In vitro study on effect of bardoxolone methyl on cisplatin-induced cellular senescence in human proximal tubular cells

Bardoxolone methyl [methyl-2-cyano-3, 12-dioxooleana-1, 9(11)dien-28-oate (CDDO-Me)], an activator of the nuclear factor erythroid-derived 2-related factor2 pathway, is a potential therapeutic candidate for the treatment of kidney diseases. However, its effect against cellular senescence remains unclear. This study aimed to investigate whether CDDO-Me protects cells against cisplatin-induced cellular senescence using an in vitro model. The human renal proximal tubular epithelial cell line HK-2 was treated with cisplatin for 6 h, followed by treatment with or without CDDO-Me (0.1 or 0.2 μmol/L). Senescence markers were analyzed using western blotting and real-time PCR. Apoptosis was evaluated through TUNEL staining. Cisplatin induced changes in the levels of markers specific for proliferation, cell cycle, and senescence in a time- and dose-dependent manner. Furthermore, IL-6 and IL-8 levels in the culture medium increased markedly. These data suggested that cellular senescence-like alterations occurred in HK-2 cells exposed to cisplatin. CDDO-Me treatment reversed the cisplatin-mediated alterations in the levels of cellular senescence markers. The antioxidant enzymes, HO1, NQO1, GPX1, and CAT were upregulated by CDDO-Me treatment. Furthermore, CDDO-Me treatment induced apoptosis in cisplatin-exposed HK-2 cells. Pretreatment with Ac-DEVD-CHO, the caspase inhibitor, suppressed the reversal effect of CDDO-Me against cisplatin-induced cellular senescence-like alterations. This study showed that CDDO-Me attenuated cisplatin-induced premature senescence of HK-2 cells. This beneficial effect may be related to Nrf2 activation. Our findings also showed that CDDO-Me induced apoptosis in cisplatin-treated HK-2 cells, potentially protecting the kidneys from cellular senescence. CDDO-Me appears to be a candidate treatment for acute kidney injury.


Introduction
Acute kidney injury (AKI) is a complex syndrome characterized by renal dysfunction and is associated with high morbidity and mortality worldwide. Patients with post-AKI are at an increased risk for chronic kidney disease, CKD [1][2][3]. Proximal tubule injury caused by AKI triggers several features of CKD by inducing inflammation and interstitial fibrosis [4]. Therefore, it is important to protect the proximal tubules for preventing the progression of AKI to CKD; however, the definitive evidence for this effect is lacking.
There is an association between cellular senescence in proximal tubular epithelial cells (PTCs) and CKD in the animal models of hypertension [5] or diabetes [6]. Senescence is a tumor suppressor mechanism by which cells adapt to DNA damage, oxidative stress, and telomere shortening. Senescence induces cell cycle arrest in cells exposed to various stresses 1 3 [7,8]. Senescent cells secrete pro-inflammatory molecules in order to recruit immune cells. This behavior is termed as senescence-associated secretory phenotype, SASP [9]. These pro-inflammatory molecules, including cytokines and chemokines, can lead to the clearance of senescent cells in the damaged tissue [10,11]. In contrast, the chronic secretion of these SASP factors promotes inflammation and epithelialto-mesenchymal transition, thereby reducing the functional reserve of tissues. Thus, senescence is protective in the acute phase; however, chronic senescence can be harmful to tissue homeostasis. Senescent cells undergo an anti-apoptotic state [12][13][14], leading to the constitutive secretion of SASP factors. Chronic secretion of SASP factors can spread senescence to nearby cells [15]; therefore, the removal of senescent cells by activating apoptosis seems to be a critical mechanism for protecting the proximal tubule and preventing CKD progression. In fact, much effort has been made to eliminate senescent cells using novel drugs called senolytics. These drugs remove senescent cells by inducing apoptosis [16] or by blocking their resistance to apoptosis [17,18]. Therefore, protecting PTCs from cellular senescence could prevent CKD development; however, effective treatments that target senescence are lacking.
Cisplatin is one of the most widely used anti-cancer drugs; however, its use is limited because of its nephrotoxicity, which causes AKI [29]. Cisplatin treatment is widely used as a model for AKI; it induces cellular senescence, both in vitro [30,31] and in vivo [32,33]. The aim of this study was, therefore, to investigate whether CDDO-Me protects PTCs against cisplatin-induced cellular senescence to explain the beneficial effect of CDDO-Me.

