1 Nefrologia Dialisi e Trapianto, Ospedale Regina Margherita, Torino, 2 Nefrologia e Dialisi, Azienda Ospedaliera Villa Scassi, Genova and 3 Clinica Pediatrica, Centro Medicina Nucleare Infantile, Università di Torino, Italy
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Abstract |
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Methods. Apoptosis was evaluated by terminal 3' uridine labelling. Key proteins involved in the apoptotic pathway were investigated by reverse transcription polymerase chain reaction (RTPCR) mRNA expression and immunoperoxidase staining (caspase 9, tumour suppressor protein p53, inducible cyclooxygenase COX-2).
Results. The apoptotic effects of MGly and For were dose and time dependent. GPDs at concentrations detected in TPD induced greater transcription and translation of apoptotic pathway proteins (caspase 9, p53 and COX-2) than GPDs in 3CB. This resulted in a higher apoptotic rate, which was not influenced by addition of sterile glucose. A similar enhancement of apoptosis was detected when mesothelial cells were incubated with TPD, whereas incubation in 3CB PDS resulted in less enhanced apoptosis. The 12 h incubation effect of PDS on cultured mesothelial cells was not related to advanced glycosylated end-product formation.
Conclusions. As the rate of mesothelial cell apoptosis is lower in 3CB than in TPD solutions, the 3CB appears to provide improved biocompatibility.
Keywords: peritoneal dialysis; biocompatibility; glucose degradation products; apoptosis; peritoneal fibrosis
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Introduction |
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In order to reduce GDP formation, a new three-compartment bag (3CB) has recently been conceived and developed [6]. The system allows maintenance of glucose at high concentrations and low pH, and is free from substances that catalyse the degradation of glucose during heat sterilization and storage. By mixing the compartments, the system enables patients to prepare different glucose concentrations using the same bag, obtaining a peritoneal solution that has a pH above 6 and is low in GDP levels [7].
Wieslander et al. [7] reported that concentrations of various GDPs in traditional peritoneal dialysis solutions (TPD) were much higher than in PDS obtained by separating glucose from other peritoneal dialysis components during heat sterilization and storage, as is done using the 3CB [7]. An Italian multi-centre study reported increases in CA125 in effluents using PDS obtained from the 3CB system. As Ca125 is a marker of mesothelial cell mass, the data indicate that this PDS may have better biocompatibility than conventional solutions [8].
A loss of viable mesothelial cells without signs of inflammation is the characteristic pathologic feature of peritoneal membrane use in long-term dialysed patients, and supports the hypothesis of apoptotic death over that of necrosis.
Apoptosis has recently been implicated in progressive scarring that is due to various pathologic events. Programmed cell death is comprised of an active complex machinery requiring gene transcription and translation, and is triggered by different stimuli to monotonically result in activation of specific endonucleases leading to DNA fragmentation [9]. Apoptotic cells promptly undergo phagocytosis by professional and non-professional phagocytic cells, expressing vß3 integrins that are able to recognize the phosphatidylserine residues expressed on cell surfaces during apoptotic death [9]. Apoptosis is characterized by an altered mithocondrial function leading to an abnormal intracellular redox state [8]. Oxidants are strong apoptotic inductors [10] and act by stimulating the neo-transcription of the tumour suppressor protein, p53 [9].
Carbonyl compounds are formed in uraemia as a result of enhanced oxidative stress caused by uraemic toxin. Moreover, carbonyl compounds amplify oxidant damage in a vicious circle to elicit a series of events resulting in the activation of p53 tumour suppressor protein, of the caspase system, and finally producing internucleosomal DNA breaks that are characteristic of apoptotic death.
In recent papers, GDPs incubated with human mesothelial cells induced significant depression of cell growth, viability and function [11,12]. From these observations, we speculated that the 3CB, having lower detectable amounts of GDPs [7], may be less active than TPD in inducing apoptosis of mesothelial cells in vitro.
