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 G₁ checkpoint [proliferating cell nuclear antigen (PCNA) expression], DNA synthesis (³H-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

METHODS

Animals

This study was done using 610 albino mice weighing 20 to 25 g. Animals were handled according to the National Institutes of Health Guidelines for Care of Laboratory Animals¹⁵. Mice were housed in plastic cages (Technicon, Bugugiatte, Italy) and maintained under a 12-hour light/dark cycle (light from 7 a.m. to 7 p.m.) and were fed with normal mouse chow (Maabarot, Israel) and water ad libitum.

Preparation of imprints for morphometry

The present investigation was designed on the basis of the in vivo model of population analysis of mesothelium². Imprints of the mesothelial cell monolayer were taken immediately after the animals were sacrificed by neck dislocation. After laparotomy, slides coated with 2.75% Agar (Sigma, Rejovot, Israel) were applied for 15 to 20 seconds to the anterior liver surface, peeling off the monolayer. Two imprints were taken from each animal, so that 20 slides for each experimental group could be made at each time interval.

Processing of imprints

Three critical moments in the life cycle of the mesothelial cell population were explored.

Proliferating cell nuclear antigen (PCNA) expression

This auxiliary 36 kD protein for DNA polymerase delta and epsilon, deeply involved in DNA replication and nucleotide excision repair, is required for cell cycle progression from the G₁ to the S phases¹⁶. It has been widely used as a marker of passage through the G1 checkpoint¹⁷ and positively correlates with the prevalence of mitotic activity.

Staining was performed with a kit using a biotinylated PCNA monoclonal antibody (clone PC 10) that can be used in cell samples of most species, including mouse (Streptavidin-Biotin System for PCNA staining; Zymed Lab Inc., San Francisco, CA, USA)¹⁷. For this purpose, mesothelial cell imprints taken as described earlier in this article, after being rinsed with phosphate-buffered saline (PBS; Biological Industries, Kibbutz Beit Ha'Emek, Israel), underwent fixation in 70% ethanol for 30 minutes at 4°C. Endogenous peroxidase activity was blocked with 3% H₂O₂ in methanol for 10 minutes, and then slides were rinsed in three changes of PBS for two minutes each. The next step was incubation of the monolayer with a solution containing the antibody (Biotinylated mouse anti-PCNA) for 30 minutes at room temperature. Slides were again rinsed three times with PBS for two minutes each and were covered with the streptavidin-peroxidase solution and incubated for 10 minutes at room temperature. Samples were rinsed once again three times with PBS for two minutes each and covered with the DAB reagent mixture (diaminobenzidine tetrahydrochloride, 0.6% hydrogen peroxide reagent and substrate buffer; Zymed Lab Inc.) for three to four minutes. After rinsing with distilled water, counterstain was carried out for two additional minutes with the hematoxylin reagent (Zymed Lab Inc.) and was rinsed with water, incubated in PBS for 30 minutes, and finally rinsed again with distilled water. Dehydration was performed in a graded series of ethanol (from 50 to 100%) and xylene (Frutarom, Haifa, Israel), and finally slide processing was completed with two drops of Histomount (Zymed Lab Inc.) and a cover slip.

Using this technique, nuclei in cells that are in the process of proliferating will stain brown, whereas cells in the resting state will not stain.

Autoradiography

The methodology used for this technique was based on that described by Baker¹⁸.

Mice had an intraperitoneal injection of tritriated thymidine (Thymidin methyl H3, TNC29B, 5 Ci/mmol; Rotem Industry Ltd., Beer Sheeva, Israel). The stock solution of ³H thymidine contained 48.4 g of the reagent per mL (1 mCi/mL). The working solution for intraperitoneal injection carrying 10 Ci/mL was prepared with 0.1 mL of the stock solution in 9.9 mL of HBSS (Sigma). Mice were intraperitoneally injected with 0.1 mL (1 Ci) per gram of body weight and diluted in 1.5 mL of HBSS through a 25-gauge hypodermic needle (Terumo Europe, Leuven, Belgium).

Mesothelial cell imprints were taken as already described. Slides were then rinsed twice with PBS, each time during three minutes, and fixed with a solution made up by Glacial-acetic acid (1 part; E. Merck. Darmstadt, Germany) and 100% ethanol (Frutaron, Haifa, Israel) for an additional 30 minutes.

For each experiment, background control slides were prepared for positive (slide without any tissue), negative chromography (slide with tissue exposed to light), and a slide with an imprint of the mesothelial monolayer, taken from a mouse exposed to a nontritriated thymidine dosage 100 times higher than that of ³H thymidine.

