Melatonin inhibits oxalate-induced endoplasmic reticulum stress and apoptosis in HK-2 cells by activating the AMPK pathway

Abstract

ABSTRACT Background: Deposition of various crystal and organic substances in the kidney can lead to kidney stone formation. Melatonin is an effective endogenous antioxidant that can prevent crystalluria and kidney damage due to crystal formation and aggregation. In this study, we investigated the mechanism by which melatonin inhibits endoplasmic reticulum (ER) stress and apoptosis. Methods: We treated HK-2 cells with oxalate to establish an in vitro kidney stone model, and treated these cells with different concentrations of melatonin (0, 5, 10, 20 μmol/L) and the AMP-activated protein kinase (AMPK) inhibitor Compound C. We measured levels of stress response markers including reactive oxygen species (ROS), lactate dehydrogenase (LDH), glutathione (GSH), superoxide dismutase (SOD), malondialdehyde (MDA), catalase (CAT), and factors in the stress response pathway, such as ATF6, GRP78, DDIT3, PERK, p-PERK, IRE1, p-IRE1, XBP1s, AMPK, and p-AMPK, using real time-PCR, western blot, and immunofluorescence analyzes. We measured mitochondrial membrane potential and caspases-3 activity using the CCK8, enzyme-linked immunosorbent, and flow cytometry assays to assess HK-2 cell viability and apoptosis. Results: Melatonin improved the total antioxidant capacity (T-AOC) of the HK-2 cells, as evidenced by the dose-dependent reduction in apoptosis, ROS levels, and protein expression of ATF6, GRP78, DDIT3, p-PERK, p-IRE1, XBP1s, caspase-12, cleaved caspase-3 and cleaved caspase-9. Addition of the AMPK inhibitor, Compound C, partially reversed the protective effect of melatonin. Conclusion: Our study revealed that the protective effects of melatonin on oxalate-induced ER stress and apoptosis is partly dependent on AMPK activation in HK-2 cells. These findings provide insight into the prevention and treatment of kidney stones.