Nitrate attenuates cisplatin-induced acute kidney injury by promotion of mitophagy and reduction of oxidative stress

Cisplatin, an anticancer drug, has limited its clinical application due to its severe nephrotoxicity, such as acute kidney injury (AKI). Damaged or dysfunctional mitochondria caused by cisplatin are toxic to the cell by producing reactive oxygen species and releasing cell death factors. Mitophagy is the mechanism of selective degradation of these damaged mitochondria via autophagy, that is critical to cellular homeostasis and viability. In this study, the protective functions of inorganic nitrate against cisplatin-induced nephrotoxicity are assessed. Our results in vitro show that nitrate significantly reduced the apoptosis of HK2 or NRK52E cells induced by cisplatin treatment. Furthermore, dietary nitrate notably alleviates the tubular and glomerular damages as well as the loss of renal function in cisplatin-induced AKI mice models. These protective effects are closely related to downregulation of cell apoptosis and reduction of reactive oxygen species (ROS) generation. Mechanistically, inorganic nitrate treatment promotes the activation of mitophagy mediated by the PINK1-PRKN/PARK2 pathway, which plays an important role in the maintenance of mitochondrial quality, helping renal tubular cells to survive and recover from cisplatin stress. These novel findings suggest that inorganic nitrate supplementation deserve further exploration as a potential treatment in patients with cisplatin-induced renal injury.


Introduction
Acute kidney injury (AKI), characterized by a rapid decrease in renal function, is a global public health problem that poses a significant economic burden on health finances (Gonsalez et al. 2019;Lewington et al. 2013).Cisplatin, as a first-line chemotherapeutic drugs, has commonly been used for treating numerous solid tumours, such as cervical, ovarian, head-neck, and nonsmall-cell lung cancers (Rottenberg et al. 2021;Goldberg et al. 2022;Wen et al. 2023).However, the clinical application of cisplatin is largely restricted due to its severe undesirable toxic effects, particularly nephrotoxicity, such as AKI (Miller et al. 2010;Tang et al. 2023;Klumpers et al. 2022).Recent research reports that cisplatin caused renal tubular cell injury through several factors (Volarevic et al. 2019), such as the mitochondria, which is an important involved organelle (Oh et al. 2017).Specifically, cisplatin can induce mitochondria DNA damage in renal proximal tubular cells and evoke excessive production of reactive oxygen species (ROS).These effects ultimately damage the mitochondrial redox balance, decrease membrane potential and lead to kidney injury (Ichinomiya et al. 2018;Jia et al. 2019).
Autophagy is a process of engulfing its own cytoplasmic proteins or organelles, wrapping them into vesicles and merging with the lysosome to degrade the contents of them, so as to realize the metabolic needs of the cell itself and the renewal of certain organelles.Many studies have proved that downregulation of autophagy in the kidney is associated with serious AKI because autophagy is crucial for normal homeostasis of the renal proximal tubules (Kaushal and Shah 2016).In the AKI model, loss of autophagy in the proximal tubule due to abnormalities in autophagy-related genes worsen tubular injury and renal function by exacerbating mitochondrial damage (Tang et al. 2018;Lin et al. 2019).Conversely, certain methods that promote autophagy could protect renal tubular cells from multiple stressors (Zhang et al. 2019;Liu et al. 2018).Mitophagy, as a form of selective autophagy, can selectively isolate and degrade damaged or incomplete mitochondria, is vital for maintaining the integrity of mitochondrial function and cell homeostasis (Pickles et al. 2018).Some researches reveal that stimulation of mitophagy ameliorate the mitochondrial function in cisplatin-induced AKI mice (Zhu et al. 2020).Therefore, safe and effective approaches that activate mitophagy, and reduce oxidative stress may provide a new strategy for the prevention and treatment of cisplatin-induced AKI.
Inorganic nitrate, derived from our daily diet (such as green leafy vegetables), can be converted into nitrite by symbiotic bacteria in the mouth, and then nitrite is swallowed, absorbed in the intestine and metabolized to nitrogen oxide (NO) and other bioactive nitrogen oxides in tissues (Lundberg et al. 2008;Duncan et al. 1995).Many studies have shown the antioxidant properties of nitrate which protect cells against ROS-related injury efficiently through the nitrate-nitrite-NO pathway in adipocytes of diabetes mellitus type 2 models (Ohtake et al. 2015).In the rat model of renal and cardiovascular disease induced by high-salt diet, dietary inorganic nitrate has been proved to prevent proteinuria and histological signs of kidney injury and attenuate the cardiac hypertrophy and fibrosis via decreasing the levels of ROS (Carlström et al. 2011). Moreover, Carlstrom et al. (2011) report that dietary nitrate alleviates renal ischemia-reperfusion (IR) injury, suggesting that patients with high risk of renal IR damage may benefit from a high nitrate diet (Yang et al. 2017).Consistently, our previous studies have proved that dietary inorganic nitrate exerts protective role on irradiation-induced systemic injury model by reducing ROS levels (Chang et al. 2019).However, the effect of inorganic nitrate on kidney injury in cisplatininduced AKI models and its influence on mitophagy have not been studied.
In this study, we constructed in vitro and in vivo cisplatin-induced renal injury model to explore the protective effects of inorganic nitrate on renal injury and whether these effects were relied on the activation of mitophagy.