The emulsion for high-resolution microautoradiography was prepared as follows. In the darkroom, the LM1 emulsion for high-resolution microautoradiography (Amersham International, Amersham, UK) was incubated in a water bath at 43 to 45°C for 30 minutes. The experimental slides were covered with emulsion and allowed to gel for 30 minutes. After this step, slides were kept in light-proof plastic cages containing silica gel at 4°C until development (30 days). This part of the processing was done using the D-19 Kodak developer (Eastman Kodak, Rochester, NY, USA). After fixation with Kodak fixer P-6557, slides were rinsed again with distilled water, exposed to Kodak hypo-clearing agent (Eastman Kodak), and finally rinsed with distilled water for five minutes.

Immediately, imprints were stained with a 2.5% solution of Pyronine-B (Sigma) for 30 seconds, rinsed with distilled water, dehydrated with acetone (Frutarom) for one minute, cleared with xylene (Frutarom), and covered with a cover slip. After development, particles of metallic silver liberated in the emulsion by radiation were seen as black points or microgranules. These particles were not observed in cells that did not incorporate tritriated thymidine.

Prevalence of mitosis (or mitotic index) and of apoptotic cells

For this purpose, samples of the monolayer were fixed with 70% ethanol and were stained with hematoxylin eosin (Sigma) and 1% Pyronine-B (Sigma).

Morphometric techniques

All data concerning morphometric information were obtained using the WScan Array 2/3 Image Analysis (Galai Production Ltd., Migdal Ha Emek, Israel) connected to a Zeiss standard microscope (Carl Zeiss, Oberkochen, Germany) by means of a Sony Hyper Had-Iris-RGB color camera, model DXC-151AP (Sony Electronics, Tokyo, Japan).

Two mesothelial imprints were taken from each experimental animal. The quality of exfoliated monolayers was initially evaluated by scanning each slide at low magnification. The main goal was to detect and choose observation areas free from artifactual lack of cells, resulting from poor initial attachment and/or subsequent loss during the several steps of processing. Then imprints were observed at higher magnification and projected on the computer screen. Three to five 125274.70 m² surface area fields were chosen for evaluation of the different parameters by randomization¹⁹. Since each experimental group included 10 mice, the total number of examined fields for group at each time interval ranged between 60 and 76. The number of observed cells/field oscillated between 221 and 267, which makes from 13,274 to 19,746 cells for each group at every time interval.

Definitions of morphometric parameters

For autoradiography, the labeling index (prevalence of cells incorporating ³H-thymidine)²⁰ is presented as a percentage of the total number of observed cells. Growth fraction represents the sequential level of labeling index, after reaching the plateau value²¹. Area fraction gives the ratio of the object area, in this case, area labeled by tritriated thymidine, related to the box area containing the sample. This parameter offers information about both the prevalence of stained cells as well as the extension of labeling. The integrated optical density (IOD)/area parameter gives the integrated optical density of the labeling as it is related to the nuclear area occupied by the radioactive tracer.

Proliferating cell nuclear antigen expression was quantitated as the prevalence of stained cells observed in each experimental group at every time interval, whereas that of mitosis (mitotic index) was also estimated as the percentage of mitotic cells in specimens taken at specific determined periods, as shown in the paragraph describing the experimental groups. Apoptotic cells were morphologically defined on the basis of retracted cytoplasm, condensed nuclear chromatine forming round or oval dark structures, and followed by the subsequent formation of multiple fragments of condensed nuclear material and cytoplasm. These changes concerned isolated cells without any manifestation of local inflammatory reaction²². The methodology followed for counts was the same used for autoradiography (area of microscopic fields and number of defined objects).

Experimental groups

The prevalence of mitosis in intact unexposed animals and after one intraperitoneal injection of HBSS or the 4.25% glucose solution was evaluated at 30 and 60 minutes, and 2, 3, 4 and 24 hours. Ten mice were included in each time interval for every experimental group: intact (10 mice), HBSS (60 mice), and 4.25% glucose (60 mice).

An additional experiment was designed to investigate the acute effect of one intraperitoneal injection on the prevalence of mitosis, observed after reiterated exposure of the mesothelium to the high glucose concentration solution once a day, during a follow-up period of six days. For this purpose, 10 animals were chosen as the unexposed control group, whereas 60 mice were used in the experimental group. Ten animals were sacrificed each day, during six consecutive days, two hours after being exposed to the experimental solution. Mesothelial imprints were taken, processed, and examined as already described.

The prevalence of apoptotic cells was evaluated in five additional groups of 10 mice each: intact unexposed mice (1 group), animals treated with one intraperitoneal injection of HBSS (2 groups), and mice injected with the high glucose concentration fluid (2 groups). In the four groups of injected animals, imprints were taken after two and three hours of exposure.

For autoradiographic studies, mice were divided in three groups.

