RESUMEN
BACKGROUND: Normal saline gained wide popularity in abdominal surgery as a basic compound used in intraoperative drainage of the peritoneal cavity. However, recent studies have revealed that saline solution is not quite biocompatible with the intraperitoneal enviroment and may promote peritoneal adhesions. OBJECTIVES: The aim of the study was to evaluate the function and viability of human mesothelial cells cultured in vitro in 0.9% NaCl solution from intraperitoneal lavage carried out during laparoscopic cholecytectomies. MATERIAL AND METHODS: The study included 40 consecutive patients suffering from gallstones who underwent laparoscopic cholecystectomy. Fluid was collected after intraperitoneal lavage during the surgical procedures. The samples obtained were used as a medium for in vitro incubation of primary human mesothelial cells. After 24 h the synthesis of interleukin 6 (IL-6), plasminogen activator inhibitor (PAI) and tissue plasminogen activator (tPA), as well as the index of cell proliferation were assessed in all the experimental groups. RESULTS: All the mesothelium cell cultures treated with fluid samples obtained ex vivo were characterized by elevated levels of IL-6. The highest concentrations of PAI-1 were found in groups of cells exposed to fluid with bile; similarly, tPA synthesis was extremely elevated in groups treaded with fluid containing bile and small amounts of hemolyzed blood. In contrast, cell proliferation was exceedingly high in 2 groups of cells placed in a standard culture medium and in 0.9% NaCl solution. CONCLUSIONS: Normal saline introduced into the abdominal cavity modifies the biological and physicochemical conditions of the intraperitoneal environment. The impact of 0.9% NaCl on mesothelial cells is manifested in destabilized tissue regeneration, which supposedly initiates adhesion formation.
Asunto(s)
Células Epiteliales/citología , Lavado Peritoneal , Cloruro de Sodio/farmacología , Procedimientos Quirúrgicos Operativos , Proliferación Celular/efectos de los fármacos , Medios de Cultivo/farmacología , Células Epiteliales/efectos de los fármacos , Femenino , Humanos , Interleucina-6/metabolismo , Masculino , Inhibidor 1 de Activador Plasminogénico/metabolismo , Activador de Tejido Plasminógeno/metabolismoRESUMEN
Hemodialysis induces oxidative stress causing intravascular inflammation, which may cause endothelial dysfunction. We evaluated how hemodialysis-induced changes in blood affect the function of endothelial cells in in vitro culture. Serum samples were collected from 42 uremic patients treated with hemodialysis, one before the start of dialysis and the other one at the end of session. All patients were dialysed with polysulfone dialyzer. Concentrations of the inflammatory molecules carbonyl protein and metabolites of NO synthesis were measured in blood. Additionally, the effect of the serum obtained before and after dialysis on the function of endothelial cells in in vitro culture was studied. Hemodialysis caused increase of monocyte chemoattractant protein (MCP)-1 (+17%), hepatocyte growth factor (+91%), and pentraxin-3 (+30%) concentration in serum. Concentration of carbonyl protein was decreased by 30%. Decrease of blood level of asymmetric dimethylarginine (-25%) and nitrate/nitrites (-62%) was observed. Serum obtained after hemodialysis stimulated proliferation of endothelial cells (+10%) and synthesis of MCP-1(+11%) in these cells. Hemodialysis-induced intravascular inflammation changes the function of endothelial cells, which may lead to acceleration of atherosclerosis.
Asunto(s)
Endotelio Vascular/fisiopatología , Inflamación/sangre , Diálisis Renal/efectos adversos , Uremia/sangre , Uremia/terapia , Enfermedades Vasculares/sangre , Femenino , Humanos , Masculino , Persona de Mediana Edad , Estrés Oxidativo/fisiologíaRESUMEN
BACKGROUND: Preservation of the mesothelial cells (MCs) is crucial for longevity of the peritoneal dialysis membrane. Glucose accelerates aging of MC and we tested whether N-acetylglucosamine (NAG) has an identical effect. METHODS: Replicative aging of MCs was studied during 10 passages performed every three days in cells cultured in standard medium or in medium supplemented with Glucose 30 mmol/L or NAG 30 mmol/L. Changes in population doubling time and ß-galactosidase activity were used as an index of aging and compared with other cellular parameters. RESULTS: Repeated passages of MC cause their aging, as reflected by prolongation of the population doubling time, increased ß-galactosidase activity, oxidative stress and release of cytokines. Healing of injured mesothelial monolayer is impaired in senescent cells. Glucose accelerates in vitro aging of MC, whereas NAG does not cause this effect. CONCLUSIONS: Replacement of glucose with NAG in the dialysis fluid can slow down aging of MC.