Cell culture
The human renal cell line HK2 and mouse renal cell line NRK25E cells were purchased from the National Infrastructure of Cell Line Resource (Beijing, China) and Procell Life Science&Technology Co.,Ltd (Wuhan, China).HK2 cells was incubated in MEM medium and NRK25E cells was incubated in DMEM medium, supplemented with 10% fetal bovine serum (complete medium) and 1% penicillin/streptomycin under a humidified atmosphere of 5% CO 2 at 37 °C.All cells were maintained in culture for no more than 25 continuous passages and were verified by short tandem repeat DNA fingerprinting and tested for mycoplasma before using.

Cell viability measurement and cell apoptosis analysis
Cell viability was measured using a CCK8 assay kit (Dojindo, Kumamoto, Japan).In short, 10 µL of CCK8 solution was added to each well containing 90 µL of medium.After incubating for 1 h, the absorbance was detected at 450 nm using an EnSpire label microplate reader (PerkinElmer, Waltham, MA).Cell Apoptosis Detection Kit were purchased from Solarbio (Beijing, China).The cells were digested using trypsin without EDTA and then resuspended in PBS.After centrifugation, the cells were then stained with 5 μL fluorescein isothiocyanate (FITC)-Annexin V, incubated at room temperature in a dark place, and then stained with 10 μL propidium iodide (20 μg /mL).The apoptotic cells were analyzed by flow cytometry (SBeckman Coulter, USA).

Animals and experimental models
A total of 60 male C57BL/6 mice (6-8 weeks) were obtained from the Capital Medical University, Beijing, China.This study was conducted following the National Institutes of Health Guide for the Care and Use of Laboratory Animals and was approved by the Animal Use and Care Committee of Capital Medical University (ethical code: AEEI-2018-216).The animals were acclimatised to the surrounding environment for 1 week prior to the study, maintained at a 12-h light/dark cycle, and provided regular water and food.To assess the protective role of inorganic nitrate on cisplatin-induced AKI, the mice were divided into 4 groups (normal control, Ctrl: n = 15; nitrate-treated, NaNO 3 : n = 15; cisplatin-treated, Cisp: n = 15; cisplatin + nitrate, Cisp + NaNO 3 : n = 15).The control mice were given drinking water supplemented with sodium chloride (Nacl, 2 mmol) and the nitratetreated group mice were subjected to drinking water supplemented with NaNO 3 (2 mmol).The cisplatin-treated mice were single intraperitoneal injection of cisplatin (20 mg/kg) and given drinking water supplemented with sodium chloride (2 mmol).The cisplatin + nitrate group mice were subjected to 2 mmol NaNO 3 drinking water for 1 week before cisplatin injection.The normal control and nitrate-treated group mice received saline injection with equal volume of cisplatin.At the end of the experiment, the animals were sacrificed and the blood and kidneys were collected for related testing.