Sham-injected animals (30 mice)

Mesothelial cell imprints were obtained 24 hours after one sham (needle effect) intraperitoneal injection in 10 mice and after 15 and 30 days of repeating the procedure in two other groups of 10 animals each.

Animals exposed to HBSS (80 mice)

In this group, imprints were taken 30, 60, 120, 180, 240, and 1440 minutes (24 hours) after one intraperitoneal injection of the fluid, as well as after 15 and 30 days of repeating the procedure in two other groups of 10 animals each. In these last two groups, samples were taken two hours after the last injection.

Animals exposed to 4.25% glucose fluid (80 mice)

These animals were treated using the same experimental protocol used for those exposed to the glucose-free HBSS except that they were injected with the glucose-enriched fluid.

Proliferating cell nuclear antigen expression was investigated in three additional groups of mice: intact unexposed controls (10 mice), mice exposed to HBSS (80 mice), and the group of mice treated with the high glucose concentration solution (80 mice).

The experimental protocol and schedule used in this part of the study were the same applied to investigate the incorporation of tritriated thymidine.

Statistical methods

Data are presented as mean SD. Analysis of data presented in the nominal scale (prevalence and percentages) was done using the two-tailed Fisher's exact test, applying the criteria of the minimal required sample size (0.95 power), calculated for = error (type I and type II error)²³. ( error: finding a statistically significant difference when analyzing rqo populations of data that are actually identical; error: inferring that both populations are not significantly different when, from a statistical point of view, we cannot find an actual significant difference). Means of data measured on an interval scale were initially compared with the one-way analysis of variance. The level of significance was fixed at P < 0.05. As a second step, the appropriate sample size was calculated on the basis of power = 0.95²³, calculated for = error. Experimental groups showing a number of cases within the limits of power = 0.95 were compared with the nonparametric Dunnet test for multiple comparisons against a single control²⁴. This statistical approach was chosen because the frequency distribution of data, concerning the parameter IOD/area obtained from mice exposed to the high glucose solution, failed to pass the Kolmogorov-Smirnov normality test^25,26.

RESULTS

Exposure of the mesothelial monolayer to the high glucose concentration solution (4.25% equivalent to 236 mmol/L) induced an initial marked increase in the prevalence of mitosis Figure 1, with a short-lived maximal peak observed two hours after the intraperitoneal injection. This acute acceleration of the mechanisms activating the mitotic process was most probably affected by cells that had already completed the phase of synthesis (end of S phase or G₂) before being exposed to high glucose. Indeed, Figure 2 shows that around 85% of mitotic cells were in prophase and metaphase. This stimulation, however, lasted for a short period of time. After a four-hour exposure, the prevalence of cells in mitosis dropped to values not significantly different from those observed in intact unexposed mice, as well as in those treated with the HBSS. This more modest mitotic activity was also observed 24 hours after the intraperitoneal injection of the high-glucose concentration fluid Figure 1.

Figure 1.

A sequential comparison of prevalence of mitosis (mitotic index) observed in two experimental groups of mice, treated with one injection of Hank's balanced salt solution (HBSS) and the high glucose concentration fluid. Peaks of mitosis observed at two and three hours after the intraperitoneal injection of the glucose-enriched solution are significantly higher than the corresponding values showed by the HBSS control group, as well as values seen in the control group of mice at zero time. Notice that 24 hours after the injection, the glucose-induced stimulation vanished (*P < 0.05; ***P < 0.001). Symbols are: () intact; () HBSS; () 4.25% glucose.

Full figure and legend (7K)

Figure 2.

Stages of mitosis after two hours of exposure to high glucose (N = 161 mitotic cells). Around 85% of the observed cells in mitosis in mice exposed to high glucose concentration for two hours were in prophase.

Full figure and legend (14K)

Data obtained from mice that were injected once a day during six consecutive days with the glucose-enriched solution revealed that the initial and short-lived stimulation of mitotic activity lasted for only the first three days of exposure. Then it collapsed by the fourth day and reached values near zero after six days of repeating the experimental intervention Figure 3.

Figure 3.

Prevalence of mitosis after one daily injection for six consecutive days. Intact () mice showed no change, and 4.25% glucose () mice showed a stimulation of the mitotic process that lasted for only three days that appears to be exhausted by the fourth day of exposure (***P < 0.001).

Full figure and legend (8K)

The experiment evaluated the prevalence of cells incorporating tritriated thymidine points at an additional aspect of the early accelerating effect of high-glucose concentration on the cell cycle. As shown in Figure 4, cells that should have been in the G₁ phase before the injection had a significant increase in the prevalence of those entering into S phase as early as after 30 minutes of exposure, compared with cell numbers obtained from mice treated with HBSS Figure 4. This peak activity was sustained for the first 24 hours. The plateau indicating the growth fraction level was reached at three hours in the glucose-treated animals (around 10% of cells incorporating ³H thymidine), whereas in the intact group, this value was observed between 4 and 24 hours of exposure Figure 4.

