Polyacetylene glycoside attenuates ischemic kidney injury by co-inhibiting inflammation, mitochondria dysfunction and lipotoxicity

Accepted Manuscript Polyacetylene glycoside attenuates ischemic kidney injury by coinhibiting inflammation, mitochondria dysfunction and lipotoxicity

Yijie Zhou, Dan Du, Shuyun Liu, Meng Zhao, Yujia Yuan, Lan Li, Younan Chen, Yanrong Lu, Jingqiu Cheng, Jingping Liu PII: DOI: Reference:

S0024-3205(18)30249-2 doi:10.1016/j.lfs.2018.05.009 LFS 15703

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Life Sciences

Received date: Revised date: Accepted date:

12 March 2018 29 April 2018 3 May 2018

Please cite this article as: Yijie Zhou, Dan Du, Shuyun Liu, Meng Zhao, Yujia Yuan, Lan Li, Younan Chen, Yanrong Lu, Jingqiu Cheng, Jingping Liu , Polyacetylene glycoside attenuates ischemic kidney injury by co-inhibiting inflammation, mitochondria dysfunction and lipotoxicity. The address for the corresponding author was captured as affiliation for all authors. Please check if appropriate. Lfs(2017), doi:10.1016/ j.lfs.2018.05.009

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ACCEPTED MANUSCRIPT

Polyacetylene glycoside attenuates ischemic kidney injury by co-inhibiting inflammation, mitochondria dysfunction and lipotoxicity Yijie Zhou1a, Dan Du2a, Shuyun Liu1, Meng Zhao1, Yujia Yuan1, Lan Li1, Younan Chen1, Yanrong Lu1, Jingqiu Cheng1* and Jingping Liu1* Laboratory of Transplant Engineering and Immunology, Regenerative Medicine Research Center,

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West China-Washington Mitochondria and Metabolism Center, West China Hospital, Sichuan

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West China Hospital, Sichuan University, Chengdu, China

University, Chengdu, China Co-first authors

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*Corresponding author: Jingping Liu

Laboratory of Transplant Engineering and Immunology, Regenerative Medicine Research Center,

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West China Hospital, Sichuan University, Chengdu 610041, China

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Tel: +86-28-85164029, E-mail: [email protected] *Corresponding author: Jingqiu Cheng

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Laboratory of Transplant Engineering and Immunology, Regenerative Medicine Research Center,

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West China Hospital, Sichuan University, Chengdu 610041, China Tel: +86-28-85164029, E-mail: [email protected]

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Abstract Aims: Ischemic acute kidney injury (AKI) is a serious clinical problem and no efficient therapeutics is available in clinic now. Natural polyacetylene glycosides (PGAs) had shown antioxidant and anti-inflammatory properties, but their effects on kidney injury have not been

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evaluated. This study aimed to investigate the protective effect of PGA on ischemic kidney injury

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in renal tubular epithelial cells (TECs) and mice.

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Main methods: Hypoxic HK-2 cells and renal ischemia/reperfusion injury (IRI) mice were treated with PGA from Coreopsis tinctoria, and the cell viability, renal function, apoptosis,

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inflammation, mitochondrial injury, lipids metabolism were analyzed.

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Key findings: In vitro results showed that PGA improved cell viability and reduced oxidative stress, pro-apoptotic / pro-inflammatory factors expression and NFκB activation in TECs under

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hypoxia/reperfusion (H/R). Moreover, PGA reduced mitochondria oxidative stress and improved

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ATP production, ΔΨm and mitochondria biogenesis, and inhibited lipids uptake, biosynthesis and accumulation in hypoxic TECs. In vivo, PGA significantly attenuated kidney injury and reduced

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blood urea nitrogen (BUN), serum creatinine (CREA) and urinary albumin (Alb), and increased

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creatinine clearance (CC) in IRI mice. PGA also decreased cell apoptosis, mitochondria oxidative stress, inflammatory response and lipid droplets accumulation, and promoted ATP generation in kidney of IRI mice.

