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25 February 2019 : Laboratory Research  

Pyridoxamine Treatment of HK-2 Human Proximal Tubular Epithelial Cells Reduces Oxidative Stress and the Inhibition of Autophagy Induced by High Glucose Levels

Ying Wang12ABDE, Ying Li1FG*, Zhiping Yang3C, Ziqiang Wang4BF, Jiang Chang5ABE, Tao Zhang1D, Yanqing Chi1D, Ning Han1BD, Kunxiao Zhao1CD

DOI: 10.12659/MSM.914799

Med Sci Monit 2019; 25:1480-1488

Abstract

BACKGROUND: Diabetic nephropathy is a predominant cause of renal failure, which is an important chronic complication of diabetes. Pyridoxamine (PM) has been reported to protect renal tubular epithelial cells against oxidative damage and delay or inhibit the development and generation of glucose-induced renal insufficiency at the early stage of disease. In this study, we attempted to explore the protection mechanism of PM on human proximal tubular epithelial cells (HK-2 cells) induced by high glucose.

MATERIAL AND METHODS: HK-2 cells were cultivated by high glucose medium in the absence or presence of PM. Cell Counting Kit-8 was used to investigate the most appropriate drug concentration of PM by detecting the cell viability of HK-2 cells. The expression of autophagy-related protein Beclin-1, LC-3II, and p62 was measured by western blot analysis, reverse transcription‑quantitative polymerase chain reaction (RT‑qPCR), and immunofluorescence. The expression and localization of Beclin-1 and p62 were also detected via immunofluorescence. The intracellular reactive oxygen species generation was detected using the reactive oxygen species assay kit. The effects of PM on antioxidant defenses were evaluated with glutathione peroxidase (GPx), manganese superoxide dismutase (MnSOD) activity, and glutathione/glutathione disulfide (GSH/GSSG) ratio.

RESULTS: High glucose levels were able to upregulate the expression of oxidative stress associated protein and inhibit autophagy‑associated changes verified by western blotting, RT‑qPCR and immunofluorescence. Administration of PM reversed the high glucose‑induced low-expressed Beclin-1 and LC-3II, and overexpressed p62 and intracellular reactive oxygen species levels. Furthermore, non-enzymatic antioxidant defenses and enzymatic antioxidant defenses were turned on by the application of PM.

CONCLUSIONS: Treatment with PM could reverse high glucose-induced inhibition of autophagy and oxidative stress.

Keywords: Glucose, Pyridoxamine, Cell Line, Glutathione Peroxidase, Kidney Tubules, Proximal, Oxidation-Reduction

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Medical Science Monitor eISSN: 1643-3750
Medical Science Monitor eISSN: 1643-3750