Different effects of amino acid-based and glucose-based dialysate from peritoneal dialysis patients on mesothelial cell ultrastructure and function

Tak-Mao Chan1,, Jack Kok-Hung Leung1, Yuling Sun2,3, Kar-Neng Lai1, Ryan Chi-Wai Tsang1 and Susan Yung1

1 Division of Nephrology, Department of Medicine, The University of Hong Kong, Queen Mary Hospital, Hong Kong SAR and 2 Department of Nephrology, First Affiliated Hospital, Sun Yat-Sen University, Guangzhou 510080, China 3 Present address: Division of Nephrology, Department of Medicine, the University of Hong Kong, Queen Mary Hospital, Hong Kong SAR, China



   Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Background. Peritoneal dialysis fluid (PDF) containing amino acids has been introduced recently aiming to improve the nutritional status of PD patients. Dextrose-based PDFs have been implicated in progressive functional and structural deterioration of the peritoneal membrane. Limited data are currently available regarding the effect of amino acid-based PDF on the function and ultrastructure of human peritoneal mesothelial cells (HPMCs), which play a critical role in peritoneal membrane pathophysiology.

Methods. We investigated the effects of two commercially available PDFs, which utilized dextrose (1.5% Dianeal) or amino acids (1.1% Nutrineal) as the osmotic agent, obtained from patients after a 4 h dwell, on HPMC proliferation (MTT assay and cell counting) and viability [lactate dehydrogenase (LDH)release], interleukin-6 (IL-6) secretion (commercial enzyme-linked immunosorbent assay) and ultrastructure (scanning and transmission electron microscopy).

Results. Exposure of HPMCs to 1.5% Dianeal reduced cell proliferation, total cellular protein synthesis, IL-6 secretion and cell attachment, but prolonged the cell doubling time on recovery, and increased LDH release (P<0.001, P<0.001, P<0.0001, P<0.0001, P<0.001 and P<0.001, respectively). The 1.1% Nutrineal reduced HPMC proliferation (P<0.001) and increased IL-6 secretion (P<0.0001), but did not affect cell attachment, LDH release, protein synthesis or cell doubling time. Ultrastructural studies of HPMCs exposed to Dianeal showed cell flattening, increased cell surface area, reduced microvilli, and intracellular organelles compatible with dysfunctional mitochondria. In contrast, the ultrastructural morphology of HPMCs was relatively preserved after incubation with Nutrineal.

Conclusions. Our results showed that HPMC ultrastructure, viability and protein synthesis were better preserved with amino acid-based PDF, compared with conventional dextrose-based PDF. The significance of IL-6 induction by Nutrineal remains to be elucidated.

Keywords: amino acid; glucose; interleukin-6; mesothelial cell; peritoneal dialysis



   Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Peritoneal dialysis (PD) is an important treatment modality for patients with end-stage renal failure. Conventional PD fluids (PDFs) contain dextrose at variable concentrations to provide the osmotic drive for ultrafiltration, a low pH to prevent glucose caramelization, and lactate to correct metabolic acidosis. During heat sterilization of conventional PDF, glucose degradation products (GDPs) are also generated. All these attributes are detrimental to the structural and functional integrity of the peritoneal membrane, thereby compromising the long-term clinical outcome. In vivo studies in long-term PD have demonstrated diabetiform alterations of the peritoneum, which included re-duplication of the peritoneal membrane, increased synthesis and deposition of matrix proteins within the submesothelium, and progressive subendothelial hyalinization, with narrowing or obliteration of the vascular lumen [1]. These structural changes are accompanied by functional abnormalities, such as ultrafiltration failure or reduced solute clearance [1]. In addition to the induction of pro-inflammatory and/or pro-fibrotic cytokines, animal and in vitro studies have highlighted the adverse effects of glucose-based PDFs on resident peritoneal cells [2,3]. The absorption of glucose from conventional PDFs also leads to dyslipidaemia, obesity and increased satiety. In view of the adverse effects of dextrose-based PDFs, the search for alternative osmotic agents has continued.

