Valery Wajsbrot

High Glucose Induces a Hypertrophic, Senescent Mesothelial Cell Phenotype after Long in vivo Exposure

Previous studies, done using our mouse model for population analysis of the mesothelium, showed evidence indicating that in vivo, long-term exposure (up to 30 days) of the peritoneum to high-glucose (4.25% D-glucose) concentration dialysis solutions resulted in a hypertrophic mesothelial phenotype characterized by increased cell surface area, multinucleation, low proliferative capabilities, reduced cell viability, and enhanced enzymatic activity. These elements that define a senescent population of cells were not related to the pH of the fluid and its osmolality, or to the presence of buffer lactate. The present study was designed to explore the adverse effects of a lactate-free, filter-sterilized, high-D-glucose concentration solution (4.25%) at normal pH and prepared in Hanks’ buffered salt solution after 2 h, 15 and 30 days of once a day intraperitoneal injection. Analysis of our observations indicate that in vivo exposure of the mesothelium to a high-glucose concentration induced a decreased density of the cell population, made up by larger and multinucleated cells, the viability of which was significantly lower than that observed in intact unexposed mice. The prevalence of mitosis showed an early and short-lived acceleration (up to 3 days), followed by values near zero during the rest of the follow-up period. So far, the main effect of the high-glucose concentration appears to result not from a mechanism of cytotoxicity, but from a substantial change in the life cycle of the exposed cell population, leading to their premature senescence and death in apoptosis. We hypothesize that this outcome may well be mediated by sustained oxidative stress derived from both a reduced production of scavengers, as well as the increased generation of oxygen-reactive species.

Icodextrin-induced lipid peroxidation disrupts the mesothelial cell cycle engine.

Fluids commonly used for peritoneal dialysis hold poor biocompatibility vis a vis the peritoneal membrane, basically due to the presence of osmotic agents. When rat mesothelium was exposed to glucose-enriched dialysis solutions for 2 h in vivo, an early and short-lived acceleration of cell life cycle was observed, which, after 30 d of exposure, resulted in a depopulated monolayer of senescent cells. These changes appear to result from persistent oxidative stress due to continuous exposure to high concentration of glucose and to substances generated by the Maillard reaction. Long-term exposure (30 d) of the peritoneal mesothelium to 7.5% icodextrin resulted in a depopulated monolayer consisting mostly of senescent cells, which, additionally, showed atypical nuclear changes and atypical mitosis suggesting DNA damage. These changes coincided with substantial lipid peroxidation, starting immediately after the introduction of the icodextrin solution into the rats abdominal cavity. So far, the currently used osmotic agents in peritoneal dialysis fluids induce substantial oxidative injury to the exposed monolayer in vivo. Use of high concentrations of glucose results in premature senescence of the exposed cell population. The 7.5% icodextrin dialysis fluid induces through lipid peroxidation substantial genomic damage, which, in turn, sets the biological mechanisms leading to protective cellular suicide in motion.

The Cytochemical Profile of Visceral Mesothelium under the Influence of Lactated-Hyperosmolar Peritoneal Dialysis Solutions

Male albino mice had one daily intraperitoneal injection of 4.25 g/100 ml glucose concentration fluid for peritoneal dialysis at pH 5.0-5.2, for a period of 30 days. At the end of the experimental periods, mesothelial cell imprints were taken from the peritoneal layer of the anterior liver surface. Histochemical staining of imprints obtained from mice exposed to the peritoneal dialysis fluid showed a consistently increased activity of: (a) enzymes associated with the cell membrane: Na-K-ATP-ase, alkaline phosphatase and 5-nucleotidase; (b) cytoplasmic enzymes: acid phosphatase and cytochrome oxidase, and (c) a modestly increased activity of glucose-6-phosphatase. These changes, which are not far from those observed in activated mesothelial cells, suggest that exposure of mesothelial cells to high glucose concentrations of PD fluid is associated with increased production and disposal of energy to be used for maintaining the constancy of the cellular environment and, probably, for fuelling the transcellular transport of solutes of large molecular size.

Protective effect of pyruvate upon cultured mesothelial cells exposed to 2 mM hydrogen peroxide

Rat peritoneal mesothelial cells in culture have the capability of generating hydrogen peroxide. Exposure of these cells to glucose-enriched, lactated-buffered fluids for peritoneal dialysis significantly increases the production of H2O2. Increased liberation of oxygen radicals also involves the risk of damaging the peritoneal membrane. Pyruvate being a natural oxidant scavenger abundantly present in mammalian cells, we hypothesized that its protective effects facing H2O2 can eventually be of relevance for the mesothelial monolayer of patients on long-term peritoneal dialysis. So far, we designed an experimental study in which rat peritoneal mesothelial cells in culture were exposed to 2 mM H2O2. Cell damage was estimated in terms of decreased capability of the mitochondrial dehydrogenases to reduce MTT. Addition of 2 mM sodium pyruvate to the medium prevented the negative effect of hydrogen peroxide. The MTT/protein values for the control group were 0.00357 ± 0.00075. The ratio after exposure to 2 mM H2O2 was 0.00217 ± 0.00028, whereas that detected in cells incubated in H2O2 plus pyruvate was 0.00325 ± 0.0082 (p < 0.05). These results indicate that pyruvate protected rat peritoneal mesothelial cells in culture against oxidant injury. These data are one more piece of evidence pointing at pyruvate as a potentially useful buffer for peritoneal dialysis solutions.

