陆军军医大学学报 (Mar 2023)
Amifostine enhances the resistance of renal proximal tubular epithelial cells to cisplatin by upregulating PKM2
Abstract
Objective To explore the molecular mechanism of amifostine in relieving cisplatin nephrotoxicity. Methods CCK-8 assay was used to detect the inhibitory effect of amifostine on cisplatin cytotoxicity in HK-2 and primary renal proximal tubular epithelial cells. The expression of PKM2 was analyzed by online database tabula muris. The proportion of apoptotic cells was detected by flow cytometry. The expression of PKM2 gene was detected by fluorescence quantitative PCR and Western blotting. PKM2 gene expression was knocked down by small RNA interference. Results Amifostine significantly enhanced the resistance of renal proximal tubular epithelial cells to cisplatin toxicity. In cisplatin treated cells, with the increase of the concentration of amifostine, the cell viability was increased from 0.37 to 0.45 to 0.77 (P < 0.001). In addition, with the increase of amifostine concentration, PKM2 gene expression in HK-2 cells was increased in turn. The expression of PKM2 gene was increased to 8.76 times higher at 1 μmol/L of amifostine (P < 0.001). PKM2 gene expression in HK-2 cells was increased with the increase of amifostine concentration. When PKM2 gene expression was depleted in HK-2 cells, and the protective effect of amifostine on cisplatin cytotoxicity was significantly inhibited. The proportion of apoptotic cells was reduced and the levels of apoptotic markers Cleaved-PARP and Cleaved-Caspase3 induced by cisplatin were significantly inhibited by amifostine. Single-cell data analysis indicated that the expression of PKM2 in renal proximal tubular epithelial cells was the lowest compared with other cell subsets in renal tissue. Conclusion Amifostine inhibits cell apoptosis induced by cisplatin by up-regulating PKM2 in renal proximal tubular epithelial cells, and thus alleviates the cytotoxicity of cisplatin.
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