To this aim, we tested the mesothelial cell apoptotic rate induced by scalar doses of relevant GDPs (MGly and For) incubated at different times. Moreover, we extended the analysis by testing each GDP detected previously in 3CB and TPD PDS at the concentrations reported by Wieslander et al. [7]. Finally, we incubated mesothelial cells in both commercial 3CB and TPD PDS in a Transwell system mimicking peritoneal dialysis. To obtain a deeper insight into the apoptotic pathways we evaluated the gene transcription and translation of some key molecules, including caspase 9, the tumour suppressor protein p53, and the inducible isoform of cyclo-oxygenase (COX-2).
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Subjects and methods |
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The additional effect of glucose on GDP effects was investigated by supplementing the culture medium with filter sterilized glucose solutions to obtain final glucose concentrations of 1.36%, which matches commercial PDS.
To test commercial PDS, mesothelial cells were incubated for 12 h in Transwell plates (Lab-Tek, Miles Scientific Inc., Naperville, IL, US), while avoiding PDS dilution in culture medium and while mimicking conditions of peritoneal dialysis (schematically represented in Figure 1) with commercial 3CB or TPD PDS (either at final 1.36% glucose concentration) by following previously described procedures to test mesothelial cells with PDS [13]. Transwell plates are transparent, microporous filter culture devices (24.5 mm diameter, with a membrane thickness of 10 mm, pore size 3.0 mm). The growth medium was first added to the lower reservoir followed by addition of the Transwell. Then, the mesothelial cells were seeded into the upper reservoir. The microporous membrane was completely sealed by mesothelial cells within 24 h. Prior to addition of PDS, unattached cells were removed by rinsing the Transwells with culture medium. PDS (either 3CB or TPD) were then added in the upper reservoir of the Transwell. This device has been shown to allow an equilibration between the two solution compartments during the 12 h period [13].
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Preparation and characterization of human mesothelial cells
Human peritoneal specimens were obtained from four consenting non-uraemic patients undergoing abdominal surgery for non-neoplastic diseases. Peritoneal samples were washed 3x in PBS and pieces of 1 cm2 were incubated in 2 ml trypsin/EDTA (0.125%/0.01%) (Sigma, St Louis, MO, USA) for 15 min at 37°C in continuous rotation. The suspension was centrifuged at 180 g at 4°C for 5 min. The cells obtained were re-suspended and then plated in 25 cm2 culture flasks in RPMI 1640 medium (Sigma) supplemented with 20% fetal calf serum (FCS), penicillin (100 U/ml), streptomycin (100 µg/ml), L-glutamine (2 mM) (all reagents from Sigma). The medium was supplemented with 5% D-valine to inhibit contaminating fibroblast growth. Cells were gently rinsed to remove unattached cells and propagated to obtain 90% confluence prior to being harvested and sub-cultured. The purity of mesothelial cell culture was assessed by immunoperoxidase using positive staining for cytokeratin, vimentin and the negativity for factor VIII. In the culture experiments, mesothelial cell purity attained 98%.
Before use, cells were kept in a quiescent (G0S1) state by culturing them in 0.1% FCS. The experiments were performed in media supplemented with 20% FCS.
Description of GDPs and relative concentrations
The GDPs and their concentrations, detailed in Table 1, were the same as detected by Wieslander et al. [7] in 3CB and TPD PDS. All reagents were purchased from Sigma (Sigma Chemical Co.).
To avoid evaporation of volatile aldehydes, culture plates were sealed with Parafilm A (American Can Co., Greenwich, CT, USA). A time course (from 2 to 96 h of incubation) at graded doses (MGly from 1.4 to 70 µM and For from 0.7 to 19 µM) was performed.
