+)-ATPase was assessed in the presence of amiloride. Expression of Na+/K(+)-ATPase alpha 1,alpha 2 and beta 1-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 uptake (cpm/10(4) 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 1mM amiloride reduced the activity of Na+/K(+)-ATPase but did not block the stimulatory effect of insulin. RT/PCR analysis revealed that HPMC constitutively expressed alpha 1- and beta 1-subunit mRNAs while the alpha 2-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 alpha 1, alpha 2 or beta 1-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.

Cells, Cultured↗