Dissolved molecular hydrogen (H2) in Peritoneal Dialysis (PD) solutions preserves mesothelial cells and peritoneal membrane integrity

Abstract

Background: Peritoneal dialysis (PD) is used as renal replacement therapy in patients with end-stage kidney disease. However, peritoneal membrane failure remains problematic and constitutes a critical cause of PD discontinuation. Recent studies have revealed the unique biological action of molecular hydrogen (H₂) as an anti-oxidant, which ameliorates tissue injury. In the present study, we aimed to examine the effects of H₂ on the peritoneal membrane of experimental PD rats.

Method: Eight-week-old male Sprague-Dawley rats were divided into the following groups (n = 8-11 each) receiving different test solutions: control group (no treatment), PD group (commercially available lactate-based neutral 2.5% glucose PD solution), and H₂PD group (PD solution with dissolved H₂ at 400 ppb). Furthermore, the influence of iron (FeCl₃: 5 μM: inducer of oxidative cellular injury) in the respective PD solutions was also examined (Fe-PD and Fe-H₂PD groups). The H₂PD solution was manufactured by bathing a PD bag in H₂-oversaturated water created by electrolysis of the water. Twenty mL of the test solutions were intraperitoneally injected once a day for 10 days. Parietal peritoneum samples and cells collected from the peritoneal surface following treatment with trypsin were subjected to analysis.

Results: In the PD group as compared to controls, a mild but significant sub-mesothelial thickening was observed, with increase in the number of cells in the peritoneal surface tissue that were positive for apoptosis, proliferation and vimentin, as seen by immunostaining. There were significantly fewer of such changes in the H₂PD group, in which there was a dominant presence of M2 (CD163+) macrophages in the peritoneum. The Fe-PD group showed a significant loss of mesothelial cells with sub-mesothelial thickening, these changes being ameliorated in the Fe-H₂PD group.

Conclusion: H₂-dissolved PD solutions could preserve mesothelial cells and peritoneal membrane integrity in PD rats. Clinical application of H₂ in PD could be a novel strategy for protection of peritoneal tissue during PD treatment.

Keywords: Biocompatibility; Electrolyzed water; Macrophage; Mesothelial cell; Molecular hydrogen; PD solution.

Fig. 1

Procedure for producing H₂-dissolved peritoneal dialysis solution by the bathing method. a Time-course of changes in dissolved H₂ levels in electrolyzed water (*) and the solution in the PD bag (#). b Time-course of changes in dissolved H₂ levels in the solution in the PD bag when placed in air. Water was electrolyzed by Nafion (Synthetic polymer membrane; DuPont, Wilmington, DE) to generate highly dissolved H₂ water, with the concentration of H₂ exceeding 1.5 ppm. Then, the PD bag was bathed in the water to allow H₂ to shift into the PD solution by diffusion (a). Dissolved hydrogen is lost rapidly after loading once it is exposed to room air (b). Thus, the H₂-dissolved PD solution was subjected to experiments within 1 h of 12-h bathing, to ensure a high level of H₂ in the PD solution (>400 ppm)

Fig. 2

Representative histological findings in the peritoneum: Masson and immunohistochemistry staining in PD and H₂PD groups. Mesenchymal marker: vimentin; proliferative marker: Ki-67; apoptosis marker: M30 CytoDeath; total macrophage marker: CD68+; M1 macrophage: CD80+; M2 macrophage: CD163+

Fig. 3

Quantitative analysis of peritoneal thickness and immunohistochemical staining of the peritoneum in PD and H₂PD groups. Peritoneal thickness (a), and immunostainings of mesenchymal marker: vimentin (b), proliferative marker: Ki-67 (c), apoptosis marker: M30 CytoDeath (d), and total macrophage marker: CD68+, M1 macrophage: CD80+, and M2 macrophage: CD163+ (e), and ratio of M1 /M2 (positive cells number per field) (f). * p < 0.05

Fig. 4

Results of real-time PCR of peritoneal cells in PD and H₂PD groups. * p < 0.05 vs. control VEGF: Vascular endothelial growth factor, BCL2: B-cell lymphoma 2, αSMA: Smooth muscle actin A, BAX: BCL-2-like protein 4, BAD: BCL-2-associated death promoter

Fig. 5

Representative DNA Agilent array of peritoneal cells. Relative differences in representative gene expressions between H₂PD and PD groups

Fig. 6

Comparison of Fe-PD and Fe-H₂PD groups. Representative findings of Masson and immunohistochemical staining (CD68) (a), and quantitative analysis of peritoneal morphology and immunohistochemical staining of the peritoneum in the respective groups (b-g) are shown. Peritoneal thickness (b), ratio of shed cells in the peritoneal surface (c), immunostainings of mesenchymal marker: vimentin (d), apoptosis marker: M30 CytoDeath (e), proliferative marker: Ki-67 (f), and total macrophage marker: CD68+, M1 macrophage: CD80+, and M2 macrophage: CD163+ (e), respectively. * p < 0.05