Asunto(s)
Acetilglucosamina/farmacología , Senescencia Celular/efectos de los fármacos , Soluciones para Diálisis/farmacología , Células Epiteliales/efectos de los fármacos , Glucosa/farmacología , Epiplón/efectos de los fármacos , Análisis de Varianza , Proliferación Celular/efectos de los fármacos , Células Cultivadas , Soluciones para Diálisis/química , Células Epiteliales/metabolismo , Humanos , Ácido Hialurónico/metabolismo , Interleucina-6/metabolismo , Epiplón/metabolismo , Estrés Oxidativo/efectos de los fármacos , Diálisis Peritoneal , Especies Reactivas de Oxígeno/metabolismo , Factores de Tiempo , Factor de Necrosis Tumoral alfa/metabolismo , Cicatrización de Heridas/efectos de los fármacos , beta-Galactosidasa/metabolismoRESUMEN
BACKGROUND: Senescent endothelial cells acquire functional properties that make the vascular wall more prone to atherosclerotic changes. We tested whether senescence of the endothelial cells maintained in in vitro culture can be moderated by their simultaneous exposure to sulodexide. MATERIAL/METHODS: Replicative aging of the endothelial cells was studied during their 15 passages performed every 4 days in cells cultured in standard medium or in medium supplemented with sulodexide 0.5 LRU/mL. Changes in population doubling time and beta-galactosidase activity were used as indexes of aging and compared with other cellular parameters. RESULTS: Repeated passages of endothelial cells induce their senescence, as reflected by prolongation of the population doubling time, increased beta-galactosidase activity, oxidative stress and release of cytokines. Healing of the injured endothelial monolayer is impaired in senescent cells. Sulodexide partially prevents oxidative stress and totally eliminates other senescence-related changes such as increased release of MCP-1, lengthening of the population doubling time, and impaired healing of the cellular monolayer after its mechanical injury. CONCLUSIONS: Sulodexide prevented cellular senescence in cultured endothelial cells, moderating features of the cellular senescence in endothelial cells in in vitro conditions, which potentially may have practical application. The administration of sulodexide could potentially be used in prevention of atherosclerotic changes.
Asunto(s)
Senescencia Celular/efectos de los fármacos , Células Endoteliales/citología , Células Endoteliales/efectos de los fármacos , Glicosaminoglicanos/farmacología , Células Endoteliales/metabolismo , Radicales Libres/metabolismo , Humanos , Interleucina-6/biosíntesis , Espacio Intracelular/efectos de los fármacos , Espacio Intracelular/metabolismo , Factores de TiempoRESUMEN
Sulodexide is a mixture of heparin and dermatan sulfate with antithrombotic and profibrynolytic activity. Individual reports suggest the anti-inflammatory action of sulodexin. The goal of this study was to evaluate the effect of sulodexide on the release of the inflammatory mediators from endothelium in normal conditions and in cells chronically exposed to glucose. The experiments were performed on in vitro cultured human umbilical endothelial cells kept for 7 days in standard medium or in the same medium but supplemented with glucose 30 mmol/L. Sulodexide was added to the culture medium in concentrations of 0.125 lipase releasing unit (LRU)/mL, 0.25 LRU/mL, and 0.5 LRU/mL Spontaneous generation of oxygen-derived free radicals and the release of monocyte chemotactic protein-1 (MCP-1) and interleukin-6 (IL-6) from the studied cells was evaluated. Additionally, the healing of the injured mesothelium was studied in the presence of sulodexide and glucose. Sulodexide caused the inhibition of the intracellular generation of free radicals in a dose-dependent manner (maximally by 32%, P < 0.01), as well as the inhibition of MCP-1 (maximally by 60%, P < 0.001) and IL-6 (maximally by 69%, P < 0.01). Cells cultured in a medium with glucose 30 mmol/L generated more free radicals (+20%, P < 0.05) and released more MCP-1 (+113%, P < 0.001) and IL-6 (+26%, P < 0.05). Cell monolayers treated with glucose had a decreased ability to heal after mechanical injury (-28%, P < 0.001). All these glucose effects were reversed when cells were exposed to sulodexide simultaneously. The results of our study demonstrate a significant anti-inflammatory action of sulodexide in the endothelial cells and a protective effect of that drug against glucose cytotoxicity.
