Genipin attenuates cisplatin-induced nephrotoxicity by counteracting oxidative stress, inflammation, and apoptosis

Biomedicine & Pharmacotherapy 93 (2017) 1083–1097

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Original article

Genipin attenuates cisplatin-induced nephrotoxicity by counteracting oxidative stress, inflammation, and apoptosis Eglal Mahgouba , Shanmugam Muthu Kumaraswamyc, Kamal Hassan Kadera,1, Balaji Venkataramana , Shreesh Ojhaa , Ernest Adeghateb , Mohanraj Rajesha,* a Department of Pharmacology and Therapeutics, College of Medicine and Health Sciences, United Arab Emirates University, Al Ain 17666, United Arab Emirates b Department of Anatomy, College of Medicine and Health Sciences, United Arab Emirates University, Al Ain 17666, United Arab Emirates c Department of Pharmacology, Yong Loo Lin School of Medicine, National University of Singapore, 17600 Singapore, Singapore

A R T I C L E I N F O

Article history: Received 25 May 2017 Received in revised form 26 June 2017 Accepted 5 July 2017 Keywords: Genipin Cisplatin Nephrotoxicity Oxidative stress Inflammation Apoptosis

A B S T R A C T

Cisplatin (CP) is a potent and widely used chemotherapeutic agent. However, the clinical benefits of CP are compromised because it elicits nephrotoxicity and ototoxicity. In this study, we investigated the nephroprotective effects of the phytochemical genipin (GP) isolated from the gardenia (Gardenia jasminoides) fruit, using a murine model of CP-induced nephropathy. GP pretreatment attenuated the CPinduced renal tissue injury by diminishing the serum blood urea nitrogen, creatinine, and cystatin C levels, as well as those of kidney injury molecule-1. In addition, GP attenuated the CP-induced oxidative/ nitrative stress by suppressing the activation of NADPH oxidase, augmenting the endogenous antioxidant defense system, and diminishing the accumulation of 4-hydroxynonenal and 3-nitrotyrosine in renal tissues. Furthermore, reduced levels of proinflammatory cytokines such as tumor necrosis factor-alpha and interleukin-1 beta indicated that CP-induced renal inflammation was mitigated upon the treatment with GP. GP also attenuated the CP-induced activation of mitogen-activated protein kinases, excessive activities of caspase-3/7 and poly(ADP-ribose) polymerase, DNA fragmentation, and apoptosis. When administered 12 h after the onset of kidney injury, GP showed a therapeutic effect by ameliorating CPinduced nephrotoxicity. Moreover, GP synergistically enhanced the CP-induced cell death of T24 human bladder cancer cells. Collectively, our data indicate that GP attenuated the CP-induced renal tissue injury by abrogating oxidative/nitrative stress and inflammation and by blocking cell death pathways, thereby improving the renal function. Thus, our results suggest that the use of GP may be a promising new protective strategy against cisplatin-induced nephrotoxicity. © 2017 Elsevier Masson SAS. All rights reserved.

1. Introduction Cis-diamminedichloroplatinum II [cisplatin (CP)] is widely used as a chemotherapeutic drug. In particular, CP has been found to be highly efficient in the treatment of testicular germ cell cancers [1,2]. Although it is a well-known and powerful antiproliferative drug, CP usage is accompanied by moderate to severe nephrotoxicity, ototoxicity, and neurotoxicity [3]. Clinical studies have revealed that approximately 30–50% of the patients treated with CP could develop hearing loss and 14–57% experienced neurotoxicity. Furthermore, 70% of the patients treated with CP experienced nephrotoxicity [4–6]. Despite these side effects, the use of CP

remains a standard chemotherapy regimen for the treatment of cancers arising from the head and neck, as well as testicular, cervical, ovarian, and bladder tumors. This can be due to the fact that other platinum-based drugs failed to provide a solid clinical efficacy [7–10]. Nephrotoxicity is the most serious dose-limiting side effect of CP therapy [11 ototoxicity and nephrotoxicity following high-dose cisplatin and amifostine]. CP-induced nephrotoxicity can manifest with various types of symptoms such as acute kidney injury (AKI), hypomagnesemia, Fanconi-like syndrome, distal renal tubular acidosis, hypocalcemia, renal salt wasting, the renal concentrating defect, hyperuricemia, transient proteinuria, and erythropoietin

* Corresponding author. E-mail address: [email protected] (M. Rajesh). Present address: Department of Basic Medical Sciences, Mohammed Bin Rashid University of Medicine and Health Sciences, Dubai 505055, United Arab Emirates.

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http://dx.doi.org/10.1016/j.biopha.2017.07.018 0753-3322/© 2017 Elsevier Masson SAS. All rights reserved.