Cell culture
The human renal proximal tubular epithelial cell line HK-2 was cultured in Dulbecco's Modified Eagle's Medium: Nutrient Mixture F-12 (DMEM/F12, Fujifilm Wako Chemical Co., Osaka, Japan) supplemented with 10% fetal bovine serum (FBS, Biosera, Inc., Nuaille, France) and 1% penicillin-streptomycin, as previously described [34]. Cells were treated with 0-50 µmol/L cisplatin in low-glucose DMEM containing 0.1 mg/mL human serum albumin for 6 h. The medium was then replaced with fresh medium with or without 0.1-0.2 μmol/L CDDO-Me (Sigma-Aldrich Co., St. Louis, MO, USA). The cells were treated with 5 or 50 µmol/L of the caspase inhibitor, Ac-DEVD-CHO (Selleckchem, Houston, TX, USA), for 60 min before adding CDDO-Me to the medium. Cells were cultured for 24-72 h, and proteins or mRNA were extracted at the indicated points.

Real-time reverse transcription-PCR (RT-qPCR)
Total RNA and cDNA from HK-2 cells were prepared using the ISOGEN-II (Nippon Gene, Tokyo) and Prime Script RT-PCR kits (Takara Bio, Shiga), according to the manufacturer's instructions. The primers used for qPCR are shown in Supplementary Table 1. qPCR was performed on a 7500 Real-Time PCR System (Applied Biosystems) in a 96-well reaction plate using Power SYBR Green. The mRNA expression of the target genes was normalized to that of GAPDH, using the delta-delta C t method.

Cell cycle analysis using flow cytometry
HK-2 cells were harvested, washed, and resuspended in phosphate buffered saline. Cells were then fixed with 70% ethanol and stored at 4 °C overnight. Subsequently, cells were incubated with 100 µg/mL RNase for 30 min at 37 °C and stained with 5 µg/mL propidium iodide (PI) for 10 min. Flow cytometry analyses were performed using a BD FAC-SCalibur (BD Biosciences). Cell cycle phase distributions were determined using Modfit LT software version 3.0 (Verity Software House, Topsham, ME, USA).

Determination of cytokine levels by ELISA
Following exposure to cisplatin, supernatants were collected from the culture medium. Cytokine levels were determined using ELISA kits for IL-6 and IL-8 (R&D Systems, Minneapolis, MN, USA) as per the manufacturer's instructions.

TdT-mediated dUTP-biotin nick end labeling (TUNEL) staining
TUNEL staining was performed using a commercial kit (Takara Bio), following each treatment described above. The percentage of apoptotic cells was determined by counting the TUNEL-positive cells and the total number of cells (nucleus) in four to six photomicrographs (× 200 magnification, approximately 1500 cells) by a blinded observer.
This experiment was performed thrice, on different days. The apoptosis rate was expressed as the means ± standard deviation (SD) from three independent experiments.

Statistical analysis
All data are reported as the mean ± SD. The RT-qPCR and WB analysis data are presented as the fold change relative to the controls (untreated cells). The RT-qPCR data were evaluated using Students t-test to assess differences between the control and cisplatin-treated cells. Other data were analyzed using analysis of variance (ANOVA) with post hoc comparisons using the Student-Newman-Keuls method. P values < 0.05 were considered statistically significant.

Cisplatin treatment induces cellular senescence-like alterations in HK-2 cells
First, we examined whether cisplatin exposure induces cellular senescence in HK-2 cells. Cisplatin treatment induced alterations in the expression of proliferation, cell cycle, and senescence markers in a time-and dose-dependent manner (Fig. 1). The expression of Ki-67, a proliferation marker, significantly increased after exposure to 20 μmol/L cisplatin; it decreased after exposure to 50 μmol/L cisplatin compared to that after exposure to 20 μmol/L cisplatin. The expression of the cell cycle markers pRb, Rb, cyclin D, and cyclin A increased in cisplatin-exposed HK-2 cells.  Fig. 2A-D). Furthermore, the mRNA levels of IL-6 and IL-8 increased markedly (Fig. 2E, F). An increase in the cytokine levels was observed in the culture medium of cells exposed to 50 μmol/L cisplatin (Fig. 2G, H). These data suggest that cisplatin treatment induced proliferation, DNA damage, and subsequent cellular senescence-like alterations in HK-2 cells.

CDDO-Me reverses cisplatin-induced cellular senescence-like alterations in HK-2 cells
CDDO-Me treatment normalized the cisplatin-induced alterations in the expression of the cellular senescence markers Ki-67, cyclin A, cyclin D, pRb/Rb, and p16 INK4a in HK-2 cells (Fig. 3). Cell cycle analysis using cytometry revealed that CDDO-Me treatment ameliorated cisplatininduced cell cycle abnormalities (Fig. 4). Cisplatin markedly decreased the percentage of cells in the G1 phase and increased that in the S phase. The cells in the G2 phase also tended to increase after cisplatin treatment. However, CDDO-Me treatment increased the number of G1-phase cells and decreased that of S-and G2-phase cells.

