IQUIFIB   02644
INSTITUTO DE QUIMICA Y FISICOQUIMICA BIOLOGICAS "PROF. ALEJANDRO C. PALADINI"
Unidad Ejecutora - UE
congresos y reuniones científicas
Título:
Sphingomyelin metabolism involvement in epithelial-mesenchymal transition (EMT) process in renal collecting ducts during aging
Autor/es:
BRANDAN YAMILA; FAVALE NICOLAS; MARQUEZ MARIA GABRIELA; GUAYTIMA EDITH; SANTACREU BRUNO; LUCILA GISELA PESCIO; STERIN SPEZIALE NORMA
Lugar:
Virtual
Reunión:
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
Institución organizadora:
SOCIEDAD ARGENTINA DE INVESTIGACIONES EN BIOQUÍMICA Y BIOLOGÍA MOLECULAR (SAIB)
Resumen:
Renal function declines progressively with age, and EMT -a process in which cells lose their epithelial phenotype and acquire the characteristics of the mesenchymal cells- has been suggested as a mechanism that drives renal fibrosis, with the consequent loss of tissue functions. In previous works, we demonstrated that the inhibition of sphingomyelin (SM) synthase 1, induces an EMT process in collecting duct (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 (8 months) and aged-rats (15 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 implication of SM metabolism in the occurrence of EMT in renal papilla CD cells during aging. To analyze the SM content, CD were isolated from renal papilla and dried lipid extracts were separated by TLC. Different concentrations of a SM standard (C12-SM) were used to calculate the SM amount. The quantitative analysis showed a decrease in SM content in CD cells isolated from renal papilla of middle- and aged-rats. Taking into account that cells in culture behave as in intact tissue, primary cultures of CD cells isolated from renal papilla of young, middle-aged and aged-rats were performed. We evaluated the expression of SMS1 mRNA by qRT-PCR in cultured CD cells. Contrary to what we expected, significant increase in SMS1 mRNA was found in aged-rats. So, the decrease in the amount of SM in CD from older rats was not due to a decrease in SMS1 expression. To evaluate the activity of the enzymes responsible for the SM synthesis, CD cells were incubated in the presence of C6-NBD-ceramide at 37ºC for 1 h to determine total SMS activity, quantifying the amount of NBD-SM converted from NBD-ceramide. The SM fluorescence intensity of the sample extracted from middle-age rats was lower than that extracted from young rats denoting a decrease in SMS activity in older rats. In order to analyze whether the decrease in the amount of SM was due to an increase in its degradation, we evaluated the activity of sphingomyelinases (SMase), which catalyzes the hydrolysis of SM to ceramide. We used as inhibitor of acid SMase (aSMase) amitriptyline and, as inhibitor of neutral SMase (nSMase) GW4869, and we evaluated the percentage of CD cells that expressed α-SMA. We observed that the treatment with the nSMase inhibitor, but not with the aSMase inhibitor, significantly reduced α-SMA expression in CD cells in older rats. So, the decrease in SM content observed in CD cells during aging may be due to a combination of a decreased SMS activity and an increase in SM degradation mediated by nSMase. Altogether, we propose that the sphingolipid metabolism play a central role as a modulator of the fate of renal papilla CD cells during aging.