Effect of hyperosmolality upon the mesothelial monolayer exposed in vivo and in situ to a mannitol-enriched dialysis solution

Studies done using the in vivo mouse model of population analysis of mesothelium showed that dialysis solutions containing high concentrations of glucose induced the development of a hypertrophic phenotype. Since these changes were neither related to the low pH nor to the presence of lactate buffer, we hypothesized that the presence of glucose was at the origin of the observed alterations. Theoretical analysis of the problem points to three possible mechanisms: hyperosmolality; metabolic changes derived from the high-glucose concentration itself, and/or the presence of products derived from the nonenzymatic degradation of glucose. The present study was designed to demonstrate or rule out the eventual effect of hyperosmolality upon the monolayer, applying the in vivo mouse model of population analysis of mesothelium. For this purpose, morphometric observations made in mice injected once a day during 30 consecutive days with a filter-sterilized 4.25% solution of mannitol (233.29 mM) were compared with those seen in intact mice and in a previously reported group of animals exposed to heat-sterilized fluid, having an equimolar concentration of glucose (235.9 mM), and the same osmolality (486 mosm/l) and electrolyte concentrations. The main findings observed in the mannitol-treated mice during the period of exposure included increased cell size and cytoplasmic surface area, as well as decreased cell viability. The regenerative capabilities of the exposed mesothelium remained intact. After a recovery period of 7 days, the aforementioned parameters reverted to normal values. This pattern is significantly different from the hypertrophic, senescent and low regenerative phenotype observed in mice treated with the high-glucose concentration solution. We conclude that, at least in the in vivo and in situ setup, the detrimental effects of hyperosmolality alone upon the exposed mesothelium are quite limited and fully reversible within a recovery period of 7 days.

A short review of experimental peritoneal sclerosis: from mice to men.

Peritoneal sclerosis has been induced in rodents in vivo by exposing the membrane to a variety of experimental interventions: asbestos, 0.1% chlorexidine, iron dextran, glucose degradation products, AGE deposits derived from uremia per se, sodium hypochlorite, lypopolysaccharide, low pH, pure water, silica or zymosan. With a few exceptions (pure water, chlorhexidine and low pH), the other substances mentioned operate setting out different degrees of oxidative stress. This short review describes several experimental interventions in rodents, aimed at acute exfoliation or long-term, sustained injury of the mesothelial monolayer performed by means of intraperitoneal injections of different oxidant agents. Acute exfoliation induced by deoxycholate resulted in a depopulated monolayer coincident with immediate alteration of the peritoneal permeability, evidenced by increased urea D/P ratio, higher glucose absorption rate, elevated albumin losses in the effluent and significant reduction of the ultrafiltration rate. In the long term (30 days), these manifestations of membrane failure persisted and coincided with substantial peritoneal sclerosis. Peritoneal sclerosis was also induced by IP injections of 0.125% trypsin and 6.6 mM/L solution of formaldehyde. Using the doughnut rat model of mesothelial regeneration, exposure to 4.25% glucose or 7.5% icodextrin solutions severely hampered repopulation of the monolayer, which was replaced by a thick sheet of fibrous tissue. It is concluded that peritoneal sclerosis derives mostly from sustained oxidative injury to the peritoneal membrane. Loss of the mesothelial monolayer is the first step in the chain of events leading to this complication.

Peritoneal Transport after Long-Term Exposure to Icodextrin in Rats

Background: Icodextrin, an effective osmotic substance that has been proposed as an alternative agent for peritoneal dialysis induces ultrafiltration over long dwells. This study examines the peritoneal transport after exposure to Icodextrin in rats. Methods: Animals were divided in 4 groups and injected daily for 30 days with Icodextrin 7.5 % (n = 14), Glucose 4.25 % (n = 19) or glucose 4.25% plus Icodextrin 7.5 % (n = 13). Rats of the control group (n = 15) were not exposed. A 4-hour permeability study was performed using glucose at days 1, 30 and 60. At days 2, 31 and 61 the same animals were injected with Icodextrin. Results: Slopes of effluent sodium at day 30 were significantly higher (p < 0.001) in the glucose (0.006 ± 0.016), Icodextrin (0.013 ± 0.014) and mixed groups (0.012 ± 0.017) than in the control group (–0.041 ± 0.021). Urea D/P ratio was not significantly different in the 4 groups. After 30 days, glucose effluent levels were significantly lower (p < 0.001) in the glucose (701 ± 278 mg/dl), Icodextrin (552 ± 209 mg/dl) and mixed groups (587 ± 344 mg/dl) than in control rats (1519 ± 413 mg/dl). Effluent protein (mg/l) in the mixed group (1,555 ± 357) was significantly higher (p < 0.001) than control (376 ± 33), glucose (1,015 ± 232) and Icodextrin (765 ± 75) groups at day 30. Conclusion: The long-term use of Icodextrin does not affect small molecule transport, but induces changes in the peritoneal protein excretion, especially when Icodextrin and glucose are injected together.