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♦ BackgroundThe problem of mesothelial cell injury derived from the use of peritoneal dialysis solutions has been explored deeply. Conversely, the eventual detrimental effects upon mesothelial cell regeneration have awaked less investigative efforts than those focused on injury.♦ ObjectiveTo evaluate in the in vivo and in situ rat “doughnut” model of mesothelial repopulation, the eventual effect of peritoneal lavage with Hank's Balanced Salt Solution (HBSS) as well as that of 4.25% glucose and 7.5% icodextrin dialysis solutions.♦ Experimental Animals100 Sprague–Dawley albino rats were included in the study. Animals were divided into five groups of 20 rats each: group 1: control at zero time; group 2: sham-injected rats; group 3: rats exposed to HBSS; group 4: rats treated with 4.25% glucose peritoneal dialysis solution; group 5: rats injected with 7.5% icodextrin.♦ MethodsSelective exfoliation of a ring of mesothelium (width 0.8 mm, diameter 4 mm) covering the anterior surface of the liver was performed in 80 animals. The control zero-time group was used to evaluate the normal density distribution of the mesothelial cells forming the monolayer. The other groups were treated by means of daily sham injections or intraperitoneal infusion of each experimental solution for a period of 30 consecutive days. After a recovery period of 15 days, imprints and biopsies from the monolayer covering the exfoliated area were taken and processed for light microscopy.♦ ResultsMacroscopic observation of the abdominal cavity at the end of the 15-day recovery period showed that the prevalence of fibrotic adhesions between the peritoneal exfoliated area and the neighboring diaphragm was 10% for the sham-injected group, 5% for the HBSS-exposed animals, 85% for the rats injected with 4.25% glucose, and 95% for the icodextrin-treated group. Prevalence of fibrous adhesions in sham-injected animals and rats exposed to HBSS were devoid of statistically significant differences. Conversely, comparison of these groups with results observed in animals treated with the osmotic agents was significant, at the p < 0.0039 level. Regarding density distribution of mesothelial cells observed in imprints, there were no significant differences between the control zero-time and the sham-injected group. This parameter was marginally lower ( p < 0.05) in the HBSS-treated rats. Imprints were not taken from animals exposed to glucose or icodextrin because a dense layer of connective tissue replaced the exfoliated mesothelial area.♦ ConclusionsObservations made in this study support the contention that both osmotic agents, 4.25% glucose and 7.5% icodextrin, substantially restrain the normal process of mesothelial cell repopulation and induce repair by means of connective tissue. The underlying mechanism is most likely sustained oxidative stress.

We explored the acute and long-term effects of short-lived, intense oxidative stress on peritoneal permeability and structure, induced with intraperitoneal injection of the oxidant agent deoxycholate, in rats. Ten minutes after the experimental intervention, peritoneal dialysis, performed over an exposure time of 60 minutes, revealed an increased urea dialysate/plasma ratio, greater glucose absorption, increased albumin losses in the effluent dialysate, and a reduced ultrafiltration rate. Mesothelial-cell imprints taken from the anterior liver surface indicated a substantially decreased density in the cell population. After the recovery period of 30 days, all alterations were still evident. Additionally, macroscopic and histologic observations made at this time interval detected peritoneal fibrosis and sclerosis, characterized by peritoneal adhesions, wrapping of intestinal loops, and the presence of a layer of fibrous tissue dressing the cavitary aspect of the liver peritoneal envelope. This report describes a reproducible experimental model of peritoneal fibrosis induced by acute oxidative injury. On the basis of these findings, it may be speculated that functional and structural alterations observed in patients are related to long-term continuous exposure of the monolayer to oxidative injury resulting from the high concentrations of d-glucose present in peritoneal dialysis solutions.

This study evaluates albuminuria and peritoneal permeability to albumin in control and diabetic rats, as well as in diabetic animals treated with subcutaneously injected aminoguanidine hydrochloride (Ag) (5 mg/100 g/day), during a follow-up period of 6 months. Aminoguanidine effectively prevented albuminuria and albumin extravasation in the mesenteric interstitial tissue (control, 0.43 +/- 0.11 microg EB/100 g of dry tissue, Ag, 0.60 +/- 0.44; untreated diabetic animals, 1.22 +/- 0.73; control and Ag group vs untreated diabetic rats, P0.001). Albumin D/P ratio of the aminoguanidine-exposed rats (0.017 +/- 0.011) was higher than that of controls (0.008 +/- 0.002), but significantly lower (P0.001) than values observed in the untreated group of animals (0.046 +/- 0.003). Administration of aminoguanidine preserved both submesothelial and subendothelial electronegative charges. For capillary basement membrane (BM), control at zero time, 32 +/- 4 AS/microm BM; control at 6 months, 33.4; aminoguanidine-treated rats, 35 +/- 2. For submesothelial BM, control at zero time, 33 +/- 3; control at 6 months, 32 +/- 3; aminoguanidine-treated rats, 35 +/- 3. Splitting and thickening of both basement membranes were not prevented by the therapeutic intervention. We conclude that the shielding effect of aminoguanidine upon the permselectivity capabilities of the endothelial and mesothelial monolayers appears to be connected, basically to the preservation of anionic fixed charges.