Figure 4.

Percentages of ³H thymidine-labeled cells (labeling index) observed sequentially in the sham () and two experimental groups [()HBSS and () high-glucose–exposed mice] for intervals up to 30 days. Notice the early peak seen in the high-glucose group that reached a plateau (growth fraction) after two to four hours of exposure, whereas in the HBSS-treated animals, the plateau appeared between 4 and 24 hours. After 30 days, the labeling index detected in mice injected with the glucose-enriched fluid was nil and significantly lower than the corresponding value traced in the HBSS group (***P < 0.001).

Full figure and legend (9K)

Data obtained after evaluation of area of cells incorporating tritriated thymidine, expressed as a fraction of the total examined area (parameter area fraction at 2, 3 and 4 h), confirmed the information given by cell counts Figure 5.

Figure 5.

Frequency distribution of the area fraction parameter. A plot of the data clearly separates the group exposed to the high glucose concentration for four hours from the other experimental mice (***P < 0.001). Failure of data to pass the Kolmogorov-Smirnov normality test (P < 0.001) indicates that the frequency distribution of all groups significantly varied from the pattern given by the numbers obtained from a normally distributed population of data. This manifestation reflects the expected and characteristic heterogeneity of a cell population reacting, in vivo and in situ, to the experimental intervention. In other words, at variance with the synchronized standpoint peculiar of the in vitro setup, a cell population in vivo covers a whole range of stages within each individual cell life cycle. Symbols are: () HBSS after 2 to 3 and 4 hours; () 4.25% glucose after 2 hours; () 4.25% glucose after 3 hours; () 4.25% glucose after 4 hours.

Full figure and legend (20K)

The prevalence of cells incorporating tritriated thymidine in the glucose-exposed mesothelial population, after 15 days of exposure, was significantly lower than that seen in those taken from mice treated with sham injections or by means of HBSS, whereas after 30 days, the proportion of cells in the S phase was near zero Figure 4. It should be noted that the prevalence of cells incorporating thymidine in both control groups (sham and HBSS) was not significantly different from each other after 1, 15, and 30 daily injections Figure 4.

At any given time, cells incorporating tritriated thymidine showed a whole range of labeling by the radionuclide Figure 5. This pattern derives from the fact that in the in vivo situation, the population of cells is heterogeneous regarding the specific moment of the cell cycle at which they were put in contact with the tracer. This characteristic is at variance with that seen in cultured, synchronized cells where, by definition, most cells (around 80% in the first cycle) are blocked and therefore accumulate at a specific stage of their cell cycle²⁷. Consequently, we evaluated the intensity of labeling for each group at every time interval using the integrated optical density (IOD). As shown in Figure 6, the highest IOD values observed in the HBSS–treated groups were below 2.5 units, whereas in imprints taken from mice exposed to the glucose-enriched solution, the values for three and four hours were higher (for the four-hour group reaching a level of significance, P < 0.001; Figure 6), suggesting a shortened and accelerated phase of synthesis. Thus, not only the prevalence, but also the intensity of staining were markedly enhanced by the high concentration of glucose.

Figure 6.

The integrated optical density (IOD)/area frequency parameter was significantly higher in mice treated with high glucose at four-hour intervals compared with the other experimental groups at each time interval (***P < 0.001). Symbols are: () HBSS after 2 to 3 and 4 hours; () 4.25% glucose after 2 hours; () 4.25% glucose after 3 hours; () 4.25% glucose after 4 hours.

Full figure and legend (24K)

Figure 7 summarizes our observations on PCNA expression, describing a significantly increased prevalence of cells passing through the G₁ checkpoint when under the influence of glucose in high concentration, compared with that detected in mice exposed to HBSS. This enhanced proliferative activity was short lived, reaching low levels 24 hours after one intraperitoneal injection, was quite moderate after 15 days of treatment, and exhausted at 30 days.

Figure 7.

Percent of PCNA stained cells. PCNA expression sequentially observed in mice exposed to HBSS is compared to those treated with the high glucose concentration fluid. The proportion of cells positively reacting to the immunocytochemical staining was significantly higher (P < 0.001) at the two-, three-, and four-hour time intervals, reaching values near zero after 30 intraperitoneal injections (P < 0.01 compared with the corresponding values evaluated in mice exposed to HBSS). Symbols are: () HBSS; () 4.25% glucose; () intact.