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deficiency [12]. The most serious and life-threatening side effect is AKI, which occurs in 20–30% of the patients treated with CP [13]. Vigorous hydration with saline and simultaneous administration of mannitol before, during, and after CP administration have been shown to reduce the risk of CP-induced nephrotoxicity [13,14]. Amifostine (an organic thiophosphate) also reduces the CPinduced nephrotoxicity. The amifostine mechanism of renoprotection involves scavenging of oxygen free radicals via generation of glutathione (GSH) [14,15]. In addition, erythropoietin has been shown to confer the protection against CP-induced nephrotoxicity [16]. Besides its essential role as a hematopoietic agent, erythropoietin inhibits the apoptotic cell death, enhances the tubular epithelial regeneration, and promotes the renal functional recovery in hypoxic or ischemic acute renal injury [16]. Salicylates are anti-inflammatory agents used in the treatment of various inflammatory ailments. Salicylates exhibit anti-inflammatory activities by inhibiting cyclooxygenase activity and prostaglandin synthesis. Furthermore, high doses of salicylates are able to stabilize the inhibitor of kappa B (IkB) enzyme and to reduce the transcriptional activity of nuclear factor-kappa B (NFkB), which consequently attenuates the generation of tumor necrosis factor-alpha (TNF-a) and reduces the renal inflammatory response during CP-induced nephrotoxicity [13,17]. However, these approaches have shown a limited clinical success since CP-induced nephrotoxicity could not be abrogated. Therefore, there is an urgent need to develop agents that can confer renoprotection without compromising the anticancer activity of CP. Phytochemicals are bioactive non-nutrient plant compounds, which are widely distributed in the plant kingdom. Regular consumption of fruits and vegetables has been associated with reduced risk for the development of major chronic diseases such as metabolic syndrome as well as cardiovascular and inflammatory diseases. Presently, more than 5000 phytochemicals have been identified. Despite this progress, the pharmacology and health benefits of most phytochemicals have hitherto been unknown [18]. However, the U.S. Food and Drug Administration has approved the anticancer drug paclitaxel and antimalarial drug artemisinin, phytochemicals derived from Taxus brevifolia and Artemisia annua, respectively [19,20]. In addition, antidiabetic drugs belonging to the chemical class of biguanides were originally isolated from Galega officinalis [21]. These developments provide an indication that phytochemicals have a tremendous translational potential for the treatment of various diseases. Genipin (GP) is a phytochemical extracted from the fruit of Gardenia jasminoides. This plant belongs to the coffee family (Rubiaceae) and is native to subtropical countries of Africa, Asia, and Pacific islands [22]. In traditional Chinese medical practice, GP formulations are used for the treatment of inflammationassociated pain, hypertension, and hepatic disorders [23]. Recently, chemopreventive effects of GP have also been recognized [23]. GP is a terpenoid molecule widely used in the pharmaceutical industry as a cross-linking agent in the synthesis of various biopolymers and as a drug delivery agent. Because of its natural availability and low cytotoxicity, GP has been pursued for the development of novel cross-linking agents. In addition, GP also serves as the backbone for the synthesis of various alkaloids in synthetic medicinal chemistry [24]. Previous studies have indicated that GP possesses anti-inflammatory and antioxidant properties [25]. Furthermore, GP has been shown to ameliorate hepatic ischemia–reperfusion injury, steatosis, autoimmune hepatitis, and fibrosis in rodents [26], [27–29]. Similarly, GP has been reported to attenuate the myocardial tissue injury [30] and to suppress neuroinflammation [31]. In addition, GP inhibited hyperglycemiainduced renal tissue injury by diminishing oxidative stress and inflammation [32].

