Role of sphingosine kinase activity in the collecting duct formation during postnatal renal development

GUAYTIMA EDITH; BRANDAN YAMILA; SAAVEDRA CAMPILLAY JULIETA; STERIN SPEZIALE NORMA; MARQUEZ M GABRIELA

Salta

Congreso; LV REUNIÓN ANUAL DE LA SOCIEDAD ARGENTINA DE INVESTIGACIONES EN BIOQUÍMICA Y BIOLOGÍA MOLECULAR (SAIB) - PABMB; 2019

In mammals, nephrogenesis is completed postnatally. A characteristic of mammalian kidneys is the hypertonicity of the interstitial fluid in the renal papilla, which induces and maintains the differentiation of collecting duct (CD) cells. The osmolality of the renal papilla is much lower in neonatal rats than in adults and increases dramatically after weaning. Previously, we demonstrated that the sphingosine kinase (SK)/sphingosine 1 phosphate (S1P) pathway is developmentally regulated, leading sphingolipid metabolism to the formation of S1P in the neonatal period, which is consistent with the immature-proliferative stage of neonatal rat renal papilla. Thus, the aim of this work is to study the involvement of SK activity during postnatal CD development. Taking into account that the cells in culture behave as in intact tissue, primary cultures of CD cells isolated from the renal papilla of 10-day-old rats were performed. During the postnatal development, the renal papilla gradually acquires a hypertonic (HT) medium. To mimic this physiological condition, cultured CD cells were subjected to gradual increases of NaCl concentration until it reached the final concentration of 200 mM. D,L-threo-dihydrosphingosine (tDHS) was used as an SK activity inhibitor. CD cell morphology was analyzed in isotonic (ISO) and HT medium by phase-contrast microscopy and F-actin distribution, and the degree of differentiation was evaluated by immunofluorescence analyzing adherens junction (AJ) (E-cadherin, β- and α-catenin) and the presence of primary cilium. In ISO medium, cultured cells displayed an elongated morphology with an irregular distribution and intensity of AJ protein immunostaining, proper of immature epithelial cells. By contrast, HT medium induced a more mature cell phenotype, reflected by a morphological change from an elongated to a hexagonal shape, and a uniform and enhanced positive signal for AJ proteins. Likewise, a greater number of CD cells with primary cilium was observed. When cells were treated with tDHS 24h before being subjected to HT, cell-cell adhesion was impaired, denoted by an irregular intensity and distribution of AJ protein immunostaining. To evaluate the effect of the inhibition of SK activity in already differentiated CD cells, tDHS was added to the culture after the cells were subjected to HT medium. In this condition, the epithelial morphology was not altered. Altogether, these results suggest that SK activity is necessary for the acquisition of the epithelial differentiated phenotype of CD cells in the physiological HT environment of the renal papilla, during the postnatal development.