Nephroprotective Role of Chrysophanol in Hypoxia/Reoxygenation-Induced Renal Cell Damage via Apoptosis, ER Stress, and Ferroptosis

Acute kidney injury (AKI) is caused by hypoxia-reoxygenation (H/R), which is a kidney injury produced by a variety of causes, resulting in the remaining portion of the kidney function being unable to maintain the balance for performing the tasks of waste excretion metabolism, and electrolyte and acid-base balance. Many studies have reported the use of Chinese medicine to slow down the progression and alleviate the complications of chronic renal failure. Chrysophanol is a component of Rheum officinale Baill, a traditional Chinese medicine that has been clinically used to treat renal disease. We aimed to study the nephroprotective effect of chrysophanol on hypoxia/ reoxygenation (H/R)-induced cell damage. The results showed that chrysophanol prevented H/R-induced apoptosis via downregulation of cleaved Caspase-3, p-JNK, and Bax but upregulation of Bcl-2 expression. In contrast, chrysophanol attenuated H/R-induced endoplasmic reticulum (ER) stress via the downregulation of CHOP and p-IRE1α expression. Our data demonstrated that chrysophanol alleviated H/R-induced lipid ROS accumulation and ferroptosis. Therefore, we propose that chrysophanol may have a protective effect against AKI by regulating apoptosis, ER stress, and ferroptosis.


Introduction
It is well known that acute kidney injury (AKI) is a clinical syndrome characterized by a rapid loss of renal function that may further develop into chronic kidney disease (CKD) or even end-stage renal disease (ESRD) [1]. Many studies have reported the use of Chinese Biomedicines 2021, 9,1283 2 of 12 medicine to slow down the progression and alleviate the complications of acute renal failure, thereby improving quality of life [2,3]. In a cisplatin-induced AKI mouse model, wogonin reversed abrupt kidney dysfunction by substantially suppressing the increased levels of serum creatinine and blood urea nitrogen (BUN) to almost normal levels [4]. According to the literature on Chinese herbal intervention in AKI, Chinese herbs have been reported to play a critical role in the management of AKI by promoting repair and regeneration, enhancing extrarenal clearance of uremic toxins, and preventing progression to CKD [5,6]. Therefore, determining the best possible treatment may be a good strategy to attenuate or prevent renal injury in AKI.
Endoplasmic reticulum (ER) stress is the major cause of renal cell damage during the progression of AKI by renal I/R [14]. Furthermore, excessive reactive oxygen species (ROS) that cause oxidative stress may contribute to the increased susceptibility of the aging kidney in prolonging ER stress-induced acute injury [15]. Mounting evidence indicates that ER stress contributes to glomerular and tubular damage in patients with acute and chronic kidney disease [16]. Therefore, modulation and normalization of ER stress in kidney cells using pharmacological agents may be a promising therapeutic approach for preventing the progression of kidney disease [17]. On the other hand, an increasing number of studies have significantly implicated ferroptosis, a unique type of regulated cell death that is characterized by a large amount of iron accumulation and lipid peroxidation during the cell-death process, in the development of acute kidney disease [18][19][20]. Altogether, targeting the homeostasis of ferroptosis may provide a new target for therapeutic intervention in AKI [21]. Shu et al. reported that I/R led to kidney injury via the underlying mechanism of ER stress as indicated by the increased expression of the C/EBP homologous protein (CHOP). Additionally, inhibition of ER stress prevents renal tubular epithelial cell apoptosis, inflammation, and autophagy caused by I/R [22].
Recent studies have revealed an emerging role of ferroptosis in the pathophysiological processes of AKI, which can eventually lead to acute renal failure (ARF) [19,23,24]. Yang and Stockwell suggested that the downregulation of GPX4 may be more sensitive to ferroptosis, whereas inhibition of ferroptosis was detected upon the upregulation of GPX4 in vitro [25]. Furthermore, SLC7A11 is a key regulator of ferroptosis [18]. It has also been reported that inhibiting SLC7A11 activity induces ROS accumulation and triggers ferroptosis [26].
In the present study, we investigated whether chrysophanol attenuated cell death in HK-2 cells exposed to H/R injury and exerted renal protection via modulation of apoptosis, ER stress, and ferroptosis. The antibodies used for immunofluorescence staining and Western blotting were as follows: rabbit polyclonal antibodies against cleaved Caspase-3 (Cell Signaling Technology
Hypoxia/reoxygenation conditions were created using a previously described method with some modifications [27,28]. Briefly, cells were grown on 6-or 24-well plastic dishes in a hypoxia chamber and equilibrated for 30 min with humidified gas containing 1% oxygen, 5% CO 2 , and 94% nitrogen (Hypoxic incubator APM-30D, Astec, Tokyo, Japan). The cell lines were maintained under hypoxic conditions for 15 h and then additionally incubated under normoxic conditions for another 2 h. The normal group consisted of cells grown under normal (21%) oxygen conditions for the same duration.