Full figure and legend (9K)

The prevalence of apoptotic cells in unexposed mice was around 1% (1 0). Values seen in both groups treated with HBSS were not far from that observed in intact cells (1 0% at 2 hours and 1 0 after 3 hours). In turn, mice injected with the high glucose concentration fluid showed a substantially increased prevalence of apoptosis at both time intervals (3 1% and 4 1% at 2 and 3 hours, respectively, P < 0.001).

DISCUSSION

The three evaluated parameters of cell proliferation, PCNA expression, thymidine incorporation and mitotic index, show information dealing with three different moments in the life cycle of the mesothelial population: the G₁, S, and M phases. They coincide, indicating that an early and short-lived acceleration of the cell cycle with a longer exposure to the high glucose concentration was followed by a depletion of the growth capabilities of the exposed monolayer. In other words, cells consumed all of their proliferative potential during the period of early acceleration. The significantly higher IOD detected in cells exposed during four hours to the high-glucose solution is a further argument pointing at the early acceleration of the mean cell cycle induced by glucose. The end result of this change cannot be other than that of a prematurely senescent, low-density population of large, multinucleated and nonproliferating cells, unable to respond to mitogenic stimulation²⁸. According to the data obtained from the autoradiographic studies, it appears that the proliferative potential of mesothelial cell populations obtained from intact unexposed mice was limited to three or four rounds of mitosis during their life span Figure 2. This sequence is graphically depicted in Figure 8, which also offers our interpretation of the phenomenon of the glucose-induced life cycle acceleration.

Figure 8.

Our interpretation of the glucose-induced changes on the life cycle of the living and in situ-exposed mesothelial cell population. Normally, cells undergo two to three rounds of mitosis (upper part of the graphic) before reaching terminal differentiation and death in apoptosis. Early stimulation derived from exposure to a high concentration of glucose (lower part) induces acceleration of the cell cycle, multinucleation, accelerated senescence, and early activation of the mechanisms leading to apoptosis.

Full figure and legend (39K)

This situation, already observed in a variety of cultured cells exposed to a high concentration of glucose^{5,11,12,13,29}, the only outcome of which is an early cell death, has also been described in a mesothelial monolayer that was depopulated after long-term in situ and in vivo exposure to a high concentration of the osmotic agent^2,3,10. This point was heralded by the loss of cell junctions and microvilli already detected in human patients on long-term peritoneal dialysis^30,31, a clear sign of impending apoptosis³². The present study, to our knowledge, is the first done in a cell population exposed in vivo to a high concentration of glucose, and shows the early steps of the process leading to accelerated cell death.

Selected genes, hormones, growth factors, and cytokines regulate the cell cycle^33,34,35,36. The number of cells per unit of surface area in populations obtained from mammalian tissues is maintained by growth-stimulating and growth-inhibiting mechanisms, which are inhibitory signals superimposed over growth stimuli. Within this context, homeostasis is the net result of a highly balanced network of growth-stimulating and growth-inhibitory signals³⁷, including the still nebulous phenomenon of contact inhibition³⁸. The observations reported in the present study suggest that in vivo exposure of the mesothelial monolayer to a high glucose concentration disrupts this delicate homeostatic equilibrium, after 30 days resulting in a population of senescent cells²⁸ that are blocked in the G1 and G2/M phases of development³⁹. This point is revealed by the absence, at that time, of the stimulation initially induced by the same high concentration of glucose, since the mitotic index is extremely low, as well as the incorporation of thymidine and the expression of PCNA⁴⁰.

Which could be the mechanism(s) behind this early stimulation induced by a high glucose concentration? Several groups of investigators have demonstrated that low levels of oxidants potentiate growth signals and stimulate proliferation of different cell types. However, higher concentrations and longer exposure to the same compounds can block cell growth, consequently inducing both premature senescence and activation of the mechanisms leading to cell death in apoptosis. The prime mover behind this phenomenon can be explained on the basis of the response to DNA damage, derived from a progressive shortening of telomeres present at the end of chromosomes occurring at each mitosis. This process, known as the end-replication problem⁴¹, is considered one of the basic mechanisms of cellular aging and death³⁹, and is ascribed to oxidative damage to the telomere DNA⁴².

It has been shown that intact, cultured mesothelial cells acutely exposed to a high glucose concentration (24 mmol/L) manifest a significantly increased liberation of hydrogen peroxide⁴³. Furthermore, in high concentration (24 mmol/L) D-glucose induced a substantial impairment of hydrogen peroxide degradation by human endothelial and mesothelial cells. However, this phenomenon was not detected when both types of cells were incubated in equimolar concentrations of L-glucose and raffinose^44,45.