Significance: Our results proved that PGA ameliorated ischemic kidney injury via synergic anti-inflammation, mitochondria protection and anti-lipotoxicity actions, and it might be a promising multi-target therapy for ischemic AKI. Keywords: acute kidney injury; inflammation; polyacetylenes glycoside; lipotoxicity; 2

ACCEPTED MANUSCRIPT mitochondria Abbreviations AKI, acute kidney injury; ATP5a1, ATP synthase, H+ transporting, mitochondrial F1 complex, alpha 1; Bax, BCL2 associated X; BUN, blood urea nitrogen, CREA, serum creatinine; CC,

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creatinine clearance; CCK-8, cell counting kit-8; CD36, cluster of differentiation 36; CPT-1,

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Carnitine palmitoyl-transferase 1; DAPI, 4',6-diamidino-2-phenylindole; FASN, fatty acid

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synthase; GAPDH, glyceraldehyde 3 phosphate dehydrogenase; HK2, Human renal proximal tubule epithelial cell line; H/R, hypoxia/reperfusion; ICAM-1, intercellular adhesion molecule-1;

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IL-1β, interleukin-1β; MCP-1, monocyte chemoattractant protein-1; NF-κB, nuclear factor-κB;

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PPAR-α, peroxisome proliferator activated receptor alpha; PGA, polyacetylenes glycosides; PGC-1α, Peroxisome proliferator-activated receptor-γ co-activator 1-α; ROS, reactive oxygen

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species; SCD1, stearoyl-CoA desaturase-1; TECs, renal tubular epithelial cells; TNF-α, tumor

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necrosis factor α.

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1. Introduction Acute kidney injury (AKI) is a serious worldwide clinical problem characterized by a rapid decrease in kidney function with high mortality and morbidity, and ischemia is one of the main causes for AKI [1-3]. Moreover, incomplete recovery of AKI further induces the development of

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chronic kidney disease (CKD) [3]. The pathophysiology of AKI is complex and many factors such

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as oxidative stress, inflammation and apoptosis are involved [1]. Currently, numerous single-target

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therapies for AKI such as antioxidants and anti-inflammatory agents have been developed, but their renal outcomes need to be improved to meet clinical demands. Thus, novel effective agents

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for AKI are still desired.

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It was well-documented that mitochondrial dysfunction played an essential role in the initiation and progression of AKI [4]. Previous study reported a persistent disruption of mitochondrial

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homeostasis in renal tubules after ischemia-reperfusion (I/R) injury [5]. Hypoxia induced cytosolic

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calcium overload, mitochondrial stress and energy depletion in the renal tubular cells [4-6] . Mitochondrial injury during AKI further impaired energy-dependent renal repair mechanism, and

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led to oxidative stress and cell death in hypoxic renal tubular cells and kidney [5-7]. In addition,

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renal lipid metabolism disorder was also involved in renal fibrosis after AKI. Previous studies found obvious lipids including fatty acids (FA) deposition in the renal tubular cells of AKI mice and patients [8-10], and suggested that lipotoxicity played an important role in the development of renal fibrosis after AKI [8]. The accumulated FAs in kidney during H/R further induced sustained mitochondrial depolarization and energetic deficit in renal tubules after AKI [10]. Therefore, these studies suggested that renal mitochondria injury and lipid metabolism disorder might serve as potential therapeutic targets for AKI. 4

ACCEPTED MANUSCRIPT Polyacetylene belongs to a class of compounds characterized by the presence of two or more carbon-carbon triple bonds in the carbonic skeleton [11]. Natural polyacetylene and its derivatives such as polyacetylene glucoside (PGA) were widely distributed in various food and medical plants, and they exerted multiple bioactivities such as anti-inflammatory, anti-microbial and anti-allergic

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effects [12,13]. It had been reported that PGA from purple carrots reduced LPS-induced nitric

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oxide (NO), iNOS and pro-inflammatory cytokines such as IL-6, IL-1β and TNF-α production in

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macrophage and endothelial cells [14]. We had previously isolated a new linear C14 PGA from Coreopsis tinctoria that was traditionally used as tea-like beverage to prevent cardiovascular

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diseases in Chinese folk medicine, and proved that it reduced lipids accumulation in 3T3-L1

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adipocytes [15]. In addition, PGA from Coreopsis tinctoria showed anti-inflammatory activity via inhibiting COX-2 in macrophages [12]. Although the anti-inflammatory and anti-lipogenic role of

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PGA were reported, its effect on AKI has not been fully illustrated.

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In this study, we purposed to evaluate the renal protective role of natural PGA isolated from Coreopsis tinctoria on H/R-induced TECs injury and IRI-induced AKI mice, and its potential

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effective mechanism were further revealed.

