MO683EXPRESSION OF PARACELLULAR JUNCTION COMPONENTS AND TRANSCELLULAR TRANSPORTERS IN HEALTH, CKD5 AND PERITONEAL DIALYSIS
Authors: Marinovic Iva , Bartosova Maria , Lévai Eszter , Ridinger David , Schaefer Betti , Zhang Conghui , Herzog Rebecca , Sallay Peter , Vondrak Karel , Bestvater Felix , Hausmann Michael , Kratochwill Klaus , Zarogiannis Sotirios G , Schmitt Claus 
Published: May 2021

Abstract

Background and Aims
Tight junction (TJ) proteins have been suggested as molecular correlates for peritoneal semi-permeability and dialytic transport function in patients on peritoneal dialysis. Junction abundance in healthy individuals, in those with CKD5 and in patients on PD has not been described yet, the relation with peritoneal solute transport is unknown.

Method
Junction and transporter expression was analysed in multi-omics data sets from microdissected omental arterioles in children with normal renal function, CKD5 and on PD with low and high glucose degradation product (GDP) content (n=6/group). Parietal peritoneal tight junction proteins CLDN-1,-2,-3,-4,-5,-15, the adapter protein of claudins to actin cytoskeleton protein, zonula occludens-1 (ZO-1), the tricellular junction protein tricellulin (TriC), and transcellular transporters for sodium (ENaC), glucose (SGLT-1) and phosphate (PIT-1) were quantified in 40 non-CKD individuals, 20 children with CKD5 and 20 and 15 children on low- and high-GDP PD by quantitative, digital immunohistochemistry. Findings were correlated to 2-hour peritoneal equilibration test data obtained within 6 months of biopsy sampling (n=23). Primary human umbilical vein endothelial cells (HUVEC) were used to study the effects of single PD compounds on transepithelial electrical resistance (TER) and molecular size-dependent paracellular transport capacity. Co-stained monolayers were visualized by confocal microscopy. Single junction molecule localization and clustering were analysed by super resolution microscopy.

Results
Transcriptome and proteome pathway enrichment analysis of arteriolar junction and membrane protein demonstrated regulation in CKD5 versus health, and differential regulation by low- and high-GDP PD versus CKD5. In the parietal peritoneum all junctions and cellular transporters were expressed in endothelial and mesothelial cells. Pore forming CLDN-2, -4 and -15 were localized also in submesothelial immune cells. Parietal peritoneal junction abundance was age-dependent and also modified by CKD5 and PD. Mesothelial and endothelial abundance of the selective cation/water channel CLDN-2 increased in patients on low- and high-GDP PD fluids. Adaptor protein ZO-1 was upregulated in low GDP-PD versus CKD5, while sealing proteins CLDN -3 and -5 were downregulated. D/P creatinine, D/P phosphate, D/D0 glucose were similar in CKD5 and PD groups. D/P creatinine correlated with mesothelial CLDN-15, with arteriolar CLDN-2 and TriC and with endothelial ENaC. D/P phosphate correlated with endothelial CLDN-15, D/D0 glucose with mesothelial CLDN-4 and arteriolar CLDN-2. Capillary ZO-1 correlated with 24-h ultrafiltration standardized to body surface area and dialytic glucose exposure. In vitro, TER was decreased by low pH, glucose and 0.5µM methylglyoxal after 5h. Alanyl-glutamine (AlaGln) dose-dependently increased TER, and reduced 10kDa and 70kDa solute at 24mM, increased the abundance of ZO-1 and CLDN5 at cell-cell contacts, and on nanoscale clustering of the pore-forming CLDN2 and CLDN5.

Conclusion
Abundance of parietal peritoneal sealing and pore forming junctions and transcellular solute transporters varies with cell type and age and is differentially regulated by PD and associated with dialytic transport function. Our preliminary analyses illustrate the role of junctions and cellular transporters for solute transport across the peritoneal mesothelial and endothelial cell barrier. In-depth understanding of specific molecular functions should provide targets for modulation to improve efficacy of PD.