Implication of sphingolipids in epithelial-mesenchymal transition process in renal collecting ducts of aged rats

BRANDAN YAMILA; GUAYTIMA EDITH; PESCIO LUCILA; FAVALE NICOLAS; STERIN-SPEZIALE NORMA B; MARQUEZ M GABRIELA

Salta

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

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

The epithelial-mesenchymal transition (EMT) is a process in which the cells lose their epithelial phenotype and acquire the characteristics of the mesenchymal cells, which includes loss of cell-cell binding. Renal function declines progressively with age, and the EMT process has been suggested as a mechanism that drives renal fibrosis, with the consequent loss of tissue functions, which occurs mainly in old age. In previous works, we demonstrated that the inhibition of sphingomyelin synthase 1, the enzyme responsible for the synthesis of sphingomyelin (SM) at the Golgi Ap level, induces a EMT process in CD cells from renal papilla of young rats (70 days-old). We also demonstrated that the EMT occurs spontaneously in renal papillary CD cells of middle-aged rats (6-8 months), denoted by an impairment of cell-cell adhesion, a higher number of CD cells expressing the mesenchymal protein vimentin, and the de novo synthesis of α-smooth muscle actin (α-SMA), another mesenchymal biomarker. These results motivated us to study the possible implication of sphingolipids, and in particular SM, in the occurrence of EMT in renal papilla CD cells during aging. Taking into account that the cells in culture behave as in intact tissue, primary cultures of CD cells isolated from renal papilla of young and middle-aged rats were performed. Since the occurrence of the EMT process was observed in rats from 6 month old, we performed a recovery experiment using the exogenous addition of 10 μM C12-SM to primary cultured CD cells from middle-aged rats. For this purpose, we simultaneously evaluated the intercellular adhesions by α- catenin immunostaining, and the expression of the mesenchymal biomarker α-SMA. After the addition of exogenous SM, the intercellular spaces between the CD cells disappeared, and α- catenin lined the lateral cell membranes, reflecting the presence of mature adherens junctions. Moreover, the percentage of CD cells that express α-SMA decreased (young vs middle-aged, p=0.0006). We also analyzed the total SM content in CD cells isolated from young, middle-aged and aged-rats (15 months) by thin layer chromatography (TLC) and densitometry. Surprisingly, although we observed an EMT in CD cells from middle-aged rats, the quantitative results showed a decrease in SM content only in CD cells isolated from renal papilla of aged rats (young vs aged-rats, p= 0.0030). Taking into account our previous and present results, we conclude that the epithelial-mesenchymal phenotypic conversion that spontaneously occurs in CD cells during aging is reversible, and can be reverted by the exogenous addition of SM. Altogether, we propose that the sphingolipid metabolism play a central role as modulator of the fate of renal papilla CD cells during aging.