Terminal uridine nick 3' end labelling (TUNEL) method
After a 5 min incubation with 1% paraformaldehyde, cells were fixed in 2:1 v/v ethanol/acetic acid for 10 min at room temperature. After three washings with PBS, cells were incubated with 100 U/ml terminal deoxyuridine transferase, 0.5 µg/ml biotinylated uridine in 1 M potassium cacodylate and 125 mM TrisHC1, 2.5 mM cobalt chloride (Boeringher, Mannheim, Germany), at pH 6.6 for 1 h at 37°C in a humidified chamber. After washes, a 1:40 solution of fluoresceinated streptavidin (Boeringher) was incubated for 30 min at room temperature. Slides were counter-stained with 0.3 µg/ml propidium iodine (Sigma) in PBS for 1 min at room temperature. An epi-fluorescent microscope (Ernst Leitz, Inc., Rockleigh, NJ, USA) was used to detect apoptotic cells, which were quantified by counting the number of fluorescein-positive cells relative to the total number of cells in at least 10 microscopic fields. For each experiment 200 cells were counted.
Cell viability
Cell viability was ensured by Trypan Blue staining, and by the effective gene transcription and protein translation of house-keeping genes.
Immunoperoxidase staining for caspase 9 and p53
For immunoperoxidase studies cells were cultured in chamber slide plates (Lab-Tek, Miles Scientific Inc., Naperville, IL, USA) and fixed after extensive washing with phosphate buffer 0.15 M, pH 7.4 (PBS). Cells were stained using a standard protocol. Briefly, cells were air dried and fixed in chilled acetone for 10 min. Endogenous peroxidase activity was inhibited by incubation for 30 min in 0.025% H2O2 in methanol. Endogenous biotin activity was inhibited by sequential 30 min exposures to avidin D and biotin blocking solutions (Vector Laboratories Inc., Burlingame, CA, USA). The sections were placed in dilute goat serum as blocking agents, followed by application of 1:40 rabbit IgG anti p53 (Dako, Milan, Italy), or 1:100 rabbit anti-caspase 9 (Santa Cruz Bio-technologies, Santa Cruz, CA, USA) or normal rabbit IgG at the same final concentrations for 30 min; antibodies were diluted in PBS containing 10% goat serum. After washing, the slides were developed with the Vectastain ABC rabbit IgG detection kit (Vector Laboratories). According to the manufacturer's directions, slides were sequentially exposed to biotinylated goat anti-rabbit IgG for 1 h, then streptavidinperoxidase for 30 min, and finally 5,5'diaminobenzidine in 0.05 M Tris buffer at pH 7.2, and hydrogen peroxide 3x10-5% for 5 min. Three washes in PBS were performed between each step. The cells, after counter-staining with haematoxylin and mounting under coverslips, were qualitatively evaluated for staining.
RNA extraction and RTPCR analysis for tumour suppressor p53 mRNA expression
For mRNA, extraction mesothelial cells were washed thrice with sterile PBS, trypsinized and stored in the RNA STAT-60 (TEL-TEST B, Inc., Friendswood, TX, USA) at -80°C until use. Total RNA was extracted from cell cultures using the RNA STAT-60 (TEL-TEST B, Inc.). Cells grown in monolayers, with or without in vitro challenge, were lysed directly in a culture flask by adding extraction reagent (which contains guanidinium thiocyanate and phenol). The resultant homogenates were incubated for 5 min at room temperature to permit complete dissociation of nucleoprotein complexes. Next, 0.2 ml chloroform was added per ml of extraction reagent; the mixture was shaken vigorously for 15 s, and incubated for 23 min at 4°C. Centrifugation separated the homogenates into two phases: a lower red phenolchloroform phase and a colorless upper aqueous phase containing RNA. The aqueous upper phase was transferred to a fresh tube, and 0.5 ml isopropanol was added per 1 ml of the extraction reagent used for homogenization. This mixture was incubated at room temperature for 10 min and then centrifuged for 10 min at 12 000 g. Supernatants were removed, and the RNA pellet was washed once with 75% ethanol by vortexing and subsequent centrifugation at 7500 g for 5 min at 4°C. The extracted RNA in the pellet was air dried and dissolved in DEPCH2O for use in RTPCR analysis.