CDDO-Me upregulates the expression of antioxidant enzymes
To evaluate the mechanisms by which CDDO-Me protected the cisplatin-exposed HK-2 cells against cellular senescence-like alterations, we evaluated whether the expression of antioxidant enzyme gene were upregulated under the present conditions (Fig. 5). The mRNA of antioxidant enzymes, heme oxygenase-1 (HO1), NAD(P)H:quinone oxidoreductase (NQO1), glutathione peroxidase 1 (GPX1), and catalase (CAT ), were upregulated by CDDO-Me treatment. However, CDDO-Me treatment did not significantly increase the mRNA expression of superoxide dismutase 1 (SOD1).

CDDO-Me induces apoptosis in cisplatin-treated HK-2 cells
The effect of CDDO-Me on apoptosis was evaluated because CDDO-Me treatment significantly decreased the cell number in cisplatin-exposed HK-2 cells (Fig. 6A). CDDO-Me induced apoptosis in cisplatin-exposed HK-2 cells; however, only cisplatin treatment also induced apoptosis, as observed using TUNEL staining (Fig. 6B, C). WB analysis confirmed that CDDO-Me treatment induced apoptosis, decreased the level of pro-caspase-3, and increased the level of cleaved caspase-3 and p62/SQSTM1 in HK-2 cells (Fig. 7A, B). reverses cisplatin-induced cellular senescence-like alterations via apoptosis, cisplatin-exposed cells were treated with Ac-DEVD-CHO, a caspase inhibitor, before the CDDO-Me treatment. Ac-DEVD-CHO treatment inhibited the decrease in Ki-67, cyclin A, cyclin D, and p16 INK4a expression levels induced by CDDO-Me (Fig. 7C, D).

