#5837 MOLECULAR AND FUNCTIONAL CHARACTERIZATION OF THE PERITONEAL MESOTHELIAL AND ENDOTHELIAL CELL BARRIER
Authors: Marinovic Iva , Bartosova Maria , Lévai Eszter , Herzog Rebecca , Hausmann Michael , Zhang Conghui , Pap Domonkos , Zarogiannis Sotirios , Kratochwill Klaus , Schmitt Claus Peter 
Published: June 2023

Abstract

Background and Aims
Solute transport across cellular levels is mediated by paracellular and transcellular pores, channels and carriers. Knowledge on their cell specific expression, regulation and function in peritoneal dialysis (PD) is limited.

Method
Polarized primary (HPMC) and immortalized human peritoneal mesothelial (MeT-5A), microvascular (HCMEC) and umbilical vein endothelial (HUVEC) cells underwent RNA sequencing and gene enrichment analysis. Key findings were confirmed by western blotting (WB), confocal and single molecule localization microscopy (SMLM). Arteriolar transcriptome and proteome datasets of non-CKD, CKD5 and PD children (n = 6/group) underwent targeted transport pathways analysis. Key transporter proteins were quantified in parietal peritoneum (n = 20–30/group) and related to peritoneal transport rates available in 23 children. Barrier function was studied in vitro (transepithelial electrical resistance, TER, and molecular weight dependent flux), ex vivo and in vivo.

Results
Junction, transmembrane and transcytotic transporter expression was highly cell type specific on RNA and protein level. Sealing Claudin (CLDN)1 was only expressed in mesothelial cells, sealing CLDN5 in endothelial cells. TER which reflects functional junction status, was 50% lower in HCMEC compared to HUVEC and the two mesothelial cell lines; 4- and 10-kDa dextran permeability was higher in HCMEC. At nanoscale, SMLM yielded highest distance of junction molecules in HCMEC and different spatial organisation, reflecting the low TER. In sheep peritoneum, removal of the mesothelium abolished tissue TER. In mice, short-term LPS exposure to modify mesothelial permeability resulted in faster transperitoneal 4- and 70-kDa dextran transport, suggesting a specific barrier function of the mesothelium. In human parietal peritoneum, total endothelial surface area per section was age dependently 1.5- to 2-fold higher than the respective mesothelial surface area, and further increased with double-chamber PD fluid, due to major hypervascularisation. Tight junction proteins CLDN-1 to -5, and -15, ZO-1, occludin and tricellulin, and transcellular transporter ENaC, PIT1, and SGLT1 were detected in mesothelial and arteriolar endothelial cells. In CKD mesothelial CLDN-1 and arteriolar CLDN-2 and -3 were more abundant than in non-CKD controls, and PD patients had highest mesothelial and arteriolar CLDN-1 and mesothelial CLDN-2, lower mesothelial and arteriolar claudin-3 and lower arteriolar ENaC. D/P creatinine and D/D0 glucose correlated with arteriolar CLDN-2 and with mesothelial CLDN-4 and -15, which are pore forming junctions, and for creatinine with mesothelial PIT-1, a sodium/phosphate co-transporter.

Conclusion
We provide the first in-depth analysis of peritoneal determinants of solute transport. The molecular expression pattern of the mesothelial and endothelial cell barrier and transporter proteins differs substantially. Disruption of the mesothelial layer increases peritoneal solute absorption rate. In the human peritoneum, peritoneal transporter status is modified by CKD and PD, and pore forming junction proteins are associated with dialytic solute transport function. These represent a promising target for therapeutic intervention.