However, there have been no reports on the nephroprotective activity of GP against CP-induced renal toxicity. Hence, we conducted this study to investigate the GP nephroprotective potential using a murine model of CP-induced nephrotoxicity. 2. Materials and methods 2.1. Animals and drug treatment All animal experimentation protocols adhered to the National Institutes of Health (Bethesda, MD, USA) guidelines for responsible use of laboratory animals and were approved by the Institutional Animal Care and Use Committee of United Arab Emirates University [approval # A5-13]. C57BL/6J male mice (6–8-weekold) were purchased from The Jackson Laboratory (Bar Harbor, ME, USA). The animals were maintained in a temperature-controlled facility, with a 12-h light/dark cycle, and were provided access to water and food ad libitum. GP was obtained from Santa Cruz Biotechnology (Dallas, TX, USA). CP was purchased from Sigma– Aldrich (St. Louis, MO, USA). GP (1–10 mg/kg) was administered via intraperitoneal injections either 2 h before or 12 h after CP administration. The animals were sacrificed 72 h after a single dose of CP (20 mg/kg; intraperitoneally) under anesthesia with tribromomethanol (0.2 mL/10 g body weight). Blood samples were collected by retro-orbital puncture, and the mice were later euthanized by cervical dislocation. After confirming the death of the animals, they were dissected. The kidneys were removed and snap-frozen in liquid nitrogen for biochemical analysis or placed in 10% neutral buffered formalin (Electron Microscopy Sciences, Hatfield, PA, USA) for histological evaluations. Unless specified, all fine reagents were obtained from Sigma–Aldrich. 2.2. Determination of renal function Serum levels of blood urea nitrogen (BUN) and creatinine (Cr) were determined using a VetTest chemistry analyzer (IDEXX Laboratories, Hoofddorp, Netherlands). Test kits were procured from IDEXX Laboratories, and samples were analyzec as per protocol supplied by the manufacturer [33]. 2.3. Determination of serum cystatin C Levels of cystatin C in serum samples were determined using a commercially available enzyme-linked immunosorbent assay (ELISA) kit (R&D Systems, Minneapolis, MN, USA) as per protocol supplied with the kit. In brief, 50 mL of the assay diluent was added to a 96-well microtiter plate, followed by the addition of 50 mL of serum samples or standards, and the plate was incubated for 2 h at room temperature (RT). After washing the wells, 100 mL of the cystatin C conjugate solution was added, and the plate was incubated at RT for 2 h. After washing, the wells were probed with 100 mL of the substrate solution, and absorbance was determined at 450 nm using a microtiter plate reader (Molecular Devices, Sunnyvale, CA, USA). Values were expressed as ng/mL. 2.4. Determination of kidney injury molecule-1 in renal tissues Renal tissues were homogenized with tissue lysis buffer (Thermo Fisher Scientific, Paisley, UK). The protein content was determined in the homogenates using the Lowry reagent (Bio-Rad, Hercules, CA, USA). Levels of kidney injury molecule-1 (KIM-1) in renal tissues were determined using a commercially available kit (R&D Systems), and the protocol was identical to that used for cystatin C measurements. Values were expressed as pg/mg protein.

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2.5. Histological evaluation of CP-induced renal injury

2.6. Determination of NADPH oxidase activity

After fixation of renal tissues in 10% neutral buffered formalin for 72 h, the samples were processed, embedded in paraffin, and sectioned. After deparaffinization, slides were placed in distilled water until further processing of the sections. Periodic acid–Schiff (PAS) staining of the renal sections was performed using a kit purchased from American MasterTech (Lodi, CA, USA). The sections were immersed in 0.5% periodic acid, incubated for 7 min, and washed in running tap water for 1 min. Next, the sections were covered with Schiff solution and incubated for 15 min, followed by washing in running tap water for 5 min. Then, the sections were counterstained with Mayer’s hematoxylin for 2 min and washed in running water for 1 min. Finally, the sections were dehydrated in absolute alcohol and xylene for 1 min and permanently mounted with cover slips. The slides were air-dried and observed under a light microscope (Carl Zeiss, Germany). Tubular damage in PASstained sections was examined under the microscope (200
magnification) and scored based on the percentage of cortical tubules showing epithelial necrosis: 0, normal; 1, <10%; 2, 10–25%; 3, 26–75%; and 4, >75%. Tubular necrosis was characterized by the loss of the proximal tubular brush border, blebbing of apical membranes, epithelial detachment from the basement membrane, or intraluminal hyaline cast formation [33].

Renal tissues were washed in ice-cold phosphate-buffered saline (PBS) and homogenized in 20 mM KH2PO4 buffer (pH 7.0) containing a protease and phosphatase inhibitor cocktail tablet (Calbiochem). NADPH oxidase activity of the renal homogenates was measured using a method described earlier [34]. In brief, photon emission from the chromogenic substrate lucigenin was measured every 15 s for 10 min in a luminometer (Molecular Devices) as a function of acceptance of electron/O2 generated by the NADPH oxidase complex. The composition of the NADPH oxidase assay buffer was as follows: 250 mM 4-(2-hydroxyethyl)1-piperazineethanesulfonic acid (pH 7.4), 120 mM NaCl, 5.9 mM KCl, 1.2 mM MgSO4, 1.75 mM CaCl2, 11 mM glucose, 0.5 mM ethylenediaminetetraacetic acid, 100 mM NADH, and 5 mM lucigenin. The data were converted to relative light units per minute per milligram of protein and expressed as% activity. 2.7. Determination of superoxide dismutase activity Superoxide dismutase (SOD) activity was determined in renal tissues using an assay kit and the protocol obtained from Trevigen, Inc. (Gaithersburg, MD, USA). In this assay, O2 generated from the conversion of xanthine to uric acid and H2O2 by xanthine oxidase reduces WST-1 (colorless) to WST-1 formazan (chromogen), which absorbs light at 450 nm. SOD present in the samples would reduce

Fig. 1. Genipin (GP) was administered to mice at the indicated doses 2 h prior to cisplatin (CP) administration. The animals were sacrificed after 72 h. [A] Serum levels of blood urea nitrogen (BUN) in the respective groups of animals. [B] Serum levels of creatinine in the respective groups of animals. n = 6/group; #p < 0.001 vs. vehicle (Veh)/GP; *p < 0.01 vs. CP; @p < 0.001 vs. CP.

