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. 2023 Jan 1;31(1):97-107.
doi: 10.4062/biomolther.2022.054. Epub 2022 Sep 13.

Melatonin Attenuates Mitochondrial Damage in Aristolochic Acid-Induced Acute Kidney Injury

Jian Sun  1 , Jinjin Pan  1 , Qinlong Liu  1 , Jizhong Cheng  2 , Qing Tang  1 , Yuke Ji  1 , Ke Cheng  1 , Rui Wang  1 , Liang Liu  1 , Dingyou Wang  1 , Na Wu  1 , Xu Zheng  1 , Junxia Li  1 , Xueyan Zhang  1 , Zhilong Zhu  1 , Yanchun Ding  1 , Feng Zheng  1 , Jia Li  3 , Ying Zhang  4 , Yuhui Yuan  1
Affiliations

Melatonin Attenuates Mitochondrial Damage in Aristolochic Acid-Induced Acute Kidney Injury

Jian Sun et al. Biomol Ther (Seoul). .

Abstract

Aristolochic acid (AA), extracted from Aristolochiaceae plants, plays an essential role in traditional herbal medicines and is used for different diseases. However, AA has been found to be nephrotoxic and is known to cause aristolochic acid nephropathy (AAN). AA-induced acute kidney injury (AKI) is a syndrome in AAN with a high morbidity that manifests mitochondrial damage as a key part of its pathological progression. Melatonin primarily serves as a mitochondria-targeted antioxidant. However, its mitochondrial protective role in AA-induced AKI is barely reported. In this study, mice were administrated 2.5 mg/kg AA to induce AKI. Melatonin reduced the increase in Upro and Scr and attenuated the necrosis and atrophy of renal proximal tubules in mice exposed to AA. Melatonin suppressed ROS generation, MDA levels and iNOS expression and increased SOD activities in vivo and in vitro. Intriguingly, the in vivo study revealed that melatonin decreased mitochondrial fragmentation in renal proximal tubular cells and increased ATP levels in kidney tissues in response to AA. In vitro, melatonin restored the mitochondrial membrane potential (MMP) in NRK-52E and HK-2 cells and led to an elevation in ATP levels. Confocal immunofluorescence data showed that puncta containing Mito-tracker and GFP-LC3A/B were reduced, thereby impeding the mitophagy of tubular epithelial cells. Furthermore, melatonin decreased LC3A/B-II expression and increased p62 expression. The apoptosis of tubular epithelial cells induced by AA was decreased. Therefore, our findings revealed that melatonin could prevent AA-induced AKI by attenuating mitochondrial damage, which may provide a potential therapeutic method for renal AA toxicity.

Keywords: Acute kidney injury; Aristolochic acid; Melatonin; Mitochondrial damage; Mitophagy; Oxidative stress.

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Conflict of interest statement

CONFLICT OF INTEREST

All authors declare that they have no conflict of interest.