As stated in a previous study in our laboratory¹⁰, it may well be hypothesized that D-glucose metabolism and the subsequent increased generation, coupled to the already described reduced degradation of reactive oxygen species in chronic uremia as well as in continuous ambulatory peritoneal dialysis (CAPD) patients^46,47, are at the origin of the altered life cycle of the exposed mesothelial population detected in our experimental animals. This speculation, however, remains to be tested by additional experimental research. If proven correct, the entire approach for new formulations of biocompatible dialysis solutions will change dramatically.

Most of the available information dealing with the topic of biocompatibility of peritoneal dialysis solutions was obtained from in vitro studies in which cultured, synchronized mesothelial cells were acutely exposed for short periods of time to different experimental solutions^48,49. All of the aforementioned studies coincided in ascribing substantial acute cytotoxic effects to glucose-based dialysis solutions, derived from low pH, lactated buffer, and/or high osmolar concentration. The present study, the last of a series of experiments done using the mesothelial cell population exposed in vivo for long periods of time to glucose-enriched fluid, offers a quite different view. Basically, hypertrophic, differentiated, senescent-like growth-arrested cells lay on a depopulated monolayer resulting from a substantial change of the cells' life cycle⁵⁰. These cells appear morphologically and probably functionally far distant from the unexposed, intact, cultured mesothelial cells, synchronized at the very beginning of their vital cycle. In this sense, observations made in the in vitro setup, using young and synchronized cells, acutely exposed to dialysis solutions, can hardly be extrapolated to the in vivo experimental and/or clinical situation of CAPD¹⁰.

