Polydatin Attenuates Cisplatin-Induced Acute Kidney Injury via SIRT6-Mediated Autophagy Activation

In the treatment of malignant tumors, the effectiveness of cisplatin (CP) is limited by its nephrotoxicity, leading to cisplatin-induced acute kidney injury (CP-AKI). Polydatin (PD) has been demonstrated to regulate autophagy in tumors, sepsis, and diabetes. We have recently confirmed that PD attenuated CP-AKI by inhibiting ferroptosis, but it is not clear whether PD can regulate autophagy to protect from CP-AKI. The purpose of this study was to investigate the effect of PD on autophagy in CP-treated HK-2 cells and CP-AKI mouse models, exploring the role of sirtuin 6 (SIRT6) upregulated by PD. In this study, the blocking of autophagy flux was observed in both CP-treated HK-2 cells in vitro and CP-AKI mouse models in vivo, whereas this blocking was reversed by PD, which was characterized by the increase of autophagy microtubule-associated protein light chain 3 II expression and autophagolysosome/autophagosome ratio and the decrease of p62 expression. Furthermore, PD also significantly increased the expression of SIRT6 in vivo and in vitro. The protective effect of PD manifested by the stimulating of autophagy flux, with the reducing of inflammatory response and oxidative stress, which included downregulation of tumor necrosis factor-α and interleukin-1β, decreased activity of myeloperoxidase and content of malondialdehyde, and increased activity of superoxide dismutase and level of glutathione, both in vivo and in vitro, was reversed by either inhibition of autophagy flux by chloroquine or downregulation of SIRT6 by OSS-128167. Taken together, the present findings provide the first evidence demonstrating that PD exhibited nephroprotective effects on CP-AKI by restoring SIRT6-mediated autophagy flux mechanisms.


Introduction
As the mainstay in the treatment of various types of tumors, the application of cisplatin (CP) is hindered by dose limitation due to its nephrotoxicity, such as acute kidney injury (AKI) in 20%-30% of patients [1]. However, so far, there is still a lack of effective drugs to treat Cis-AKI clinically. The pathogenesis of CP-induced AKI (CP-AKI) is complex and multifactorial, including autophagy, which has been proved to exert a nephroprotective role in experimental models of CP-AKI by using both pharmacological and genetic approaches in recent years [2]. Autophagy has always been regarded as a double-edged sword in tumor therapy [1,3], so when targeting autophagy as a nephroprotective strategy in CP-AKI, its effect on tumors should be considered.
Polydatin (PD, C 20 H 22 O 8 ), a glucoside of resveratrol, known as a natural active ingredient extracted from Polygonum cuspidatum Sieb. et Zucc., is used for both medication and food, which plays a multitarget protective role in AKI by antiapoptosis, anti-inflammation, antioxidative stress, antiferroptosis, and regulation of autophagy [4][5][6][7][8][9][10][11][12]. What is more, PD has also been proved to play an antitumor effect by inhibiting cell viability, migration, and invasion of different tumor cells [13][14][15]. PD has been clinically used to treat multiple diseases, showing its safety in human application [16][17][18], which suggests that it can be used as a promising alternative compound for treating CP-AKI in cancer patients.
Sirtuin 6 (SIRT6) belongs to a nicotinamide adenine dinucleotide (NAD + )-dependent deacetylase, which plays a protective role in different organ injuries by regulating autophagy [31]. Overexpression of SIRT6 can alleviate CP-AKI [32], but the relationship between SIRT6 and autophagy in CP-AKI remains a mystery. Recently, PD has been found to protect septic myocardial injury by promoting SIRT6-activated autophagy [19]. Our previous studies confirmed the renal protective effect of PD through antioxidative stress and antiferroptosis in I/R-induced AKI (I/R-AKI) and CP-AKI [4,9,11]. Based on the above evidence, we hypothesized that PD may attenuate CP-AKI through SIRT6-mediated autophagy.
Therefore, this study intends to investigate the correlation between autophagy and SIRT6 activation in the nephroprotection of PD in CP-AKI model both in vivo and in vitro, which may provide reliable evidence for the research and development of nephroprotective drugs in the clinical treatment of AKI. 2.2. Cell Culture and Treatment. Human proximal tubular epithelial cells (HK-2 cells), obtained from the China Center for Type Culture Collection (GDC0152, Wuhan, China), were cultured in DMEM medium containing 10% FBS and 100 units of antibiotics (streptomycin and penicillin) per ml at 37°C. The exponentially growing HK-2 cells were inoculated in six-well culture plate with 2 to 4 × 10 5 cells/well and cultured for 1 day before each experiment. After treating HK-2 cells with CP in dose gradient (5 μM, 10 μM, and 20 μM) and time gradient (6 h, 12 h, 18 h, and 24 h), according to the protein expression of LC3 II and p62, the cells treated with CP in a dose of 20 μM for 24 h were determined for the follow-up experiment. To investigate the effect of CP on autophagy of HK-2 cells, the cells were pretreated with CQ (20 μM) or RAP (50 nM) for 4 h before induction with CP (20 μM) for 24 h. To evaluate the role of PD on autophagy, HK-2 cells were pretreated with PD in a dose gradient (20 μM, 40 μM, and 80 μM) for 2 h before induction with CP (20 μM) for 24 h. According to the change of the protein expression of LC3 II and p62, and cell apoptosis rate in HK-2 cells, a 40 μM dose of PD was determined as the best dose. Therefore, the following experiments divided the cells into four groups: control group, CP (20 μM) group, CP + PD (40 μM) group, and CP + PD + CQ (20 μM) group. To clarify the role of SRIT6 in PD on the autophagy flux of CP-treated HK-2 cells, OSS-128167 (50 μM), a SIRT6 selective inhibitor, was used instead of CQ to pre-treat the cells for 0.5 h before induction with CP (20 μM) for 24 h. Experimental design in vitro is shown in Figure 1.  [11].