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2. Materials and Methods

2.1. Cell culture and treatment Human renal proximal tubule epithelial cell line (HK2) were cultured in Dulbecco’s modified Eagle’s medium (DMEM, Invitrogen) supplemented with 10% fetal bovine serum (Gibco, CA, USA), 50 U/ml penicillin and 50 µg/ml streptomycin in a humidified atmosphere at 37°C with 5% CO2. To induce H/R injury, HK2 cells were incubated in a hypoxic chamber (≤1% O2, 5% CO2, 94% N2) at 37°C for 24 h and followed with reoxygenation (21%O2, 5% CO2, 74% N2) for 2 h. The 5

ACCEPTED MANUSCRIPT linear C14 PGA was isolated from Coreopsis tinctoria as previously described [15], and its chemical structure was shown in Fig. S1. HK-2 cells were treated with 80 μM PGA in the H/R experiments (n=3 per group). 2.2. Cell viability assay

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Cells were seeded in 96-well plate and incubated in H/R condition and treated with PGA. After

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treatment, CCK8 solution (Dojindo, Kumamoto, Japan) was added and incubated with cells at

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37°C for 2 h. The absorbance at 450 nm was measured by microplate reader (BioTek Instruments

group to the control group.

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2.3. Cell Annexin V-FITC / PI staining

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Inc, USA). The cell viability was calculated by normalizing optical density of the experimental

Cell apoptosis was detected by using Annexin V-FITC and propidiumiodide (PI) staining kit

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(BD Biosciences, USA). Briefly, cells were washed with PBS and stained with 5 μl of Annexin

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V-FITC and 10 μl of PI solution in binding buffer for 15 min at room temperature in the dark. The stained cells were then observed by fluorescence microscope (Axio Observer D1, Carl Zeiss,

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Germany). The apoptotic rates were expressed as the percentage of apoptotic cells normalized to

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total cells in the sections. The experiment was repeated three times independently, and ten randomly selected fields per slides were recorded each time. 2.4. Measurement of intracellular ROS 2’7’-Dichlorofluorescein diacetate (DCFH-DA, Sigma, USA), Dihydroethidium (DHE, Thermo Fisher Scientific, USA) and MitoSOX (Molecular Probes, Thermo Fisher Scientific) were used to measure intracellular ROS. After treatment, cells were incubated with 10 μM DCFH-DA / DHE at 37°C for 30 min, or 5 μM MitoSOX at 37°C for 10 min. After washing with PBS, digital images 6

ACCEPTED MANUSCRIPT were captured by fluorescent microscope (Axio Observer D1, Carl Zeiss, Germany). For images analysis, three slides of different samples in each group were chose, and ten randomly selected fields per slides were recorded, and the mean fluorescence intensity in each image was analyzed by NIH Image J software.

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2.5. Quantitative real-time PCR (qPCR)

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Total RNA was extracted by Trizol (Gibco, Life Technologies, CA, USA) and

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reverse-transcribed into cDNA by iScript cDNA synthesis kit (Bio-Rad, USA). Real-time polymerase chain reaction (Real time-PCR) was performed on CFX96 real-time PCR detection

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system (Bio-Rad, USA) with SYBR Green (Bio-Rad, USA). The mice primers including cluster of

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differentiation 36 (CD36), fatty acid synthase (FASN) and stearoyl-CoA desaturase-1 (SCD1) were purchased from GeneCopoeia Company (Rockville, MD, USA), and the sequences of other

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primers were listed in Table S1. qPCR data were analyzed by Bio-Rad CFX Manager software,

reference gene.

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2.6. Western blot

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and the relative change of mRNA was calculated by delta-delta Ct method with GAPDH as

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Cells and kidney tissues were lysed in radioimmuno-precipitation assay (RIPA) buffer supplemented with protease and phosphatase inhibitors (Calbiochem, CA, USA). Protein concentration was determined by BCA Assay Kit (CWBIO, China). Equal amount of protein was subjected to electrophoresis on 12% SDS-PAGE, and then transferred to PVDF membranes (Merck Millipore, USA). PVDF membranes were blocked with 5% non-fat milk and incubated with primary antibodies against Bax (1:1000, 2772S, CST, USA), Bcl-2 (1:500, A2212, ABclonal, USA), ICAM-1 (1:1000, 10831-1-AP, Proteintech, USA), IL-1β (1:1000, A1112, ABclonal, USA), 7

ACCEPTED MANUSCRIPT p-NFκB (1:1000, 3033S, CST, USA), NFκB (1:500, 4764S, CST, USA), PGC-1α (1:1000, 20658-1-AP, Proteintech, USA), ATP5a1 (1:1000, A5884, ABclonal, USA), CPT1A (1:1000, 15184-1-AP, Proteintech, USA) and PPARα (1:1000, 15540-1-AP, Proteintech, USA) overnight at 4 °C. After washing with PBST, PVDF membranes were incubated with HRP-conjugated

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secondary antibody (1:2000, AS014, ABclonal, USA) at 37 °C for 1 h. Protein bands were

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detected by chemiluminescence kit (Millipore, USA) and quantified by NIH Image J software.

