Molecular mechanisms underlying resveratrol effect on renal osmoprotection: modulation of COX-2 expression

Abstract

Resveratrol (RSV) is a polyphenol naturally present in several plants. Nowadays it is sold as an over-the-counter dietarysupplement due to its antioxidant, anti-inflammatory and antitumoral effects. Paradoxically, it has been documented that RSVmay also present pro-oxidizing and pro-proliferative effects. In fact, some studies suggest that RSV treatment can result inopposite effects depending on the cell type, its concentration, or the treatment time. Particularly in renal tissue, animal injurymodels described RSV beneficial effects, while studies with chronic intake of RSV observed nephrotoxicity. Hence, RSVeffects on renal tissue are still controversial. Due to the urinary concentrating mechanism, renal medullary interstitium presentsan elevated osmolality that can abruptly change depending on the hydric state of the body, reaching values up to 800-1200mOsm/kg H2O. To survive in this environment, renal cells activate protective pathways. We have demonstrated that renalepithelial cell line MDCK undergoes an adaptive process during the first 24h of hyperosmolarity, in which the transcription ofthe osmoprotective gene cyclooxygenase 2 (COX-2) is activated, among others. After 48h these cells are already adapted andbegin to differentiate, acquiring a polarized epithelium morphology. In this work we evaluate RSV effect on adaption anddifferentiation mechanisms, focusing particularly on COX-2 role. To do this, MDCK cells were pretreated with differentconcentrations of RSV (1, 5, 10, 25 µM) and cultured in hyperosmolar medium (~512 mOsm/kg H2O) for 24 and 48h. Cellswere harvested to obtain cell number and viability. Cell cycle, immunofluorescence (IF), western blot and RT-PCR analysiswere performed. We found that RSV significantly decreased cell number in a concentration-dependent manner at 24 and 48h.Cell cycle analysis revealed that RSV increased S-phase and Sub-G0 cell population. In addition, treated cells did not reachtypical epithelium morphology. COX-2 mRNA and protein levels were surprisingly upregulated by RSV at 24 and 48h, andIF revealed an accumulation of the protein in cytoplasmic granules. To investigate the pathways leading to this upregulation,we indirectly evaluated TonEBP, NF-κB and ERK1/2 pathways, which are activated by hyperosmolarity; and SIRT1implication, a target of RSV. TonEBP target genes mRNA did not show any significant change under RSV treatment, whileNF-κB target gene mRNA presented an increase similar to that of COX-2 mRNA. Moreover, NF-κB IF revealed an increasein its nuclear localization. Regarding ERK1/2, treatment with ERK1/2 selective inhibitor (U0126) completely blocked COX2 protein expression. These results suggest that in renal cells RSV pretreatment decreased cell number and impeded typicalcell morphology acquisition; but it increased COX-2 expression, possibly through NF-κB and ERK1/2 activation.