NR4A1 suppresses cisplatin-induced ferroptosis in renal proximal tubular epithelial cells by up-regulating the expression of NRF2 / 中华肾脏病杂志

ABSTRACT

Objective:To explore the role and mechanism of nuclear receptor subfamily 4 group A member 1 ( NR4A1) in suppressing cisplatin nephrotoxicity. Methods:The expression of NR4A1 gene in renal cell subpopulations was analyzed using the "Tabula-muris" single cell transcriptome sequencing database. NR4A1 gene was over-expressed by lentivirus infection in HK-2 cell line and primary renal proximal tubular epithelial cells. Cell counting kit-8 was used to detect the cytotoxicity of cisplatin. The cell death ratio was analyzed using propidium iodide (PI) staining by flow cytometry. The expression of NR4A1 and nuclear factor erythroid 2-related factor 2 ( NRF2) was detected by real-time fluorescent quantitative PCR and Western blotting. Ferroptosis was analyzed by detecting the contents of malondialdehyde (MDA), oxidized glutathione (GSSG) and lipid reactive oxygen species (ROS). Results:The single cell transcriptome sequencing database showed that NR4A1 gene was the lowest expression in renal proximal tubular epithelial cell subsets. Cisplatin (50 μmol/L or 100 μmol/L) could significantly induce MDA, GSSG and lipid ROS production in renal proximal tubular epithelial cells (all P<0.01), and higher cisplatin concentration accompanied with a more increase of MDA, GSSG and lipid ROS. Compared with the control HK-2 cells, the lipid ROS content and iron ion content of HK-2 cells over-expressing NR4A1 were significantly lower (all P<0.01), and the over-expression of NR4A1 inhibited cisplatin-induced cytotoxicity and ferroptosis in renal proximal tubular epithelial cells. Mechanistically, NR4A1 up-regulated the expression of anti-ferroptosis gene NRF2 in proximal renal tubular epithelial cells ( P<0.01). Furthermore, single cell data analysis showed that, similar to the expression of NR4A1 in renal tissue subsets, NRF2 was also the lowest in renal proximal tubular epithelial cells. Conclusions:Cisplatin can induce ferroptosis of renal proximal tubular epithelial cells in a dose-dependent manner. NR4A1 can inhibit cisplatin-induced ferroptosis by up-regulating NRF2 in renal proximal tubular epithelial cells, thereby alleviating the cytotoxicity of cisplatin.