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the O2 concentration and inhibit the generation of WST-1 formazan. Therefore, the reduction in WST-1 formazan was proportional to the SOD activity in the tissues. Values were expressed as U/mg protein.

peroxidase (HRP)-conjugated secondary antibody, and the plate was incubated for 1 h at RT. After washing the wells, a substrate solution was added, and absorbance was measured at 450 nm. The 4-HNE content in the samples was expressed as nmol/mg protein.

2.8. Determination of GSH content

2.10. Determination of 3-nitrotyrosine

GSH levels in tissues were determined using a kit obtained from Trevigen, Inc. This kit uses the enzymatic recycling method for the quantification of GSH. GSH reductase reduces oxidized glutathione to GSH. The sulfhydryl group of GSH reacts with 5,50 -dithiobis(2nitrobenzoic acid) to generate a yellow chromogen, 5-thio-2nitrobenzoic acid (TNB), which absorbs at a peak of 405 nm. The rate of TNB production was directly proportional to the concentration of GSH in tissues. The GSH content was expressed as mmol/ mg protein.

Nitrotyrosine (3-NY) has been considered a footprint of peroxynitrite formation [35]. The 3-NY content in renal tissue homogenates was determined using an ELISA kit procured from Hycult Biotech (Uden, Netherlands).

2.9. Determination of 4-hydroxynonenal Lipid peroxides, which are decomposed to form more complex and reactive compounds such as malondialdehyde and 4hydroxynonenal (4-HNE), are unstable indicators of the oxidative stress in cells. Determination of the end products of lipid peroxidation is one of the most widely accepted assays for oxidative damage. 4-HNE was determined in renal tissues using an ELISA kit procured from Cell Biolabs, Inc. (San Diego, CA, USA) and the protocol supplied with the kit. First, an HNE conjugate was coated on an ELISA plate. Unknown HNE protein samples and HNE standards were then added to the HNE conjugate-coated ELISA plate. After incubation for 1 h at RT, an anti-4-HNE polyclonal antibody was added, followed by the addition of a horseradish

2.11. Determination of NF-kB activation NF-kB activation was determined in renal tissues using a commercially available sandwich ELISA kit procured from Cell Signaling Technology (Danvers, MA, USA) following the protocol supplied by the manufacturer. 2.12. Determination of proinflammatory cytokines Levels of proinflammatory cytokines such as TNF-a and interleukin (IL)-1b were determined in renal tissue homogenates using commercially available ELISA kits (R&D Systems). In brief, 50 mL of an assay diluent was added to a 96-well microtiter plate, followed by the addition of 50 mL of serum samples or standards, and the plate was incubated for 2 h at RT. After washing the wells, 100 mL of conjugate solution was added, and the plate was incubated at RT for 2 h. After washing, the wells were probed with 100 mL of a substrate solution, and absorbance was determined at

Fig. 2. [A] Serum levels of cystatin C and [B] renal tissue levels of kidney injury molecule-1 (KIM-1) as determined by an enzyme-linked immunosorbent assay. n = 6/group; #p < 0.001 vs. vehicle (Veh)/genipin (GP); *p < 0.001 vs. cisplatin (CP).

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Fig. 3. [A] Representative images of periodic acid–Schiff (PAS)-stained paraffin-embedded kidney sections from the respective groups of animals. Asterisk denotes tubular necrosis with cast formation and arrowhead denotes tubular epithelial anoikis. Magnification = 200
. [B] shows the quantification of tubular injury, n = 6/group; # p < 0.001 vs. veh/GP; *p < 0.001 vs. CP.

450 nm using a microtiter plate reader (Molecular Devices). Values were expressed as pg/mg protein. 2.13. Determination of mitogen-activated protein kinase activation by western blot analysis Activation of c-Jun N-terminal kinase (JNK) and p38 mitogenactivated protein kinase (MAPK) was determined in renal tissues by a western immunoblot assay using antibodies purchased from Cell Signaling Technology. Detailed description of the western immunoblot assay protocol can be found in our previous report [36].

2.14. Determination of caspase-3 activity Caspase-3 activity was determined in renal tissue homogenates using a kit purchased from Promega (Madison, WI, USA). Caspase-3 is a protease that specifically cleaves after the C-terminal aspartate residue of the DEVD sequence. In this assay, 100 mL of a substrate solution (Z-DEVD–rhodamine 100) was mixed with 100 mL of a renal tissue homogenate in a microtiter plate, and the plate was incubated for 1 h at RT. The substrate was cleaved by caspase-3 present in the samples to produce fluorescent rhodamine. Fluorescence was measured at an excitation wavelength of 499 nm and an emission wavelength of 521 nm using a microtiter plate reader (Molecular Devices) in a fluorescence reading mode.

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Fig. 4. Genipin (GP) attenuates cisplatin (CP)-induced oxidative stress in renal tissues. [A] NADPH oxidase activity; [B] glutathione (GSH) content; [C] superoxide dismutase (SOD) activity; [D] 4-hydroxynonenal (4-HNE) levels; and [E] 3-nitrotyrosine (3-NY) levels in the respective groups. n = 6/group; #p < 0.001 vs. vehicle (Veh)/GP; *p < 0.01 vs. CP.