Previous studies done in our model of population analysis of mesothelium (PAM) showed that longterm in vivo exposure of the mesothelial monolayer to glucose-enriched peritoneal dialysis (PD) solutions resulted in a substantial change in cell phenotype. Basically, the monolayer becomes composed of large and hypertrophic multi-nucleated cells, showing increased enzyme activity at the plasmalemmal, cytoplasmic, and nuclear compartments (1). In addition, cells show substantially reduced regenerative capabilities, as indicated by a low prevalence of mitosis, despite decreased cell viability. This shift from normal homeostasis results in a depopulated monolayer (Figure 1). These changes were linked to the concentration of glucose, as indicated by the dose-dependent effect, and were unrelated either to the low pH and high osmolarity of the solution, or to the presence of lactate or bicarbonate buffer. This view of the problem was confirmed in an additional study using a 4.25% glucose concentration in filter-sterilized fluid, prepared using the much more physiological and compatible Hanks Balanced Salt Solution (HBSS) (2). The new investigation corroborated the concept that sustained in vivo exposure of the monolayer to high concentrations of glucose resulted in a depopulated mesothelium composed mostly of senescent cells near the end of their life cycle. Senescence was defined on the basis of hypertrophy, multi-nucleation, low density of the population, substantially reduced prevalence of mitosis (Figure 2), and low viability (Figure 3), as well as on an increased number of cells in apoptosis. METHODS AND RESULTS

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.

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.

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.

Repopulation of the mesothelial monolayer after focal exfoliation, having the monolayer in vivo and in situ exposed to dialysis solutions, has not been thoroughly investigated. This study describes repopulation of a 'doughnut' like mesothelial ring exfoliated from the anterior liver surface of rats.Animals were divided into 5 groups of 20 rats each. Group 1 - control unexposed animals: mesothelial cell imprints were taken after 1 (5 rats), 5 (5 rats), and 15 (10 rats) days following the procedure of exfoliation. Group 2 - sham injected animals. Group 3 - rats IP injected once a day, during 30 consecutive days with Hank's balanced salt solution. Groups 4 and 5 - same experimental protocol, but injecting 4.25% glucose single bag or 7.5% Icodextrin PDF. Imprints and/or biopsies were taken after a recovery period of 15 days, counted from the last IP injection.Density distribution of mesothelial cells in group 2 was not significantly different from that seen in unexposed rats, whereas that seen in group 3 Hank's balanced salt solution was marginally but significantly lower (p0.05) from that seen in controls. Eighty five percent of rats injected with 4.25% glucose developed fibrous adhesions, binding up together the exfoliated liver surface and the diaphragmatic muscle. For Icodextrin treated rats, the prevalence of fibrous adhesions was 95%.The 'doughnut' experimental model appears as a promising tool for in vivo and in situ investigation of mesothelial repopulation. Both osmotic agents substantially restrain mesothelial repopulation, leading to repair by connective tissue.

Repopulation of the mesothelial monolayer after focal exfoliation, having the monolayer in vivo and in situ exposed to dialysis solutions, has not been thoroughly investigated. This study describes

