A NEW IN VIVO MULTI-PHOTON MICROSCOPY BASED APPROACH TO STUDY THE PERITONEAL MEMBRANE CHANGES INDUCED BY PERITONEAL DIALYSIS

Abstract BACKGROUND AND AIMS Peritoneal dialysis (PD) is a renal replacement therapy that allows the elimination of metabolic waste products and excess body fluid through the peritoneal membrane. The exposure to PD solution contributes to membrane aging and fibrosis resulting in ultrafiltration and clearance failure. The glucose, used as an osmotic factor, in PD dialysate triggers several processes involved in the pathogenesis of peritoneal fibrosis, angiogenesis and epithelial to mesenchymal transition. Studies on natural extracts, such as Oleuropein (Ole), a powerful antioxidant with remarkable antifibrotic and protective effects on the peritoneal membrane, are currently being validated. This study aims to develop a method based on multi-photon microscopy to study the physiology of the peritoneal membrane during dialysis exchange and to validate in vivo the effects of the Ole in animal models of fibrosis undergoing dialysis treatment. METHOD Multi-photon microscopy allows in vivo evaluation of the microcirculation that supply the peritoneum and also to study the framework of mesothelial cells and their underlying layer of collagen fibers that contributes to the sub-mesothelial space. We have implemented the surgical procedure in order to optimize the stability of a flap of parietal peritoneum to directly observe at the scope. With this approach the peritoneal membrane is evaluable at baseline condition and during exposure to dialysate solutions. RESULTS Our method allows building a three-dimensional render of the peritoneal membrane, with the evaluation of all the single layers without the use of specific markers. In this way we could assess specifically the phenomena induced by the fibrotic process: the thickening of the sub-mesothelial interstitium and the greater density of the vascular network. Furthermore, in vivo measurements of flow in the vessels of microcirculation (arterioles, capillaries, post-capillary venules) determine that exposure to hypotonic solutions increases significantly the flow in large-diameter vessels due to better permeability and hemodilution. Finally in order to evaluate the possibility to detect morphological and functional changes in pathogenic model we have used the well-established model of peritoneal fibrosis derived by 15 days’ long exposure of the peritoneal membrane with 3.86% glucose dialysate. Whth this approach we could detect the significant increase in the parameters of cellularity, vascularization, fibrosis and thickening of collagen fibers. In order to test the sensibility of our approach to the evaluation of the peritoneal membrane senescence parameters, we tested the effect of Ole in preventing the damage induced by high glucose–containing dialysate. Ole reduced both the thickness and the organization of the collagen fibers and the vascular network, including the number of branch points. CONCLUSION The developed method has potential for a dynamic and reliable in vivo approach. Studies are ongoing to validate the effects of drugs and dialysates with different osmotic and electrolytic compositions on the dialysis capacities of the peritoneal membrane and on the blood flow in peritoneal capillaries. This method offers great potential for testing new pharmacological approaches aimed at preserving the structural and functional integrity of the peritoneum and for the validation of substances, such as natural extracts with beneficial effects against the damage induced in the long term by conventional dialysis solutions.