RTPCR was performed using specific oligonucleotides that detect the p53 and COX-2 transcripts. The p53 sense and antisense primers were: 5'-CTGAGGTTGGCTCTGACTGTAC-3' and 5'-CTCATTCAGCTCTCGGAACATCT-3', respectively; COX-2 sense and antisense primers: 5'-TTCAAATGAGATTGTGGGAAAATTGCT-3' and 5'-AGATCATCTCTGCCTGAGTATCTT-3', respectively (TIB MOLBIOL CBA, Genoa, Italy). The RTPCR was performed by using a commercial kit (Gene Amp RNA-PCR, Perkin-Elmer, Roche Molecular System Inc., Branchburg, NJ, USA) following strictly the instructions furnished by the manufacturer. Briefly, 2 µg of total RNA were added to a master mix for reverse transcription to obtain a final concentration of 5 mM MgCl2, 1 mM dNTPs, 1 U RNase inhibitor, 2.5 µM of Oligo(T)16 and 2.5 U of MuLV reverse transcriptase. The tubes were allowed to incubate at room temperature for 10 min, allowing the extension of Oligo(T)16 by reverse transcriptase.
RT was performed at 42°C for 45 min and at 99°C for 5 min on a Gene Amp PCR System 9700 (PE Applied Biosystem, Foster City, NJ, USA). The reaction mixture was cooled at 4°C, then 20 µl of reverse transcription products were amplified by 0.0125 U Taq DNA polymerase in a mix reaction containing 2 mM MgCl2 and 0.15 µM of the sense and antisense primers. The PCR profile for both p53 and COX-2 consisted of a 5 min initial denaturation at 94°C, followed by 40 cycles of 30 s of denaturation at 94°C, 1 min of annealing at 56°C, 1 min of polymerization at 72°C, and finally a 7 min extension at 72°C. Ten microlitres of the PCR amplification products were separated by electrophoresis at 100 V for 1 h in 1% agarose gel in TBE buffer (45 mM Trisborate, 1 mM EDTA, pH 8). Primers specific for GAPDH were used in each experiment to normalize the results: sense and antisense primers were 5'-CGGAGTCAACGGATTTGGTCGTAT-3'; and 5'-AGCCTTCTCCATGGTGGTGAAGAC-3', respectively. The gels were exposed to a UV scanner using the Gel DOC 2000 (Bio-Rad, Software Quantity One, Hercules, CA, USA).
Both p53 and COX-2 mRNA, normalized for the housekeeping gene GAPDH mRNA, were expressed as percentage increase vs unconditioned cells. Four experiments for each condition were performed in order to allow a statistical analysis of densitometry data.
Statistical analysis
The values reported in the results represent means±SD of four independent experiments from four different peritoneal cell donors, each done in triplicate to obtain a sufficient quantity of data for statistical analysis. RTPCR was done on four individual experiments. Statistical significance was analysed by one-way analysis of variance (ANOVA) using a post-hoc analysis with Dunnet's multiple comparison test when appropriate. Values of P<0.05 were considered statistically significant.
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Results |
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Pre-treatment of mesothelial cells with rabbit anti-human RAGE failed to modify the apoptotic rate induced by TPD or 3CB PDS (decrease in percent apoptotic cells of 2±2.3% and 0.5±0.5%, respectively, P not significant).
Culture media supplemented with 1.36% sterile glucose failed to modify the apoptotic rate induced by MGly (1.470 µM) and For (0.719 µM) indicating a lack of additive effect (data not shown, P not significant). Even though glucose undergoes auto-oxidation with final production of carbonyl compounds, the short time of our experiments (12 h) did not allow a sufficient production of GDPs to modify the apoptotic rate induced by directly supplemented GPDs.
Caspase 9 and p53 expression
Caspase 9 and p53 expression in human mesothelial cells conditioned with GDPs at the concentrations detected in TPD and 3CB fluids showed similar profiles to the modulation of apoptosis (Figure 6) with a higher percentage of caspase 9 and p53 positively stained mesothelial cells when GDPs were used at the concentrations present in TPD solutions compared with concentrations in 3CB PDS (P<0.01). The commercial TPD PDS were more active than 3CB in inducing the neo-expression of caspase 9 and p53, which are involved in the apoptotic pathway in mesothelial cells. The expression of caspase 9 and p53 in unconditoned mesothelial cells was negligible and was rarely present in trace amounts.