Sample harvest
The mice were anesthetized with isoflurane and blood were isolated from the ophthalmic vein.Whole blood with 2 mmol EDTA was centrifuged immediately with 6000 × g at 4 °C for 15 min to obtain the serum and stored at -80 °C.The kidneys were weighed and the tissues were mixed with 0.9% NaCl solution and homogenized by a tissue homogenate breaker, and then the tissue homogenate was centrifuged at 6500 × g for 15 min at 4 °C.The supernatant of kidney tissue was collected to test the levels of superoxide dismutase (SOD), catalase (CAT), glutathione (GSH) and malondialdehyde (MDA) using commercial kits.The mice kidneys were harvested and fixed in 4% neutral paraformaldehyde, and then embedding with paraffin or optimal cutting temperature compound.The remaining kidney tissues were stored at -80 °C for other detection.

Serum biochemistry analysis
Plasma samples from mice were centrifuged with 1000 × g for 15 min, and then serum creatinine (Crea) and blood urea nitrogen (BUN) were measured by Zhong Yan Biolab Company (Shenzhen, China).

p-H2AX and TUNEL staining
Phosphorylated-H2A.X variant histone (p-H2AX) was detected as previously described (Chang et al. 2019).Kidney tissue section were incubated with primary antip-H2AX antibody for 1 h at room temperature, then stained with secondary antibody (Table S1), and counterstained with DAPI (1,002,339,028; Sigma-Aldrich).Cell apoptosis was measured by a TUNEL apoptosis detection kits according to the manufacturer's instructions (C1098; Beyotime Institute of Biotechnology).Images were obtained from a confocal system and a digital camera (SP8; Leica), and the positive cells were calculated by the ImageJ software.

Immunofluorescence
After treating with cisplatin and/or nitrate, HK2 cells cultured in 6-well plate were further incubated with 200 nM MitoTracker Deep Red FM (M22426, Invitrogen) in 37° C incubator for 30 min.After staining, wash the cells in fresh, pre-warmed growth medium, then carefully remove the medium covering the cells, washed with ice-cold PBS, fixed with 4% paraformaldehyde (SigmaAldrich, P6148) and permeabilized with 0.5% Triton X-100 (MP Blomedicals,194,854). Cells were then blocked with 2% FCS and followed by incubation with anti-LC3B antibody (CST, 83506S; diluted 1:100) overnight at 4 °C.Next day cells were washed with TBST and incubated with goat anti-mouse IgG Alexa Fluor 488-labeled secondary antibody (2,120,125, Invitrogen) for 1 h at room temperature.Finally, the images were captured using confocal microscopy (Leica DM6000 CS, Germany).

Autophagic flux detection assay
Autophagic flux activity was detected by the mCherry-GFP-LC3B reporter.HK2 cells were transfected with adenovirus expressing mCherry-GFP-LC3B (C3011, Beyotime Biotechnology) according to the manufacturer's protocol.At 24 h post-transfection, the cells were treated with cisplatin and/or nitrate.The cells were fixed with 4% paraformaldehyde for 1 h, and washed three times with PBS.Autophagic flux was analysed by detecting the fluorescent intensity of autophagosomes and autolysosomes using confocal system (Leica DM6000 CS, Germany).DAPI staining was used to visualize the nucleus.The ratio of the fluorescence of mCherry + puncta to the fluorescence of GFP + puncta is used as the autophagic index.More than 10 cells were counted for each condition.