Asunto(s)
Células Endoteliales/efectos de los fármacos , Células Endoteliales/patología , Glucosa/antagonistas & inhibidores , Glucosa/toxicidad , Glicosaminoglicanos/farmacología , Inflamación/patología , Muerte Celular/efectos de los fármacos , Células Cultivadas , Quimiocina CCL2/metabolismo , Radicales Libres/metabolismo , Humanos , Interleucina-6/metabolismo , Espacio Intracelular/efectos de los fármacos , Espacio Intracelular/metabolismo , Cicatrización de Heridas/efectos de los fármacosRESUMEN
BACKGROUND: Peritoneal adhesions are the most common complication of the abdominal surgery. Normal saline is frequently used to rinse the peritoneal cavity during abdominal surgery, although there is no well-established data describing effect of such procedure on the process of formation of peritoneal adhesions. METHODS: Effect of 0.9% NaCl solution on viability, oxidative stress, and fibrinolytic activity of human peritoneal mesothelial cells maintained in in vitro culture was evaluated. RESULTS: Exposure of mesothelial cells to 0.9% NaCl induces oxidative stress, derangement of their structure with subsequent increased release of tissue factor (+75%) and plasminogen activator inhibitor-1 (+19%), and simultaneous suppression of tissue plasminogen activator release (-39%). In effect, ration tissue plasminogen activator/plasminogen activator inhibitor-1 was reduced in 0.9% NaCl-treated cells by 50%. Pretreatment of cells with precursor of glutathione synthesis: L-2-oxothiazolidine-4-carboxylic acid prevented these changes. CONCLUSIONS: Oxidative stress in the peritoneal mesothelium caused by 0.9% NaCl activates their procoagulant activity and impairs fibrinolytic properties of these cells. These effects disqualify 0.9% NaCl as rinsing solution during abdominal surgery.
Asunto(s)
Células Epiteliales/efectos de los fármacos , Epiplón/citología , Estrés Oxidativo/efectos de los fármacos , Cloruro de Sodio/toxicidad , Células Cultivadas/efectos de los fármacos , Células Cultivadas/metabolismo , Contraindicaciones , Células Epiteliales/metabolismo , Fibrinólisis/efectos de los fármacos , Glutatión/metabolismo , Humanos , Concentración Osmolar , Cavidad Peritoneal , Enfermedades Peritoneales/inducido químicamente , Enfermedades Peritoneales/prevención & control , Ácido Pirrolidona Carboxílico/farmacología , Especies Reactivas de Oxígeno/metabolismo , Soluciones/efectos adversos , Irrigación Terapéutica , Tiazolidinas/farmacología , Tromboplastina/metabolismo , Adherencias Tisulares/inducido químicamente , Adherencias Tisulares/prevención & controlRESUMEN
Glucose is commonly used as an osmotic solute in peritoneal dialysis fluids despite vast knowledge about deleterious peritoneal and systemic effects of that solute. N-acetylglucosamine (NAG) is a solute of the comparable size to glucose, with strong anti-inflammatory properties. We compared the chronic in vitro effect of both solutes on phenotype of peritoneal mesothelial cells. Experiments were performed of primary cultures of human peritoneal mesothelial cells, which were cultured over 4 weeks in medium supplemented either with glucose 45 mmol/L (GLU) or with NAG 45 mmol/L (NAG). Generation of reactive oxygen species (ROS) in cells was studied, as well as their ability to proliferate, synthesis of cytokines, fibronectin, and factors regulating peritoneal fibrinolysis. Cells cultured in the presence of glucose 45 mmol/L generated more ROS (+73% vs control, P < 0.01), whereas NAG did not stimulate generation of ROS. GLU caused hypertrophy of mesothelial cells (+53% vs control, P < 0.001) and prolonged their population doubling time (+16% vs control, P < 0.01); NAG did