References

1. Gotloib, L, Shostak, A, Wajsbrot, V & Kushnier, R: The cytochemical profile of visceral mesothelium under the influence of lactated-hyperosmolar peritoneal dialysis solutions. Nephron 1995 69:466–471,  | PubMed | ISI | ChemPort |
2. Gotloib, L, Wajsbrut, V, Shostak, A & Kushnier, R: Acute and long-term changes observed in imprints of mouse mesothelium exposed to glucose-enriched, lactated, buffered dialysis solutions. Nephron 1995 70:466–477,  | PubMed | ISI | ChemPort |
3. Gotloib, L & Shostak, A: Large mesothelial cells in peritoneal dialysis: A sign of degeneration or adaptation? Perit Dial Int 1996 16:118–120,  | PubMed | ISI | ChemPort |
4. Angello, JC, Pendergrass, WR, Norwood, TH & Prothero, J: Cell enlargement: One possible mechanism underlying cellular senescence. J Cell Physiol 1989 140:288–294,  | Article | PubMed | ISI | ChemPort |
5. Morocutti, A, Earle, KA, Sethi, M, Piras, G, Pal, K, Richards, D, Rodeman, P & Viberti, G: Premature senescence of skin fibroblasts from insulin-dependent diabetic patients with kidney disease. Kidney Int 1996 50:250–256,  | PubMed | ISI | ChemPort |
6. Gotloib, L, Wajsbrot, V, Shostak, A & Kushnier, R: Population analysis of mesothelium in situ and in vivo exposed to bicarbonate-buffered peritoneal dialysis fluid. Nephron 1996 73:219–227,  | PubMed | ISI | ChemPort |
7. Wajsbrot, V, Shostak, A, Gotloib, L & Kushnier, R: Biocompatibility of a glucose-free, acidic lactated solution for peritoneal dialysis evaluated by population analysis of mesothelium. Nephron 1998 79:322–332,  | Article | PubMed | ISI | ChemPort |
8. Gotloib, L, Wajsbrot, V, Shostak, A & Kushnier, R: Effect of hyperosmolarity upon the mesothelial monolayer exposed in-vivo and in-situ to a mannitol enriched solution. Nephron 1999 81:301–309,  | Article | PubMed | ISI | ChemPort |
9. Gotloib, L, Shostak, A & Wajsbrot, V: Detrimental effects of peritoneal dialysis solutions upon in vivo and in situ exposed mesothelium. Perit Dial Int 1997 17(Suppl 2):S13–S16,  | PubMed | ISI |
10. Gotloib, L, Shostak, A, Wajsbrot, V & Kushnier, R: High glucose induces an hypertrophic, senescent mesothelial cell phenotype after long, in-vivo exposure. Nephron 1999 82:164–167,  | Article | PubMed | ISI | ChemPort |
11. Lorenzi, M, Cagliero, E & Toledo, S: Glucose toxicity for human endothelial cells in culture: Delayed replication, disturbed cell cycle, and accelerated death. Diabetes 1985 34:621–627,  | PubMed | ISI | ChemPort |
12. Sibbit, WlJr, Mills, RG, Bibler, CF, Eaton, RP, Griffey, RH & Vander-Jagt, DL: Glucose inhibition of human fibroblast proliferation and response to growth factors is prevented by inhibitors of aldose reductase. Mech Ageing Dev 1989 47:265–279,  | PubMed |
13. Ziyadeh, FN, Snipes, ER, Watanabe, M, Alvarez, RJ, Goldfarb, S & Haverty, TP: High glucose induces cell hypertrophy and stimulates collagen gene transcription in proximal tubule. Am J Physiol 1990 259:F704–F714,  | PubMed | ISI | ChemPort |
14. Shostak, A, Pivnik, K & Gotloib, L: Daily short exposure of cultured mesothelial cells to lactated, high-glucose, low pH peritoneal dialysis fluid induces a low-profile regenerative steady state. Nephrol Dial Transplant 1996 11:608–612,  | PubMed | ISI | ChemPort |
15. Institute of Laboratory Animal Resources:Guide for the Care and Use of Laboratory Animals [DHEW Publications no. (NIH) 72-73]. 1972 Washington D.C., National Institutes of Health,
16. Prosperi, E: Multiple roles of the proliferating cell nuclear antigen: DNA replication, repair and cell cycle control. Prog Cell Cycle Res 1997 3:193–210,  | PubMed | ChemPort |
17. Bravo, R, Frank, R, Blundell, PA & McDonald-Bravo, H: Cyclin/PCNA is the auxiliary protein of DNA polymerase-delta. Nature 1987 326:515–517,  | Article | PubMed | ISI | ChemPort |
18. Baker, JRJ:Autoradiography: A Comprehensive Review. 1989, New York, Oxford University Press, Royal Microscopic Society, pp 17–34
19. Rohlf, FJ & Sokal, RR:Statistical Tables. 1981, San Francisco, W.H. Freeman and Company, pp 71–75
20. Westermark, B: The deficient density-dependent growth control of human malignant glioma cells and virus-transformed glia-like cells in culture. Int J Cancer 1973 12:438–451,  | PubMed | ISI | ChemPort |
21. Freshney, RI:Culture of Animal Cells (1987, 2nd ed). New York, J Wiley and Sons, Inc., pp 241–244
22. Van de Schepop, HAM, De Jong, JS, Van Diest, PJ & Baak, JPA: Counting of apoptotic cells: A methodological study in invasive breast cancer. J Clin Pathol Mol Pathol 1996 49:M214–M217,
23. Glantz, SA:Primer of Biostatistics. 1992, New York, McGraw-Hill, pp 155–187
24. Glantz, SA:Primer of Biostatistics. 1992, New York, McGraw-Hill, pp 91–109
25. Pascali, VL, Moscetti, A, Dobosz, M, Pescarmona, M & D'aloja, E: Errors in sizing bands of hypervariable DNA profiles on autoradiograms: Are they Gaussian? Electrophoresis 1992 13:341–345,  | Article | PubMed | ISI | ChemPort |
26. Weil, CS: Statistical analysis and normality of selected hematologic and clinical chemistry measurements used in toxicologic studies. Arch Toxicol (1982 Suppl 5):237–253,
27. Freshney, RI: Culture of Animal Cells (1990, 2nd ed). New York, Wiley-Liss, p 324