Malnutrition is a powerful predictor of poor clinical outcome in patients on PD. The recent introduction of amino acid-based PDFs has been associated with modest nutritional benefits. Results from in vitro studies suggest that amino acid-based PDFs may be more biocompatible compared with dextrose-based PDFs with respect to their effects on the viability and function of mononuclear leukocytes and mesothelial cells [4,5]. However, these studies have either exposed peritoneal cells to fresh unused PDF, or have involved ex vivo culture of peritoneal cells without further exposure to PDF, and therefore represented artificial settings rather than the clinical scenario. At each exchange of dialysate, PDF from a new bag is mixed immediately with a residual volume of 195–255 ml in the peritoneal cavity [6]. The composition of the infused PDF then changes progressively during intraperitoneal dwell, with neutralization of its cytotoxicity occurring within 15 min of administration [7]. Prolonged exposure of peritoneal cells in vitro to fresh PDF therefore does not reflect the true in vivo state. Since the equilibration of pH is achieved within 15 min after infusion [7], and the duration of dwell can be up to 6–10 h, it is pertinent to examine the effects of spent PDF, with which the resident peritoneal cells are in prolonged contact. It is now well established that the mesothelium is not only a structural barrier. It participates in fluid and solute transport during PD, and plays a pivotal role in intraperitoneal inflammatory responses. In this context, chemical insult to peritoneal mesothelial cells can induce the synthesis of cytokines, chemokines and growth factors, and is associated with accumulation and deposition of matrix proteins [8]. Cultured human peritoneal mesothelial cells (HPMCs) provide a relevant in vitro model to study the effect of PD solutions on cell proliferation and function, since previous studies have demonstrated that they possessed the same immunohistochemical markers as peritoneal mesothelial stem cells. We have assessed the effects of spent dextrose-based and amino acid-based PDFs (1.5% Dianeal and 1.1% Nutrineal, respectively) on the proliferation, viability, protein synthesis, cell doubling time after recovery, secretion of interleukin-6 (IL-6) and the ultrastructure of HPMCs. Our data demonstrated marked differences between the two PDFs.



   Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
All chemicals were of the highest purity and were purchased from Sigma (China South, Hong Kong) unless otherwise stated. Tissue culture flasks, multiwell plates and polyethylene terephthalate (PET) track-etched membranes (0.4 µm diameter pore size) were purchased from Falcon (Becton Dickinson, Hong Kong), Thermanox plastic coverslips from ProSciTech (Gene Company, Hong Kong), and culture media and supplements from Invitrogen Technologies (Hong Kong). The cytotoxicity kit based on the release of lactate dehydrogenase (LDH) was purchased from Roche (Gene Company, Hong Kong). The IL-6 duoset enzyme-linked immunosorbent assay (ELISA) development system was purchased from R&D (Onwon Trading Inc., Hong Kong). The different types of PDF were obtained from Baxter Healthcare Corporation (Deerfield, IL, USA), namely 1.5% Dianeal (dextrose-based) and 1.1% Nutrineal (amino acid-based), the composition of which is shown in Tables 1Go and 2Go. A Millipore resistance meter was purchased from Minicell ERS, Millipore Inc., Bedford, MA.


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Table 1.  Composition of 1.5% Dianeal and 1.1% Nutrineal

 

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Table 2.  The amino acid constituents in 1.1% Nutrineal

 
We examined the effects of spent PDFs on the proliferation, viability, selected aspects of cell function and ultrastructure of HPMCs. Cell proliferation was assessed by both the MTT assay and direct cell counting. Cell viability was determined by LDH release. Total cell protein synthesis and cell doubling time were also measured. IL-6 secretion by HPMCs was measured using a specific ELISA. Ultrastructural morphology was studied using scanning and transmission electron microscopy.

Spent PDF samples and patients
PDFs were obtained from seven patients using Dianeal only, two patients using Nutrineal only, and five patients using both Dianeal and Nutrineal. Nutrineal was administered as one morning dwell daily. Table 3Go shows the biochemical characteristics of each bag of spent PDF collected. Dianeal (n=12) or Nutrineal (n=7) were obtained after an intraperitoneal dwell of 4 h, centrifuged at 1000 g to remove cell debris, filtered-sterilized, and stored at -70°C before commencement of the experiments. The pH of spent PDF samples was 7.2–7.6.