2.15. Determination of poly(ADP-ribose) polymerase activity

2.16. Determination of DNA fragmentation

Poly(ADP-ribose) polymerase (PARP) activity was determined in renal tissues using reagents obtained from Trevigen, Inc. This assay measures the incorporation of biotinylated poly(ADP-ribose) in histones pre-adsorbed on 96-well microtiter strips. In brief, 50 mL/well of PARP buffer was added to a microtiter plate to rehydrate histones for 30 min at RT. The contents of the wells were aspirated, followed by the addition of 25 mL of a diluted renal tissue homogenate and 25 mL of assay buffer, and the plate was incubated at RT for 60 min. This was followed by washing and probing of the wells with 50 mL of a diluted streptavidin–HRP conjugate, and then the plate was incubated at RT for 60 min. After washing the wells, 50 mL of a substrate solution was added, and the plate was incubated in the dark for 15 min. The reaction was stopped by adding 50 mL of 0.2 N HCl, and the absorbance was measured at 450 nm.

The assay is based on quantitative sandwich ELISA with mouse monoclonal antibodies against DNA and histones (Sigma–Aldrich), which facilitates specific determination of mono- and oligonucleosomes in total tissue homogenates. Tissue homogenates (20 mL) were placed into streptavidin-coated wells. Then, 80 mL of anti-histone-biotin and anti-DNA-peroxidase was added, and the plate was incubated at RT for 2 h. Unbound components were removed by washing, followed by the addition of 100 mL of a 2,20 azino-di-(3-ethylbenzthiazoline sulfonic acid) solution, and the absorbance was measured at 405 nm. 2.17. Terminal deoxynucleotidyl transferase dUTP nick end labeling staining Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining was performed using a kit obtained from Sigma– Aldrich. Cleavage of genomic DNA during apoptosis yields double-

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Fig. 5. Genipin (GP) attenuates cisplatin (CP)-induced nuclear factor-kappa B (NF-kB) activation and proinflammatory cytokine expression in renal tissues. [A] NF-kB activity; [B] tumor necrosis factor-alpha (TNF-a) and [B] interleukin-1b (IL-1b) levels in the respective groups of samples. n = 6/group; #p < 0.001 vs. vehicle (Veh)/GP; *p < 0.01 vs. CP.

stranded, low-molecular-weight DNA fragments as well as singlestrand breaks in high-molecular-weight DNA. These DNA strand breaks can be identified by labeling free 30 -OH termini with modified nucleotides in an enzymatic reaction. Paraffin sections were dewaxed; then, the slides were placed in a plastic Coplin jar containing 50 mL of 0.1 M citrate buffer, pH 6.0 (Sigma–Aldrich),

and microwaved (750 W) for 1 min. The slides were immediately cooled by adding 80 mL of distilled water and then transferred to a Coplin jar containing PBS. The sections were immersed for 30 min at RT in Tris–HCl (0.1 M, pH 7.5) containing 3% bovine serum albumin. After careful washing of the slides with PBS, 50 mL of the TUNEL reaction mixture was carefully overlaid, and the sections

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Fig. 6. Genipin (GP) attenuates cisplatin (CP)-induced mitogen-activated protein kinase (MAPK) activation in renal tissues. [A] Representative western immunoblot analysis of the activation of p38 MAPK and c-Jun N-terminal kinase (JNK) in the respective groups of animals. [B] and [C] Quantification of the MAPK activation. n = 6/group; #p < 0.001 vs. vehicle (Veh)/GP; *p < 0.001 vs. CP.

were incubated at 37  C for 60 min in a humidified side staining chamber (Electron Microscopy Sciences). After washing with PBS, the sections were mounted in a glycerol medium and observed under a fluorescence microscope using a 515–565-nm filter (fluorescein isothiocyanate filter; EVOS FL, Thermo Fisher Scientific, Paisley, UK). Images were acquired at a final magnification of 100
. 2.18. Cell culture and determination of cell viability A CP-sensitive human bladder carcinoma cell line (T-24) was kindly provided by Dr. Ratha Mahendran, Department of Surgery, National University of Singapore. Cells were cultured in RPMI 1640 medium containing 10% fetal bovine serum, as described previously [37]. Cells were treated with various concentrations of either CP or GP alone for 24–72 h to determine respective 50% inhibitory concentration (IC50) values using a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay kit, as described previously [38]. Thereafter, based on the IC50 values, a combined effect of CP and GP on cell viability was determined. 2.19. Flow cytometric analysis of the cell cycle To determine the effects of GP, CP, and their combination on the cell cycle distribution, cells were exposed to GP or CP alone and to a combination of CP and GP for 72 h. Thereafter, the cells were washed, fixed with 70% ethanol, and incubated for 2 h at 80  C with 0.1% RNase A (Thermo Fisher Scientific, Waltham, MA, USA) in PBS. The cells were then washed, resuspended, and stained in PBS containing 50 mg/mL propidium iodide for 30 min at RT. Cell distribution across the cell cycle was analyzed using a CyAn ADP flow cytometer (DakoCytomation) as described previously [39].

