During postnatal renal development, sphingosine 1-phosphate acts either intracellularly or extracellularly in collecting duct cells depending on their differentiation state

GUAYTIMA, EDITH DEL VALLE; FAVALE, NICOLÁS OCTAVIO; SIREROL ROSALERS FACUNDO; ROMERO VICTOR; CARRIZO JULIETA; STERIN-SPEZIALE NORMA B; MARQUEZ MARIA GABRIELA

Rosario

Congreso; LIX REUNIÓN ANUAL DE LA SOCIEDAD ARGENTINA DE INVESTIGACIÓN BIOQUÍMICA Y BIOLOGÍA MOLECULAR (SAIB); 2023

SOCIEDAD ARGENTINA DE INVESTIGACIÓN BIOQUÍMICA Y BIOLOGÍA MOLECULAR (SAIB)

Renal development is a complex event that is completed postnatally in mammals. A characteristic of mammalian kidneys is the hypertonicity of the interstitial fluid in the renal papilla, which during postnatal development induces and maintains the differentiation of collecting duct (CD) cells. Previously, our group has demonstrated that regulation of sphingosine kinase (SK) expression and activity leads to sphingosine 1-phosphate (S1P) formation in the neonatal period, consistent with the immature-proliferative stage of neonatal papilla CD cells. S1P can act intracellularly (iS1P) as a second messenger or extracellularly (eS1P) as a ligand of five different G protein-coupled receptors (S1PR1-5). Thus, the aim of our current work is to deepen the understanding regarding the involvement of SK/S1P pathway in CD cell differentiation induced by external hypertonicity (HT), during postnatal development. We performed primary cultures of papillary CD cells isolated from 10-day-old rats with 10% fetal calf serum, which contains S1P. To mimic the physiological condition, cells were subjected to gradual increases of NaCl concentration. Treatment with D,L-threo-dihydrosphingosine (t-DHS), an SK activity inhibitor, and pharmacological inhibition of S1PR1, 2 and 3 were performed. In primary developing CD cell cultures, different populations of cells regarding their differentiation state were found. We observed that, to initiate the differentiation process induced by HT, CD cells need the activity of SK- that is iS1P-, since cells subjected to HT in the presence of t-DHS exhibited impairment in cell-cell adhesion structures. In this experimental condition, cell death was observed in areas where more immature cells were present. When cells were exposed to t-DHS after having been subjected to HT condition, their characteristic differentiated epithelial phenotype was preserved, suggesting that CD cells do not need iS1P once their differentiated state was acquired. On the other hand, cells subjected to HT in the presence of the S1PR1-3 antagonists exhibited a more differentiated epithelial phenotype. Interestingly, the presence of circular cellular structures containing central apoptotic cells were also observed, surrounded by cells that do not express epithelial cell markers such as ZO-1, α- and β-catenin, and E-cadherin. These structures might constitute the initial event of the tubular lumen formation, although it cannot be accomplished in the 2D culture conditions. These last results suggest that CD cells need to desensitize S1PR1-3 to achieve a more differentiated state induced by HT, being eS1P not necessary. Therefore, during the postnatal development S1P could act intracellularly or extracellularly, depending on the differentiation state of CD cells. Altogether, S1P might modulate through several cellular events the formation of the epithelial cell sheet, that finally would give rise to the renal papilla collecting ducts.