Aristolochic acid (AA) and its derivatives, isolated from the Aristolochiaceae plant family, are a group of nitrophenanthrene carboxylic acids (Balachandran et al., 2005; Yang et al., 2013). Applications prepared from Aristolochiaceae plants, including AA, have been used for the treatment of diverse diseases, such as arthritis, gout, rheumatism, hypertension, urinary tract infection, and festering wounds (Debelle et al., 2008; Anger et al., 2020). However, these applications are reported to be nephrotoxic. Aristolochic acid nephropathy (AAN) is a common nephropathy caused by AA (Chen et al., 2012; Wang et al., 2015). In AAN, most patients rapidly deteriorate to end-stage renal disease (ESRD) (Luciano and Perazella, 2015). Patients with AAN exhibit increased serum creatinine (Scr), severe anemia, rapid tubulointerstitial injury, loss of renal proximal tubules, and tubule atrophy (Priestap et al., 2012). Recently, AA has been shown to cause acute kidney injury (AKI) and chronic kidney disease (CKD), which are often used for research on the transition from AKI to CKD (Wang et al., 2020). Interstitial fibrosis is the chronic pathological sign of CKD (Fu et al., 2018). AKI is a syndrome characterized by a sudden decline in renal function. Despite the fact that AKI is a reversible illness, patients who recover from AKI have a higher tendency for developing CKD (Jones et al., 2012). Thus, the investigation of AA-induced AKI has important clinical significance.

Proximal tubular epithelial cells are considered the primary target of AA (Chevalier, 2016). Researches have implicated AA causes mitochondrial damage, resulting in the apoptosis and necrosis of tubular epithelial cells (Romanov et al., 2015; Anger et al., 2020). Reactive oxygen species (ROS) play critical roles in this pathological progression. Damaged mitochondria increase ROS generation, contributing to oxidative stress, which in turn causes further damage to mitochondria (Tang et al., 2015). Mitochondrial damage promotes the release of proapoptotic factors, such as cytochrome c, to activate the cell apoptosis pathway (Szeto et al., 2017). Recently, the prevention of AAN has been limited to glucocorticoids. Moreover, most patients with ESRD still require renal replacement therapy, such as dialysis or kidney transplantation (Luciano and Perazella, 2015). Therefore, finding protective agents to prevent AA-induced AKI has become an urgent problem to be solved.

Melatonin (N-acetyl-5-methoxytryptamine), a circadian hormone, is mainly secreted by the pineal gland during the night (Singh and Jadhav, 2014). It has acquired a variety of activities at different stages of evolution in mammals, such as immunomodulatory, antiproliferative, antioxidative, anti-inflammatory, and mitochondrial protective activities (El-Sokkary et al., 2002; Tan et al., 2010; Han et al., 2019). Abnormalities in melatonin are associated with cancer, hypertension, diabetes, and neurodegenerative disease (Cardinali et al., 2008; Ganguly et al., 2010; Hardeland, 2012). We previously reported that melatonin attenuated pulmonary hypertension in rats and inhibited the proliferation of breast cancer cells (Jin et al., 2014; Wang et al., 2018; Wang et al., 2022). Mitochondria are the major organelles of melatonin synthesis (Tan and Hardeland, 2021). It is worth noting that melatonin, a potent protector of mitochondria, decreases ROS production, maintains the mitochondrial membrane potential (MMP), and elevates mitochondrial fusion, thereby protecting mitochondria (Tan et al., 2016). Studies have revealed that melatonin-pretreated mitochondria inhibit oxidative stress, inflammation, cellular stress and mitochondrial damage in acute liver ischemia‒reperfusion injury (Ko et al., 2020). Even though previous work has reported that melatonin exhibites a protective effect against AAN in a CKD mouse model via inhibiting oxidative stress and inflammation, its mechanisms in AA- induced AKI were not clarified (Kim et al., 2019). In the present study, we aimed to explore whether melatonin can prevent AA-induced AKI via attenuating mitochondrial damage in vivo and in vitro.