not cause significant changes in these parameters. Healing of mesothelial monolayer after mechanical injury was impaired in GLU treated cells: (-48% vs control, P < 0.001 and -40% vs NAG, P < 0.05). Synthesis of Il-6, vascular endothelial growth factor (VEGF), transforming growth factor beta (TGFbeta), and fibronectin was higher in GLU group as compared with control: + 86%, P < 0.001, +38%, P < 0.05, +51%, P < 0.001, +38%, P < 0.05, respectively. In the presence of NAG, these parameters were comparable with the control group, but at the same time NAG stimulated synthesis of hyaluronan: +116% versus control, P < 0.001 and + 96% versus GLU, P < 0.01. Treatment with GLU resulted in decline of tissue plasminogen activator/plasminogen activator inhibitor-1 (t-PA/PAI-1) ratio by 23% versus control, P < 0.001, whereas NAG increased that parameter by 43%, P < 0.01 versus control. Glucose, contrary to NAG, induces oxidative stress and proinflammatory and profibrotic changes in mesothelial cells. NAG seems to be more biocompatible osmotic solute than glucose.
Asunto(s)
Acetilglucosamina/administración & dosificación , Antiinflamatorios/administración & dosificación , Soluciones para Diálisis/administración & dosificación , Células Epiteliales/efectos de los fármacos , Glucosa/administración & dosificación , Fenotipo , Animales , Materiales Biocompatibles , Línea Celular , Proliferación Celular/efectos de los fármacos , Células Cultivadas , Citocinas/metabolismo , Esquema de Medicación , Células Epiteliales/patología , Fibronectinas/metabolismo , Glucosa/química , Humanos , Ácido Hialurónico/metabolismo , Concentración Osmolar , Estrés Oxidativo , Cavidad Peritoneal/citología , Diálisis Peritoneal/métodos , Especies Reactivas de Oxígeno/metabolismo , Activador de Tejido Plasminógeno/metabolismo , Factor de Crecimiento Transformador beta/metabolismo , Factor A de Crecimiento Endotelial Vascular/metabolismoRESUMEN
Intravenous iron supplementation is commonly used in uremic patients treated with peritoneal dialysis. Infusion of iron compounds results in various systemic noxious effects, mainly because of its prooxidant and proinflammatory actions. The authors studied how the intravenous infusion of iron sucrose (IS) affects intraperitoneal homeostasis in rats undergoing acute peritoneal dialysis. Experiments were performed on Wistar rats, which were infused intravenously with IS in a dose 10 mg/kg body weight or with normal saline in the controls. Simultaneously, 4-hour acute peritoneal dialysis was started. At the end of the dialysis, systemic and peritoneal inflammatory reaction and peritoneal permeability were evaluated. Compared with controls, rats exposed to IS showed increased dialysate iron concentration by +70%, P<0.001, and in the differential cell count, more eosinophils (+113%, P<0.05) and fewer macrophages (-16%, P<0.05) existed. In in vitro conditions, macrophages obtained from IS-treated rats released more tumor necrosis factor-alpha (TNF-alpha; +173%, P<0.05) upon stimulation with endotoxin. Additionally increased (+73%, P<0.01) dialysate elastase activity was found in IS-treated animals. Rats infused with IS demonstrated increased peritoneal permeability to total protein (+60%, P<0.001) as compared with control animals. When rats with simultaneous peritonitis received intravenous IS, ex vivo isolated peritoneal leukocytes generated more free radicals (+73%, P<0.05) than did cells harvested from control animals. It has been concluded that intravenous infusion of IS affects the intraperitoneal homeostasis in rats, moving it toward the inflammatory state. These changes may contribute to peritoneal damage.