Cell Viability Assay
Cell viability was measured using the WST-1 assay. Cells were seeded at a density of 5 × 10 4 cells/mL in 24-well plates and cultured in phenol red-free DMEM containing 0.5% heat-inactivated FBS for 24 h. Then, the cells were incubated with the indicated concentrations of chrysophanol (30 µM) for 24 h. The WST-1 reagent was then added to the medium and the cells were incubated at 37 • C for 2 h. The absorbance was measured at 450 nm using a microplate reader (Bio-Rad, Hercules, CA, USA).

Western Blotting
The cells 1 × 10 6 were harvested and lysed according to a protocol described in our previous study [29]. Briefly, cells were collected, washed three times with PBS and lysed using RIPA lysis buffer (Pierce, Rockford, IL, USA), containing 1% Sigma protease cocktail, for 30 min at 4 • C. The lysates were centrifuged at 10,000× g at 4 • C to obtain solubilized cellular proteins. The supernatant protein concentration was measured using a bicinchoninic acid (BCA) protein assay (Pierce, Rockford, IL, USA). Proteins were separated by 8%, 10% or 12% SDS-PAGE and electro-transferred onto a polyvinylidene fluoride membrane. Blots were probed with specific primary antibodies and followed by HRPconjugated goat anti-rabbit IgG (1:3000-1:5000) or HRP-conjugated goat anti-mouse IgG (1:5000) (Zymed, San Francisco, CA, USA). After washing with PBS containing 0.5% Tween-20, peroxidase activity was assessed using enhanced chemiluminescence (ECL; PerkinElmer Life Science, Hopkinton, MA, USA). The same membrane was re-probed with a monoclonal antibody directed against GAPDH or β-actin as a loading control (1:5000; GeneTex, Irvine, CA, USA). The intensities of the reaction bands were analyzed with the UVP Biospectrum (UVP, LLC Upland, CA, USA). The primary antibodies used are mentioned in Section 2.1 (Reagents and antibodies).

Lipid ROS Detection
Cells were incubated with 2 µM C11-BODIPY 581/591 (Thermo Fisher Scientific, Waltham, MA, USA) in culture medium for 1 h and then washed with phosphate-buffered saline (PBS). After trypsinization, cells were collected and processed for flow cytometry (BD Biosciences, San Jose, CA, USA) at an excitation wavelength of 488 nm and an emission wavelength of 517-527 nm.

Nuclear Fraction Extraction
The nuclear fraction was extracted from 1 × 10 6 HK-2 cells according to the protocol of a previous study [30]. Briefly, nuclear fraction was extracted from cells. The cells were collected and resuspended in a hypotonic buffer (10 mM HEPES, pH 7.9; 10 mM KCl; 1.5 mM MgCl 2 ; 0.2 mM PMSF; 20 µg/mL aprotinin; 0.5 mM DTT; and 0.5% NP-40) on ice for 15 min. After centrifuging at 6000× g for 15 min at 4 • C, the pellet was collected and then washed with basal buffer (hypotonic buffer without 0.5% NP-40). After centrifuging again at 6000× g for 15 min at 4 • C, the pellet was collected and resuspended in a hypertonic buffer (20 mM HEPES, pH 7.9; 400 mM KCl; 1.5 mM MgCl 2 ; 0.2 mM PMSF; 20 µg/mL aprotinin; 0.5 mM DTT; 0.2 mM EDTA; 10% glycerol) at room temperature for 30 min. After centrifuging at 10,000× g for 30 min at 4 • C, the nuclear fraction contained in the supernatant was collected.