2.20. Statistical analysis Values are expressed are the mean  standard error of the mean. One-way analysis of variance, followed by the Tukey’s posthoc test, was performed to compare groups. Probability values of p < 0.05 were considered significant. Analysis was performed using the GraphPad Prism 5 software (GraphPad Software, La Jolla, CA, USA). 3. Results 3.1. GP treatment attenuated CP-induced renal tissue injury Animals were treated as described in methods section and sacrificed after 72 h. Clinical markers for renal tissue injury, such as the levels of BUN and Cr, were determined in serum samples. As shown in Fig. 1, CP treatment resulted in significant renal tissue injury, which was characterized by elevated levels of BUN (Fig. 1A) and Cr (Fig. 1B). However, GP elicited dose-dependent nephroprotective effects and ameliorated the CP-induced nephrotoxicity (Fig. 1). The maximum nephroprotective activity of GP was observed at 10 mg/kg. Therefore, this concentration was used in subsequent animal experiments. Further, we evaluated the nephroprotective effects of GP against CP-induced nephrotoxicity by determining specific biomarkers of AKI, such as cystatin C and KIM-1, in serum and renal tissue samples. Our data indicated that CP treatment significantly elevated the levels of cystatin C (Fig. 2A) and KIM-1 (Fig. 2B). Upon treatment with GP (10 mg/kg), the levels of cystatin C and KIM-1 were reduced, indicating the amelioration of CP-induced nephrotoxicity (Fig. 2).

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Fig. 7. Genipin (GP) attenuates cisplatin (CP)-induced apoptotic cell death in renal tissues. [A] Caspase-3 activity, [B] poly(ADP-ribose) polymerase (PARP) activity, and [C] DNA fragmentation in the respective groups. n = 6/group; #p < 0.001 vs. vehicle (Veh)/GP; *p < 0.001 vs. CP.

3.2. GP treatment protected the renal histoarchitecture from CPinduced renal injury Effects of different treatment modalities on renal histological features were evaluated by PAS staining. CP administration induced severe and widespread tubular necrosis with dilatation, marked degeneration, intraluminal epithelial anoikis, and cast formation (Fig. 3A) Furthermore, CP treatment was also characterized by infiltration of immune cells, which is a characteristic

feature of inflammation. However, treatment of the animals with GP significantly improved the histological architecture by attenuating the tubular necrosis and inflammation (Fig. 3A, B). 3.3. GP ameliorated CP-induced oxidative stress in renal tissues Oxidative stress is one of the key mechanisms by which CP perpetuates the renal tissue injury [40]. Therefore, we investigated various endogenous markers of oxidative stress and antioxidant

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Fig. 8. [A] Representative terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining images in the respective groups of animals. White arrowhead indicates TUNEL-positive cells. Magnification = 100
. [B] Quantification of TUNEL positive cells in the respective group. n = 6/group; #p < 0.001 vs. vehicle (Veh)/GP; *p < 0.001 vs. CP.

defense systems in renal tissues. First, we determined the levels of an O2-generating enzyme (NADPH oxidase), which has been shown to play a pivotal role in CP-induced renal damage [41]. Our data revealed that CP significantly induced the NADPH oxidase activity (Fig. 4A), which, however, was mitigated upon GP treatment. SOD, a major endogenous defender against oxidative damage, was diminished by CP, but its activity was restored by GP (Fig. 4C). A similar trend was observed for the renal content of GSH (Fig. 4B). The superoxide anion (O2) is a powerful oxidant that rapidly reacts with membrane lipids and produces lipid peroxides, which affects the structure and function of lipids [42]. Our results indicated that the levels of 4-HNE were elevated in renal tissues of the CP-treated animals but were attenuated by GP (Fig. 4D).

the accumulation of 3-NY in renal tissues, which was ameliorated upon GP treatment (Fig. 4E). 3.5. GP inhibited CP-induced renal inflammation NF-kB acts as a key switch in the modulation of inflammatory cytokines and renal tissue injury [43]. Since our earlier histological observation indicated that GP inhibited the renal inflammation, we further confirmed this effect by quantification of proinflammatory cytokines (TNF-a and IL-1b) and determination of NF-kB activation in renal tissues by ELISA. As expected, CP provoked significant NF-kB activation (Fig. 5A) and expression of proinflammatory cytokines; however, these effects were diminished when the animals were treated with GP (Fig. 5B, C).