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2.7. Immunofluorescence (IF) staining

Cells and frozen kidney tissue sections were fixed with 4% paraformaldehyde in PBS for 10

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min at room temperature, and then washed with PBS and permeabilized with 0.3% Triton X-100

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for 10 min. After blocking in 1% BSA for 60 min, cells were incubated with rabbit anti-NFκB antibody (1:200, 4764S, CST, USA), rabbit anti-ICAM-1 antibody (1:200, 10831-1-AP,

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Proteintech, USA), rabbit anti-IL-1β antibody (1:200, A1112, ABclonal, USA), rabbit anti-Bax

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antibody (1:200, 2772S, CST, USA), rabbit anti-PGC1α antibody (1:200, 20658-1-AP, Proteintech, USA), rabbit anti-ATP5a1 antibody (1:200, A5884, ABclonal, USA), rabbit anti-CPT1A antibody

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(1:200, 15184-1-AP, Proteintech, USA) and rabbit anti-PPARα antibody (1:200, 15540-1-AP,

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Proteintech, USA) overnight at 4 °C. After washing with PBS, cells were incubated with FITC or TRITC conjugated secondary antibody (1:500, Abcam, UK) for 1 h at 37 °C. Nuclei were visualized by staining with DAPI (1:1000, Sigma, USA). Ten visual fields in three sections of each group were randomly selected and digital images were captured by fluorescent microscope (Imager Z2, Zeiss, Germany). The mean fluorescence intensity of images was semi-quantitative analyzed by NIH Image J software. 2.8. Measurement of cellular and tissue ATP 8

ACCEPTED MANUSCRIPT The ATP level was determined by ATP Bioluminescence Assay Kit (Beyotime, China) according to the manufacturer’s protocol. Briefly, cells and fresh kidney tissues were harvested and lysed in a lysis buffer, followed by centrifugation at 12,000 g for 5 min at 4°C. The level of ATP was determined by mixing 100 μl of the supernatant with 100 μl of luciferase reagent, which

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catalyzed the luminescence production from ATP and luciferin. The emitted luminescence was

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measured by microplate luminometer (Synergy4 Mx, Bio-Tek, USA).

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2.9. Mitochondrial membrane potential assay

Mitochondrial membrane potential (ΔΨm) was measured by tetramethylrhodamine methyl ester

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(TMRM, Invitrogen, USA). After treatment, the cells were incubated with 100 nM TMRM for 20

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min at 37 °C. After washing with PBS, the digital images of stained cells were captured by fluorescent microscope (Eclipse TS100, Nikon, Japan). Ten randomly selected fields in three

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stained cells were record, and the mean fluorescent intensity was analyzed by NIH Image J

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software.

2.10. Cellular Oil-Red O (ORO) staining

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To mimic renal lipid metabolism in vitro, HK-2 cells were cultured in media supplemented with

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palmitic acid (50 μM, Aladdin, China) dissolved in BSA solution. After treatment, cells were washed with PBS and fixed with 10% (v/v) formalin for 10 min at room temperature. After fixation, the cells were washed with PBS and 60% (v/v) isopropanol, and then stained with a filtered Oil-Red solution (Nanjing Jiancheng Bioengineering Institute, China) for 30 min and observed by light microscope (Eclipse TS100, Nikon, Japan). To quantify the intracellular lipids, stained lipid droplets in cells were dissolved in isopropanol for 10 min, and the absorbance at 545 nm was measured. 9

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2.11. Mouse renal IRI model and treatment All animal experiments were approved and conducted according to the guidelines of Animal Care and Use Committee of Sichuan University. Adult male C57BI/6J mice (20-25 g) were purchased from Experimental Animal Center of Sichuan University (Chengdu, China). Animals

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were housed in individual cage with controlled temperature, humidity and 12 h cycles of light and

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darkness, and fed with standard chow and tap water ad libitum. Animals were random divided into

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four groups: Control (sham, n=6), IRI (n=6), IRI + DMSO (vehicle, n=6) and IRI + PGA (n=6). Animals were anesthetized by 1% pentobarbital sodium, and placed in a supine position on a

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warming pad at 37°C for midline laparotomy. The kidney IRI injury was induced by clamping the

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renal pedicles for 30 min using non-traumatic vascular clips, while sham group received laparotomy without ischemia. After surgery, IRI mice were i.p. injected with 100 μl DMSO (2.5 %,

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dissolved in PBS) or PGA (50 mg/kg, dissolved in DMSO/PBS), respectively. Mice in each group

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were sacrificed by overdose of anesthesia at day 3 after surgery, and kidney tissue and serum were collected for further tests.