Immunofluorescence Staining
The cells were fixed in 3% formaldehyde at room temperature for 30 min, blocked with PBS containing 3% FBS at room temperature for 1 h, and incubated with anti-p65 antibody and β-catenin at 4 • C for 16 h, followed by incubation with FITC-labeled secondary antibody (Jackson ImmunoResearch Laboratories, West Grove, PA, USA) at room temperature for 1 h. The nuclei were stained with diamidino-2-phenylindole (DAPI, Molecular Probes) at room temperature for 15 min, which is a DNA groove-binding dye, and examined using a Leica TCS SP5 laser scanning microscope (Leica, Bensheim, Germany).

Statistical Analyses
All data were analyzed using one-way or two-way analysis of variance (ANOVA). Differences were considered statistically significant when the p-values were less than 0.05 (* p < 0.05, ** p < 0.01).

Chrysophanol Regulated ER Stress and Ferroptosis Pathway under H/R Conditions
To investigate the effect of chrysophanol on H/R-induced ER stress, we used Western blotting to examine the expression of CHOP and phosphorylated phosho-IRE1α

Chrysophanol Regulated ER Stress and Ferroptosis Pathway under H/R Conditions
To investigate the effect of chrysophanol on H/R-induced ER stress, we used Western blotting to examine the expression of CHOP and phosphorylated phosho-IRE1α (p-IRE1α) in the presence and absence of chrysophanol under H/R conditions. The results indicated that H/R increased the expression of CHOP and p-IRE1α (Figure 2A

Chrysophanol Regulated ER Stress and Ferroptosis Pathway under H/R Conditions
To investigate the effect of chrysophanol on H/R-induced ER stress, we used Western blotting to examine the expression of CHOP and phosphorylated phosho-IRE1α    Figure 3E). Taken together, we suggest that chrysophanol regulates ER stress and the ferroptosis pathway under H/R conditions. chelator). Results showed that the DFO attenuated the increase (Figure 3C,D) and the decrease in cell viability under H/R conditions ( Figure 3E). Taken together, we suggest that chrysophanol regulates ER stress and the ferroptosis pathway under H/R conditions.

Chrysophanol Treatment Attenuated p-JNK Expression and NF-κB Nuclear Translocation under H/R Conditions
Our results demonstrated that H/R increased the expression of p-JNK (Figure 2A  Error bars represent the standard deviation from three independent replicates. n = 3. * p < 0.05.

Chrysophanol Treatment Attenuated p-JNK Expression and NF-κB Nuclear Translocation under H/R Conditions
Our results demonstrated that H/R increased the expression of p-JNK (Figure 2A Figure 4B. Additionally, to further confirm the effect of chrysophanol on β-catenin and NF-κB p65 nuclear translocation under H/R conditions, we performed immunofluorescence staining ( Figure 4C,D and Supplementary Materials Figure S1). Moreover, results also demonstrated that chrysophanol alleviated the increase in IL-6 expression under H/R conditions ( Figure 4E). These results indicated that chrysophanol treatment attenuated p-JNK expression and NF-κB nuclear translocation under H/R conditions. NF-κB p65 nuclear translocation under H/R conditions, we performed immunofluorescence staining ( Figure 4C,D and Supplementary Materials Figure S1). Moreover, results also demonstrated that chrysophanol alleviated the increase in IL-6 expression under H/R conditions ( Figure 4E). These results indicated that chrysophanol treatment attenuated p-JNK expression and NF-κB nuclear translocation under H/R conditions.