Western Blot
To estimate the effect of autophagy flux blocking on renal protection of PD in CP-AKI mice, the animals were divided into the following four groups (n = 6-8 each): control group, CP (20 mg/kg, i.p.) group, CP + PD (40 mg/kg, i.p.) group, and CP + PD + CQ (60 mg/kg, i.p.) group [33]. To evaluate the role of SRIT6 in PD on the autophagy flux in CP-AKI mice, OSS-128167 (50 mg/kg, i.p.) [34] was used to replace CQ in the previous groups (n = 6-8 each). Mice were injected with CP once; PD or CQ was given 1 h before and 24 h after CP. OSS-128167 was administered only 1 h before CP. Experimental design in vivo is shown in Figure 1. The whole blood and kidneys were collected when animals were ethically killed by dislocating their spines at 48 h after CP injection.  3 Oxidative Medicine and Cellular Longevity of kidney weight (g) and 50 mmol/l phosphate buffer (pH 7.4) volume ðmlÞ = 1 : 9. Centrifuge at 1500 g for 20 min and collect the supernatant as the sample to be tested immediately.
2.10. Enzyme-Linked Immunosorbent Assay (ELISA). The levels of tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β) in renal tissue homogenate were evaluated with a commercially available ELISA kit (R&D Systems, USA), according to the manufacturer's recommendation. All standards and samples were measured in duplicate.
2.11. Measurement of Renal Oxidative Indexes. The kidney supernatant was used to measure the activity of MPO and SOD, and the content of MDA and GSH followed the commercial kit instructions by using a spectrophotometer (Spectrophotometer DU640, Beckman Coulter, Fullerton, CA). All levels were expressed as U/mg protein, nmol/mg protein, or mg/g protein, respectively.

Renal Histopathology and Terminal Deoxynucleotidyl
Transferase dUTP Nick-End Labeling (TUNEL) Assay. Fresh kidney tissue was rinsed with frozen stroke physiological saline, fixed in 10% neutral buffered formalin overnight, embedded in paraffin, and cut into 4-μm-thick sections for hematoxylin-eosin (H&E) staining and TUNEL fluorescent staining according to the manufacturer's instructions. Renal tubular epithelial cells with the following histopathological changes were considered injured: loss of brush border, tubular dilation and disruption, cast formation, and cell lysis. The kidney tissue damage was performed by renal pathologists in a blinded fashion and scored by calculating the percentage of damaged tubules: 0 none; 1, <25%; 2, 25-50%; 3, 50-75%; and 4, >75%.
The scores of at least 10 randomly selected areas per mouse kidney were averaged and used as the scores of the individual mouse kidneys.
2.13. Statistical Analysis. All data are presented as mean ± standard deviation (SD) of at least three independent experiments. Statistical analysis of data was performed with SPSS 19, and P values were determined using Student's t -test and one-way analysis of variance (ANOVA) for two independent samples. Immunofluorescence and grayscale analysis of western blot bands were performed semiquantitative analysis using ImageJ. P < 0:05 was statistically significant.

