A Novel Formulation of Glucose-Sparing Peritoneal Dialysis Solutions with l-Carnitine Improves Biocompatibility on Human Mesothelial Cells

Abstract

The main reason why peritoneal dialysis (PD) still has limited use in the management of patients with end-stage renal disease (ESRD) lies in the fact that the currently used glucose-based PD solutions are not completely biocompatible and determine, over time, the degeneration of the peritoneal membrane (PM) and consequent loss of ultrafiltration (UF). Here we evaluated the biocompatibility of a novel formulation of dialytic solutions, in which a substantial amount of glucose is replaced by two osmometabolic agents, xylitol and l-carnitine. The effect of this novel formulation on cell viability, the integrity of the mesothelial barrier and secretion of pro-inflammatory cytokines was evaluated on human mesothelial cells grown on cell culture inserts and exposed to the PD solution only at the apical side, mimicking the condition of a PD dwell. The results were compared to those obtained after exposure to a panel of dialytic solutions commonly used in clinical practice. We report here compelling evidence that this novel formulation shows better performance in terms of higher cell viability, better preservation of the integrity of the mesothelial layer and reduced release of pro-inflammatory cytokines. This new formulation could represent a step forward towards obtaining PD solutions with high biocompatibility.

Keywords: glucose-sparing; l-carnitine; mesothelium; peritoneal dialysis; peritoneal dialysis solution; xylitol.

Figure 1

Effects of PD solutions on cell viability and TEER in HMC. Cells were exposed to low, medium and high (L, M, H) osmolarity glucose-based solutions (Physioneal 40^®, Dianeal PD4^®, BicaVera^®), to glucose-free solutions (Extraneal^®, Nutrineal^®), and to low, medium and high (L, M, H) osmolarity glucose-sparing XyloCore solutions (± l-carnitine) for 8 h. (a) MTT assay was performed to evaluate cell viability. Cells incubated with Prigrow I medium were used as a control, and their cell viability set to 100%. Staurosporine (5 µM) served as a negative control of cell viability. (b) TEER was measured on each monolayer before and after incubation with PD solutions. Reductions of TEER (ΔTEER) were expressed as % of the value measured before exposure to the PD solutions. Significance was analyzed using a one-way ANOVA. Values are expressed as mean ± SEM. (n = 5). * p < 0.05; ** p < 0.01; *** p < 0.001 indicate statistical significance respect to control cells by one-way analysis of variance (ANOVA) with Dunnett’s multiple comparison test; # p < 0.05; ## p < 0.01; ### p < 0.001 values calculated by one-way analysis of variance (ANOVA) with Sidak’s multiple comparisons test, indicating how much the results differ within the same formulation as the glucose concentration varies. § p < 0.05; §§ p < 0.01; §§§ p < 0.001; values calculated by one-way analysis of variance (ANOVA) with Sidak’s multiple comparisons test, indicating how much the results differ from other treatment with the same concentration of glucose and same osmolarity. $ p < 0.05 values calculated in HMC incubated ± l-carnitine by t-test.

Figure 2

Immunofluorescence analysis of tight junction marker Zo-1 in HMC, grown on permeable supports and exposed for 8 h to different PD solutions at the apical side. HMC monolayers grown on permeable supports were incubated for 8 h at the apical side with high osmolarity glucose-based PD solutions (Physioneal 40^®, Dianeal PD4^® and BicaVera^®), glucose-free Extraneal^®/Nutrineal^® and high osmolarity glucose-sparing PD solutions (± l-carnitine). Cells were immunostained with anti Zo-1 antibodies (green) and scanned by laser confocal microscopy. Nuclei were stained with propidium iodide (red). Scale bars = 10 μm.

Figure 3

Cytokines, chemokines and growth factors released from the apical side by HMC incubated with high osmolarity PD solutions. A heatmap shows the result of a quantitative 27-plex Luminex assay. The concentration (pg/ml) increases as the color changes from red to violet.