Asunto(s)
Compuestos Férricos/administración & dosificación , Hematínicos/administración & dosificación , Diálisis Peritoneal , Peritoneo/efectos de los fármacos , Animales , Células Cultivadas , Soluciones para Diálisis/química , Quimioterapia Combinada , Eosinófilos/efectos de los fármacos , Eosinófilos/patología , Sacarato de Óxido Férrico , Radicales Libres/metabolismo , Ácido Glucárico , Infusiones Intravenosas , Hierro/análisis , Leucocitos/efectos de los fármacos , Leucocitos/metabolismo , Lipopolisacáridos/farmacología , Macrófagos Peritoneales/efectos de los fármacos , Macrófagos Peritoneales/metabolismo , Macrófagos Peritoneales/patología , Masculino , Elastasa Pancreática/metabolismo , Peritoneo/metabolismo , Peritoneo/patología , Peritonitis/metabolismo , Peritonitis/patología , Permeabilidad/efectos de los fármacos , Ratas , Ratas Wistar , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
BACKGROUND/AIMS: Peritonitis is one of the complications of peritoneal dialysis. We demonstrate the systemic and intraperitoneal anti-inflammatory action of sulodexide given systemically. METHODS: Dialysis was performed in male Wistar rats with acute peritonitis induced by addition of endotoxin to the fluid. Sulodexide (10 mg/kg b.w.) was used acutely as supplement to the dialysis fluid or chronically, during 7 days preceding the study by intramuscular (i.m.) injection. RESULTS: In rats given i.m. sulodexide the dialysate cell count was lower by 45% (p < 0.001) versus untreated rats with peritonitis. Dialysate elastase activity in i.m. sulodexide-treated rats was lower by 22% (p < 0.05) compared to peritonitis. In rats treated with i.m. sulodexide the increase of plasma tumor necrosis factor-alpha was reduced by 53% (p < 0.002). Pretreatment with i.m. sulodexide reduced transperitoneal loss of total protein and albumin during peritonitis by 26% (p < 0.002) and by 16% (p < 0.05), respectively. CONCLUSION: Sulodexide given systemically reduces the intraperitoneal and vascular inflammatory response during acute peritonitis in rats.
Asunto(s)
Glicosaminoglicanos/farmacología , Inflamación/tratamiento farmacológico , Diálisis Peritoneal/efectos adversos , Peritonitis/tratamiento farmacológico , Enfermedad Aguda , Animales , Antiinflamatorios , Glicosaminoglicanos/uso terapéutico , Inflamación/etiología , Masculino , Peritonitis/patología , Ratas , Ratas Wistar , Resultado del TratamientoRESUMEN
Treatment of anemia in uremic patients requires simultaneous supplementation of erythropoietin and iron. Because of the impaired iron absorption from the gastrointestinal tract in conditions of renal insufficiency, intravenous supplementation is a treatment of choice in such conditions. Iron compounds used for intravenous supplementation induce several systemic side effects, and therefore, we studied the effect of chronic exposure to iron sucrose in rats on renal function. Experiments were performed on male Wistar rats, which were infused intraperitoneally every 4 days, for 28 days with iron sucrose in a dose 1 mg/kg bw or 10 mg/kg bw diluted in 20 mL of the dialysis fluid. Control animals were infused with plain dialysis fluid. Renal function was evaluated at the beginning and at the end of the study. Additionally morphology of the kidneys was evaluated in all animals after 28 days of the study. Chronic exposure of rats to iron sucrose resulted in increased accumulation of PAS-positive material in their glomeruli: + 38% at Fe 1 mg/kg bw P < 0.05 and + 42% at Fe 10 mg/kg/bw P < 0.01 and collagen in the peritubular area: + 40% at Fe 1 mg/kg bw P < 0.005 and + 77% at Fe 10 mg/kg/bw P < 0.001. Only renal clearance of urea was decreased by 53%, P < 0.01 in rats exposed to iron sucrose at a dose of 10 mg/kg bw. Chronic exposure of rats to iron sucrose results in morphologic changes of the kidney; however, mild impairment in renal function was observed only at the highest (10 mg Fe/kg bw) concentration of iron sucrose.