Transmission Electron Microscopy (TEM)
To observe the morphology of autophagosomes at different stages, HK2 cells were fixed with 2% paraformaldehyde and 2.5% glutaraldehyde and post fixed with 1% OsO4 in 0.1 mol/L cacodylate buffer for 2 h.The samples were then cut into 0.12-μm sections after embedded, and stained with 0.2% lead citrate and 1% uranium acetate.To morphologically demonstrate the structure of mitochondria in renal tubular epithelial cells of cisplatin-induced AKI mice models, kidney tissues from mice were cut into 1 mm × 1 mm × 1 mm sized patch and fixed in paraformaldehyde and glutaraldehyde, post fixed in osmium tetroxide, dehydrated in ethanol, subjected to resin penetration and embedded in Epon.Ultrathin sections were stained with uranyl acetate (E.Merck, Darmstadt, Germany) and lead citrate (Sigma-Aldrich, St. Louis, MO, USA).Transmission electron microscope (HT7700, HITACHI, Japan) was used for observation.All analysis was performed blind to the observer.

Western blot analysis
Western blot was carried out as described previously (Ma et al. 2017).In short, the cell and tissue were harvested and homogenized with RIPA buffer.Samples were incubated with primary antibodies overnight at 4 °C and then incubated with horseradish peroxidase-linked secondary antibodies (Table S1).Immunoreactive bands were detected by NIH Image 1.62 (National Institutes of Health, Bethesda MD).Quantification of protein expression was performed using Image J software.

ROS measurement
ROS detection was accomplished following the procedures of Chang et al. (Chang et al. 2019).Briefly, kidneys were harvested and immediately frozen, sectioned at 10-μm thickness.According to the ROS Fluorescein Assay Kit (GMS10016.6,Genmed Scientifics, Inc) instructions, the sections were incubated with stains for 20 min at 37℃, then washed with cleaning liquid, incubated with DAPI and observed under a confocal system and a digital camera.The number of ROS-positive cells were calculated by the ImageJ software (10 random fields per section, × 400).

Statistics analysis
Data were presented as the mean ± standard error of the mean (SEM) and analysed by GraphPad Prism version 6.0 (GraphPad Software, San Diego, CA, USA).Continuous variables were analysed by analysis of variance or t-test, and nonparametric test was used if the data did not assume Gaussian distribution.One-way analysis of variance was performed to compare differences when more than two groups.P < 0.05 was considered statistically significant, and two-tailed P-value was used in analyses.

Nitrate suppresses cisplatin-induced renal cell apoptosis in vitro
To assess the role of nitrate (NaNO 3 ) in cisplatintreated renal cells, cisplatin-treated HK2 or NRK52E cells were incubated with or without nitrate.Firstly, the result of CCK8 assay indicated that HK2 cell viability was descended in a concentration-and timedependent manner after cisplatin treatment (Fig. 1A).Secondly, we explored the optimal acting concentration of nitrate with sodium chloride as an osmotic control and found a significant decrease in cell viability at 50 mmol/L (Fig. 1B).Therefore, we chose cisplatin for 20 μmol/L, nitrate for 20 mmol/L and action for 48 h as the conditions for the subsequent cell experiments.Subsequently, the cisplatin-treated HK2 cell viability was increased to 0.69 ± 0.04-fold from 0.52 ± 0.05-fold after nitrate incubation (Fig. 1C).Similar results were obtained in NRK52E cell and the cisplatin-treated NRK52E cell viability was ascended to 0.73 ± 0.07-fold from 0.46 ± 0.06-fold under the action of nitrate (Fig. 1D).Moreover, the number of apoptotic cells caused by cisplatin was notably decreased after nitrate treatment (Fig. 1E and F).Consistent with attenuating cell apoptosis, the elevated expression of Bax and decreased expression of Bcl-2 induced by cisplatin treatment were reduced and increased after nitrate incubation, respectively (Fig. 1G-I).These data indicate that nitrate inhibit cisplatin-induced renal cell apoptosis in vitro.

