Alanyl-Glutamine Restores Tight Junction Organization after Disruption by a Conventional Peritoneal Dialysis Fluid

Abstract

Understanding and targeting the molecular basis of peritoneal solute and protein transport is essential to improve peritoneal dialysis (PD) efficacy and patient outcome. Supplementation of PD fluids (PDF) with alanyl-glutamine (AlaGln) increased small solute transport and reduced peritoneal protein loss in a recent clinical trial. Transepithelial resistance and 10 kDa and 70 kDa dextran transport were measured in primary human endothelial cells (HUVEC) exposed to conventional acidic, glucose degradation products (GDP) containing PDF (CPDF) and to low GDP containing PDF (LPDF) with and without AlaGln. Zonula occludens-1 (ZO-1) and claudin-5 were quantified by Western blot and immunofluorescence and in mice exposed to saline and CPDF for 7 weeks by digital imaging analyses. Spatial clustering of ZO-1 molecules was assessed by single molecule localization microscopy. AlaGln increased transepithelial resistance, and in CPDF exposed HUVEC decreased dextran transport rates and preserved claudin-5 and ZO-1 abundance. Endothelial clustering of membrane bound ZO-1 was higher in CPDF supplemented with AlaGln. In mice, arteriolar endothelial claudin-5 was reduced in CPDF, but restored with AlaGln, while mesothelial claudin-5 abundance was unchanged. AlaGln supplementation seals the peritoneal endothelial barrier, and when supplemented to conventional PD fluid increases claudin-5 and ZO-1 abundance and clustering of ZO-1 in the endothelial cell membrane.

Peritoneal Dialysis Fluid Supplementation with Alanyl-Glutamine Attenuates Conventional Dialysis Fluid-Mediated Endothelial Cell Injury by Restoring Perturbed Cytoprotective Responses

“…Although the molecular mechanisms by which AlaGln exerts its cytoprotective actions are not yet fully elucidated, this study corroborates recent data on endothelial specific peritoneal structural alterations, including cytoskeletal and cellular junction rearrangement as potential pathways influenced by AlaGln to maintain peritoneal barrier function [54].…”

Mechanisms of Peritoneal Fibrosis: Focus on Immune Cells–Peritoneal Stroma Interactions

“…A recent discovery is the immunomodulatory effect of alanyl-glutamine (AlaGln) supplementation in PD solutions. This treatment seems to ameliorate peritoneal inflammation status and to improve healthy peritoneum biomarkers as well as tight junction organization and functionality ( 64 , 65 ).…”

An Experimental Workflow for Studying Barrier Integrity, Permeability, and Tight Junction Composition and Localization in a Single Endothelial Cell Monolayer: Proof of Concept

“…In most cases, these data have been produced when the monolayers are subjected to transmission electron microscopy (TEM) or freeze fracture electron microscopy (FFEM) in order to visualize the ultrastructural morphological changes in the interface of adjacent cells with high resolution, but in this case an evaluation of specific TJ proteins is not possible as a totally different sample preparation procedure is required [ 23 , 24 ]. Recent advances in single molecule localization microscopy (SMLM) have provided opportunities for a more in-depth assessment of TJ changes with high spatial resolution as recently illustrated in studies involving ZO-1 in human endothelial cells [ 25 ]. This approach allows for the use of monolayers grown in filters after being subjected to TER, dextran leakage measurements, and immunostaining for specific TJ molecules [ 25 , 26 ].…”

“…Recent advances in single molecule localization microscopy (SMLM) have provided opportunities for a more in-depth assessment of TJ changes with high spatial resolution as recently illustrated in studies involving ZO-1 in human endothelial cells [ 25 ]. This approach allows for the use of monolayers grown in filters after being subjected to TER, dextran leakage measurements, and immunostaining for specific TJ molecules [ 25 , 26 ]. Combining these methods in one experimental setting should circumvent inter-experimental variations and allow for combined analyses of specific TJ expression and localization, functional studies, and single molecule cluster analyses in a matched manner.…”

“…In this paper we provide an experimental workflow that combines functional studies (TER and dextran permeability measurements) with immunofluorescence and with SMLM probing for ZO-1 and CLDN5 in the same human endothelial cell monolayer. To provide proof of the concept, we treated the endothelial cells with Alanyl-Glutamine (AlaGln), a dipeptide that tightens the paracellular barrier [ 25 ].…”