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Table 3.  Comparison of PDF constituents present in spent 1.5% Dianeal and 1.1% Nutrineal

 
Culture of HPMCs
HPMCs were obtained by enzymatic digestion of omental specimens, from non-uraemic patients with informed consent at the time of abdominal surgery, and cultured in Medium 199 (M199) containing 5 mM D-glucose supplemented with 10% fetal calf serum (FCS) as previously described [9]. All experiments were performed on cells of the second passage that had been growth-arrested for 72 h. After synchronization of the cell cycle, HPMCs were cultured in parallel with M199 containing either 5 (physiological) or 30 mM D-glucose (dextrose concentration after intraperitoneal equilibration of Dianeal), supplemented with 0.1% FCS (the protein concentration present in spent Dianeal and Nutrineal was 0.099±0.03 and 0.11±0.037%, respectively, Table 3Go), spent Dianeal or Nutrineal for time periods up to 72 h. In experiments to assess cellular proliferation, LDH release or protein synthesis, cells were cultured in 96-well plates. For direct cell counting or measurement of IL-6 secretion, cells were cultured in 24-well tissue culture plates. For cell morphology studies, HPMCs were cultured on Thermanox coverslips or PET membranes. Cells were cultured on PET membranes for polarity of IL-6 secretion studies. The integrity of the mesothelial monolayer was confirmed by measuring the transmesothelial electrical resistance of the HPMC monolayer [10].

Assessment of cell proliferation
HPMCs were seeded into 96-well plates at a density of 10 000 cells/cm2 and cultured in M199 supplemented with 10% FCS for 24 h. Thereafter, the cells were washed with phosphate-buffered saline (PBS) and incubated with M199 supplemented with 10% FCS containing (i) 5 mM D-glucose; (ii) 30 mM D-glucose; or spent samples of (iii) 1.5% Dianeal, or (iv) 1.1% Nutrineal. At selected time periods (0.5–5 days), cell proliferation was assessed by the addition of MTT (final concentration 0.5 mg/ml) to HPMCs during the final 4 h of the time point at 37°C. The formazan product generated was solubilized overnight with 10% SDS in 0.01 M HCl, and the absorbance recorded at 520 nm with a reference wavelength of 690 nm.

LDH release
Confluent HPMCs were cultured under control and experimental conditions in 96-well tissue culture plates for selective time periods up to 48 h. Supernatants were collected, centrifuged for 10 min at 2000 g and assessed for LDH release using a commercially available cytotoxicity kit according to the manufacturer's instructions. The cytotoxicity of control and experimental samples was expressed as the percentage of LDH release compared with the total intracellular LDH content, the latter determined by lysis of representative cell monolayers using 2% Triton X-100 (v/v).

Determination of total cellular protein concentration
HPMCs cultured in triplicate in 96-well plates under control or experimental conditions were lysed with 4 M urea buffer, 20 mM sodium acetate, pH 6.0 containing 1% (v/v) Triton X-100 (50 µl). The protein concentration in each sample was determined using a modified Lowry assay according to the manufacturer's instructions (BioRad, Hong Kong).

Assessment of cell attachment and cell doubling time
Confluent HPMCs were cultured in 24-well tissue culture plates under control and experimental conditions for 24–72 h. Cell attachment was examined by direct cell counting at selective time points as described by Witowski and colleagues [3]. Cells were harvested by the addition of 0.05% (w/v) trypsin/0.02% (w/v) EDTA (300 µl) for 5 min at 37°C, neutralized by the addition of M199 containing 10% FCS, and the number of cells counted directly using a Neubauer chamber (China South, Hong Kong). Trypsinized HPMCs subsequently were re-seeded at an equal density of 10 000 cells/cm2 into new 24-well tissue culture plates in M199 containing 10% FCS. After 12 h, cell attachment was assessed, non-adherent HPMCs were removed, and adherent cells were washed twice with PBS, trypsinized and counted as above. In parallel experiments, HPMCs were continued in culture for 24 h prior to assessment of cell attachment by direct cell counting. Cell doubling time was calculated with the following equation: Go


(001)
where {Delta} time represents the incubation time, and counts A and B represent the number of HPMCs at the beginning and end of the incubation period, respectively [11].

Determination of IL-6 concentration in culture supernatant
The IL-6 concentration in supernatants obtained from HPMCs cultured under control and experimental conditions for time periods up to 72 h was measured using a commercial ELISA kit according to the manufacturer's instructions. Lower and upper limits of the assay were 5 and 300 pg/ml, respectively.