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2.12. Biochemical measurement

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Clinical biochemistry analysis of creatinine (CREA), urea nitrogen (BUN) and urinary albumin (Alb) from mice was performed on an Automatic Biochemistry Analyzer (Cobas Integra 400 plus, Roche) by using appropriate kits. Creatinine clearance (CC) was calculated as (urine volume × urine CREA) / serum CREA according to the previous reported [16]. 2.13. Histological examination The kidney removed from mice were fixed in 4% paraformaldehyde, embedded in paraffin and sliced into 5 μm sections. Paraffin renal sections were stained with H&E by standard protocols. 10

ACCEPTED MANUSCRIPT Digital images of HE sections were acquired by light microscope, and the areas of necrotic tubules were quantified by Image J software as described above. 2.14. Kidney cell apoptosis TUNEL assay The apoptotic cells in frozen kidney sections were detected by TUNEL staining kit (Promega,

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USA) according to the manufacturer’s instruction, and the nucleus was labeled by Hoechest 33258

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(Sigma, USA). The stained kidney sections were observed by fluorescence microscope (Imager Z2,

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Zeiss, Germany). The rate of apoptotic cells in each group was calculated by average of TUNEL-positive apoptotic cells in ten fields from each kidney sample.

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2.15. Kidney lipids Nile red staining

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The frozen renal sections were fixed in 4% paraformaldehyde and stained with Nile red (MCE, USA) at a final working concentration of 10 μg/ml in acetone/water (1:9) mixture. Nile red stained

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renal tissue sections were visualized by fluorescent microscope (Imager Z2, Zeiss, Germany) at

Image J software.

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Ex485/Em525 nm. The mean fluorescence intensity of images from each sample was analyzed by

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2.16. Statistical analysis

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All data expressed as mean ± SD were analyzed by one or two-way ANOVA test with Tukey’s post hoc analysis using SPSS software (version 11.5, IBM Corporation, USA), and p<0.05 was considered as significant difference. 3. Results 3.1. PGA alleviated H/R-induced cell apoptosis and oxidative stress in TECs CCK8 results showed that PGA enhanced HK-2 cell viability in a dose-dependent manner (20-150 μM) under H/R, and DMSO (vehicle) did not affect the viability of hypoxic TECs (Fig. 11

ACCEPTED MANUSCRIPT 1A). PGA exerted best protective role on TECs viability at 80-150 μM under H/R stress, but there was no significant difference between 80,100 and 150 μM groups. Thus, the lower and effective concentration (80 μM) was used in following cell experiments. H/R induced apoptosis in HK-2 cells, whereas PGA reduced apoptotic rates of TECs during H/R (Fig. 1B-C). PGA also reduced

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pro-apoptotic Bax and Fas expression, and enhanced anti-apoptotic Bcl-2 expression (Fig. 1D-E).

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In addition, PGA inhibited intracellular ROS generation in HK-2 cells under H/R (Fig. 1F).

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Fig. 1 Effects of PGA on cell viability, apoptosis and oxidative stress in HK-2 cells during H/R. (A) Cell viability was determined by CCK8 assay. (B) Cell apoptosis was detected by Annexin V-FITC/PI staining (Scale bar = 25 μm). (C) Quantification analysis of cell apoptotic rates determined by Annexin V-FITC/PI staining. (D) Real-time PCR analysis of Fas and Bax mRNA. (E) Western blot and quantification analysis of BAX and Bcl-2 protein level. (F) Intracellular ROS measured by DHE and DCF staining (Scale bar = 25μm), *p<0.05, H/R group vs control, #p<0.05, PGA group vs H/R group, (n=3 per group). 12

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3.2. PGA reduced inflammatory factors expression in TECs under H/R The qPCR data showed that PGA inhibited ICAM-1, TNF-α, IL-6 and HMGB1 expression in TECs under H/R (Fig. 2A). Western blot (Fig. 2B) and IF staining (Fig. 2D) results proved that PGA reduced ICAM-1 and IL-1β protein in TECs under H/R, while DMSO could not inhibit

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IL-1β in H/R-treated TECs (Fig. S2). PGA also reduced p-NFκB protein in H/R-treated TECs (Fig.

