#1288 Bioinformatic analysis of phosphate transporters in peritoneal cells and tissues and phosphate transport kinetics in vitro
Authors: Du Zhiwei , Bartosova Maria , Marinovic Iva , Lévai Eszter , Al-Saeedi Mohammed , Lucante Theo , Zaloszyc Ariane , Begin Sylvie , Herzog Rebecca , Kratochwill Klaus , Zarogiannis Sotirios G , Schmitt Claus Peter 
Published: May 2024

Abstract

Background and Aims
Hyperphosphatemia is frequent in peritoneal dialysis (PD) patients due to limited dialytic clearance, and significantly increases cardiovascular risk. Molecular mechanisms of phosphate removal across the peritoneal membrane remain unclear, PD specific in vitro models for phosphate transport and modulator studies are not established.

Method
Phosphate transporter expression was assessed by RNAseq in mesothelial cells (HPMC, MeT5A) and human venous and microvascular endothelial cells (HUVEC, HCMEC). Transcriptome profiles of microdissected omental arterioles from 10 children with normal kidney function (NKF), 15 with chronic kidney disease (CKD5), and 10 children on PD were analyzed for phosphate transporter expression. Specific phosphate transporter localization and abundance in parietal peritoneum membranes and in the cell lines were assessed by immunostaining. In Transwell system, a phosphate transport model across polarized monolayers was established. Cell viability and integrity were verified by MTT assay and transepithelial resistance measurement (TER).

Results
Out of nine identified phosphate transporters, PiT1 (SLC20A1) and PiT2 (SLC20A2) exhibited high expression in all four cell lines, while SLC34A2 was specific in HPMC and SLC34A3 in MeT5A. In human arterioles, four phosphate transporters, SLC34A1, PiT1, PiT2, and SLC17A1, were detected. Arteriolar PiT1 was two-fold more abundant in PD patients treated with low glucose degradation product (GDP) fluids compared to those with CKD5 and NKF. In parietal peritoneal membranes, PiT1 was abundant in mesothelial and endothelial cells, with no difference between children with NKF, CKD5 and low GDP PD treatment. Concentration and time dependent phosphate kinetics across polarized mesothelial cells were studied in Transwells. Upon increased phosphate concentration in medium (1 mM and 2 mM vs. 0.1 mM), cells didn´t show signs of impaired viability, reduced TER or junction dysregulation (ZO-1 abundance) for up to 12 h. Independent of the added phosphate concentration, 60% of expected equilibrium was achieved after 12 h. Addition of PFA (SLC20/34 sodium-phosphate cotransporter inhibitor) alone or in combination with Tenapanor (an NHE3 blocker inhibiting paracellular phosphate transport) reduced phosphate transport across mesothelial cells by 10% and 20%, respectively.

Conclusion
We describe phosphate transporter expression and localization in peritoneal cells, in arterioles and parietal peritoneal tissues, and the impact of CKD5 and PD. The Transwell system allowed for quantification of phosphate transport across cell barriers and demonstrated the role of peritoneal sodium-phosphate cotransporter and of the paracellular route for phosphate transport across the mesothelial cell barrier. Further, yet unknown phosphate transport mechanisms should be active.