Examination of HPMC ultrastructural morphology
Scanning electron microscopy. HPMCs were cultured on Thermanox coverslips or PET membranes under control and experimental conditions for 24 h, fixed in 0.1 M sodium cacodylate buffer (pH 7.4), containing 2.5% (v/v) glutaraldehyde, overnight at 4°C. After dehydration in an ascending series of ethanol, samples were subjected to critical point drying with CO2, and membranes were mounted onto aluminium stubs and coated with gold palladium, before they were examined with a Cambridge S440 scanning electron microscope (Leica Cambridge, Hong Kong). Preliminary studies showed no difference in HPMC morphology when cultured on either substratum.

Transmission electron microscopy. HPMCs were cultured on Thermanox coverslips or PET membranes under control and experimental conditions as for SEM, post-fixed in 1% osmium tetroxide, dehydrated in an ascending series of alcohol and propylene oxide, and embedded in Epon resin. Ultrathin sections were cut with a diamond knife, stained with uranyl acetate and lead citrate, and examined with a Philips EM208S microscope (Philips, Hong Kong).

Statistical analysis
Results are expressed as mean±SD. All experiments were performed four times using HPMCs from four separate donors, unless otherwise stated. Statistical analyses were performed using SPSS version 10. Data from experimental groups and controls were compared using analysis of variance (ANOVA) and Bonferroni's multiple comparison procedure. P<0.05 was considered statistically significant.



   Results
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Effect of PDF on HPMC proliferation, cell viability and protein synthesis
HPMC proliferation and viability were assessed under control and experimental conditions using the MTT assay and LDH release, respectively. Our preliminary experiments using serially diluted samples demonstrated that neither spent Dianeal nor Nutrineal PDF interfered with the LDH assay compared with M199 control.

HPMC proliferation was almost completely inhibited after incubation with 30 mM D-glucose for 3 days (48.27±4.2, 42.85±5.8 and 38.46±7.1% of control cells at t=3, 4 and 5 days, respectively, P<0.001 for all compared with controls). Similar findings were observed with spent Dianeal (34.4±4.8, 30.9±8.1 and 28.8±3.1% of control cells at t=3, 4, and 5 days, respectively; P<0.001 for all compared with controls) (Figure 1Go). Inhibition of HPMC proliferation was significantly less with Nutrineal (51.7±5.3, 47.6±5.2 and 55.3±7.2% of control cells for Nutrineal, at t=3, 4, and 5 days, respectively; P<0.001 for all samples compared with controls at the same time point).



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Fig. 1.  The effect of 30 mM D-glucose (open circles), spent Dianeal (filled circles) or Nutrineal (filled squares) on HPMC proliferation, as assessed by the MTT assay. HPMC proliferation was inhibited most significantly by 30 mM D-glucose and Dianeal, and to a lesser extent by Nutrineal, compared with control (5 mM D-glucose, open squares). Data are expressed as mean±SD of four individual experiments. *P<0.001 compared with control at identical time points.

 
Cytotoxicity was observed only with 30 mM D-glucose and spent Dianeal, which induced LDH release in a time-dependent manner (2.1- and 3.4-fold increase, respectively, compared with control after 24 h incubation, P<0.001 for both samples; Figure 2Go). Dianeal, but not 30 mM D-glucose, also inhibited cell protein synthesis (5.01±0.12 vs 2.75±0.53 µg protein/104 cells, control vs Dianeal, P<0.001; Figure 3Go). Nutrineal did not induce significant LDH release or alter total cellular protein synthesis.



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Fig. 2.  The effect of 30 mM D-glucose, spent Dianeal or Nutrineal on LDH release by HPMCs compared with control (open squares). Data are expressed as a percentage of total releasable LDH. Significant LDH release was observed following exposure to 30 mM D-glucose (open circles) and Dianeal (filled circles), but not Nutrineal (filled squares). Data represent the mean±SD. *P<0.001 compared with control at identical time points.

 


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Fig. 3.  The effect of 30 mM D-glucose concentration, spent Dianeal or Nutrineal on total cellular protein synthesis. HPMCs were incubated in 5 (control) or 30 mM D-glucose, or spent PDF for 24 h. Total cellular protein was extracted and determined using a modified Lowry assay. Data represent the mean±SD.