28. Cristofalo, VJ, Gerhard, GS & Pignolo, RJ: Molecular biology of aging. Surg Clin North Am 1994 74:1–21,  | PubMed | ISI | ChemPort |
29. Stout, RW: Glucose inhibits replication of cultured human endothelial cells. Diabetologia 1982 23:436–439,  | PubMed | ISI | ChemPort |
30. Di Paolo, N, Sacchi, G, De Mia, M, Gaggiotti, E, Capotondo, L, Rossi, P, Bernini, M, Pucci, AM, Ibba, L & Sabatelli, P: Morphology of the peritoneal membrane during continuous ambulatory peritoneal dialysis. Nephron 1986 44:204–211,  | PubMed | ChemPort |
31. Gotloib, L, Shostak, A, Bar Sella, P & Kohan, R: Continuous mesothelial injury and regeneration during long-term peritoneal dialysis. Perit Dial Bull 1987 7–3:148–155,
32. Bowen, ID, Mullarkey, K & Morgan, SM: Programmed cell death during metamorphosis in the blow-fly Calliphora vomitoria. Microsc Res Tech 1996 34:202–217,  | Article | PubMed | ISI | ChemPort |
33. Khaoustov, VI, Ozer, A, Smith, JR, Noda, A, Mearns, M, Krishnan, B, Slagle, BL & Yoffe, B: Induction of senescent cell-derived inhibitor of DNA synthesis gene, SDII, in hepatoblastoma (HepG2) cells arrested in the G2-phase of the cell cycle by 9-nitocramptothecin. Lab Invest 1995 73:118–127,  | PubMed | ISI | ChemPort |
34. Cox, LS: Multiple pathways control cell growth and transformation: Overlapping and independent activities of p53 and p21Cip1/WAF1/Sdi1. J Pathol 1997 183:134–140,  | Article | PubMed | ISI | ChemPort |
35. Shibanuma, M, Mochizuki, E, Maniwa, R, Mashimo, J, Nishiya, N, Imai, S, Takano, T, Oshimura, M & Nose, K: Induction of senescence-like phenotypes by forced expression of hic-5, which encodes a novel LIM motif protein, in immortalized human fibroblasts. Mol Cell Biol 1997 17:1224–1235,  | PubMed | ISI | ChemPort |
36. Gallimore, PH, Lecane, PS, Hann, B & Grand, RJ: Differentiation is inhibited and a senescence pathway is activated when simian virus 40 tsA 58-transformed human retinoblasts are grown at the restrictive temperature. Cell Growth Differ 1997 8:763–771,  | PubMed | ISI | ChemPort |
37. Gradl, G, Faust, D, Oesch, F & Weiser, RJ: Density-dependent regulation of cell growth by contactinhibin and the contactinhibin receptor. Curr Biol 1995 5:526–535,  | Article | PubMed | ISI | ChemPort |
38. Von-Zglinicki, T & Saretzki, G: Molecular mechanisms of senescence in cell culture. Z Gerontol Geriatr 1997 30:24–28,  | PubMed | ChemPort |
39. Vaziri, H: Critical telomere shortening regulated by the ataxia-telangiectasia gene acts as a DNA damage signal leading to activation of p53 protein and limited life-span of human diploid fibroblasts: A review. Biochemistry (Mosc) 1997 62:1306–1310,  | PubMed | ISI | ChemPort |
40. Chang, CD, Phillips, P, Lipson, KE, Cristofalo, VJ & Baserga, R: Senescent human fibroblasts have a post-transcriptional block in the expression of the proliferating cell nuclear antigen gene. J Biol Chem 1991 266:8663–8666,  | PubMed | ISI | ChemPort |
41. Olovnikov, AM: Telomeres, telomerase, and aging: Origin of the theory. Exp Gerontol 1996 31:443–448,  | Article | PubMed | ISI | ChemPort |
42. Furumoto, K, Inoue, E, Nagao, N, Hiyama, E & Miwa, N: Age dependent telomere shortening is slowed down by enrichment of intracellular vitamin C via suppression of oxidative stress. Life Sci 1998 63:935–948,  | Article | PubMed | ISI | ChemPort |
43. Shostak, A, Pivnik, E & Gotloib, L: Cultured rat mesothelial cells generate hydrogen peroxide: A new player in peritoneal defense? J Am Soc Nephrol 1996 7:2371–2378,  | PubMed | ISI | ChemPort |
44. Kashiwagi, A, Asahina, T, Ikebuchi, M, Tanaka, Y, Takagi, Y, Nishio, Y, Kikkawa, R & Shigeta, Y: Abnormal glutathione metabolism and increased cytotoxicity caused by H2O2 in human umbilical vein endothelial cells cultured in high glucose medium. Diabetologia 1994 37:264–269,  | PubMed | ISI | ChemPort |
45. Breborowicz, A, Witowski, J, Wieczorowska, K, Martis, L, Serkes, KD & Oreopoulos, DG: Toxicity of free radicals to mesothelial cells and peritoneal membrane. Nephron 1993 65:62–66,  | PubMed | ISI | ChemPort |
46. Shainkin-Kestenbaum, R, Giatt, Y & Berlyne, GM: The presence and toxicity of guamidinopropionic acid in uremia. Kidney Int 1975 7:302–305,
47. Taccone-Gallucci, M, Giardini, O, Lubrano, R, Mazzarella, V, Bandino, D, Khashan, S, Mannarino, O, Elli, M, Cozzari, M & Buoncristiani, U: Red blood cell membrane lipid peroxidation in continuous ambulatory peritoneal dialysis patients. Am J Nephrol 1986 6:92–95,  | PubMed | ChemPort |
48. Breborowicz, A, Rodela, H & Oreopoulos, DG: Toxicity of osmotic solutes on human mesothelial cells in vitro. Kidney Int 1992 41:1280–1285,  | PubMed | ISI | ChemPort |
49. Topley, N, Kaur, D, Petersen, MM, Jorres, A, Passlick-Deetjen, J, Coles, GA & Williams, JD: Biocompatibility of bicarbonate buffered peritoneal dialysis fluids: Influence on mesothelial cell and neutrophil function. Kidney Int 1996 49:1447–1456,  | PubMed | ISI | ChemPort |
50. Chen, Q & Ames, BN: Senescence-like growth arrest induced by hydrogen peroxide in human diploid fibroblast F65 cells. Proc Natl Acad Sci USA 1994 91:4130–4134,  | PubMed | ChemPort |