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2C), and inhibited the nuclear translocation of NFκB p65 induced by H/R (Fig. 2D).

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Fig. 2 Effects of PGA on inflammatory response in HK-2 cells during H/R. (A) Real-time PCR analysis of HMGB1, ICAM-1, TNF-α and IL-6 mRNA levels. (B) Western blot and quantification analysis of ICAM-1 and IL-1β protein levels. (C) Western blot and quantification analysis of p-NFκB level. (D) IF staining of ICAM-1, IL-1β and NFκB p65 in HK-2 cells (Scale bar = 25 μm), *p<0.05, H/R group vs control, #p<0.05, PGA group vs H/R group, (n=3 per group). 3.3. PGA restored mitochondria function in TECs under H/R stress H/R induced severe mitochondria injury in TECs, while PGA significantly enhanced cellular 13

ACCEPTED MANUSCRIPT ATP (Fig. 3A) and mitochondrial membrane potential (Fig. 3B), and reduced mtROS in TECs under H/R (Fig. 3B). In addition, PGA reversed the decline of PGC-1α, ATP5a1, COX5b and SDHb in hypoxic TECs (Fig. 3C). Western blot results confirmed that PGA increased PGC-1α and ATP5a1 protein level in H/R-treated TECs (Fig. 3D), and DMSO had no effect on ATP5a1

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expression (Fig. S2).

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Fig. 3 Effects of PGA on mitochondrial injury in HK-2 cells during H/R. (A) Measurement of intracellular ATP level. (B) mtROS and mitochondrial membrane potential were determined by MitoSOX Red and TMRM staining, respectively (Scale bar = 25 μm). (C) Real-time PCR analysis of PGC-1α, ATP5a1, COX5b and SDHb mRNA. (D) Western blot and quantification analysis of PGC-1α and ATP5a1 protein, *p<0.05, H/R group vs control, #p<0.05, PGA group vs H/R group, (n=3 per group). 3.4. PGA reduced intracellular lipid accumulation in TECs under H/R 14

ACCEPTED MANUSCRIPT H/R promoted lipid droplets formation in TECs, while PGA decreased the lipid accumulation in hypoxic TECs (Fig. 4A-B). H/R increased CD36, SCD1 and FASN, and inhibited CPT1 in TECs, while PGA reduced CD36, SCD1 and FASN but enhanced CPT1 in hypoxic TECs (Fig. 4C). Western blot and IF staining results showed that PGA up-regulated PPARα and CPT1A protein in

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hypoxic TECs (Fig. 4D-E), and DMSO failed to rescue CPT1A in TECs under H/R (Fig. S2).

Fig. 4 PGA decreased lipid metabolism disorder in HK-2 cells during H/R. (A) Measurement of

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intracellular lipids by ORO staining (Scale bar= 25 μm). (B) Quantification analysis of lipids detected by ORO staining. (C) Real-time PCR analysis of CD36, FASN, SCD1 and CPT1 mRNA. (D) Western blot and quantification analysis of CPT1A and PPARα protein level. (E) IF staining of CPT1A and PPARα in HK-2 cells (Scale bar = 25μm), *p<0.05, H/R group vs control, #p<0.05, PGA group vs H/R group, (n=3 per group). 3.5. PGA attenuated renal injury and tubular apoptosis in IRI mice

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ACCEPTED MANUSCRIPT IRI mice showed severe kidney injury as indicated by increased level of serum BUN / CREA, urinary albumin (Fig. 5A) and tubular necrosis (Fig. 5C-D), and decreased creatinine clearance (Fig. 5A) compared to controls. By contrast, PGA reduced serum BUN / CREA, urinary albumin and necrotic tubules, and reversed decline of creatinine clearance in IRI mice. Moreover, PGA

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decreased the level of tubular cells apoptosis and pro-apoptotic Bax and Fas, and enhanced

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anti-apoptotic Bcl-2 expression in IRI mice (Fig. 5B-E). DMSO showed no protective effect on

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renal injuries in IRI mice (Fig. 5A-E).