 

Cell attachment and cell doubling time
Confluent HPMCs were incubated for up to 72 h under control or experimental conditions, and cell attachment was examined by direct cell counting at selective time points (Figure 4Go). Exposure of HPMC to either 30 mM D-glucose or Dianeal for longer than 24 h resulted in a time-dependent reduction in cell attachment (46 120±5480 vs 33 210±4219 cells, P<0.001, and 44 320±2650 vs 29 309±5320 cells, P<0.001, for 5 vs 30 mM D-glucose at 48 and 72 h, respectively; 46 120±5480 vs 29 870±1540 cells, P<0.001, and 44 320±2650 vs 23 500±5380 cells, P<0.001, for 5 mM D-glucose vs Dianeal at 48 and 72 h, respectively). Exposure to Nutrineal reduced HPMC attachment to 74.8±3.6% of control cells after 24 h (P<0.001), without further effects thereafter (Figure 4Go). Similar cell attachment was observed with Dianeal or Nutrineal after 48 h of exposure. Thereafter, cells exposed to Dianeal demonstrated reduced attachment.



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Fig. 4.  The effect of 30 mM D-glucose (open circles), spent Dianeal (filled circles), Nutrineal (filled squares) or 5 mM D-glucose (control, open squares) on HPMC attachment. HPMCs were exposed to these media for periods up to 72 h. At selective time points, cells were trypsinized, neutralized with M199 containing 10% FCS and counted. Data are expressed as the mean±SD. *P<0.001, compared with control at identical time points.

 
HPMCs pre-exposed to 30 mM D-glucose, Dianeal or Nutrineal for 24, 48 or 72 h were re-seeded into new culture wells at identical cell density (10 000/cm2), and allowed to recover in M199 supplemented with 10% FCS. The number of cells after 12 or 24 h of recovery did not differ among cells previously exposed to control or experimental conditions for up to 48 h (Figure 5Go, upper and middle panels). HPMCs pre-exposed to 30 mM D-glucose or Dianeal, but not Nutrineal, for 72 h showed significantly reduced number during recovery (P<0.001, P<0.0001 and P=0.947, respectively; Figure 5Go, lower panel). Mean cell doubling time was increased for HPMCs pre-exposed to 30 mM D-glucose or Dianeal for 48 or 72 h (Table 4Go, cell doubling times 52.5±3.4 vs 67.4±5.3 h, P<0.001, and 53.3±6.1 vs 75.3±8.3 h, P<0.001, for 5 vs 30 mM D-glucose after 48 and 72 h of pre-exposure, respectively; 52.5±3.4 vs 70.7±6.9 h, P<0.001, and 53.3±6.1 vs 90.2±11.0 h, P<0.001, for 5 mM D-glucose vs Dianeal after 48 and 72 h of pre-exposure, respectively).



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Fig. 5.  Assessment of cell proliferation and attachment after 12 (open squares) or 24 h (filled squares) of recovery, following prior incubation with 5 mM D-glucose, 30 mM D-glucose, spent Dianeal or Nutrineal for 24 (upper panel), 48 (middle panel) or 72 h (lower panel). The number of attached cells at recovery was significantly reduced after exposure to 30 mM D-glucose or spent Dianeal for 72 h. Data represent the mean±SD. *P<0.001 and **P<0.0001 compared with 5 mM D-glucose.

 

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Table 4.  Comparison of mean cell doubling time of HPMCs after exposure to control and experimental conditions

 

Secretion of IL-6 by HPMC
The combination of 30 mM D-glucose and spent 1.5% Dianeal significantly suppressed IL-6 secretion compared with 5 mM D-glucose (P<0.001 for 30 mM D-glucose vs control at all time points from 12 to 72 h, and P<0.0001 for Dianeal vs control at all time points from 12 to 72 h; Figure 6AGo). Nutrineal increased IL-6 secretion by HPMCs in a time-dependent manner beginning at 24 h (P<0.0001 for all subsequent time points). The secretion of IL-6 was predominantly basolateral (Figure 6BGo).



