Sphingosine-1-phosphate receptor 2 (S1PR2) is essential in epithelial renal cells adaptation and differentiation

ROMERO DJ; MOSCA JM; BENSUSAN S; PALAVECINO A; PESCIO LG; FAVALE NO

Congreso; SAIB LVIII; 2022

Renal collecting duct cells are physiologically subject to an interstitial hypertonic environment, which is a key signal for renal physiology. High interstitial osmolality contributes to the differentiation of tubular structures and to the maturation of the urinary concentrating system in the developing kidney. We have demonstrated that, in hypertonic media, epithelial collecting duct cells (MDCK) acquire a fully differentiated epithelial cell phenotype. Epithelial cell differentiation is a process that involves mesenchymal-epithelial transition (MET) and includes cell cycle arrest, cell-cell junction maturation and changes in cell migration capacity. Sphingosine 1-phosphate (S1P) is a bioactive sphingolipid resulting from the phosphorylation of sphingosine by sphingosine kinases (SKs) which is involved in processes such as cell proliferation, growth, migration and differentiation. S1P can act both intracellularly as a second messenger or extracellularly as a ligand of 5 different G protein-coupled receptors (S1PR1-5). Only S1PR1, 2 and 3 are expressed in renal epithelial cells. In the present work, we evaluated the role of the S1P/S1PR2 pathway MDCK cell adaptation to hypertonic media and MET/differentiation. First, we evaluated the expression and localization of S1PR2 during epithelial differentiation and adaptation to hypertonic media. We found an increase in the expression of S1PR2 RNA and protein levels in differentiated cells (subjected to hypertonicity). Moreover, immunofluorescence studies showed that S1PR2 was progressively enriched on the plasma membrane during cell differentiation. We then evaluated the effect of S1PR2 antagonist JTE-013 on the acquisition of an epithelial differentiated phenotype. Immunofluorescence studies showed that S1PR2 (but not S1PR1 or S1PR3) prevented adherens junction maturation and F-actin cortex formation, with a decrease in the protein levels of E-cadherin, beta-catenin, and alpha-catenin. Furthermore, cells were unable to adapt to hypertonic stress. Later we used a specific S1PR2 siRNA to corroborate our results. We observed that knockdown resembled the effect observed with the antagonist. In order to corroborate these results, we obtained a MDCK-S1PR2-KO line by CRISPR/Cas9 technology. MDCK-S1PR2-KO cells were unable to acquire a differentiated phenotype or adapt to a hypertonic medium. For these reasons, we performed a recovery assay where MDCK-S1PR2-KO cells were transfected with a S1PR2-EGFP plasmid. We observed that transfected cells recovered their adaptive capacity and presented a differentiated phenotype. In addition, recovery was blocked by JTE-013. These findings highlight the central role of S1P/S1PR2 in renal epithelial cell differentiation and tissue preservation in hypertonic environmentinduced stress.