Asunto(s)
Compuestos Férricos/farmacología , Riñón/fisiología , Albuminuria , Animales , Peso Corporal/efectos de los fármacos , Creatinina/sangre , Creatinina/orina , Diuresis/efectos de los fármacos , Sacarato de Óxido Férrico , Ácido Glucárico , Riñón/efectos de los fármacos , Masculino , Ratas , Ratas Wistar , Urea/metabolismoRESUMEN
BACKGROUND: Results of clinical studies suggest that peritoneal dialysis (PD) is less harmful to the residual renal function than haemodialysis. However, we have no objective data describing the potential injuring effect of PD to kidney. We studied in rats after unilateral nephrectomy changes in renal structure and function after 12 weeks exposure to standard, glucose-based PD fluid. METHODS: One month after removing one kidney PD catheters were implanted in rats and during the following 12 weeks, twice a day, animals were infused with 20 ml of 3.9% glucose dialysis fluid containing high concentration of glucose degradation products. Rats not infused with the dialysis fluid served as control (CON). At the beginning and after 12 weeks of the study renal creatinine clearance, urinary excretion of albumin, N-acetyl-beta-glucosaminidase (NAG) and cytokines were measured. Concentration of malondialdehyde (MDA), advanced glycation end products (AGEs) and monocyte chemoattractant protein-1 (MCP-1) were measured in serum samples. Morphology of the kidneys was evaluated in the light microscope. RESULTS: After 12 weeks exposure to the dialysis fluid serum MDA, AGEs and MCP levels were increased as compared with CON by 80%, P < 0.002, 29%, P < 0.05 and 71%, P < 0.005, respectively. Renal clearance of creatinine was comparable in both groups, but urinary excretion of albumin was increased by 55% in control group and by 160% in the studied group, P < 0.001; whereas urinary excretion of NAG was not changed in control group but increased by 125% in the studied group, P < 0.01. Increase of the remnant kidney's weight was higher (+77%, P < 0.01) in the CON group, but accumulation of the extramesangial matrix in glomeruli and collagen in the peritubular space was stronger in the studied group by 69%, P < 0.0001 and 274%, P < 0.0001, respectively. CONCLUSION: Chronic exposure of rats to the glucose-based dialysis fluid causes morphological changes in the renal glomeruli similar to diabetic nephropathy. Albuminuria increases what may accelerate progression of the kidney damage.
Asunto(s)
Riñón/patología , Riñón/fisiopatología , Diálisis Peritoneal/efectos adversos , Animales , Masculino , Ratas , Ratas WistarRESUMEN
Peritoneum is a serous membrane with a significant fibrinolytic potential, playing an important role in the abdominal response to trauma. Peritoneum takes part in the formation and degradation of postoperative adhesions. The sequence of changes during the adhesion formation is indispensable in the healing of peritoneal trauma. Presented paper describes the short historical update of mesothelial research and review of contemporary knowledge over the peritoneal function with special regard to its fibrinolytic activity. The factors influencing the fibrinolytic capacity of peritoneum were discussed, as well as present pathways of research on the prevention of postoperative adhesion formation.
Asunto(s)
Productos de Degradación de Fibrina-Fibrinógeno/metabolismo , Fibrina/metabolismo , Fibrinólisis/fisiología , Peritoneo/fisiopatología , Adherencias Tisulares/fisiopatología , Activador de Tejido Plasminógeno/metabolismo , Animales , Líquido Ascítico/metabolismo , Epitelio/metabolismo , Humanos , Peritoneo/cirugía , Inhibidor 1 de Activador Plasminogénico/metabolismo , Complicaciones Posoperatorias/fisiopatología , Complicaciones Posoperatorias/prevención & control , Ratas , Adherencias Tisulares/metabolismo , Adherencias Tisulares/prevención & control , Cicatrización de Heridas/fisiologíaRESUMEN
BACKGROUND: CA125 is commonly used as an index of the mesothelial cell mass in patients treated with peritoneal dialysis. However, we have no data that show a direct relationship between the number of mesothelial cells, their functional properties, and the amount of CA125 produced in these cells. METHODS: Experiments were performed on primary in vitro cultures of human peritoneal mesothelial cells obtained from 32 donors of various ages and of both sexes. Spontaneous release of CA125 from the confluent mesothelial cells was measured and correlated with the number of cells in monolayers and with their functional properties. We also studied acute effects of cytokines (IL-1beta, TNF-alpha, and INF-gamma) and the chronic effects of glucose (45 mM) on the CA125 content in mesothelial cells and the release of this antigen from their cytosol. RESULTS: Cells from older donors released more CA125, but we found no correlation between the number of cells and the amount of CA125 released from their cytosol. The synthesis of CA125 in mesothelial cells does not correlate with the amount of monocyte chemoattractant protein 1 or interleukin-6 produced in these cells. Acute exposure to cytokines did not modify CA125 content or its release from mesothelial cells. Chronic exposure of mesothelial cells for 4 weeks to glucose (45 mM) decreased the CA125 content of their cytosol and the release of this antigen into the culture medium. Mannitol, at the same concentration and under the same conditions, did not produce these effects, namely a decrease in the CA125 content in the cytosol or its release into the cultural medium. CONCLUSIONS: The amount of CA125 released from mesothelial cells is not a good index of their number or their functional properties, because the CA125 release depends not only on the number of cells, but also on their properties. Furthermore, the process is affected by the age of the cell donor and environmental factors such as a high glucose content. The results of this study show the limitations of CA125 as an index of the mesothelial cell mass and viability.
