Hyperbranched polyglycerol is superior to glucose for long-term preservation of peritoneal membrane in a rat model of chronic peritoneal dialysis

Abstract

Background: Replacing glucose with a better biocompatible osmotic agent in peritoneal dialysis (PD) solutions is needed in PD clinic. We previously demonstrated the potential of hyperbranched polyglycerol (HPG) as a replacement for glucose. This study further investigated the long-term effects of chronic exposure to HPG as compared to a glucose-based conventional PD solution on peritoneal membrane (PM) structure and function in rats.

Methods: Adult male Wistar rats received once-daily intraperitoneal injection of 10 mL of HPG solution (1 kDa, HPG 6%) compared to Physioneal™ 40 (PYS, glucose 2.27%) or electrolyte solution (Control) for 3 months. The overall health conditions were determined by blood chemistry analysis. The PM function was determined by ultrafiltration, and its injury by histological and transcriptome-based pathway analyses.

Results: Here, we showed that there was no difference in the blood chemistry between rats receiving the HPG and the Control, while PYS increased serum alkaline phosphatase, globulin and creatinine and decreased serum albumin. Unlike PYS, HPG did not significantly attenuate PM function, which was associated with smaller change in both the structure and the angiogenesis of the PM and less cells expressing vascular endothelial growth factor, α-smooth muscle actin and MAC387 (macrophage marker). The pathway analysis revealed that there were more inflammatory signaling pathways functioning in the PM of PYS group than those of HPG or Control, which included the signaling for cytokine production in both macrophages and T cells, interleukin (IL)-6, IL-10, Toll-like receptors, triggering receptor expressed on myeloid cells 1 and high mobility group box 1.

Conclusions: The results from this experimental study indicate the superiority of HPG to glucose in the preservation of the peritoneum function and structure during the long-term PD treatment, suggesting the potential of HPG as a novel osmotic agent for PD.

Keywords: Biocompatibility; Hyperbranched polyglycerol; Long-term PD; PD solution; Peritoneal membrane.

Fig. 1

The chemical changes in the blood of rats after 3 months of intraperitoneal injection of PD solutions. Male Wistar rats received once-daily intraperitoneal injection of Control (n = 7), PYS (n = 7) or HPG (n = 8), or needle puncture as Sham Control (n = 3). The blood chemistry test was performed three times during 3-month period of treatment (once at the end of each month). a Creatinine (Cre) (PYS vs. Control: p = 0.0231, HPG vs. Control: p = 0.8439, HPG vs. PYS: p = 0.0269). b Alkaline phosphate (ALP) (PYS vs. Control: p = 0.0074, HPG vs. Control: p = 0.4325, HPG vs. PYS: p = 0.0204). c Globulin (Glob) (PYS vs. Control: p = 0.0141, HPG vs. Control: p = 0.7534, HPG vs. PYS: p = 0.0171). d Albumin (ALB) (PYS vs. Control: p = 0.003, HPG vs. Control: p = 0.7796, HPG vs. PYS: p = 0.0403). e ALB/Glob ratio (PYS vs. Control: p = 0.0022, HPG vs. Control: p = 0.5636, HPG vs. PYS: p = 0.0114). Data were presented as mean ± standard derivation (SD) of each group, and were statistically analyzed using two-way ANOVA

Fig. 2

HPG has better preservation of peritoneal membrane function. The ultrafiltration as a parameter of peritoneal function was measured after 3 months of daily exposure of peritoneal cavity to the PD solutions. Each rat received intraperitoneal injection of 30 mL of PYS. After 4 h of dwell time, the fluid was recovered from the peritoneal cavity using a syringe, and its volume was measured. Data were presented as mean ± SD of each group (Sham: n = 3; Control: n = 7; PYS: n = 7; HPG: n = 8), and were statistically analyzed using two-tailed t test

Fig. 3

HPG has better protection of peritoneal membrane structure. At the end of 3-month treatment, peritoneal tissue sections (2 sections/rat) were taken from rats (Sham: n = 3; Control: n = 7; PYS: n = 7; HPG: n = 8) after 4 h of PYS exposure for UF test. a A typical microscopic image of the peritoneal tissue sections in each group, and were stained using either H&E (top panel) or Masson’s trichrome method (bottom panel). H&E stain showing the thickness of the peritoneal membrane indicated by the distance between two arrows. Nuclear dark blue stain cellular infiltrates; blue arrows blood vessels; black thin bar 100 µm. Masson’s trichrome stain showing collagen deposition (dark blue). b The thickness of submesothelial layer in each tissue section was measured using the Digital Image Hub software. Data were presented as mean ± SD of each group, and were statistically analyzed using two-tailed t test. c Angiogenesis in the peritoneal membrane was represented by the number of the blood vessels and capillaries per millimeter. Data were presented as mean ± SD of each group, and were statistically analyzed using two-tailed t test

Fig. 4

HPG induces less VEGF production, less myofibroblast differentiation and lower macrophage activation. The expression of VEGF, α-SMA and MAC387 in the peritoneal tissue sections was examined using a routine immunohistochemical method. Data were a typical microscopic view of the peritoneal tissue sections in each group. a VEGF was detected using mouse monoclonal anti-VEGF antibody from Novus. Left graph a typical microscopic view, Dark brown stain VEGF-expressing cells (pointed by red arrows), Bv blood vessels, M muscle, black small bar 10 µm. Right graph VEGF-expressing cells per 200 μm PM length in cross sections. Data were presented as mean ± SD (n = 6) and were analyzed using t test. b α-SMA (a myofibroblast marker) was detected using mouse monoclonal anti-α-SMA antibody from Sigma-Aldrich. Left graph a typical microscopic view, Dark brown stain α-SMA-expressing cells or myofibroblasts (pointed by red arrows), Bv blood vessels, M muscle, black small bar 10 µm. Right graph α-SMA-expressing cells per 200 μm PM length in cross sections. Data were presented as mean ± SD (n = 6) and were analyzed using t test. c Macrophages were detected using mouse monoclonal anti-MAC387 antibody from Santa Cruz Biotech. Left graph a typical microscopic view, Dark brown stain MAC387-expressing cells or macrophages (pointed by red arrows), M muscle, black small bar 10 µm. Right graph MAC387-expressing cells per 200 μm PM length in cross sections. Data were presented as mean ± SD (n = 6) and were analyzed using t test

Fig. 5

Changes in transcriptome in the PM of rats receiving daily i.p. injection of electrolyte Control, PYS or HPG solution. Venn diagram analysis of microarray data (four separate samples in each group, n = 4; one rat in Sham provided two samples) of Control, PYS or HPG was performed as compared to those of Sham or of PYS or HPG as compared to Control using the Agilent Gene Spring software. Only the transcripts that were significantly changed (p ≤ 0.05; FC ≥ 2.0) as compared to Sham or Control group were included and presented in this analysis. Top panel The changes of gene expression in Control, PYS or HPG using Sham as a reference. Bottom panel The change of gene expression in PYS or HPG using Control as a reference. Positive FC up-regulated, negative FC down-regulated