Fig. 5 PGA improved kidney function in IRI mice. (A) Serum CREA, BUN, urinary albumin and creatinine clearance at 3 days after surgery. (B) Real-time PCR analysis of Fas and Bax mRNA. (C) H&E and TUNEL staining of kidney tissues from mice (Scale bar = 25 μm). (D) The percentage of necrotic tubules and TUNEL+ apoptotic cells at in mice 3 days after IRI. (E) Western blot and quantification analysis of BAX and Bcl-2 protein level. *p<0.05, IRI group vs 16

ACCEPTED MANUSCRIPT control, #p<0.05, PGA group vs IRI group, (n=6 per group). 3.6. PGA ameliorated renal inflammatory responses in IRI mice IRI mice showed increased pro-inflammatory factors including HMGB1, ICAM-1and IL-1β mRNA, and ICAM-1 and IL-1β protein expression compared to controls (Fig. 6A-D). In contrast,

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PGA inhibited HMGB1, ICAM-1and IL-1β mRNA, and ICAM-1 and IL-1β protein levels

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factors expression in the kidney of IRI mice (Fig. 6A-D).

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compared to IRI groups (Fig. 6A-D). DMSO alone could not inhibit these pro-inflammatory

Fig.6 PGA reduced inflammatory response in kidneys after IRI. (A) Real-time PCR analysis of HMGB1, ICAM-1 and IL-1β mRNA levels. (B) Western blot analysis of ICAM-1 and IL-1β protein. (C) Quantification analysis of ICAM-1 and IL-1β protein levels. (D) IF staining of IL-1β in kidneys (Scale bar = 25 μm), *p<0.05, IRI group vs control, #p<0.05, PGA group vs IRI group, (n=6 per group). 3.7. PGA rescued renal mitochondrial dysfunction in IRI mice 17

ACCEPTED MANUSCRIPT IRI mice showed increased renal mitochondria oxidative stress and impaired function compared to controls (Fig. 7), while PGA significantly reduced mtROS (Fig. 7A), restored ATP production (Fig. 7B), and enhanced renal mitochondria biogenesis proteins PGC-1α and ATP5a1 expression in IRI mice (Fig. 7C-F). Meanwhile, DMSO did not significantly improve renal mitochondria

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function in IRI mice (Fig. 7).

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Fig.7 PGA rescued kidney mitochondrial function after IRI. (A) mtROS was determined by MitoSOX Red staining (Scale bar = 25 μm). (B) Measurement of kidney ATP level. (C) Real-time PCR analysis of PGC-1α and ATP5a1 mRNA. (D-E) Western blot and quantification analysis of PGC-1α and ATP5a1 protein levels. (F) IF staining of ATP5a1 in kidney from mice (Scale bar = 25 μm). *p<0.05, IRI group vs control, #p<0.05, PGA group vs IRI group, (n=6 per group). 3.8. PGA prevented renal lipids deposition in IRI mice Nile red staining results showed increased lipid contents accumulation in the kidney tubules of 18

ACCEPTED MANUSCRIPT IRI and IRI + DMSO groups compared to control group, whereas PGA significantly reduced the renal lipid deposition in IRI mice (Fig. 8A). In addition, our data showed decline of PPARα and CTP1A mRNA and protein in the kidney of IRI mice, while PGA group had higher level of

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PPARα and CTP1A than those of IRI and IRI + DMSO groups (Fig. 8B-E).

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Fig.8 PGA decreased renal lipid accumulation in mice after IRI. (A) Nile red staining and

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quantification analysis of lipids in kidney. (B) Real-time PCR analysis of PPARα and CPT1 mRNA. (C) Western blot analysis of PPARα and CPT1A proteins. (D) Quantification analysis of PPARα and CPT1A proteins. (E) IF staining of PPARα in kidneys (Scale bar = 25μm). *p<0.05, IRI group vs control, #p<0.05, PGA group vs IRI group, (n=6 per group). 4. Discussion IRI led to severe kidney injury associated with of morbidity and mortality, and increased the risk of CKD in patients [17]. Currently, there are no effective therapeutic agents available for AKI 19

ACCEPTED MANUSCRIPT in clinic [2]. Therefore, novel effective agents for AKI are urgently needed. In this study, we aim to evaluate the protective role of natural PGA on I/R-induced injury in vitro and vivo. Oxidative stress, inflammation and cell apoptosis were well-known features in kidney after AKI [1]. Renal tubules were the initial regions affected by I/R injury, which triggered the release of

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inflammatory factors and subsequently recruited pro-inflammatory cells such as neutrophils and

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macrophages infiltration into the tubulointerstitium, and in turn led to cell death and further

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development of renal fibrosis after AKI [1]. NFκB pathway played a key role in inflammation by regulating pro-inflammatory cytokines, chemokines and cell adhesion molecules expression [18].