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p53 and COX-2 gene expression
GDPs elicited the transcription of the gene encoding for p53. The concentrations of GDPs in TPD solutions strongly activated the transcription of p53 mRNA compared with the concentrations present in 3CB PDS (Figures 8 and 9
). Accordingly, p53 mRNA transcription was significantly more enhanced by TPD than by 3CB PSD.
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Discussion |
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To investigate the modulation of either PDS or pure aldehydes on mesothelial cell apoptosis, we decided to use mesothelial cells from healthy subjects instead of uraemic patients to avoid possible underlying damage due to uraemic toxins and naturally occurring AGEs. The effects we found would eventually be amplified in patients undergoing regular PD treatment, as their mesothelial cells should be relatively depleted in protective scavengers.
Peritoneal fibrosis with loss of ultrafiltration capability represents one of the major cause of peritoneal dialysis patient drop-out. The pathogenetic mechanisms responsible for peritoneal fibrosis are still largely unknown and are under investigation. In addition to peritonitis, much recent attention has been devoted to the role of biocompatibility of PDS. The negative effects of PDS, pH, and lactate buffer [14] have been extensively evaluated, and studies attempting to elucidate the negative effects of glucose are still ongoing. An anti-proliferative effect of glucose per se is well known and has been ascribed to a specific inhibition of cyclin-dependent kinases responsible for cell-cycle arrest in the G0S1 phase [15]. In our experiments, culture media supplemented with 1.36% sterile glucose failed to modify the apoptotic rate induced by MGly and For, indicating a lack of additive effect. Even though glucose undergoes auto-oxidation to produce carbonyl compounds, the short time of our experiments (12 h) did not allow a production of GDPs sufficient to modify the apoptotic rate induced by directly supplemented GPDs.
At high concentrations glucose may alter the intracellular redox state and this effect is mediated by a phosphokinase C activation (polyol pathway) or by altering the NADH/NAD ratio responsible for pseudo hypoxia conditions (esosomonophosphate-shunt). The pro-oxidant activity exhibited by glucose is enhanced by the tendency of glucose at high concentration to undergo auto-oxidation [16], a process that is accelerated and catalysed by high temperature and neutral pH, such as during heat sterilization of glucose containing-PDS [7]. The glucose oxidation results in formation of the so called carbonylic stress products, having an aldehyde biochemical structure and strong oxidative effects [17]. Aldehydes are able to induce DNA oxidative damage that could represent the starting point of the apoptotic process. Moreover, a modification of the intracellular redox state with increases in oxidants and eventual reductions in scavenger molecules or enzymes (such as gluthatione, gluthatione peroxidase, superoxidodismutase, characteristic of uraemia), through activation of different phosphorylation systems may induce IkB phosphorylation and consequent activation and nuclear translocation of the transcriptional factor NFkB. This factors uses consensus sequences initiating the transcription of different genes, such as TGF-ß, COX-2, nitric oxide synthase and collagens involved in the progressive scarring processes.
Apoptosis has been recently implicated in the silent cell loss occurring in different scarring processes [9]. The redox imbalance is pivotal in the process of apoptosis, inducing modifications in genes (such as Bcl-2, Bad, Bax, p53) involved in programmed cell death [9,18,19]. Oxidative agents provoke apoptosis, and scavengers such as superoxide dismutase or glutathione prevent apoptotic death.
We demonstrated that GPDs are able to induce the neo-transcription of p53 and that the effect is dose-dependent. In fact, we demonstrated that GDPs at the concentrations present in TPD were significantly more active than the lower concentrations of GDPs present in 3CB, and that the commercially available TPD PDS significantly up-regulated p53 gene expression when compared with 3CB. The effective translation of p53 mRNA was demonstrated by the higher percentage of p53 positive mesothelial cells in immunoperoxidase induced by GDPs in TPD fluids with respect to the percentage of positive cells following treatment with GDPs at the concentrations in 3CB fluids. Similarly, COX-2 gene transcription paralleled the modulation of p53 induced by GDPs in TPD fluids and 3CB and by commercial TPD fluids and 3CB.
