Background
Peritoneal dialysis (PD) is a well-established life-saving renal replacement therapy for individuals suffering from end-stage renal disease (ESRD) [
1]. In PD the waste products and excess water are removed from the body by using a hypertonic solution via the peritoneal membrane (PM). At present, crystalloid glucose at concentrations of 0.55 to 4.25% is the most commonly used osmotic agent in PD solutions such as Dianeal from Baxter (Deerfield, IL, USA). However, the incidence of ultrafiltration (UF) failure among PD patients increases with time on glucose-based PD required for effective new PD [
2,
3], and UF failure is one of main reasons for PD technique failure [
3]. Numerous studies have demonstrated that inclusion of glucose in the conventional PD solutions largely contributes to peritoneal membrane (PM) damage and UF failure [
4‐
8], and its high level or long-term peritoneal load is positively correlated with an increase in all-cause and cardiovascular disease mortality, low residual renal function (RRF) and dialysate to plasma ratio of creatinine in PD patients [
9,
10]. Thus, it has been proposed that a non-glucose based biocompatible osmotic agent in a PD solution may preserve long-term integrity of PM structure and function, resulting in a delay in PD technique failure.
Recently we have shown that hyperbranched polyglycerol (HPG) based PD solutions at concentrations of 2.5–15% (w/v) increases the solution osmolality to 294–424 mOsm/kg and can maintain neutral solution pH (6.6–7.4) [
11‐
13]. In a rat model of acute PD, HPG-based PD solutions produce similar or better fluid and waste removal while preserving the PM function compared to either Dianeal (2.5% glucose) or Physioneal (PYS) (2.27% glucose) [
11,
12]. Very recently, we have also demonstrated that HPG is superior to glucose for long-term preservation of the PM in a rat model of chronic PD [
13,
14]. All these preclinical studies may suggest that HPG is a promising alternative to glucose in PD. However, the safety profiling of this polymer has not been fully assessed. In the current study, the objective was to investigate the global biological responses (as a measure of biocompatibility) of human peritoneal mesothelial cells (HPMCs) using a whole-transcriptome analysis after repeated exposure to HPG-based PD solution as compared to glucose-based conventional PYS.
Methods
Hypertonic PD solutions and cells
HPG (1 kDa) was synthesized as described previously [
11,
12]. A hypertonic HPG PD solution (denoted as HPG here, approximately 402 mOsmol/kg, pH 7.4) was prepared by dissolving HPG (1 kDa, 6% w/v) in a buffered electrolye solution that had the same composition as that of PYS without glucose: sodium chloride (538 mg/100 mL), sodium lactate (168 mg/100 mL), calcium chloride dehydrate (18.4 mg/100 mL), magnesium chloride hexahydrate (5.1 mg/100 mL) and sodium bicarbonate (210 mg/100 mL) [
11,
13,
14]. PYS (2.27% glucose, 395 mOsmol/L, pH 7.4) was purchased from Baxter Healthcare Co. (Deerfield, IL, USA).
The immortalized HPMCs were generated from primary HPMCs by immortalizing it with origin-deficient SV40 DNA and grown and maintained in complete K1
+/+ medium as described previously [
12]. Jurkat cells, a human leukemic T cell line, were purchased from the American Type Culture Collection (Manassas, VA, USA), and grown and maintained in RPMI 1640 medium containing 10% fetal bovine serum (FBS).
Repeated exposure of cells with hypertonic PD solutions
Immortalized HPMCs (0.25 × 106 cells/well) were seeded in 24-well plates in complete K1 medium for 18 h, followed by 6 h in either PYS or HPG solution (total 24 h or a day) in a humidified 5% CO2 incubator at 37 °C. This treatment cycle (18-h medium/6-h hypertonic solution) was continuously repeated for six more days/times.
Determination of HPMC growth or death rate
At the end of 6 h treatment with either PYS or HPG in each treatment cycle, both cell death or growth of cultured HPMCs was determined by lactate dehydrogenase (LDH) release using LDH assay kit (Roche Applied Science, Laval, QC, Canada) following the manufacturers’ protocol. In brief, after each time of exposure to HPG or PYS at 37 °C under a 5% CO2 atmosphere, supernatants were collected from the wells, followed by complete lysis of remaining cells in a 2% Triton X-100 solution. The levels of LDH in both the supernatant and the cell lysate were measured at each time point. The cell death rate was calculated: % = LDHs/(LDHs + LDHce) × 100, whereas growth or survival rate: % = LDHce/LDH0 × 100; where LDHs represented the LDH level in the supernatant and LDHce the LDH level in the cell lysate at indicated time point, and LDH0 indicated the total LDH level of seeded cells in untreated cell monolayer (0 h time point).
Five experimental groups were included in this study: untreated control at day 0, PYS at day 3 and 7, and HPG at day 3 and 7. Three separate cell samples were collected from each group. In brief, after 6 h of exposure to HPG or PYS, cells were detached with a Trypsin-EDTA solution and pelleted by centrifugation. Cell pellets were snap-frozen with liquid nitrogen first, and then stored in -80 °C until use. Total RNA was extracted from the cell samples by using mirVana™ isolation kit (Ambion, Austin, TX, USA), and only the RNA samples with ≥8 of RNA Integrity Number (RIN) were used for below microarray analysis.