Autophagy Flux Was Restrained in Cisplatin-Treated
HK-2 Cells. Autophagy flux detection is considered as the "gold standard" to evaluate autophagy level [35], so we analyzed the effect of CP on the expression of autophagy-related genes LC3 and p62 in HK-2 cells. The results of western blot showed that CP led to the upregulation of the LC3 II and p62 protein expression in HK-2 cells in a dose-dependent manner, and this change was most significant at the dose of 20 μM CP (Figures 2(a) and 2(b)). Therefore, we chose 20 μM CP to treat the cells and observed the effects of different culture times (6, 12, 18, and 24 h) on the autophagy flux of HK-2 cells. Similarly, LC3 II and p62 expression were also increased in a time-dependent manner by CP, and this change was most significant at 24 h of culture, about 3.48and 4.17-fold as compared to the control group (Figures 2(c) and 2(d)). These findings indicated that the autophagy degradation of HK-2 cells was interfered by CP, and the subsequent experiments in this study were carried out when the HK-2 cells were treated with CP at a dose of 20 μM for 24 h. Then, we introduced CQ (an autophagy inhibitor) or RAP (an autophagy activator) into CP-treated HK-2 cells and evaluated the changes of autophagy flux by the above protein expression assay and mRFP-GFP-LC3 transfection method. WB results showed that the LC3 II and p62 expression increased by CP were promoted by CQ and decreased by RAP (Figures 2(e) and 2(f)). mRFP-GFP-LC3 is a widely used autophagic indicator with a yellow LC3 signal when in the autophagosome (with both GFP and RFP signals) and a red LC3 signal when in the autophagolysosome due to the acidic milieu that quenches GFP signal. As shown in Figures 2(g) and 2(h), compared with the control, CP induced the increase of yellow puncta (named YFP), suggesting the blockage of autophagy flux from the autophagosome to the autophagolysosome. Furthermore, the CP-increased YFP/RFP ratio was further aggravated by CQ and reversed by RAP (Figures 2(g) and 2(h)). Concomitantly, the FACS analysis of Annexin V and PI staining showed that the increased apoptosis induced by CP was worsened by CQ and alleviated by RAP (Figures 2(i) and 2(j)). These findings suggested that CP-induced autophagy flux blocking had adverse effects on HK-2 cells.  e)). Similarly, the mRFP-GFP-LC3 transfection method showed that compared with CP alone, PD significantly induced the increase of red puncta, but PD combined with CQ significantly increased the yellow puncta (with both GFP and RFP signals), suggesting that PD enhanced not 4 Oxidative Medicine and Cellular Longevity    Increasing evidence shows that SIRT6-mediated autophagy has a positive effect on cell survival [31]. To confirm whether SIRT6 is involved in the cytoprotective effect of PD against CP, the CP-induced HK-2 cells were treated with PD combined with or without OSS-128167, an inhibitor of SIRT6, to evaluate the relationship between SIRT6 and autophagy flux.   Supplementary Table 1(a)) staining showed that all three doses of PD dramatically reduced tubular damage and cell apoptosis, and PD-M had the best effect. These results suggested that PD can accelerate the recovery of damaged autophagy flux and renal function in CP-AKI mice, and the dosage of 40 mg/kg PD is the best. Therefore, the subsequent experiments in this study were all performed following this treatment.