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Meanwhile, oxidative stress had a vital relationship with the inflammation via activation of NFκB

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pathway [19]. In this study, our data proved that PGA had protective role on AKI, which could reduce cellular ROS and apoptosis in TECs under H/R and improved renal function in IRI mice.

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Moreover, PGA suppressed NFκB activation and inflammatory factors expression in hypoxic

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TECs and ameliorated renal macrophages infiltration and pro-inflammatory cytokines release in the IRI mice. The anti-inflammatory effect of PGA had been previously reported, which inhibited

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the release of inflammatory factors and NFκB signaling in macrophages and endothelial cells

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[14,20]. Therefore, our results indicated that PGA ameliorated ischemic kidney injury partly due to its direct anti-inflammation action. Mitochondria played essential roles in mediating cellular energy metabolism, survival and apoptotic pathways response to stress stimuli [21,22]. Previous studies found that I/R caused persistent mitochondria injury and energy deletion in tubular cells after AKI [5]. It had been proposed that mitochondria protection was a promising strategy for ischemic kidney and cardiovascular diseases [22], and mitochondria-targeted antioxidants such as SS31 promoted 20

ACCEPTED MANUSCRIPT mitochondria recovery after ischemia and thus reduced AKI in rodent model [23]. Our results showed severe mitochondria dysfunction as indicated by elevated mtROS, and reduced ATP generation, ΔΨm and mitochondria biogenesis genes in TECs under H/R and kidney of IRI mice. By contrast, PGA effectively alleviated mitochondrial injuries and restored energy metabolism in

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vitro and in vivo. Peroxisome proliferator-activated receptor-γ co-activator 1-α (PGC1α) was a

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master regulator of mitochondrial biogenesis, and activation of PGC1α had shown to promote

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kidney recovery after IRI in mice [24]. Our data showed that PGA promoted PGC1α expression in hypoxic TECs and kidney of IRI mice. Thus, our results suggested that PGA preserved ischemic

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renal injury via enhancing mitochondria function and PGC-1α pathway.

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Previous studies had found increased lipids including FAs accumulation in hypoxic renal tubules, and suggested that overloaded FAs induced-lipotoxicity contributed to the development of

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kidney fibrosis after AKI [8,10,25]. In this study, we observed elevated lipids deposition

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associated with up-regulation of lipogenic genes such as SCD1, CD36 and FASN in hypoxic TECs and kidney of IRI mice, while PGA reduced tubular lipids accumulation and lipogenic

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genes expression. These results suggested that PGA could block the abnormal FA uptake and

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biosynthesis in TECs under I/R. FAs were important mitochondrial substrates in proximal tubules, and could be oxidized (β-oxidation) to meet renal metabolic demand [8,10]. Carnitine palmitoyl-transferase 1 (CPT1) was a rate-limiting enzyme of β-oxidation in the step of FAs entry into mitochondria[26]. Dysfunction of CPT-1 and FAs β-oxidation contributed to hypoxia-induced lipids accumulation in hepatocytes [26]. PPARα was a member of steroid /nuclear receptor super family, which played a key role in maintaining FAs and ATP metabolism homeostasis in kidney PTECs [27]. In this study, we found that PGA enhanced both CPT1 and PPARα expression in 21

ACCEPTED MANUSCRIPT hypoxic TECs and kidney of IRI mice, which indicated that PGA reduced I/R induced tubular lipid accumulation by promoting FAs β-oxidation. Therefore, our results suggested that PGA exerted its renoprotection via restoring lipid metabolism and inhibiting lipotoxicity in TECs after AKI.

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In summary, our study showed that I/R caused severe cell apoptosis, inflammation, oxidation

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stress, mitochondria dysfunction and lipid accumulation in hypoxic TECs and kidney of IRI mice,

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whereas PGA significantly ameliorated these adverse effects in vitro and in vivo. We further revealed that PGA inhibited inflammatory factors release by inactivating NFκB pathway, restored

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mitochondria function via promoting PGC1α signaling, and reversed lipid metabolism disorder by

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enhancing CPT1/PPARα pathways in hypoxic TECs and kidney of IRI mice. This study suggested that PGA attenuated ischemic AKI via synergic anti-inflammation, mitochondrial protection and

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Conflicts of interest

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anti-lipotoxicity mechanism.

The authors have declared no conflicts of interest.

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Acknowledgments

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This work was supported by grants from National Natural Science Foundation of China (31200754, 81571808) and China Postdoctoral Science Foundation (2012M511931). References

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