Oral administration of inorganic nitrate attenuates cisplatin-induced AKI
Considering the prospect of nitrate application in clinical practice in the future, we constructed cisplatininduced AKI mice model to further explore whether oral administration of nitrate could attenuate cisplatin-induced renal injury in vivo.Male C57BL/6N mice orally administered nitrate drinking water (2 mmol/L) one week before intraperitoneally injected with cisplatin (20 mg/kg) and sacrificed after 72 h for related testing (Fig. 2A).As expected, the weight loss of cisplatin-induced AKI mice was remarkable increased compared with the controls.Importantly, nitrate pretreatment tended to attenuate this cisplatin-induced severe undesirable toxic effects (Fig. 2B).To further validate the protective role of nitrate on cisplatininduced kidney injury, we measured mice renal function with BUN and Crea.The results indicated that cisplatin treatment caused a significant increase of BUN and Crea compare to controls, and these were prevented by nitrate pre-treatment.Baseline BUN and Crea were similar between controls and nitrate-treated mice (Fig. 2C and D).In addition, severe histological changes of renal injury were observed in the kidney of cisplatin-induced AKI model mice, including cast formation, brush border loss, vacuolisation and loss of tubular epithelial cells, whereas this renal structural damage was improved by dietary nitrate supplementation (Fig. 2E and F).
To confirm the nephroprotective effect of inorganic nitrate in cisplatin-induced AKI mice, we further measured the level of kidney injury marker-1 (KIM-1).Immunohistochemical results showed that cisplatin treatment caused a significant increase of KIM-1 levels compare to controls, and this was prevented by inorganic nitrate pre-treatment (Fig. 2G and H).The same results were obtained by western blot for detecting KIM-1 (Figure S1).Subsequently, we tested the effect of oral nitrate on apoptosis related proteins in vivo models.In line with the results of in vitro studies, western blot assay indicated that oral administration of nitrate reduced the level of pro-apoptotic Bax and cleaved caspase 3 in cisplatininduced AKI mice (Fig. 2I-L), which was consistent with the results of immunofluorescence staining of TUNEL (Fig. 2M and N).

Dietary nitrate improves DNA damage and inhibits ROS release induced by cisplatin
DNA damage and ROS production are considered to critically contribute to kidney injury (Tejchman et al. 2021).Firstly, we compared the levels of phosphorylated histone H2AX (p-H2AX), a marker of early-stage DNA damage, at different cisplatin treatment time points between the groups.After 2 or 4 h of cisplatin treatment, the levels of p-H2AX in the kidney of mice significantly increased compared to the controls, while in cisplatin-induced AKI group given nitrate, it decreased remarkably (Fig. 3A and B).Similar results were obtained in western bolt experiment (Figure S2A and B).However, the level of p-H2AX tended to be reduced in nitrate-treated AKI model mice compared to the AKI group at 6 h of cisplatin treatment, but the differences between the two groups were not statistically significant.
Next, we detected the ROS changes in the kidney following cisplatin or/and nitrate treatment.The results displayed that the number of ROS-positive cells were significant ascended at 2, 4, 6 and 72 h after cisplatin treatment compare to controls.As expected, the productions of ROS were reduced in the nitratetreated AKI model mice at 2, 4 and 72 h compare to cisplatin-treated mice, although the decrease of ROS is not statistically significant at 6 h (Fig. 3C and D).In addition, the activities of superoxide dismutase (SOD) and catalase (CAT) and together with the contents of glutathione (GSH) and malondialdehyde (MDA) in the kidney tissues were measured to assess the antioxidant  the levels of CAT, SOD and GSH, and decreased the MDA level compare to cisplatin-treated mice (Fig. 3E-H).These data prove that nitrate has a potential protective effect against cisplatin-induced AKI by improving oxidative stress.

