Involvement of sphingosine kinase activity in collecting duct cell differentiation during postnatal renal development

GUAYTIMA EDITH; BRANDAN YAMILA; STERIN-SPEZIALE N; MARQUEZ MARIA GABRIELA

Virtual

Congreso; LVI REUNIÓN ANUAL DE LA SOCIEDAD ARGENTINA DE INVESTIGACIONES EN BIOQUÍMICA Y BIOLOGÍA MOLECULAR (SAIB) y XV Asociación Civil de Microbiología General (SAMIGE); 2021

SOCIEDAD ARGENTINA DE INVESTIGACIONES EN BIOQUÍMICA Y BIOLOGÍA MOLECULAR (SAIB)

Nephrogenesis is completed postnatally in mammals. In this regard, the renal papilla osmolality is much lower in neonatal rats than in adults, increasing dramatically after weaning. Previously, we demonstrated that the developmental regulation of sphingosine kinase (SK) expression and activity leads sphingolipid metabolism to the formation of sphingosine-1-phospate (S1P) in the neonatal period, consistent with the immature-proliferative stage of neonatal renal papilla. Thus, the aim of this work was to evaluate the involvement of SK activity in renal papilla collecting duct (CD) cell differentiation during postnatal development. Taking advantage of the fact that primary cultured cells retain many characteristics of their behavior in intact tissue, primary cultures of renal papillary CD cells isolated from 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) and RNA interference for SK1 were used as an SK activity inhibitor and SK1 knockdown, respectively. Cell behavior after the different culture conditions was evaluated by immunofluorescence. We found that CD cells acquire a higher differentiation degree when subjected to an HT environment, reflected by the establishment of intercellular adhesions. SK1 knockdown, prior to HT treatment, caused a pronounced discontinuous distribution of α-catenin only in transfected cells, with a clear cell junction loss. The fully differentiated epithelial phenotype of CD cells is reflected by the presence of a primary cilium. The percentage of ciliated cells in the different experimental conditions did not show significant differences. Instead, it was evident a switch in the cilia phenotype: cells in isotonic medium have a higher number of short cilia, while elongated cilia were predominant in HT. SK activity inhibition with tDHS prevented the formation of elongated cilia, but this phenomenon was partially reverted when exogenous S1P and NaCl were added to the medium simultaneously, in the presence of the inhibitor. Altogether, these results suggest that SK activity is necessary for the acquisition and maintenance of the epithelial differentiated phenotype of CD cells in the physiological HT environment of the renal papilla. These findings highlight the importance of SK activity during the postnatal development.