Materials

Aristolochic acid I (A5512) was purchased from YUAN YE (Shanghai, China). Melatonin (M5250) was purchased from Sigma Aldrich (Shanghai, China). The human kidney proximal tubular epithelial cell line HK-2 (CRL-2190) and rat kidney proximal tubular epithelial cell line NRK-52E (CRL-1571) were kindly provided by Dr. Feng Zheng (Dalian Medical University, Dalian, China). Antibodies against LC3A/B (12741) caspase-3 (9662), and cleaved caspase-3 (9664) were obtained from Cell Signaling Technology (MA, USA). An antibody against iNOS (18985-1-AP) was purchased from Proteintech (Wuhan, China). An antibody against SQSTM1/p62 (WL02385) was purchased from Wanleibio (Shenyang, China). The ROS assay kit (DCFH-DA, S0033), superoxide dismutase (SOD) assay kit (S0101), malondialdehyde (MDA) assay kit (S0131), adenosine triphosphate (ATP) assay kit (S0026), BCA protein assay kit (P0012) and TUNEL assay kit (C1088) were purchased from Beyotime (Shanghai, China). A tissue ROS assay kit (BB470512) was purchased from Bestbio (Shanghai, China). Mito-tracker (40741ES50) and JC-1 probes (40705ES03) were purchased from YESEN (Shanghai, China). A periodic acid Schiff (PAS) kit (G1280) and Hoechst 33342 (C0030) were purchased from Solarbio (Beijing, China). A creatinine assay kit (C011-2) and urine protein (Upr) assay kit (C035-2) were purchased from Nanjing Jiancheng Bioengineering Institute (Nanjing, China).

Animal models

Twenty-four Kunming male mice (10 weeks old, 25-30 g) were purchased from the Animal Laboratory Center of Dalian Medical University. Animal experimental protocols and care methods were approved by the Animal Care and Use Ethics Committee of Dalian Medical University. The mice were randomly divided into four groups (six mice per group): (1) control group treated with vehicle; (2) melatonin group treated with melatonin; (3) AA group treated with AA; and (4) AA+melatonin group treated with AA and melatonin. For groups (3) and (4), mice were injected intraperitoneally with AA (2.5 mg/kg/day) for 3 days. For groups (2) and (4), mice were injected intraperitoneally with melatonin (30 mg/kg/day) from 3 days before AA exposure for 10 days, which was performed 30 min prior to AA injection. AA was diluted in saline with 5% dimethyl sulfoxide (DMSO, #D8370, Solarbio). Melatonin was dissolved in phosphate-buffered saline (PBS) with 10% propylene glycol (PEG, #3015708, AR, Sinopharm Chemical Reagent, Co., Ltd, Shanghai, China). The injection volume was 0.1 mL/10 g/mouse. All mice were euthanized on Day 11 after melatonin treatment. Blood and urine were collected for further investigations. Kidney tissue was fixed in 4% paraformaldehyde. Paraffin-embedded renal sections (4 µm) stained with hematoxylin-eosin (HE) and PAS were prepared for histological analysis.

Cell treatment

The NRK-52E or HK-2 cells were incubated overnight at 37°C with 5% CO₂. Then, the cells were subjected to different concentrations (0, 0.5, 2.5, 5, 10 µg/mL) of AA for 24, 48, and 72 h to assess cytotoxicity. For melatonin treatment, the cells were incubated with 2.5 µg/mL AA and 1 mM melatonin for 48 h.

Cell viability measurement

Cell viability was assessed by a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay (Wang et al., 2018). After culturing in 96-well plates (5×10³ cells per well) at 37°C with 5% CO₂ overnight, the cells were incubated with 10 μL MTT (0.5 mg/mL) for 4 h at 37°C. Then, the supernatant was discarded, 150 µL DMSO was added and the cells were shaken evenly. The absorbance was detected at 490 nm with a microplate reader (Perkin Elmer, MA, USA).

Colony formation assay

NRK-52E or HK-2 cells (5×10² cells per well) were seeded in a 6-well plate and incubated for 10-14 days at 37°C with 5% CO₂. Then, colonies were fixed with 2% paraformaldehyde, stained with a 0.05% crystal violet solution, and counted under an inverted microscope (Leica, Solms, Germany).

ROS measurement

For ROS detection in the kidneys, the tissue homogenate was centrifuged at 1,000×g for 3 min at 4°C. Then, the precipitate was discarded. 200 μL of homogenate supernatant and 2 μL of dihydroethidium (DHE) probe were added to the 96-well plate and incubated at 37°C for 30 min in the dark. The fluorescence intensity was measured by a microplate reader (Perkin Elmer). For ROS detection in vitro, the cells were incubated with DCFH-DA (10 μM) for 30 min at 37°C. Fluorescence images were obtained by a fluorescence microscope (Olympus, Tokyo, Japan).