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Fig. 6.  IL-6 secretion by HPMCs after exposure to 5 mM D-glucose, 30 mM D-glucose, spent Dianeal or Nutrineal for 12–72 h. Data represent the mean±SD. In separate experiments, cells were cultured on PET inserts until confluent, and the apical (filled squares) or basolateral (open squares) IL-6 secretion was determined. Data represent the mean±SD. *P<0.001 and **P<0.0001 compared with 5 mM D-glucose.

 

Ultrastructural morphology of HPMC
HPMCs were seeded onto PET membranes or Thermanox coverslips, cultured under control or experimental conditions for 24 h, and examined by scanning electron microscopy. Preliminary experiments did not show significant shrinkage or disruption of the monolayer using either substratum (data not shown). Control cells cultured in M199 containing 5 mM D-glucose exhibited a heterogeneous population of predominantly cobblestone-like polygonal cells, with occasional cells showing a flattened appearance, which may represent cells at confluence or a proliferative phase, respectively (Figure 7AGo). Both types of cells demonstrated numerous microvilli on the cell surface. Cells exposed to either 30 mM D-glucose or Dianeal demonstrated flattening, resulting in an increase in two-dimensional surface area. Some cells also demonstrated prominent areas devoid of microvilli (Figure 7B and CGo). The normal morphology of HPMCs was preserved after incubation with Nutrineal (Figure 7DGo).



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Fig. 7.  Ultrastructural study of HPMCs by scanning electron microscopy. HPMCs were cultured on PET membranes in 5 mM D-glucose (A), 30 mM D-glucose (B), Dianeal (C) or Nutrineal (D) for 24 h, then fixed with 2.5% glutaraldehyde. Under 5 mM D-glucose, a heterogeneous population of HPMCs was observed, comprising a predominant population of polygonal cells (thin arrow) and a minor population of cells with a larger cell surface area (asterisk). All cells were covered with numerous microvilli. Cells cultured in either 30 mM D-glucose (B) or spent Dianeal PDF (C) adopted a more flattened appearance, with areas devoid of microvilli (thick arrows). In comparison, the morphology of HPMCs incubated with Nutrineal (D) was similar to that of control. Representative photomicrographs of three individual experiments using spent Dianeal and Nutrineal from patients receiving both PDFs. Horizontal bar=10 µm.

 
Transmission electron microscopic studies showed normal microvilli, mitochondria, rough endoplasmic reticulum and Golgi apparatus in HPMCs cultured under 5 mM D-glucose (Figure 8AGo). Numerous electron-dense and globular intracellular organelles with double membrane, compatible with dysfunctional mitochondria, were noted in cells exposed to 30 mM D-glucose or Dianeal (Figure 8B, C and EGo). HPMCs incubated with Nutrineal showed no significant difference in internal organelles compared with control cells (Figure 8DGo), except for the presence of irregularly shaped intracellular organelles with granulation and a single membrane, suggestive of lyzosomes (Figure 8FGo).



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Fig. 8.  Ultrastructural study of HPMCs by transmission electron microscopy under control (5 mM D-glucose, A) and experimental conditions [30 mM D-glucose (B); spent Dianeal (C and E); and Nutrineal (D and F)]. Organelles are identified as microvilli (M), nucleus (N), rough endoplasmic reticulum (RER), Golgi apparatus (G) and mitochondria (Mi). High-power magnification of the darkened, globular organelles induced by Dianeal (*) or Nutrineal revealed that the former possessed a double membrane (E, arrowhead), while the latter (**) exhibited a single-layered membrane and internal granulation (F). Original magnification x16 000 for (A–D), x92 000 for (E and F). Representative photomicrographs of three individual experiments using spent Dianeal and Nutrineal from patients receiving both PDFs.

 
The salient findings with regard to the effects of the different PDFs and glucose on HPMCs are summarized in Table 5Go.