The problem of mesothelial cell injury derived from the use of peritoneal dialysis solutions has been explored deeply. Conversely, the eventual detrimental effects upon mesothelial cell regeneration have awaked less investigative efforts than those focused on injury.To evaluate in the in vivo and in situ rat "doughnut" model of mesothelial repopulation, the eventual effect of peritoneal lavage with Hank's Balanced Salt Solution (HBSS) as well as that of 4.25% glucose and 7.5% icodextrin dialysis solutions.100 Sprague-Dawley albino rats were included in the study. Animals were divided into five groups of 20 rats each: group 1: control at zero time; group 2: sham-injected rats; group 3: rats exposed to HBSS; group 4: rats treated with 4.25% glucose peritoneal dialysis solution; group 5: rats injected with 7.5% icodextrin.Selective exfoliation of a ring of mesothelium (width 0.8 mm, diameter 4 mm) covering the anterior surface of the liver was performed in 80 animals. The control zero-time group was used to evaluate the normal density distribution of the mesothelial cells forming the monolayer. The other groups were treated by means of daily sham injections or intraperitoneal infusion of each experimental solution for a period of 30 consecutive days. After a recovery period of 15 days, imprints and biopsies from the monolayer covering the exfoliated area were taken and processed for light microscopy.Macroscopic observation of the abdominal cavity at the end of the 15-day recovery period showed that the prevalence of fibrotic adhesions between the peritoneal exfoliated area and the neighboring diaphragm was 10% forthe sham-injected group, 5% for the HBSS-exposed animals, 85% for the rats injected with 4.25% glucose, and 95% for the icodextrin-treated group. Prevalence of fibrous adhesions in sham-injected animals and rats exposed to HBSS were devoid of statistically significant differences. Conversely, comparison of these groups with results observed in animals treated with the osmotic agents was significant, at the p0.0039 level. Regarding density distribution of mesothelial cells observed in imprints, there were no significant differences between the control zero-time and the sham-injected group. This parameter was marginally lower (p0.05) in the HBSS-treated rats. Imprints were not taken from animals exposed to glucose or icodextrin because a dense layer of connective tissue replaced the exfoliated mesothelial area.Observations made in this study support the contention that both osmotic agents, 4.25% glucose and 7.5% icodextrin, substantially restrain the normal process of mesothelial cell repopulation and induce repair by means of connective tissue. The underlying mechanism is most likely sustained oxidative stress.

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.

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 rat's 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.

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.

High glucose accelerates the life cycle of the in vivo exposed mesothelium. Background Mouse mesothelium exposed in vivo for 30 days to high glucose solutions develop morphological changes that characterize a population of cells near the end of their life span. Methods The present study was designed to explore, in mesothelial cell imprints, whether these changes could derive from an early acceleration of the cell population life cycle in mice exposed for periods of up to 30 days to a 4.25% glucose fluid (236 mmol/L/L) prepared in Hank's balanced salt solution (HBSS). Three critical points of the cell's life cycle were evaluated: the G 1 checkpoint [proliferating cell nuclear antigen (PCNA) expression], DNA synthesis ( 3 H-thymidine incorporation), and the prevalence of mitosis. Results Cell populations exposed to a high glucose concentration showed an initial acceleration of their life cycle, as sustained by a peak of mitosis at two hours, an early increase of DNA incorporation sustained during the first 24 hours, as well as a top level of PCNA expression after three to four hours. These significantly higher values, compared with the control animals treated with HBSS, collapsed after 24 hours and were nil after 30 days of exposure. Conclusions Exposure to a high glucose concentration induced an early and short-lived acceleration of the mesothelial cell cycle, and with a longer exposure this was followed by a depletion of the growth capabilities of the exposed monolayer. "The cell, the mysterious protagonist of life, is hidden obstinately in the double invisibility of smallness and homogeneity." Recollections of my Life , by SANTIAGO RAMON Y CAJAL Translated by E. Horne Craigie in Memoires of the American Philosophical Society (Vol. VIII, part II), Philadelphia, 1937, pp 526–527

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.

Population analysis of mesothelium (PAM) done using the in vivo and almost in situ technique of mesothelial cell imprints revealed that lactate-buffered solutions had detrimental effects upon cell viability, that high glucose concentration affected cytokinesis, whereas the association of both components led to a decreased density population of cells showing a larger surface area. In the present study, PAM was done on mesothelium of mice exposed to bicarbonate-buffered peritoneal dialysis fluid (BBF) with glucose concentrations of 1.5 and 4.25%, for periods of time of 2 h, 15 and 30 days, as well as after recovery intervals of 7 and 30 days. BBF did not affect mesothelial cell viability. However, the increased incidence of multinucleated cells observed with both glucose concentrations, more marked with the 4.25% solution, suggests a detrimental effect upon the mechanism of cytokinesis. Furthermore, the higher the glucose concentration, the higher the mean-cell cytoplasmic surface area and the proportion of large cells, both resulting most probably from the regulatory volume increase developed by cells continuously exposed to hyperosmolar fluids. So far, evidence presented in this study suggests once more that BBF is remarkably more compatible with a higher quality of adaptation and survival of the exposed mesothelium than the lactated fluid. The question of whether the alterations induced by the high concentration of glucose result from a specific effect of glucose, by the coincidental hyperosmolarity, or by both still remains unanswered.