MDA, SOD and ATP content measurements

The MDA, SOD, and ATP levels were measured by commercial assay kits. A detailed manipulation process was performed according to the instructions of the manufacturer. The optical density was determined by a multifunctional enzyme marker (Perkin Elmer).

Western blot analysis

The cells were collected and lysed in RIPA buffer. Protein concentrations were measured by a BCA kit (Beyotime). The proteins were separated by sodium dodecyl sulfate‒polyacrylamide gel electrophoresis as previously reported (Wang et al., 2018). The proteins were transferred to a nitrocellulose membrane for 1.5 h. Then, the membrane was blocked with 5% skim milk for 2 h at room temperature. After incubation with the primary antibodies against iNOS (1:1,000), LC3A/B (1:1,000), p26 (1:1,000), caspase 3 (1:1,000), and cleaved-caspase (1:1,000) overnight at 4°C, the membranes were exposed to the secondary antibodies for 1.5 h and analyzed by chemiluminescence (Amersham Biosciences, Buckinghamshire, UK). The images were analyzed by Image Lab software (Bio-Rad Laboratories, CA, USA).

Transmission electron microscopy (TEM) assessment

Kidneys were first fixed with glutaraldehyde and then settled in osmium tetroxide. After dehydration in ethanol and embedding in Epon, the tissues were cut into 70 nm sections. Next, the sections were subjected to uranyl acetate and lead citrate staining. The stained sections were checked at low magnification (×3,000) to locate the renal proximal tubules. Cells in the proximal tubules were viewed at a high magnification (×10,000) to observe mitochondria. Finally, the images were collected by TEM (JEM-2000EX, JEOL, Tokyo, Japan). The lengths of mitochondria were measured by NIH ImageJ tracing software (NIH, MD, USA). Approximately 50 mitochondria were measured in each cell. The percentage of cells containing less than 1% long (>1 μm) mitochondria was calculated to evaluate the levels of mitochondrial fragmentation.

Mitochondrial transmembrane potential assay

The cells were cultured in a 6-well plate and treated with AA or melatonin for 24 h, followed by incubation with the JC-1 probe (2 μM) in PBS (pH 7.4) at 37°C for 30 min in the dark. Due to the sensitivity to MMP, JC-1 accumulates in the matrix of mitochondria to form aggregates at a high MMP and emits a red fluorescence. However, if JC-1 is blocked from accumulating in the matrix of mitochondria due to a low MMP, the JC-1 monomer generates a green fluorescence. Images were obtained by a fluorescence microscope (Olympus).

Immunofluorescence colocalization measurement

The cells (1-5×10⁵ per well) were seeded on polyline-coated glass slides in a 6-well plate. Then, the cells were incubated with Mito-tracker Red for 30 min, fixed in 100% methanol for 20 min at –20°C, washed with PBS, and placed in 0.2% Triton-X-100 for 5 min. After incubation with an anti-LC3A/B (1:200) antibody at 4°C overnight, the slides were treated with the fluorescent secondary antibody (1:100, Abbkine, Wuhan, China) for 2 h. The cell nuclei were stained with DAPI (62248, Invitrogen, Carlsbad, CA, USA). The images were captured by a confocal laser scanning microscope LSM780 (Carl Zeiss, Oberkochen, Germany).

TUNEL assay

For the TUNEL assay of kidney tissues, the slides were deparaffinized, rehydrated, incubated with proteinase K free of DNase (20 μg/mL) for 20 min at 37°C, and washed with PBS. Then, TdT-labelled nucleotide mix was added, and the cells were culture at 37°C for 1 h in the dark. For the TUNEL assay of cells, the slides were assessed as described above after being fixed with 4% paraformaldehyde for 30 min. The cell nucleus was stained with DAPI. The images were captured by a fluorescence microscope, and the TUNEL positive cells were analyzed by imaging software ImageJ (NIH). The percentage of TUNEL positive cells in 500 cells of each slide was calculated.