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Table 5.  Summary of ultrastructural and functional alterations in HPMCs after exposure to 5 mM D-glucose (control), 30 mM D-glucose, spent 1.5% Dianeal or 1.1% Nutrineal

 



   Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Progressive deterioration of peritoneal transport function is a major hindrance to favourable clinical outcome in long-term PD. Such functional changes are accompanied by structural alterations of the peritoneal membrane, which include reduplication of the basement membrane, denudation of the mesothelium, deposition and accumulation of matrix proteins and modulation of the peritoneal vasculature [1]. The mesothelium represents the interface between the dialysate compartment and the submesothelial structures of the peritoneal membrane. Not only does the mesothelial monolayer serve as a mechanical barrier protecting the underlying elements from the unphysiological PDF, it also plays an active role in the inflammatory and synthetic responses upon stimulation by noxious stimuli. There is increasing evidence that perturbations in HPMC structure and function play a pivotal role in the pathogenesis of abnormalities in the peritoneal membrane during long-term PD. In this context, data to date suggest that the loss or degeneration of the mesothelium and modulation of cytokine and growth factor secretion by HPMCs are important steps in initiating thickening of the submesothelium and vasculopathy, both of which compromise peritoneal transport functions [1].

There are minimal data on the effects of amino acid-based PDF on HPMC ultrastructure and function compared with conventional dextrose-based dialysate. Since many of the detrimental cellular effects of Dianeal have been attributed to glucose, we have included glucose at 30 mM for comparison. This concentration represents the intraperitoneal glucose concentration after equilibration of the dextrose-based PDF (see Table 3Go). Our data showed that HPMC ultrastructure was altered similarly after exposure to Dianeal or 30 mM D-glucose, which included cell enlargement, flattening and a reduction of microvilli. Giant mesothelial cells have been isolated from spent dextrose-based PDFs, and defective mitosis has been implicated [12]. The reduction of microvilli could contribute to alterations in peritoneal transport properties and increased susceptibility to frictional injury in long-term PD. Mitochondria are sites of ATP synthesis and a source of energy for various cell processes. A possible mechanism leading to the adverse effects of Dianeal or 30 mM D-glucose on HPMC proliferation, viability and function could be altered mitochondrial structure and/or function, as corroborated by the finding of dysfunctional mitochondria using transmission electron microscopy. Recent studies have shown that GDPs in dextrose-based PDF could also exert detrimental effects on cell viability [3].

Previous in vitro biocompatibility studies have demonstrated adverse effects of neat dextrose-based PDF or its constituents on HPMC proliferation, membrane integrity and IL-6 secretion [24]. Our data showed that these detrimental effects persisted even after intraperitoneal equilibration of 1.5% Dianeal for several hours. Thus, it is inappropriate to presume that the bio-incompatibility and unphysiological effects of neat dextrose-based PDF can be ‘neutralized’ within 30–45 min of equilibration with the intravascular milieu. Exposure of HPMCs to spent Nutrineal did not induce LDH release, alter protein synthesis or affect cell attachment. While Breborowicz et al. had reported cytotoxic effects with individual amino acids on peritoneal mesothelial cells [13], our results were in agreement with previous studies using neat amino acid-based PDF [4,5,14].

The differential effects of various PDFs on IL-6 secretion by HPMCs is of interest. Our results showed that IL-6 secretion was reduced by Dianeal but increased by Nutrineal. Furthermore, IL-6 secretion by HPMCs was predominantly basolateral. Polarized secretion of cytokines, chemokines and growth factors has been documented in peritoneal and pleural mesothelial cells, endothelial and epithelial cells [1517]. Whilst Li et al. concluded that the apical secretion of RANTES, monocyte chemoattractant protein-1 (MCP-1) and IL-8 by HPMCs could provide a chemotactic gradient for the recruitment of leukocytes to areas of inflammation [15], other researchers have demonstrated basolateral secretion of IL-6, hepatocyte growth factor (HGF), vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF) in colorectal carcinoma cells, intestinal epithelial cells and retinal epithelial cells [16,17]. The basolateral secretion of IL-6 suggests its involvement in biological processes within the basal lamina and submesothelium. Sequestration of growth factors such as bFGF and transforming growth factor-ß1 (TGF-ß1) by proteoglycans in the extracellular milieu can protect them from proteolysis. Whether IL-6 secreted basolaterally into the subcellular matrix could be sequestered similarly remains to be investigated. IL-6 has often been regarded as a pro-inflammatory cytokine, inducible by lipopolysaccharide (LPS), tumour necrosis factor-{alpha} (TNF-{alpha}) or IL-1ß, and the level of IL-6 in peritoneal fluid has been used as an indicator of peritoneal inflammation. It is prudent to note, however, that IL-6 has also been demonstrated to possess anti-inflammatory properties such as the down-regulation of inflammatory mediators TNF-{alpha}, IL-1ß, interferon-{gamma} or granulocyte–macrophage colony-stimulating factor (GM-CSF) [18]. The exact role of IL-6 in the peritoneal membrane is therefore complex, and might vary according to the milieu of cytokines and growth factors. However, given that Nutrineal preserved the morphological and functional properties of HPMCs compared with Dianeal, we hypothesize that IL-6 in this instance acted as an anti-inflammatory mediator. Studies are on-going to confirm this. The peritoneum in many patients on long-term PD manifests variable degrees of chronic inflammation. Whether the latter could be modulated by Nutrineal can only be elucidated by histological studies.