Figures

Fig. 1
Fig. 1
Melatonin prevented AA-induced acute renal proximal tubular injury. (A) The picture of Aristolochia and the chemical structure of aristolochic acid (AA). (B) Hematoxylin-eosin (HE) and periodic acid Schiff (PAS) staining in renal sections of mice (scale bars: 50 µm). Tubular necrosis (arrow), tubular atrophy (arrowhead), and tubular casting (asterisk). (C) The treatment schedule of melatonin in AA-induced AKI mice model. (D) Coomassie blue staining detected urinary protein (Upro) at day 11 after melatonin treatment. (E) Upro and (F) serum creatinine (Scr) detection at day 11 after melatonin treatment. (G) HE and PAS staining in renal sections of mice (scale bars: 50 µm). CONT means control. Mel means melatonin. Values are means ± SEM, *p<0.05, **p<0.01, n=6.
Fig. 2
Fig. 2
Melatonin inhibited the cytotoxicity induced by AA in renal proximal tubular epithelial cells. (A, B) MTT assay of NRK-52E and HK-2 cells after AA exposure at different concentrations (0.5, 2.5, 5, 10 µg/mL) and different time points (24, 48, 72 h). (C) MTT assay of NRK-52E and HK-2 cells after melatonin (1 mM) treatment in response to AA (2.5 µg/mL). (D) HE staining of NRK-52E and HK-2 cells (scale bars: 50 µm). (E) Colony formation assay. (F) The statistic result of colony formation rate. CONT means control. Mel means melatonin. Values are means ± SEM, *p<0.05, **p<0.01, ***p<0.001, n=3.
Fig. 3
Fig. 3
Melatonin repressed the oxidative stress induced by AA in vivo and in vitro. (A) ROS detection using DHE probe in kidney tissues of mice (n=6). (B) SOD detection in serum of mice (n=6). (C) MDA detection in serum of mice (n=6). (D) DCFD-DA immunofluorescence detection of intracellular ROS levels in NRK-52E and HK-2 cells after melatonin (1mM) treatment in response to AA (2.5 µg/mL) (scale bars: 50 μm). (E) The statistic result of relative DCFH-DA fluorescence intensity in NRK-52E and HK-2 cells (n=3). (F) Western blot analysis of iNOS expression in NRK-52E and HK-2 cells. (G) The statistic result of relative expression of iNOS to GAPDH. CONT means control. Mel means melatonin. Values are means ± SEM, *p<0.05, **p<0.01, ***p<0.001.
Fig. 4
Fig. 4
Melatonin attenuated mitochondrial damage induced by AA. (A) Transmission electron microscopy (TEM) images of mitochondria in renal proximal tubular epithelial cells of mice. Mt: mitochondria, N: nucleus, yellow arrow: damaged mitochondria, white arrow: mitophagosome. (B) The statistic result of mitochondrial fragmentation (n=3). (C) Mitochondrial transmembrane potential detection by JC-1 probes in NRK-52E and HK-2 cells after melatonin (1 mM) treatment in response to AA (2.5 µg/mL) (scale bars: 20 µm) (n=3). (D) ATP levels in NRK-52E and HK-2 cells (n=4). (E) ATP levels in kidney tissues of mice (n=6). (F) Immunofluorescence colocalization detection in NRK-52E and HK-2 cells using GFP-LC3A/B antibody (green) and Mito-tracker (red) (scale bar: 5 μm). The cell nuclei were stained with DAPI (blue). (G) The statistical result of mitophagosome formation (n=3). CONT means control. Mel means melatonin. Values are means ± SEM, *p<0.05, **p<0.01, ***p<0.001.
Fig. 5
Fig. 5
Melatonin prevented the autophagy of renal proximal tubular epithelial cells induced AA. (A) Immunofluorescent detection of LC3A/B expression in NRK-52E and HK-2 cells after melatonin (1 mM) treatment after exposure AA (2.5 µg/mL) (scale bars: 20 µm). Cell nuclei were stained with DAPI (blue). (B) The statistic result of LC3A/B expression. (C) Western blot analysis of LC3A/B-I/II and P62 expression. (D, E) The statistic result of LC3A/B-I/II and P62 expression to GAPDH. CONT means control. Mel means melatonin. Values are means ± SEM, *p<0.05, **p<0.01, ***p<0.001, n=3.
Fig. 6
Fig. 6
Melatonin inhibited the apoptosis of renal proximal tubular epithelial cells induced by AA in vivo and in vitro. (A) TUNEL staining in kidney sections of mice (scale bars: 20 µm). (B) The statistic result of the percentage of TUNEL positive cells in kidney sections of mice (n=6). (C) TUNEL staining in NRK-52E and HK-2 cells after melatonin (1 mM) treatment under AA (2.5 µg/mL) exposure (scale bars: 20 µm). (D) The statistical result of the percentage of TUNEL positive cells in NRK-52E and HK-2 cells (n=3). (E) Western blot analysis of caspase-3 and cleaved caspase-3 expression. (F) The statistic result of relative expression of cleaved caspase-3 to GAPDH (n=3). CONT means control. Mel means melatonin. Values are means ± SEM, *p<0.05, **p<0.01, ***p<0.001.
Fig. 7
Fig. 7
Schematic diagram of this study. Melatonin prevents AA-induced AKI via attenuating mitochondrial damage.
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