Dietary nitrate induces autophagy in cisplatin-induced injury
To explore the molecular mechanisms by which nitrate attenuated cisplatin-induced kidney injury, and considering the importance of autophagy in tubular injury and renal function, we investigated the effect of nitrate on autophagy in cisplatin-treated HK2 cell.Consistent with previous studies, our western blot results showed a dramatic increase in LC3B-II and a significant reduction in p62, two biochemical markers of autophagy activation, in HK2 cells treated with cisplatin (Figure S3A-C, 1 vs 2 lane).These data indicated that autophagic flux were induced by cisplatin in HK2 cells.Interestingly, subsequent analysis showed that the level of LC3 II was further ascended and the level of p62 was decreased in cisplatin + NaNO 3 group compare to cisplatin-treated group (Figure S3A-C, 2 vs 4 lane).Furthermore, we determined whether autophagy was activated in cisplatin-induced AKI mice model.Renal cortical tissues were harvested for immunoblot assay and the result showed that cisplatin induced significant increases in LC3B-II and reduction in p62 (Fig. 4A-C, 1 vs 2 lane).Expectedly, we observed higher LC3B-II and lower p62 level in the cisplatin + NaNO 3 group compared with the cisplatin-induced AKI group (Fig. 4A-C, 2 vs 4 lane).Furthermore, HK2 cells were transfected with a mCherryGFP-LC3 fusion construct tool to determine autophagy flux by estimating the relative numbers of autophagosomes and autolysosomes, as indicated by yellow and red fluorescence due to the expression of mCherry and GFP.The results showed that cisplatin caused an increase in autophagic flux as indicated by the ratio of mCherry to GFP compare to control group.In cisplatin + NaNO 3 group, the further elevated ratio of mCherry to GFP suggesting a more active autophagy (Fig. 4D-E).We also monitored changes in autophagy using transmission electron microscope (TME) analysis.As shown in Fig. 4F, electron microscope data indicated that more autolysosomes were observed in cisplatin + NaNO 3 treated HK2 cells, suggesting NaNO 3 promoted autophagy flux of HK2 cell incubated with cisplatin.
To confirm the protective role of autophagy on cisplatin-treated cell damage, an autophagy inhibitor (Hydroxychloroquine, HCQ) or activator (Rapamycin; Rapa) was added to the HK2 cells.The result showed that cell viability was decreased in the cisplatin + HCQ group (Figure S3D), while increased in the cisplatin + Rapa group (Figure S3E).These data confirm that autophagy plays a protective effect on cisplatin-induced cell damage.

Nitrate induced PINK1 and PRKN/PARK2-mediated mitophagy in HK-2 cells
Given the critical role of mitophagy on mitochondrial maintenance, we explored the effect of nitrate on cisplatin-induced mitophagy.The result of western blot showed that the change of autophagy activation in HK2 cells during nitrate treatment was closed related to a marked decrease in the mitochondrial inner membrane protein TIMM23 and TOMM20 (Fig. 5A-C), suggesting activation of mitophagy or mitochondrial clearance.To further confirm mitophagy induction, we tested the colocalization of mitochondria and autophagosomes, as indicated by staining of autophagosome marker LC3B and mitochondrial marker MitoTracker.The result of confocal microscopy displayed a significant colocalization of LC3B and MitoTracker in cisplatin + NaNO 3 treated HK2 cells, indicating the formation of mitophagosomes induced by NaNO 3 .Next, we observed mitochondria morphology in the cortices of the mouse kidneys using transmission electron microscopy.Compared with the normal control group, more swollen and round mitochondria could be observed in the renal tubular cells of AKI model group mice.Subsequently, quantitative morphometric analysis revealed a decrease in the aspect ratio of mitochondria (the long axis of mitochondria/short axis of mitochondria), which is indicative of mitochondrial fragmentation.However, these phenomena were significantly alleviated after NaNO 3 administration (Fig. 5F-G).Together, these results demonstrate that nitrate is able to further activate mitophagy in cisplatin-induced AKI models.Next, to further dissect the molecular mechanism underlying nitrate-induced mitophagy in HK2 cells, we detected the effect of nitrate on PINK1-PRKN/PARK2 pathway closely associated with mitophagy.In response to cisplatin + NaNO 3 treatment, HK-2 cells showed notable elevation in PINK1 and PRKN/PARK2 levels compare to cisplatin treatment (Fig. 6A-C).Consistent with the change in HK2 cells, the protein levels of PINK1 and PRKN/PARK2 in kidney tissue from the mice were increased in cisplatin + NaNO 3 group compared with the cisplatin group (Fig. 6D-F).To further clarify the role of PINK1-PRKN/PARK2 on mitophagy in HK2 cell, we knocked down their expression with specific siRNAs.Western blot assay showed that compared with control siRNA transfected cells, the cells with either PINK1 or PRKN siRNA showed higher mitochondrial protein TIMM23 and TOMM20 subjected to cisplatin + NaNO 3 (Fig. 6G-I).Collectively, these data demonstrated that the PINK1-PRKN/PARK2 pathway is critically to tubular cell mitophagy in response to nitrate treatment under the cisplatin stress.