Chloroquine Eliminated the Nephroprotective Effect of PD on CP-AKI Mice in Terms of Recovery of Damaged
Autophagy Flux, Anti-inflammation, and Antioxidative Stress. The inhibitory effect of CQ on renal autophagy flux in CP-AKI mice in vivo has been widely confirmed [3]. To figure out the role of autophagy flux in the nephroprotection of PD, PD (40 mg/kg) and CQ (60 mg/kg) were intraperitoneally injected into CP-AKI mice 1 h before CP administration and then reinjected 24 h after CP administration (Figure 6(a)). As shown in Figures 6(b) and 6(c), in CP-AKI mice, the PD-increased degradation of p62 (a selective substrate of autophagy) was inhibited by CQ, and the expression of LC3 II was increased, suggesting that blocking lysosomal degradation by CQ abolished the recovery effect of PD on autophagy flux. Concomitantly, the protective effect of PD on body weight ( Figure 6 In AKI, besides apoptosis, the mutual crosstalk of autophagy with inflammation and oxidative stress has also been confirmed [36]. CP-AKI mice had significantly increased TNF-α (Figures 6(l)) and IL-1β (Figure 6(m)) levels in the kidney, compared to the control group. PD reduced the levels of TNF-α and IL-1β in the kidneys of CP-AKI mice, which was abolished by combined CQ treatment (Figures 6(l) and 6(m)). To validate the potential effect of PD on antioxidative stress in CP-AKI, we detected the activity of MPO (Figure 6(n)) and SOD (Figure 6(p)) and the contents of MDA (Figure 6(o)) and GSH (Figure 6(q)) in the kidneys, respectively. Compared with the control mice, the MPO activity and MDA content were significantly increased in CP-AKI mice, which were reversed in PD group, but the beneficial effect of PD was abolished by CQ (Figures 6(n) and 6(o)). Likewise, CQ abolished the    (Figure 7(a)). WB results showed that PD significantly promoted SIRT6 expression in CP-AKI mice, and this effect was clearly inhibited by OSS-128167 (Figures 7(b) and 7(c)). Meanwhile, OSS-128167 also clearly abolished the recovery effect of PD on the autophagy flux in the kidneys of CP-AKI mice (Figures 7(b) and 7(c)). In addition, OSS-128167 also diminished the protective effect of PD on body weight (Figure 7(d)), kidney index (Figure 7(e)), BUN (Figure 7(f)), Scr (Figure 7(g)), renal tubular damage (Figures 7(h) and 7(j)), and cell apoptosis (Figures 7(i) and 7(k), Supplementary Table 1(c)) in CP-AKI mice. Furthermore, OSS-128167 also significantly inhibited the anti-inflammatory and antioxidative stress effects of PD in CP-AKI mice, which showed increase in the levels of TNFα (Figure 7(l)) and IL-1β (Figure 7(m)), the MPO activity (Figure 7(n)) and MDA content (Figure 7(o)), and decrease in the SOD activity (Figure 7(p)) and GSH content (Figure 7(q)). These data suggested that the protective effect of PD on impaired autophagy flux and renal function in CP-AKI mice was at least partially related to the activation of SIRT6.

Discussion
On account of its nephrotoxicity, including AKI, CP is limited in the treatment of malignant tumors [1]. It has been proved that autophagy flux blocking is one of the mechanisms of CP nephrotoxicity, but so far there is no specific drug for AKI. We have recently confirmed the nephroprotective effect of PD in CP-AKI [4], but the role of PD in the damaged autophagy flux has not been reported in CP-AKI mice. In this study, the correlation between autophagy flux and SIRT6 in the nephroprotection of PD was investigated in in vivo and in vitro models of CP-AKI, with the following highlights: (1) It was first confirmed that PD could alleviate CP-AKI by restoring autophagy flux; (2) it was confirmed for the first time that the inhibition of SIRT6 pathway reverses the recovery effect of PD on CP-blocked autophagy flux, suggesting the potential effect of SIRT6-mediated autophagy flux on the nephroprotection of PD in CP-AKI.
Autophagy is a lysosomal degradation pathway, which acts a nephroprotective role under normal physiological conditions and when the kidney is exposed to injuries or toxins, such as CP [2]. The pharmacologic and genetic inhibition and activation of autophagy can increase and reduce renal tubular injury during CP treatment, respectively [2]. This study showed that the autophagy flux was blocked in CP-treated HK-2 cells and CP-AKI mice, which was characterized by the increase of LC3 II and p62 in WB, and the decrease of autophagolysosome/autophagosome ratio in mRFP-GFP confocal test, which were consistent with other studies [37,38]. The autophagy flux blocking and cell apoptosis induced by CP in a dose-dependent and timedependent manner were further aggravated by CQ and alleviated by RAP, suggesting that autophagy flux blocking may be associated with CP nephrotoxicity.