Denudation of the peritoneal mesothelium, a feature commonly observed in long-term PD, is related to the inability of mesothelial cells to attach to their substratum. In this context, our results demonstrated progressive cell detachment with increasing exposure time of HPMCs to Dianeal, resulting in denuded areas in the mesothelial monolayer. This impaired ability to attach to the substratum appeared initially to be a transient phenomenon which was potentially reversible, since when HPMCs were allowed to recover in M199 they exhibited attachment similar to control cells, but only when their prior exposure time to Dianeal was <48 h. The initial capability of Dianeal-treated HPMCs to recover their normal properties was corroborated by the cell doubling time studies, which showed that HPMCs exposed to Dianeal for short durations had cell doubling times comparable with control cells upon recovery. Integrins play a pivotal role in cell–cell and cell–extracellular matrix interaction. HPMCs synthesize {alpha}2ß1, {alpha}3ß1, {alpha}5, {alpha}6 and {alpha}v integrins [19,20]. Integrin-mediated cell attachment can be modulated by mechanical or chemical stimuli, through structural and/or functional changes in integrin synthesis. While excessive synthesis and deposition of matrix components in the peritoneal membrane upon exposure to high glucose concentrations have been demonstrated [8], the effect of PD on peritoneal integrin synthesis remains to be investigated.

In conclusion, we have demonstrated less severe perturbation of HPMC ultrastructure, proliferation, viability and cell attachment with amino acid-based PDF compared with conventional dextrose-based PDF. Given that spent dialysates differed only in their glucose concentration (28.15±8.43 vs 8.30±1.38 mM, P<0.0001; Table 3Go), it is likely that elevated glucose per se initiated cell injury. HPMCs cultured with amino acid-based PDF also resulted in higher IL-6 secretion, indicative of improved synthetic capacity of the cells. It is unlikely that these results could be related to inter-individual differences, since serial samples from patients who had been treated with both Dianeal and Nutrineal showed consistent observations (Table 6Go). In view of the implications in the preservation of peritoneal structure and function, long-term studies to investigate the different effects on peritoneal structure and function exerted by dextrose-based or amino acid-based PDFs are indicated.


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Table 6.  Comparison of mesothelial functional properties after exposure to spent dialysates obtained from patients who received both Dianeal and Nutrieal PDFs (n=5)

 



   Acknowledgments
 
The authors thank Drs Kent-Man Chu, Kin-Wah Chu, Sai-Man Chu, Judy Ho and other members of their surgical teams for the collection of omentum specimens. We are indebted to Ms Sui-Ling Wong and Mr Wing-Sang Lee for their technical assistance in scanning and transmission electron microscopy, and to Mr Colin Tang for statistical assistance. We would also like to thank Dr Fu-Keung Li, Dr Terence Yip, Miss Loretta Chan and the nurses of the renal division for the collection of peritoneal dialysis effluents. This study was supported by the Hong Kong Research Grants Council Earmarked Research Grant (7274/98M) and CRCG grants (10202781 and 10204234).

Conflict of interest statement. None declared.



   Notes
 
Correspondence and offprint requests to: Professor Tak-Mao Chan, Department of Medicine, The University of Hong Kong, Queen Mary Hospital, Pokfulam Road, Hong Kong SAR, China. Email: dtmchan{at}hkucc.hku.hk Back

S. Yung and T. M. Chan contributed equally to this work Back



   References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 

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Received for publication: 12.10.02
Accepted in revised form: 17. 1.03





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