Effect of resveratrol on renal COX-2 mediated osmoprotective mechanisms

FERNANDEZ TOME, MARÍA DEL CARMEN; ERJAVEC, LUCIANA C.; CASALI, CECILIA I.

Salta

Congreso; LV Annual SAIB meeting and XIV PABMB Congress; 2019

Sociedad Argentina de Investigación en Bioquímica y Biología Molecular

Resveratrol (trans-3,4′,5-trihydroxystilbene, RSV) is a stilbene polyphenol present in a large variety of plants such as mulberries, peanuts and grapes. In the last years RSV intake has increased, as it is sold as an over-the-counter dietary supplement due to its antioxidant, anti-inflammatory and antitumoral properties. However, it has also been reported that RSV may behave as a pro-oxidizing agent; thus, it may also have implication in pathology of diseases. Some studies suggest that RSV can modulate different pathways at a time, resulting in distinct biological effects depending on the cell type, its concentration or treatment time. In renal tissue, many studies describe the beneficial effects of RSV in kidney injury models; however, some studies detected nephrotoxicity in animal models subjected to chronic doses of RSV. Hence, RSV effects on renal tissue are still controversial.Renal medullary interstitium is characterized by an elevated osmolality due to the presence of high concentrations of sodium and urea. Depending on the hydric state of the body, renal interstitial osmolality can abruptly vary, reaching values up to 800-1200 mOsm/kg H2O. To survive in such an adverse environment, protective pathways are activated. We have demonstrated that renal epithelial cells (MDCK) subjected to high osmolality undergo an adaptive process during the first 24h, in which the transcription of several osmoprotective genes (sodium/myo-inositol transporter (SMIT), sodium/chloride/betaine transporter (BGT1), aldose reductase (AR), and cyclooxygenase 2 (COX-2), among others) is activated. After 48h, these cells are already adapted and acquire a polarized epithelium morphology.In the present work, we evaluate the effect of RSV on adaption and differentiation mechanisms, focusing particularly in COX-2 expression. To do this, MDCK cells were treated with different concentrations of RSV (1, 5, 10 and 25 µM) for 30 min and then cultured in hyperosmotic medium (NaCl 125 mM, 500 mOsm/kg H2O) for 24 and 48h. Cells were then harvested to obtain cell number and viability and processed for protein or RNA extraction. Cell morphology was also analyzed. Cell number recovered after RSV treatment was significantly lower in a concentration-dependent manner after 24 and 48h of hyperosmotic challenge. Cells treated with RSV did not reach typical epithelium morphology; moreover, cells treated with the highest concentrations showed a mesenchymal phenotype after 24 and 48h. mRNA expression of osmoprotective genes (COX2, BGT1, SMIT, AR) assessed by RT-PCR showed a decrease only after treatment with 25 µM of RSV, but no significant changes were found after treatment with 1, 5 or 10 µM. COX-2 protein expression was also evaluated by Western Blot and surprisingly, it was significantly upregulated by RSV treatment in a concentration-dependent manner at 24h. These results suggest that in renal cells RSV pretreatment did not affect osmoprotection but impeded monolayer differentiation.