Microarray and ingenuity pathway analyses (IPA)
A transcriptome database of each RNA sample was created by using Agilent SurePrint G3 Human GE 8 × 60 K Microarray kit (Agilent Technologies Canada Inc., Mississauga, ON) following a standardized protocol as described previously [
15]. These RNA sequencing datasets were then imported into GeneSpring GX (Agilent, Santa Clara, CA, USA), and were analyzed with 1.3 of –log
10 p value (
p = 0.05) (
t-test) and ≥ 1.5-fold change (FC) using IPA software (Ingenuity Systems, Redwood City, CA, USA) to compare the transcript profiling between groups.
The affected gene transcript profile (both up-regulated and down-regulated) in each group was imported into the “Canonical Pathway” frame of the IPA software to determine whether or not the signaling pathways were significantly regulated by each treatment (HPG or PYS at day 3 or 7) as compared to controls, such as at day 3 or 7 compared to at day 0, or at day 7 compared to day 3.
Flow cytometry analysis
The cell surface expression of HLA-DMB on HPMCs was determined by using flow cytometric analysis. In brief, cells were incubated with primary rabbit monoclonal anti-HLA-DMB antibody (ab133640, Abcam, Toronto, ON, Canada), followed by labeling with secondary fluorescein isothiocyanate (FITC)-conjugated goat anti-rabbit IgG antibody (ab97050, Abcam), or with the secondary antibody only as a FITC background stain control. Stained cells were measured using a XL flow cytometer BD FACSCanto II (BD Biosciences, Mississauga, ON, Canada). The proportion of HLA-DMB positivity in the sample was presented by means of FITC light intensity (MFI) with anti-human HLA-DMB antibody.
Western blotting analysis
Cellular levels of HLA-DMB protein in HPMCs were examined by Western blot analysis. Proteins in cellular protein extracts (50–100 μg per sample) were separated using 10% SDS-PAGE, and were then transferred onto nitrocellulose membrane. HLA-DMB protein (26–28 kDa) were identified by rabbit monoclonal anti-HLA-DMB antibody (ab133640, Abcam), and visualized by using an enhanced chemiluminescence assay (ECL: Amersham Pharmacia Biotech, Buckinghamshire, UK). Blots were re-probed using anti-Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) antibody (Epitope Biotech., Vancouver, BC, Canada) to confirm the amount of loaded protein in each sample. The expression levels of the HLA-DMB were semiquantitatively determined using a densitometry, and were presented as a ratio of the HLA-DMB protein to GAPDH on the same blots.
T cell adhesion assay
Cell adhesion of Jurket cells to monolayers of HPMCs was examined by using microscopic analysis. In brief, after repeated exposure of HPMCs with HPG, PYS or medium only (untreated control) for seven times as described above, cells (0.25 × 106 cells/well) were incubated in 24-well plates overnight, followed by incubation with Jurkat cells (0.25 × 106 cells/well) at 37 °C, 5% CO2 for 48 h. Non-adherent Jurkate cells were removed by washing four times with the culture medium. The bound Jurkat cells in each sample were counted in 10 randomly selected high power fields (hpf, 400× magnification) using a light microscope, and were averaged.
Statistical analysis
The difference between samples was analyzed by using two-tailed t-test or analysis of variance (ANOVA) (GraphPad Prism Software, GraphPad, San Diego, CA, USA) as appropriate. Data were presented as mean ± standard derivation (SD). A p value ≤0.05 was considered statistically significant.
Discussion
Long-term exposure to a glucose-based PD solution is associated with inflammation and fibrosis in the PM [
17], which consequently result in its gradual functional impairment characterized by progressive reduction in solute transport or UF failure [
18]. However, the cytotoxic mechanism of the peritoneal response to a hypertonic PD solution has not yet been fully elucidated. A previous study has demonstrated that TGF-β1/Smad signaling pathway is activated by the hypertonic solution in a chronic PD rat model, and possibly participates in the pathogenesis of PD related peritoneal fibrosis [
19]. Recent findings have suggested that epithelial-mesenchymal transition (EMT) of peritoneal mesothelial cells may play an important role in the failure of PM function [
20]. However, it is still not known that how these possible signaling pathways contribute to the inflammation and fibrosis seen in the peritoneum of PD patients. Thus, in this study, our primary goal was to reveal the biological pathways affected by a hypertonic solution in cultured HPMCs by transcriptome analysis. Since our previous studies demonstrated that the compact HPG is a superior biocompatible osmotic agent compared to glucose in rat models of PD [
11‐
14], evidenced by the fact that the HPG-based hypertonic solution induces less PM damage than glucose-based PD solutions. However, the pathways mediating the difference in the “toxicity” between the HPG- and the glucose-based hypertonic PD solutions in the peritoneum have not been fully investigated. Thus our second goal in this study was to compare the pathways affected by the HPG solution with those by PYS in HPMCs using a whole-transcriptome analysis that is an emerging and continually growing field in assessing the safety of drugs or chemical risk assessment [
21,
22]. This approach allows a more comprehensive understanding and a fuller knowledge of the “biocompatibility” of the agents/chemicals than those just based on several “biomarkers”.