Discussion
Previous reports have shown that dietary nitrate play a renoprotective role in rat model of renal disease induced by high-salt diet and renal ischemia-reperfusion injury (Carlström et al. 2011;Yang et al. 2017).Therefore, we hypothesized that dietary nitrate might have potential as an effective drug to cure cisplatin-induced AKI.In the present study we demonstrated that dietary nitrate notably reduced the tubular and glomerular injuries as well as the loss of renal function following cisplatin-induced AKI.p-H2AX is an early marker of DNA double-strand breaks, which reaches its peak at 3 h after DNA damage.Our results showed that p-H2AX reached the peak after 4 h of cisplatin treatment and decreased at 6 h.Nitrate reduced the p-H2AX levels induced by cisplatin damage at 2 and 4 h, but this reduction was not observed at 6 h, which may be related to the rapid decrease of p-H2AX within 6 h.In addition, our data showed that at the time point of 6 h, nitrate reduced the ROS production caused by cisplatin, although the difference between the two groups did not reach statistical significance.These protective effects are closely related to down-regulated apoptosis and reduced generation of ROS.This is the first description of the salutary effects of inorganic nitrate in protecting the kidney from cisplatin-induced AKI.
Autophagy is a cytoprotective mechanism to boost cellular adaptation by eliminating and recycling of damaged organelles and macromolecules under numerous stresses.Growing studies show that autophagy induction has been proved in various AKI experimental models (Kaushal and Shah 2016;Havasi and Dong 2016).The activation of autophagy alleviates renal injury, whereas inhibition of autophagy shown adverse effect, suggesting the renal protective role of autophagy in AKI disease (Jiang et al. 2010;Yang et al. 2008;Periyasamy-Thandavan et al. 2008).In line with these studies, our research shown autophagy induction in renal tubular cells during cisplatin-induced AKI using both in vitro and in vivo experimental models.Importantly, the data of this study indicate that inorganic nitrate is an autophagic inducer in cisplatin-treated cells and AKI mice models, as shown by western blot, immunofluorescence staining and transmission electron microscopy.However, the mechanism of autophagy protecting renal tubule cells in AKI is not clarified.So far, there are two schools of thought to explicate the cytoprotective effects of autophagy.First, the activation of autophagy may release anti-apoptotic protein BCL2 from BECN1 to protect cells from death (Kang et al. 2011).Second, autophagy is a known process for clearing dysfunctional organelles that are potentially cytotoxic (Mizushima and Komatsu 2011).Our results support that inorganic nitrate function as a renoprotective role through the second possibility.
Mitochondria are known as the main intracellular organelles for ROS production and mitochondria ROS (mtROS) can initiate oxidative stress and apoptosis, which eventually leading to kidney damage (Ishimoto and Inagi 2016; Yang et al. 2014).Timely clearing of impaired mitochondria can reduce ROS production and have a protective effect on cisplatin-induced AKI.Mitophagy is a form of selective autophagy where tagging damaged mitochondria for autophagic identification and degradation.In renal pathophysiology, the mechanism of mitophagy is not clarified.Variation in PINK1 and PRKN/PARK2 expression was observed in tubular cells in diabetic nephropathy experimental models (Zhan et al. 2015).Dong et al. recently reported that silencing of PINK1 or PRKN/PARK2 reduced mitophagosome formation and restored mitochondrial protein TIMM23 during ATP depletion-repletion in HK2 cells, a commonly used in vitro model of renal ischemia-reperfusion (Tang et al. 2018).In vivo, deficiency of Pink1 and Park2 caused the decrease of ubiquitination in mitochondrial proteins and the accumulation of damaged mitochondria in renal tubule cells during renal ischemia-reperfusion (Tang et al. 2018).In present study, we founded the decrease of mitochondrial membrane protein (TIMM23, TOMM20) and the increase of PINK1 and PRKN/PARK2 protein in tubular cells in both in vitro and in vivo models of cisplatin-induced AKI.This observation is not surprising, because, these data suggest that PINK1-PRKN/ PARK2 pathway of mitophagy is activated to protect against renal injury induced by cisplatin.Of note, our results showed that after inorganic nitrate treatment, the protein levels of PINK1 and PRKN/PARK2 in cisplatin-treated cells were further elevated, while the levels of TIMM23 and TOMM20 were decreased.Moreover, PINK or PRKN/PARK2 knockdown attenuated the promotion of inorganic nitrate on mitophagy identified by TIMM23 and TOMM20.Together, these results demonstrate that timely removal of impaired mitochondria via mitophagy is a pivotal mechanism of the renoprotective effect supplied by inorganic nitrate in cisplatin-induced AKI models.
In summary, this study has demonstrated that inorganic nitrate plays a key protective role in kidney damage induced by cisplatin in both in vitro and in vivo AKI models.Mechanistically, inorganic nitrate promotes the activation of mitophagy and reduces the level of oxidative stress markers.Mitophagy induced by inorganic nitrate is mainly mediated by the PINK1-PRKN/PARK2 pathway that plays a crucial role in maintaining mitochondrial quality and helping renal tubular cells to survive under cisplatin stress by clearing damaged mitochondria.It is suggested that promoting mitophagy may provide a novel therapeutic strategy for cisplatin-induced AKI.Future clinical study shall illuminate if inorganic nitrate can offer protective effects in patients with renal injury caused by cisplatin treatment.Author Songlin Wang and Wenbin Li are members of the Editorial Board for Current Medicine.The paper was handled by the other Editor and has undergone rigrous peer review process.And they were not involved in the journal's review of, or decisions related to, this manuscript.The other authors declare that they have no competing interests.