Statistical analysis

Statistical analyses were performed by GraphPad Prism 8.0 (GraphPad Software, CA, USA). Comparisons between groups were analyzed by a two-sided Student’s t test. Values are presented as the mean ± SEM. Statistically significant differences were considered significant when p<0.05.

Melatonin attenuated AA-induced AKI in mice

The appearance of Aristolochia and the chemical structure of aristolochic acid are shown in Fig. 1A. Mice were injected intraperitoneally with AA (2.5 or 5.0 mg/kg) every day for 3 days to induce AKI. HE and PAS staining were used to determine the morphological changes in kidney tissues (Fig. 1B). The results showed that renal proximal tubular necrosis and atrophy were observed in the kidney after exposure 2.5 mg/kg AA. Substantial renal proximal tubular casting was formed after exposure 5.0 mg/kg AA. The kidneys of mice in the control group showed a typical structure.

Figure 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.

Next, we chose 2.5 mg/kg AA to explore the effect of melatonin on AKI in mice. The treatment schedule is shown in Fig. 1C. As reflected by a significant increase in urinary protein (Upro) and Scr levels, AKI was observed in mice of the AA group (Fig. 1D-1F). However, melatonin (30 mg/kg) treatment repressed the increase in Scr and Upro in response to AA. Scr and Upro showed no significant change in the melatonin alone treatment group. As demonstrated in Fig. 1G, renal proximal tubular necrosis and atrophy were observed in the AA exposure group. After melatonin treatment, these morphological changes were limited to a few proximal tubules. The renal proximal tubules remained a typical structure in the melatonin alone treatment group. Interstitial fibrosis was not observed, and no abnormality was detected within the glomeruli of mice in any group. These data suggested that melatonin attenuated AA-induced AKI in mice.

Melatonin inhibited the cytotoxicity induced by AA in renal proximal tubular epithelial cells

To explore the role of melatonin in AA-induced cytotoxicity in vitro, HK-2 and NRK-52E cells were used. AA decreased cell viability in a time- and dose-dependent manner. Nevertheless, melatonin (1 mM) increased cell viability after exposure AA (2.5 µg/mL) (Fig. 2A-2C). The cell morphological results showed that melatonin reversed the cell necrosis induced by AA (Fig. 2D). Consistent with the MTT results, the colony formation results showed that melatonin enhanced the cell colony formation rate (Fig. 2E). Collectively, these data demonstrated that AA led to cytotoxicity in NRK-52E and HK-2 cells. Interestingly, melatonin preserved cell viability.

Figure 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.

Melatonin repressed the oxidative stress induced by AA in vivo and in vitro

Melatonin is a strong mitochondria targeted antioxidant (Tan et al., 2016; Reiter et al., 2017). Therefore, we next investigated the role of melatonin in AA-induced oxidative stress. In vivo, ROS production was enhanced in the kidneys of the AA group compared with those of the control group (Fig. 3A). Nevertheless, melatonin significantly inhibited the generation of ROS induced by AA. Then, SOD and MDA, as oxidative stress biomarkers were measured in the serum of mice. Melatonin increased the SOD activities and decreased the MDA levels in the AA+melatonin group (Fig. 3B, 3C). In vitro, we evaluated the ROS level in NRK-52E and HK-2 cells by DCFH-DA staining. NRK-52E and HK-2 cells incubated with AA showed a dramatic increase in the fluorescence intensity of DCFH-DA. However, melatonin reversed the increased fluorescence intensity (Fig. 3D, 3E). Additionally, iNOS expression was increased in NRK-52E and HK-2 cells after AA incubation, and melatonin inhibited the expression of iNOS (Fig. 3F, 3G). Thus, we proved that melatonin repressed oxidative stress in AA-induced AKI.

Figure 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.

Melatonin attenuated mitochondrial damage in AA-induced AKI

Given that melatonin inhibited AA-induced oxidative stress, we further explored whether this effect was related to its mitochondrial protective activity. Mitochondrial morphology in the renal proximal tubules of mice was examined by TEM (Fig. 4A, 4B). AA caused mitochondrial fragmentation and loss of cristae and mitophagosomes in the renal proximal tubular epithelial cells of mice. Melatonin treatment reversed these changes induced by AA. The mitochondria of tubular epithelial cells were normal in size and shape in the melatonin alone treatment group. In vitro, a JC-1 probe was used to evaluate the mitochondrial transmembrane potential of tubular epithelial cells (Fig. 4C). JC-1 formed red fluorescent aggregates in the mitochondrial matrix of NRK-52E and HK-2 cells in the control group. AA blocked the formation of JC-1 aggregates, leading to the generation of JC-1 monomers (green fluorescence) in the mitochondria, suggesting that AA damaged the MMP. However, melatonin reversed the changes induced by AA. The ATP levels were also assessed (Fig. 4D, 4E). AA repressed ATP levels compared with the control group in vitro and in vivo. However, melatonin increased the levels of ATP.