3.4. GP mitigated CP-induced nitrative stress in renal tissues 3.6. GP inhibited CP-induced MAPK activation Reactive nitrogen species (RNS) play a pivotal role in the pathophysiology of several diseases. ONOO is formed by coupling of O2 and NO when they are simultaneously generated by phagocytes that invade the renal parenchyma [35]. The powerful oxidant ONOO rapidly nitrates lipids, proteins, and nucleic acids, owing to the lack of an endogenous defense system that could detoxify it [35]. Our results indicated that CP significantly induced

MAPKs play crucial roles in cell proliferation, migration, and apoptosis during growth and development. In addition, they are also implicated in the pathogenesis of several human diseases. Reactive oxygen species (ROS) are potent activators of MAPKs. Since CP induces ROS generation, we evaluated the MAPK activation in renal tissues [44]. There was robust activation of p38 MAPK (Fig. 6A, B) and JNK (Fig. 6A, C) in renal tissues obtained

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Fig. 9. Genipin (GP) exhibits therapeutic effects against cisplatin (CP)-induced renal tissue injury. Mice were treated with GP (10 mg/kg) at 12 h after CP administration. [A] Serum blood urea nitrogen (BUN) levels and [B] serum creatinine levels in respective groups of animals. n = 6/group; #p < 0.001 vs. vehicle (Veh)/GP; *p < 0.001 vs. CP. [C] Representative images of periodic acid–Schiff (PAS)-stained paraffin-embedded kidney sections from the respective groups of animals. Asterisk denotes tubular necrosis with cast formation and arrowhead denotes tubular epithelial anoikis. Magnification = 200
. [D] Shows the quantification of tubular injury, n = 6/group; # p < 0.001 vs. veh/GP; *p < 0.001 vs. CP.

from the CP-treated animals; however, it was inhibited upon GP treatment. 3.7. GP diminished CP-induced apoptotic cell death The apoptotic cell death pathway is the major pathway through which renal tissue succumbs to CP toxicity [45]. Therefore, we determined various molecular determinants of apoptosis in kidney tissues (Fig. 7). There were significant increases in the caspase-3 (Fig. 7A) and PARP (Fig. 7B) activities in the animals that received CP. In addition, DNA fragmentation increased in the CP-treated animals (Fig. 7C). However, these phenotypic changes were mitigated upon GP treatment. We further confirmed the nephroprotective effects of GP, revealed by the mitigation of CP-induced apoptotic cell death, using TUNEL staining of paraffin-embedded kidney sections. There was marked apoptosis, characterized by increased numbers of TUNEL-positive cells, in the animals that were administered CP (Fig. 8A). Interestingly, GP elicited a marked nephroprotective action by mitigating the apoptosis, which was evidenced by a reduced number of TUNEL-positive cells (Fig. 8B).

BUN (Fig. 9A) and Cr (Fig. 9B). The therapeutic effect of GP, displayed as an abrogation of CP-induced nephrotoxicity, was confirmed by PAS staining of paraffin-embedded kidney sections. The results showed that GP inhibited the CP-induced tubular necrosis, degeneration, and renal inflammation (Fig. 9C, D). 3.9. GP treatment did not interfere with CP-induced cell death activity in human bladder cancer cells We first determined the IC50 values of GP and CP in a cell viability assay using T24 cells and then determined the combined effect of GP and CP on cell viability by an MTT assay. The IC50 values for GP (Fig. 10A) and CP (Fig. 10B) were determined to be 125 mM and 30 mM at the end of 72-h incubation. However, when cells were co-incubated with GP and CP at their IC50s for 72 h, there was a synergistic effect augmenting the cell death of T24 cells (Fig. 10C). Furthermore, cell cycle analysis (Fig. 11A) revealed that GP did not compromise the CP cell death activity against T24 cells. In fact, we observed enhanced cell death when cells were co-incubated with GP and CP. A similar trend was observed for DNA fragmentation upon treatment with GP and CP (Fig. 11B).

3.8. GP treatment reversed established renal tissue injury 4. Discussion We investigated the therapeutic effect of GP by studying the mitigation/reversal of the established renal tissue injury caused by CP. The data showed that GP significantly attenuated the renal tissue injury, which was evidenced by diminished levels of serum

CP is a potent chemotherapeutic agent, which causes AKI by inducing the oxidative stress, inflammation, and cell death pathways [46]. Previous preclinical studies have reported that

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Fig. 10. Effects of cisplatin (CP) and genipin (GP) alone and in combination on cell viability of human bladder cancer cells (T24) as determined by the 3-(4,5-dimethylthiazol2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Time course and concentration-dependent effects of [A] GP and [B] CP on cell viability. The IC50 values were determined to be 125 mM for GP and 30 mM for CP. [C] Cell viability upon treatments with the respective IC50s of CP and GP. n = 6/treatment; *p < 0.001 vs. vehicle (Veh); **p < 0.001 vs. GP or CP.