Figure 4. Chrysophanol reversed H/R-induced nuclear translocation of β-catenin and p65. (A)
Cytosolic and nuclear fractions were isolated as described in "materials and methods". Western blot analysis was performed to detect the subcellular localization of β-catenin and NF-κB using an antibody against the β-catenin and NF-κB subunit p65. β-actin was used as a cytosolic marker and fibrillarin as a nuclear marker. (B) The expression of β-catenin and nuclear p65 (nuclear p65) was quantified using ImageJ. All data are presented as the mean ± SD. ** p < 0.01. Immunofluorescence micrographs showed the distribution of β-catenin (C) and p65 (D).  Cytosolic and nuclear fractions were isolated as described in "materials and methods". Western blot analysis was performed to detect the subcellular localization of β-catenin and NF-κB using an antibody against the β-catenin and NF-κB subunit p65. β-actin was used as a cytosolic marker and fibrillarin as a nuclear marker. (B) The expression of β-catenin and nuclear p65 (nuclear p65) was quantified using ImageJ. All data are presented as the mean ± SD. ** p < 0.01. Immunofluorescence micrographs showed the distribution of β-catenin (C) and p65 (D). The β-catenin (C) and p65 (D) signal was intensely localized to the nucleus in HK-2 cells under H/R conditions (H/R) but was located in the cytoplasm under H/R conditions in the presence of 30 µM chrysophanol (H/R + Cho). The nuclei were counterstained with DAPI. Bar = 25 µm. (E) Chrysophanol alleviated the increasing in the IL-6 expression under H/R conditions. n = 3.

Discussion
In this study, we proposed that chrysophanol has a nephroprotective effect on renal cell damage caused by H/R. Additionally, we also suggested that apoptosis, ER stress, and ferroptosis, which are involved in H/R-induced cell death, were reversed in the presence of chrysophanol. Havasi and Borkan demonstrated that the improvement of renal cell death in AKI generated new therapeutic targets [31]. Interestingly, proximal tubular epithelial cells were reported to be highly susceptible to apoptosis, and injury at this site contributes to kidney failure [31]. Human renal proximal tubular cells (HK-2) were established as a cell model of hypoxia-reoxygenation (H/R) injury to mimic acute renal I/R injury [32], as well as our findings (Figure 1). Traditional medicines often use multi-component extracts of natural products that may be developed as therapeutic strategies to treat AKI due to their multitarget potential and established biosafety [33]. Rhus verniciflua Stokes extract was reported to prevent the progression of AKI via modulation of the Nrf2/antioxidant enzyme pathway, using in vivo and in vitro I/R injury (IRI)-induced AKI models [34]. Similarly, Taraxacum officinale has a protective effect on H/R-induced AKI via inhibition of oxidative stress, inflammation, and apoptosis in the extracellular signal-regulated kinase (ERK) and c-Jun NH2-terminal kinase (JNK) of the mitogen-activated protein kinase (MAPK) signaling pathways in vivo and in vitro [35]. Accumulating evidence has revealed that natural products or herbs may have potential for use in the clinical treatment of renal H/R injury [36][37][38], but resveratrol did not protect against H/R-induced AKI in vivo [39].
It is well known that regulating ER stress in kidney cells may provide a therapeutic target in AKI that is caused by H/R [14,16,40,41]. As shown in Figure 2A, H/R-induced ER stress was attenuated by chrysophanol treatment. Consistent with our findings, Huaier extract was found to downregulate the expression of CHOP and Bip in a thapsigargininduced ER stress model of HK-2 cells [42]; the same result was also observed in other studies [27]. On the other hand, ferroptosis is well known to play a critical role in the pathology of AKI [19]. Quercetin (QCT) was reported to inhibit ferroptosis, but not apoptosis, necrosis, or autophagy, in renal proximal tubular epithelial cells and ameliorate AKI induced by I/R or folic acid (FA) [43]. In this study, chrysophanol attenuated H/Rinduced ferroptosis via the regulation of GPX4 and SLC7A11, as seen on Western blot ( Figure 2B) and BODIPY C11 fluorescence staining (Figure 3) in HK-2 cells, similar to the results obtained in other studies [44,45]. Therefore, chrysophanol ameliorated renal cell injury with H/R, perhaps by inhibiting ferroptosis. Chrysophanol may represent a novel treatment option that improves recovery from H/R-induced renal tubular cell injury by targeting ferroptosis. It is also possible that chrysophanol acts as an antioxidant, which may consequently increase the antioxidative capacity of the cells and elevate the expression of GXP4 and SLC7A11.
NF-κB signaling was reported to play a major role in the regulation of kidney injury [46]. Tubular injury was rescued by inhibiting NF-κB signaling in the renal tubular epithelium in vivo [47]. In addition, apoptotic tubular cell death was determined via downregulation of the Wnt/β-catenin dependent pathway by Dickkopf-3 in proteinuric nephropathy [48]. However, the conflicting role of the Wnt/β-catenin signaling pathway in kidney diseases and kidney injury repair or regeneration is determined by whether kidney structure and function are "reversible" or "irreversible" after injury. Therefore, further elucidation of the mechanisms by which Wnt/β-catenin acts in AKI and CKD may offer new therapeutic options for patients with various kidney diseases [49].
Cisplatin is one of the most widely used chemotherapeutic agents for treating solid tumors, but the administration of cisplatin causes tubular cell injury and AKI [50,51]. Maltol (3-hydroxy-2-methyl-4-pyrone), found in baked products as well as red ginseng root, coffee, chicory, soybeans, bread crusts, and caramelized foods [52], was reported to serve as a valuable potential drug to prevent cisplatin-induced nephrotoxicity [53]. Ridzuan et al. summarized several studies reporting that natural products possess potent antioxidant and anti-inflammatory medicinal properties, and they can be safely used as a supplementary regime or during combination therapy against cisplatin-induced nephrotoxicity [54]. In a future study, we will investigate whether chrysophanol alleviates cisplatin-induced nephrotoxicity.