Figure 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.

Since the TEM study showed that melatonin reduced mitophagosomes in tubular epithelial cells in vivo, we assessed mitophagy in NRK-52E and HK-2 cells by Immunofluorescence colocalization detection using Mito-tracker and GFP-LC3A/B. The results showed that puncta containing Mito-tracker and GFP-LC3A/B were more abundant in the AA group, indicating increased autophagosome formation in mitochondria. Notably, melatonin reduced the puncta induced by AA (Fig. 4F, 4G). These results demonstrated that melatonin reversed the mitochondrial damage in AA-induced AKI.

Melatonin prevented the autophagy of renal proximal tubular epithelial cells induced by AA

Next, we further explored whether melatonin inhibited autophagy of tubular epithelial cells induced by AA. Immunofluorescence staining assays were performed in NRK-52E and HK-2 cells using an anti-LC3A/B antibody (Fig. 5A, 5B). LC3A/B were increased in the AA group compared with the control group, indicating that AA caused autophagosome formation in NRK-52E and HK-2 cells. Melatonin repressed the autophagy induced by AA. In accordance with the immunofluorescence results, western blot analysis showed that the autophagy-related protein LC3A/B-II was increased and that the autophagy substrate p62 was reduced after AA exposure. Melatonin reduced the protein levels of LC3A/B-II in HK-2 cells and increased the protein levels of p62 in both NRK-52E and HK-2 cells (Fig. 5C-5E). Our data confirmed that melatonin suppressed AA-activated autophagy in renal proximal tubular epithelial cells.

Figure 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.

Melatonin inhibited the apoptosis of renal proximal tubular epithelial cells caused by AA

To determine the role of melatonin in the apoptosis of renal proximal tubular epithelial cells after exposure AA, a TUNEL assay was performed on the mouse kidneys. As shown in Fig. 6A and 6B, there were fewer TUNEL-positive cells in the kidneys in the AA+melatonin group than in the AA group. In vitro, AA increased the number of TUNEL-positive in NRK-52E and HK-2 cells; however, melatonin reversed the change induced by AA (Fig. 6C, 6D). The above results were confirmed by cleaved caspase-3 expression (Fig. 6E, 6F). AA enhanced the expression of cleaved caspase-3. After melatonin treatment, cleaved caspase-3 protein levels were reduced. These findings suggested that melatonin inhibited AA-mediated apoptosis of tubular epithelial cells in vivo and in vitro.

Figure 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.

In the present study, we successfully established an AA-induced AKI mouse model characterized by increased Upr and Scr, necrosis, and atrophy in the renal proximal tubules. Melatonin remarkably prevented the biochemical and morphological changes, along with the oxidative stress caused by AA. In particular, melatonin reduced mitochondrial fragmentation, restored MMP, increased ATP levels and repressed the mitophagy of renal proximal tubular epithelial cells responding to AA. Eventually, autophagy and apoptosis of tubular epithelial cells exposed to AA were reversed, and cytotoxicity was inhibited. Therefore, our study revealed that melatonin prevents AA-induced AKI by attenuating mitochondrial damage (Fig. 7).

Figure 7. Schematic diagram of this study. Melatonin prevents AA-induced AKI via attenuating mitochondrial damage.