Asunto(s)
Antígeno Ca-125/análisis , Células Epiteliales/citología , Cavidad Peritoneal/citología , Adulto , Anciano , Anciano de 80 o más Años , Biomarcadores , Antígeno Ca-125/biosíntesis , Células Cultivadas , Células Epiteliales/fisiología , Femenino , Glucosa/farmacología , Humanos , Masculino , Persona de Mediana EdadRESUMEN
BACKGROUND: 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. METHODS: Experiments were performed on primary mesothelial cell cultures. Free radical generation in these cells after exposure to acetaldehyde (ACT), glyoxal (GLYO) or methylglyoxal (M-GLYO) was detected with a fluorescent probe. Cell viability measurements were based on release of LDH from cell cytosol, and synthesis of IL-6 and proliferation after exposure to GDP. Effects of individual GDPs and of dialysis fluid free of GDP (GDP-free PDF) or containing GDP (GDP-high PDF) on cell viability were also studied in the presence of OTZ (1 mmol/l). RESULTS: All of the GDPs as well as the autoclaved dialysis fluid caused increased free radical generation. ACT increased LDH release from the cells by 374% (P < 0.001), and this effect was abolished by OTZ. All of the GDPs inhibited cell growth (ACT, 47%, P < 0.01; GLYO, 52%, P < 0.01; M-GLYO, 26%, P < 0.05) and this effect was reversed in presence of OTZ. ACT inhibited Il-6 synthesis in mesothelial cells by 74% P < 0.01 and this effect was prevented by OTZ. GDP-high PDF but not GDP-free PDF reduced synthesis of IL-6 in mesothelial cells by 40% (P < 0.01) an effect that was reversed by OTZ. Mesothelial cell growth was more strongly inhibited by GDP-high PDF (76%, P < 0.01) than by GDP-free PDF (31%, P < 0.05). OTZ improved growth of mesothelial cells in the presence of GDP-high PDF (+150%, P < 0.01) and in presence of GDP-low PDF (+38%, P < 0.05). OTZ prevented the cytotoxic effect of GDP-high PDF on mesothelial cells. CONCLUSIONS: The GDP-induced stimulation of free radicals in mesothelial cells in the present study may provide a possible mechanism of GDP cytotoxicity. Because OTZ reduced the toxic effects of GDP on mesothelial cells, this compound may improve biocompatibility of peritoneal dialysis fluids.
Asunto(s)
Células Epiteliales/citología , Epitelio/fisiología , Glucosa/metabolismo , Glucosa/farmacología , Soluciones para Hemodiálisis/efectos adversos , Tiazoles/farmacología , División Celular/efectos de los fármacos , Células Cultivadas , Citosol/efectos de los fármacos , Citosol/enzimología , Epitelio/efectos de los fármacos , Humanos , Interleucina-6/biosíntesis , L-Lactato Deshidrogenasa/análisis , Cavidad Peritoneal , Ácido Pirrolidona Carboxílico , TiazolidinasRESUMEN
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 vitro simulation 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 microg/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 micro/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.