In vitro effect of 1-methyltryptophan isomers on epithelial-mesenchymal transition transcription factors in tubular epithelial cells after ischemia-reperfusion injury

Abstract

The compound 1-methyltryptophan (1-MT) has been shown to act protectively in renal ischemia-reperfusion injury. Toll-like receptor 4 (TLR-4) signaling is also a regular process of epithelial to mesenchymal transition (EMT) that can after Ischemia-reperfusion injury (IRI) result in as an increase of renal fibrosis. EMT is associated with specific transcription factors – Snai1, Snai2, Zeb1 and Twist. 1-MT could regulate EMT and act as antifibrotic agent. This study aimed to investigate the effect of 1-MT on EMT transcription factors in tubular epithelial cells that underwent 30 min. Renal tubular epithelial cells (TECs) were isolated from Lewis rats using a standard protocol with Fe2O3 magnetic separation and selective media as previously mentioned. ischemia and 48 hours of reperfusion. Cells were cultivated and divided into 4 groups: C-TECs– control cells, IRI-TECs – IRI-induced TECs, D-IRI-TECs – IRI-induced TECs treated with 1-methyl-D-tryptophan, L-IRI-TECs – IRI-induced TECs treated with 1-methyl-L-tryptophan. IRI was induced in all groups for 30 min by mineral oil (except for C-TECs) followed by 48 hours of reperfusion. RNA and proteins were isolated from harvested cells. Using semi-quantitative PCR (RT-sqPCR) we assessed the relative mRNA expression of EMT transcription factors Snai1, Snai2, Zeb1, and Twist. Hereby we have shown, that the treatment of ischemia-induced TECs with both 1-MT isomers lowers the expression of EMT transcription factors Snai1 and Zeb1 that were increased by ischemia and reperfusion of TECs. This could act favorably in renal IRI decreasing EMT and renal fibrosis, therefore showing the potential of 1-MT as a part of therapy in renal transplantation aimed at renal ischemia-reperfusion injury.