In vivo evaluations of the protective effects of new formulations for peritoneal dialysis adopting a powerful multi-photonmicroscopy based approach

Abstract Background and Aims Peritoneal dialysis (PD) is a renal replacement therapy that enables metabolic waste products and excess fluids to be eliminated through the peritoneal membrane. Exposure to conventional dialysates at high glucose concentrations is considered critical for the pathogenesis of peritoneal fibrosis, angiogenesis, and epithelial-mesenchymal transition (EMT). These events largely contribute to peritoneal membrane aging, resulting in ultrafiltration and clearance failure. Pre-clinical research in this field suffers from the lack of valid and reproducible in vivo systems and so far has been limited to in vitro systems of mesothelial or endothelial cell lines. In vitro studies on mesothelial cell monolayers have validated the significant protective effects of dialysates with alternative osmo-metabolic agents. With their better biocompatibility these novel formulations could provide a valid substitute for conventional solutions (1,2). The method proposed, based on multi-photon microscopy, aims to study the physiology of the peritoneal membrane during dialysis exchange and to validate the effects of biocompatible dialysates in animal models of fibrosis as observed in the course of dialysis treatment. Method We have implemented a surgical procedure to optimize the stability of a flap of parietal peritoneum to allow direct microscope observation. The innovative technology of multi-photon microscopy enables one to observe the vessels of microcirculation in vivo and provides a 3D rendering of the peritoneal membrane, giving an overview of all the single layers without the use of specific markers (Figure 1). One may also assess specific phenomena induced by the fibrotic process, such as the thickening of the sub-mesothelial interstitium, exploiting the autofluorescence signal from excited collagen fibers caused by the physical phenomenon of Second Harmonic generation. In vivo microscopy evaluation of peritoneal membrane senescence parameters was conducted in rats subjected to one daily intraperitoneal injection (10 mL/day) for 15 days with one of the following PD solutions: a commercially available glucose-based neutral-pH, low-GDP, PD solution (Physioneal 3.86%, Baxter Healthcare, Italy), and a new single-chamber low glucose PD solution containing osmo-metabolic agents such as L-carnitine and xylitol (XyloCore HS, Galenica Senese, Italy). XyloCore HS is a lactate-buffered PD solution with glucose (1.5%), L-carnitine (0.02%) and xylitol (2.0%). The osmotic strength of the two PD solutions tested was comparable. Results Treatment with XyloCore HS was associated with a significant reduction in thickness of the sub-mesothelial interstitium (p = 0.013), the density of collagen fibers (p = 0.012) and the vascular composition (p = 0.006), as well as the number of branch points (p = 0.0335), when compared to rats treated with a commercial glucose-based PD solution, Physioneal. Figure 2 shows the different thicknesses of sub-mesothelial stroma upon treatment with the PD solutions under investigation. Conclusion Previous in vitro studies have shown that XyloCore (1,2), a novel glucose-sparing PD solution currently in Phase III clinical development (ELIXIR - NCT03994471), is able to counteract the glucotoxic effects on the peritoneal membrane induced by conventional dialysates. Our pre-clinical in vivo methodology not only confirms our previous in vitro findings, but also suggests that long-term protective effects may be achieved with XyloCore. Further studies are in progress in a diabetic animal model to extend the favorable peritoneal in vivo findings of XyloCore treatment to a systemic level, possibly by improving glycemic control.