Discussion
CDDO-Me therapy improves renal function in CKD patients, in clinical trials [24,25,35]. However, the renal protective mechanism of CDDO-Me on the AKI-CKD transition remains unknown. Protecting the proximal tubules from AKI, which causes cellular senescence, is important for preventing CKD development. This study elucidated the protective effect of CDDO-Me on cisplatin-induced cellular senescence in cultured human PTCs, HK-2, which is one of the most used cells for the study of senescent PTCs [36][37][38].
In this study, WB analysis revealed an increased expression of Ki-67 in HK-2 cells following cisplatin treatment; chronic increase in proliferation induces cellular senescence in PTCs [39]. Accelerated proliferation may be one mechanism by which cellular senescence is induced in cisplatin-treated cells. In addition to a pool of proliferating cells, elevated cell cycle arrest markers were observed, indicating the presence This was demonstrated by the upregulation of cyclindependent kinase inhibitors, such as p16 INK4a , p21 Waf1/Cip1 , and phosphorylated Rb in cells exposed to cisplatin for 72 h. An increase in p21 Waf1/Cip1 and p16 INK4a induces G2/M arrest through the phosphorylation of Rb [40]. Flow cytometric analysis showed an increase in the number of S-phase cells in cisplatin-treated cultures. Thus, cisplatin treatment seemed to be associated with S-or G2/M-phase arrest by regulating p16 INK4a and/or p21 Waf1/Cip1 . Cisplatin stalls cell proliferation by damaging DNA and inhibiting DNA synthesis, which is cytotoxic during the S phase [41]. In fact, γ-H2AX, a DNA damage marker, was elevated in cisplatintreated cells. Furthermore, cisplatin treatment induced the upregulation and secretion of IL-6 and IL-8 in the medium.
These results indicate that cisplatin-exposed cells undergo premature cellular senescence, consistent with the results from previous studies [30,31].
This study showed that CDDO-Me treatment decreased senescence markers in PTCs exposed to cisplatin. Upon activation by CDDO-Me, Nrf2 translocates to the nucleus and induces the transcription of antioxidant enzyme genes via the antioxidant response element, ARE [42]. This was demonstrated by the upregulation of the antioxidant enzymes following the CDDO-Me treatment of the cisplatin-treated HK-2 cells. Aleksunes et al. elucidated using in vivo studies that cisplatin accelerates cell proliferation and secretion of IL-1β, IL-6, and TNF-α in the kidney; importantly, this phenotype was inhibited by CDDO-lm, an analog of CDDO-Me, through the increase of Nrf2 signaling [43]. Treatment with the antioxidant N-acetylcysteine ameliorates cellular senescence in PTCs induced by multiple cisplatin treatments [32]. Furthermore, an in vitro study using human PTCs showed that the upregulation of the antioxidant genes by Nrf2 enhanced cell viability [44]. These studies suggest that removing oxidative stress via the Keap1-Nrf2 pathway protects PTCs from cellular senescence. A C B Fig. 6 CDDO-Me treatment decreases the cell number in cisplatinexposed HK-2 cells by inducing apoptosis. Cisplatin (20 µmol/L)exposed HK-2 cells were cultured in medium with or without 0.2 µmol/L CDDO-Me. Cell numbers were counted at 48 h (A). n = 3, **P < 0.01. Apoptotic cells were detected in cisplatin (20 µmol/L)-exposed cultures after treatment with or without 0.2 µmol/L CDDO-Me by TUNEL staining (original magnification × 200) (B), and the number of apoptotic cells was expressed as the apoptosis rate (C). Data are presented as mean ± SD from three independent experiments. **P < 0.01 Overdosing with CDDO-Me induces apoptosis [28]. Eliminating the senescent cells by activating apoptosis has a protective effect on tissue aging [16][17][18]. Therefore, we evaluated whether CDDO-Me induced apoptosis and reversed senescence via an apoptotic mechanism. We showed that CDDOM-Me decreased the cell number, which occurs through the apoptotic mechanism. CDDO-Me treatment increased the level of cleaved caspase-3 and the number of TUNEL-positive cells. Furthermore, CDDO-Me accelerated the phosphorylation of H2AX, which occurs in response to DNA damage and induces apoptosis by mitochondrial cytochrome C release [45]. Additionally, CDDO-Me increases p62/SQSTM1 expression, which plays a critical role in both autophagy and apoptosis [46,47]. Inhibition of apoptosis suppressed the reversal effect of CDDO-Me against cisplatin-induced cellular senescence-like alterations. Therefore, CDDO-Me induces apoptosis and suppressed cellular senescence in cisplatin-treated cells. The activation of apoptosis could contribute to the removal of senescent cells and improved kidney functions. HK-2 cells were exposed to 20 µmol/L cisplatin for 6 h, followed by treatment with or without CDDO-Me (0.1 or 0.2 µmol/L). Apoptotic marker proteins were analyzed by western blotting (A), and protein bands were quantified by densitometry (B). n = 4-6, *P < 0.05, **P < 0.01 vs. control (Ctrl), # P < 0.05, ## P < 0.01 vs. cisplatin, + P < 0.05, ++ P < 0.01 vs. cisplatin + CDDO-Me (0.1 µmol/L). Cispl-atin-exposed cells were treated with 5 or 50 µmol/L Ac-DEVD-CHO, a caspase inhibitor, for 60 min prior to adding 0.2 µmol/L CDDO-Me to the medium. Senescence markers were analyzed using western blotting (C) and the expression was quantified using densitometry (D). n = 4-5, *P < 0.05, **P < 0.01 vs. cisplatin, # P < 0.05, ## P < 0.01 vs. cisplatin + CDDO-Me

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The use of only one cell line was a limitation of this study. HK-2 cells were used in this study, because senescence could be easily induced by treating the HK-2 cells with d-serine, indoxyl sulfate, and hydrogen peroxide [36][37][38]. Additionally, HK-2 cells maintain biochemical properties similar to that in the in vivo proximal tubule cells [48,49]. Therefore, this model was well-suited for this study. However, HK-2 cells lack expression of organic anion transporter 1, 3, and organic cation transporter 2, which are important for drug metabolism [50]. Therefore, drug metabolism in HK-2 might not be completely similar to that in the in vivo state. It is unclear whether CDDO-Me removes senescent cells selectively in vivo; therefore, we cannot conclude that activation of apoptosis by CDDO-Me protects against kidney injury in humans. Further in vivo studies are needed to evaluate the protective effect of CDDO-Me against cellular senescence in PTCs.
In conclusion, our results demonstrated a beneficial effect of CDDO-Me on cellular senescence in HK-2 cells. It is hypothesized that this beneficial effect is related to Nrf2 activation. CDDO-Me appears to be a candidate therapeutic for AKI. Our findings also showed that CDDO-Me induced apoptosis in cisplatin-treated HK-2 cells and potentially protects the kidneys from cellular senescence. Future studies are needed to assess the effects of CDDO-Me-induced apoptosis on cellular senescence and kidney function in AKI animal models.
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