Conclusions
In summary, the present study demonstrated that chrysophanol prevented H/Rinduced cell death via apoptosis by increasing the expression of Bax, and p-JNK but decreasing the expression of Bcl-2, as well as prolonging ER stress by increasing the expression of CHOP and p-IRE1α, finally increasing the expression of cleaved Caspase-3 ( Figure 5A). Additionally, chrysophanol attenuated the apoptosis via inhibition of apoptotic molecules, triggering the expression of anti-apoptotic molecules and inhibition of ER stress ( Figure 5A). We also suggested that H/R induced HK-2 cell death by triggering ferroptosis via lipid ROS accumulation and downregulation of anti-ferroptotic molecules, GPX4 and SLC7A11 ( Figure 5B). On the other hand, chrysophanol showed potential anti-ferroptotic effects in HK-2 cells under H/R conditions via upregulation of GPX4 and SCL7A11 and alleviating lipid ROS accumulation ( Figure 5B) supplementary regime or during combination therapy against cisplatin-induced nephrotoxicity [54]. In a future study, we will investigate whether chrysophanol alleviates cisplatin-induced nephrotoxicity.

Conclusions
In summary, the present study demonstrated that chrysophanol prevented H/R-induced cell death via apoptosis by increasing the expression of Bax, and p-JNK but decreasing the expression of Bcl-2, as well as prolonging ER stress by increasing the expression of CHOP and p-IRE1α, finally increasing the expression of cleaved Caspase-3 ( Figure  5A). Additionally, chrysophanol attenuated the apoptosis via inhibition of apoptotic molecules, triggering the expression of anti-apoptotic molecules and inhibition of ER stress ( Figure 5A). We also suggested that H/R induced HK-2 cell death by triggering ferroptosis via lipid ROS accumulation and downregulation of anti-ferroptotic molecules, GPX4 and SLC7A11 ( Figure 5B). On the other hand, chrysophanol showed potential anti-ferroptotic effects in HK-2 cells under H/R conditions via upregulation of GPX4 and SCL7A11 and alleviating lipid ROS accumulation ( Figure 5B)   Funding: The grants supported by this study include CGH-MR-A11004 from Cathay General Hospital, Taipei, Taiwan. This work was also supported by a grant from the Ministry of Science and Technology, Taiwan (109-2320-B-303-004-MY3).

Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.

Data Availability Statement:
The original data used to support the findings of this study are included in this article.