Cell death is an essential attribute of tissue damage. In this study, we showed that the levels of cell death in both HPG and PYS groups at different time points were positively correlated with the numbers of changed transcripts (both up-regulated and down-regulated) in HPMCs (Figs.
1 and
2). The IPA pathway analysis did not link the cell death induced by the hypertonic solution (either HPG or PYS) to any of these significantly affected pathways (Table
1). Instead, the “dermatan, chrondroitin or heparan sulfate biosynthesis”, “unfolded protein response”, “apoptosis signaling” and “NRF2-mediated oxidative stress response” were commonly affected by HPG at day 3 and by PYS at both time points, and in addition to more pathways by PYS at day 3 with more cell death (Tables
2 and
3). Interestingly, there was no single transcript commonly found in these pathways between HPG and PYS groups (Tables
2 and
3). These data suggest that the cell death induced by either hypertonic HPG or PYS solution may be primarily mediated by the disruption of “sulfate biosynthesis” and activation of “unfolded protein response”, “apoptosis signaling” and “NRF2-mediated oxidative stress” but their actions may be different at different time points due to regulating different transcript expression.
It is interesting to note that over the time of exposure to PYS, cell death was remarkably decreased, or cells gradually became resistant to PYS-induced cell death (Fig.
1). At the same time, we found that only one transcript – API5 (apoptosis inhibitor 5) was significantly upregulated at day 7 as compared to day 3 (Fig.
2). We also noticed significant up-regulation of PPP1R10 (protein phosphatase 1 regulatory subunit 10) at both day 3 and day 7 as compared to untreated control (Table
3). According to the GeneCards® Human Gene Database (
https://www.genecards.org/), PPP1R10 plays a role in cell cycle progression, DNA repair and apoptosis by negatively regulating the activity of protein phosphatase 1, a proapoptotic activator [
23,
24]. Thus, it is reasonable to hypothesize that up-regulation of both PPP1R10 and API5, apoptosis inhibitors, may be part of the mechanism by which cell death resistance is induced by repeated exposure to PYS in this experimental system.
Chronic inflammation is a hallmark of structural alterations and dysfunction of the PM during PD [
25], and exposure of a non-physiological, hypertonic PD solution is considered as a non-infective factor leading to chronic inflammation, macrophage infiltration and oxidative stress in the PM, resulting in UF failure in PD patients [
26]. However, the pathways by which the inflammation is initiated by a hypertonic PD solution in the PM are largely unknown. The transcriptome analysis in this study revealed many inflammation-related transcripts predominantly affected in PYS at both day 3 and day 7 (Table
4). The most notable of them were up-regulation of both HLA-DMB and MMP12, which were persistently induced by PYS at both time points (Table
4). The up-regulation of HLA-DMB protein was also confirmed by both flow cytometric and Western blot analyses (Figs.
3 and
4). HLA-DMB is the beta chain of the non-classical HLA class II heterodimer (HLA-DM) that plays a central role in the peptide loading of MHC class II molecules and initiation of an immune response to the antigen stimulation [
27,
28], and increases T cell adhesion to HPMCs (Fig.
4). This observation is supported by evidence in literature showing that HPMCs participate in antigen presentation, and T cell growth and adhesion [
29,
30], and the antigen presentation in the peritoneum stimulates resident population of effector memory T cells in PD patients [
31]. These peritoneal T cells, particularly CD8
+ cytotoxic T cells, could play a destructive role for PM damage [
32]. MMP12 is a potent elastase, and its function is involved in many inflammatory conditions, such as the inflammatory process of respiratory diseases (chronic obstructive pulmonary disease, pulmonary fibrosis and asthma) [
33], and mediation of inflammation and IL-13-induced liver fibrosis [
34] and inflammatory arthritis [
35]. In this study, we also showed increased T cell adhesion and up-regulated HLA-DMB expression in HPMCs after exposure to PYS (Fig.
4). All this evidence may indicate that both HLA-DMB and MMP12 are the local inflammatory mediators of the PM induced by exposure to hypertonic PYS or perhaps other PD solutions.
Although positive correlations were obtained in our transcriptome analysis, the present study has several limitations. Firstly, the data were collected from cultured HPMCs, which may not represent the mesothelial cells in the PM of PD patients. The findings from this study need to be verified in clinical studies as well as in other experimental systems such as preclinical animal models. Secondly, we only tested PYS in this study. The results could be different when HPMCs are treated with other types of PD solutions such as Dianeal solutions or Extraneal (Icodextrin). Thirdly, we only measured the levels of the transcripts using microarray chips, which may not exactly reflect their protein levels. Also, the pathways due to the change of the transcripts were predicted using a bioinformatics analysis tool – IPA. Further studies are required to confirm the changes in the protein levels and their impacts on cell death, survival and inflammatory initiation.