An increasing number of reports have suggested that damaged mitochondria are implicated in the pathogenesis of acute and chronic kidney diseases, including AAN, which lead to significant changes in mitochondrial morphology and function (Hall and Schuh, 2016; Tang et al., 2018; Anger et al., 2020). Reversing mitochondrial dysfunction has been shown to prevent tubular epithelial cells death and maintain renal function (Brooks et al., 2009; Ishimoto and Inagi, 2016; Suzuki et al., 2016). Mitochondria are intracellular organelles and function as the powerhouse because of their ability to generate ATP, which provides energy to support basic cell activities (Li et al., 2006). AA induces a decrease in ATP levels and mitochondrial membrane depolarization in renal proximal tubular epithelial cells (Qi et al., 2007). Melatonin, a potent protector of mitochondria, was shown to be more effective than mitochondrial-targeted antioxidative products, such as vitamin E (MitoE) and coenzyme Q10 (MitoQ) (Tan et al., 2016). Mitochondria are biological targets of melatonin, playing an antitumor role through the mitochondrial apoptosis pathway (Huo et al., 2017; Yu et al., 2018). Besides, melatonin has been shown to rescue mitochondrial function from renal fibrosis in diabetic mice and regulate mitochondrial bioenergetics to protect mesenchymal stem cells in chronic kidney disease (Han et al., 2019; Tamarindo et al., 2019). In this study, melatonin reduced fragmented mitochondria, restored MMP, and increased levels of ATP in tubular epithelial cells. In addition, autophagy has been found to promote AA-induced kidney injury (Yang et al., 2013). Mitophagy is a form of autophagy that selectively eliminates dysfunctional and damaged mitochondria to autophagosomes. However, excessive activation of mitophagy may deteriorate the pathological progression of diabetic nephropathy (Higgins and Coughlan, 2014). In the present study, we demonstrated that AA caused autophagy and mitophagy in renal proximal tubular epithelial cells in AA-induced AKI. However, melatonin reduced the expression of the autophagy marker LC3A/B-II expression and enhanced the expression of the autophagy substrate p62 expression, thereby inhibiting mitophagy and autophagy in tubular epithelial cells. All these data suggested that melatonin protected mitochondria from AA-induced damage in AKI.

Mitochondria are also a leading source of ROS in cells. Excessive ROS generated by mitochondria lead to oxidative stress, which in turn enhances ROS production and further damages mitochondria (Tang et al., 2015). Numerous reports have demonstrated that ROS are increased in renal tubular epithelial cells exposed to AA (Romanov et al., 2015; Wang et al., 2019; Zhang et al., 2019). AA-induced oxidative stress has been associated with decreased SOD2 and glutathione synthetase mRNA levels and increased MDA contents (Kim et al., 2019). In this work, melatonin reduced ROS generation, increased SOD levels, and decreased MDA levels and iNOS expression in vivo and in vitro. ROS and mitochondrial membrane depolarization also contribute to DNA damage and cell apoptosis (Thangam et al., 2014; Dai et al., 2015). Increased apoptosis has been found in AA-induced cancers (Cosyns et al., 1999; Chang et al., 2007; Simoes et al., 2008) and cultured tubular epithelial cells exposed to AA (Hsin et al., 2006; Zhou et al., 2010). Apoptosis of tubular epithelial cells is one of the central mechanisms leading to AAN (Pozdzik et al., 2008; Romanov et al., 2015). In this study, we provided evidence to support the role of damaged mitochondria in AA-induced AKI. Damaged mitochondria produce excessive ROS and release proapoptotic factors such as cytochrome c, which may ultimately result in the death of renal tubular epithelial cells (Sweetwyne et al., 2017; Szeto et al., 2017). We found that AA caused a significant increase in TUNEL-positive cells in vivo and in vitro. Melatonin inhibited AA-induced tubular epithelial cell apoptosis and decreased the expression of cleaved-caspase3.

In summary, melatonin exhibited a protective role in AA-induced AKI in vivo and in vitro. Melatonin attenuated mitochondrial damage by reducing mitochondrial fragmentation, restoring MMP, increasing ATP levels and repressing mitophagy in renal proximal tubular epithelial cells exposed to AA. Eventually, melatonin suppressed ROS generation and oxidative stress, inhibiting autophagy and apoptosis of renal tubular epithelial cells. Therefore, our findings suggest that melatonin may offer a novel therapeutic strategy and be a potential protective drug for AA-induced AKI via protecting mitochondria.

This work was funded by Dengfeng Clinical Medicine Grant Support (No. 2021024) and the National Natural Science Foundation of China (No. 81770617). We thank Dr. Feng Zheng for providing the human kidney proximal tubular epithelial cell HK-2 and rat kidney proximal tubular epithelial cell NRK-52E.

All authors declare that they have no conflict of interest.