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Oxidative Medicine and Cellular Longevity Polydatin, a natural polyphenol plant extract, has been proved to have a nephroprotective effects in different AKI and chronic kidney disease (CKD) models [21]. Deng et al. proved an important role of PD in autophagy by activating SIRT1-mediated Beclin1 deacetylation in sepsis-induced AKI [5]. Gu et al. showed that PD ameliorated autophagy imbalance in an mTORC1-dependent manner during fructose-induced podocyte injury [25]. The renal protective  effect of PD in CP-AKI has recently been confirmed by our research team [4], but it is not clear whether PD can resist CP nephrotoxicity by regulating autophagy. The results of this study clearly showed that PD can restore CP-induced autophagy flux blocking in both CP-treated HK-2 cells in vitro and CP-AKI animals in vivo, which were manifested by the increase in LC3 II and autophagolysosome/autophagosome ratio, and the decrease in p62 accumulation; however, this effect was blocked by CQ. Concomitantly, renal protective effects of PD on CP-AKI, including antiapoptosis, anti-inflammation, and antioxidative stress, were also revered by CQ, indicating that PD could at least partially protect mice against CP-AKI by restoring autophagy flux.
Cisplatin can cause multiple forms of cellular stress, such as oxidative stress, endoplasmic reticulum stress, mitochondrial damage, and mitophagy, which may be related to autophagy activation in CP-AKI [2]. It has been widely reported the role of energy signaling pathways mediated by mTOR, AMPK, and NAD + metabolism in the regulation of autophagy in CP nephrotoxicity [1]. Sirtuin 6 (SIRT6) belongs to NAD + -dependent deacetylase, which is widely expressed in mammalian organs, and regulates multiple biological processes, including inflammatory response, oxidative stress, telomere homeostasis, and autophagy [31]. The protective effect of SIRT6 activating autophagy has been proved in different organ injuries, including I/R injury [39][40][41], diabetes [42][43][44][45], and sepsis [19,46,47]. Several studies have recently demonstrated the renal protective effect of SIRT6-induced autophagy in sepsis-induced AKI [47], hypertensive cardiorenal injury [48], podocyte injury [45], and cadmium-induced renal damage [49], respectively. Li et al. [32] confirmed that overexpression of SIRT6 attenuated CP-AKI by inhibiting extracellular signal-regulated kinase 1/2 signaling; Fan et al. [50,51] confirmed that isoorientin and daphnetin can alleviate CP-AKI through antioxidative stress and antiapoptosis via activating the SIRT1/ SIRT6/nuclear factor erythroid 2-related factor 2 pathway, but the relationship between SIRT6 and autophagy has not been reported in CP-AKI. A recent report shows that PD protects against septic myocardial injury by activating SIRT6-mediated autophagy [19]. Therefore, based on the recovery effect of PD on autophagy flux blocking in CP-AKI confirmed in this study, we further evaluated whether SIRT6 was involved. As expected, in the CP-AKI models in vivo and in vitro, OSS-128167, an inhibitor of SIRT6, not only inhibited SIRT6 expression promoted by PD, but also blocked the role of PD in the recovery of autophagy flux. Meanwhile, the inhibition of SIRT6 also weakened the renal protective effects of PD on CP-AKI, including antiapoptosis,

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Oxidative Medicine and Cellular Longevity anti-inflammation, and antioxidative stress, which suggested that PD was at least partially protected from CP-AKI through the SIRT6-mediated activation of autophagy. Autophagy is protective during CP-AKI, but it is considered as a double-edged sword in cancer therapy [1,3]. The strategy of targeting autophagy in CP chemotherapy must consider the effects on both healthy and tumorous tissues, including the kidneys. Besides the protective effect on different organ injuries, the antitumor role of PD through different pathological effects including autophagy has also been confirmed [5,6]. Clinically, PD has been used to treat patients with chronic alcoholism [16], irritable bowel syndrome [17], and interstitial cystitis/bladder pain syndrome [18]. Surprisingly, PD has recently been proposed as a potential natural active drug for the treatment of coronavirus disease 2019 [52][53][54][55][56][57]. All these evidences suggest that PD may be a promising option for patients with AKI during CP chemotherapy. It is imperative to further explore the mechanism of PD in AKI and CKD, including autophagy.

Conclusions
Collectively, our study demonstrated for the first time that PD boosts autophagy flux through SIRT6 upregulation and protects renal tubular epithelial cells from oxidative stress, inflammatory response, and apoptosis, thus alleviating cisplatin-induced AKI (Figure 8). PD seems a promising inducer for restoration of autophagy flux for AKI. Furthermore, PD could be considered as a renoprotective natural compound in cisplatin-induced AKI, although further studies are required to confirm its beneficial effects in CKD development after CP chemotherapy.