GP elicited antioxidant and anti-inflammatory activities [47]. In this study, we investigated the potential nephroprotective actions of GP against CP-induced nephrotoxicity. Animals administered with CP exhibited marked tubular necrosis, degeneration, and inflammation, while treatment with GP significantly ameliorated the renal tissue injury. Various mediators such as ROS, proinflammatory cytokines, and adhesion molecules can activate leukocytes. We observed reduced renal inflammation, which corroborated diminished leukocyte infiltration to the site of tissue injury [48,49]. GSH is a pivotal endogenous antioxidant molecule that maintains redox homeostasis in cells and tissues [50], and protects biomolecules from oxidative tissue damage by scavenging ROS [50]. Previous studies have reported the depletion of GSH by CP treatment via formation of direct adducts [51]. Decreased GSH levels in renal tissues further perpetuate the CP-induced renal damage. Herein, we observed that CP-induced GSH depletion was inhibited by GP treatment. Our findings are consistent with the data from previous reports showing that GP could restore the cellular thiol pool. A diminished endogenous antioxidant reserve capacity is often observed in patients receiving cancer chemotherapy. In particular, this scenario is more pronounced in subjects on CP regimens [5]. ROS can be generated by endogenous or exogenous sources. In particular, NADPH oxidase has been implicated as a key source of CP-induced O2 in renal tissues [41]. It is pertinent to note that NADPH oxidase (phagocyte oxidase) is highly expressed in

leukocytes, particularly in macrophages and neutrophils [41]. In our present study, we observed that GP inhibited the NADPH oxidase activation by CP, probably via mitigation of leukocyte activation. Levels of 4-HNE reflect the ROS-mediated lipid peroxidation. A previous study has shown that SOD plays a pivotal role in limiting the ROS-induced tissue damage [52]. GSH participates in the regeneration of cellular lipid molecules via reacylation of cell membrane components [52]. Therefore, to investigate the antioxidant effects of GP against CP-induced renal tissue damage, we determined the GSH levels, SOD activity, and 4HNE content. We observed that GP significantly inhibited the oxidative stress by restoring the GSH levels and SOD activity and by diminishing the 4-HNE accumulation. ONOO is an important mediator implicated in RNS-mediated tissue injury. ONOO generation is accelerated in cells and tissues that lack an endogenous defense system able to detoxify this noxious and highly reactive oxidant molecule [53,54]. Another factor contributing to the enhanced generation of ONOO is simultaneous production of NO and O2 by activated leukocytes [55]. Therefore, it is pertinent to note that genetic ablation of inducible nitric oxide synthase or treatment with chemical catalysts of ONOO decomposition prevent the CP-induced renal tissue damage [56,57]. Our findings are in agreement with these observations since GP inhibited the accumulation of 3-NY in CPtreated animals. Previous studies have indicated that production of proinflammatory cytokines is mediated by NF-kB activation [58]. In this

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Fig. 11. Effects of cisplatin (CP) and genipin (GP) on cell cycle progression [A] and DNA fragmentation [B] in human bladder cancer cells (T24). Cells were incubated with the corresponding IC50s of GP and CP alone or in combination for 72 h. n = 6/treatment; *p < 0.001 vs. vehicle (Veh); **p < 0.001 vs. GP or CP.

study, we observed that GP attenuated the proinflammatory cytokine expression in renal tissues. However, the exact role of NFkB in this context needs to be further explored. ROS have been reported to be the key mediators involved in the CP-induced activation of MAPKs [59]. CP has been shown to stimulate the generation of the hydroxyl radical [60]. Production of the hydroxyl radical in renal tissues upon CP treatment has been demonstrated to be due to the release of iron from the heme group of cytochrome P450 2E1 [60]. Furthermore, previous studies have reported that genetic ablation of TNF-a resulted in the decreased ROS generation, MAPK activation, and cell death pathway activation by CP in renal tissues [36,61]. Our findings agree with these observations because GP attenuated the leukocyte activation, ROS generation, and MAPK activation. CP has been shown to target neoplastic and naïve cells, resulting in the DNA damage. To counteract the DNA damage, DNA repair enzymes are recruited. PARP is a ubiquitous nuclear enzyme that actively participates in DNA repair mechanisms [62]. However, during enhanced tissue injury, hyperactivation of PARP results in the depletion of cellular ATP levels, which activates the extrinsic pathway of apoptosis [63]. Interestingly, either pharmacological inhibition or genetic ablation of PARP protected mice from CPinduced renal tissue damage [37]. In our present study, we also

observed that GP inhibited the extrinsic pathway of cell death by inhibiting PARP, activation of caspase-3, and DNA fragmentation. Moreover, GP was able to show a therapeutic effect by reversing the established renal tissue injury induced by CP. We also observed that GP did not compromise the CP cell death actions in human bladder cancer cells. On the contrary, when GP was combined with CP, it synergistically enhanced the cell death of human bladder cancer cells. In summary, the findings from the present study indicate that the inhibition of oxidative stress, inflammation, and apoptosis pathways by GP could offer a novel strategy to counteract the CPinduced AKI. Conflict of interest The authors declare no conflict of interests. Acknowledgements MR, SO, and EA were supported by intramural funds from the College of Medicine and Health Sciences and Office of Research and Graduate Studies of United Arab Emirates University.

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