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See figure on next page.)Fig. 2 Oral administration of inorganic nitrate protects the kidney against cisplatin-induced AKI.A Schematic of the experimental design for nitrate administration of cisplatin-induced AKI mice model.B Body weight changes of mice at the end of the experiment.C, D Serum BUN and Crea levels were quantified in each group (5 mice in each group).E Representative histological changes in hematoxylin-eosin (H&E) and periodic acid-schiff (PAS) stained of mice kidney sections at 72 h after cisplatin injection.The yellow arrow indicates normal appearance of tubules; the black arrow indicates cytoplasmic vacuolization and desquamation of tubular epithelium.Scale bars: 50 μm.F Tubular damage score.G Representative images of KIM-1 IHC staining in kidney sections.Scale bars: 50 μm.H Percentage of KIM-1 staining positive cells.I-L The protein levels of Bax, Caspase 3 and Cleaved Caspase 3 in renal cortical tissues of mice were evaluated by western blotting.Representative images (I) of western blotting and three independent experiments to quantify Bax (J), Caspase 3 (K) and Cleaved Caspase 3 (L).M Representative micrographs showing TUNEL staining.N The quantitative analyses of apoptotic cell.Data are shown as the means ± SEM and analyzed by one-way ANOVA with Tukey's test.* P < 0.05, ** P < 0.01, *** P < 0.001 vs. Ctrl; # P < 0.05, ## P < 0.01, ### P < 0.001 vs. Cisp; NS, no significance (Ctrl, control; NaNO 3 , Nitrate; Cisp, cisplatin; Cisp + NaNO 3 , cisplatin + Nitrate) effect of nitrate in cisplatin-treated AKI mice.The results showed that cisplatin significantly decreased the activities of kidney SOD and CAT, reduced the GSH content and increased the MDA content compare to those of controls.Importantly, nitrate treatment in cisplatin-induced AKI model mice markedly increased