The translated p53 protein is known to modulate the expression of the WAF1/CIP1 gene [9], which in turn induces the synthesis of the regulator protein p21. Finally, p21 inhibits the activity of cyclin E. Apoptosis then occurs following the consequent inhibition of the delta DNA polymerase isoform, the most active enzyme involved in DNA replication.
The finding that caspase 9 expression occurs in immunoperoxidase strongly supports the hypothesis of a pro-apoptotic role for GDPs. Further supporting this was the higher percentage of caspase 9 positive mesothelial cells when treated with GDPs at concentrations in TPD or in commercial TPD PDS compared with GDPs in 3CB and in commercial 3CB PDS. In this regard, it is of note that the percentage of caspase 9 positive mesothelial cells was slightly higher than that of p53 positive cells. This finding could be explained by the timing of activation of the different pro-apoptotic molecules, as caspase 9 is activated earlier than p53 during the apoptotic cascade. TUNEL analysis revealed that caspase 9 expression, p53 gene transcription and translation, and COX-2 de novo synthesis result in an increased percentage of apoptotic cells. GDPs concentrations detected in TPD and in commercial TPD PDS induced significant increases in the percentage of TUNEL positive mesothelial cells compared with GDP concentrations found in 3CB PDS.
Two recent papers investigated the effects of pure GDPs and of commercial PDS on mesothelial cell viability, growth and synthetic activity [11,12]. While the data reported on pure GDPs were in agreement with our apoptosis results [11], they failed to show significant inhibition of mesothelial cell proliferation when cells were incubated during short-term experiments in freshly prepared glucose solutions, either heat sterilized or pore membrane filtered [11]. We were able to perform our experiments with commercial PDS by adopting the Transwell device (Figure 1), which mimics peritoneal dialysis conditions. This device exposes one cell surface to culture medium and the other to commercial PDS. We tested commerial PDS that had been stocked for months before the use and was likely to contain naturally formed GDPs. For these reasons we believe that our current experimental conditions more closely reproduced in vivo PD conditions. The long-term experiments performed by Witowski et al. [12], showing a gradual loss of cell viability, decreased adhesion, and cell shedding after prolonged incubation of mesothelial cells with GDPs (up to 36 days), fit very well with our data of enhanced apoptosis.
We cannot directly compare the apoptotic rate induced by single GDPs with that induced by commercial PDS, as we used different culture procedures. We directly supplemented the culture medium of mesothelial cells grown in plastic flasks with pure GDPs whereas commercial PDS were tested on mesothelial cells in Transwell devices.
The significantly higher apoptotic rate induced by TPD compared with 3CB PDS is in agreement with previous reports showing that mesothelial cells from patients using 3CB formed a confluent layer in a significantly shorter time than mesothelial cells from patients using TPD [20]. This finding also agrees with the increased levels of CA125, a marker of mesothelial cell mass in peritoneal dialysis effluent, in 3CB compared with TPD PSD [8].
Our data support the hypothesis of a mesothelial cell layer loss induced by GPDs at the high concentrations that are detected in TPD solutions. The resulting exposure of matrix components and vascular endothelial cells to the oxidative action of GDPs would produce in peritoneal barrier damage. The acceleration of non-enzymatic AGE formation (Namiki's pathway) by GDPs further favours peritoneal fibrosis and loss of ultrafiltration capability. Being 3CB PDS and their corresponding GPDs concentrations less active in inducing apoptosis of mesothelial cells, a better biocompatibility of this new formulation of PDS is expected in respect to enhancement of peritoneal fibrosis in long-term dialyzed patients.
Conflict of interest statement. None declared.
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Notes |
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References |
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