c mice

Biomedicine & Pharmacotherapy 100 (2018) 304–315

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Ferulic acid protects lipopolysaccharide-induced acute kidney injury by suppressing inflammatory events and upregulating antioxidant defenses in Balb/c mice

T

Salma Mukhtar Mira,b, Halley Gora Ravuria, Raj Kumar Pradhana, Sairam Narrac, ⁎ Jerald Mahesh Kumarc, Madhusudana Kunchaa, Sanjit Kanjilalb,d, Ramakrishna Sistlaa,b, a

Pharmacology and Toxicology Division, CSIR-Indian Institute of Chemical Technology (IICT), Hyderabad, 500 007, India Academy of Scientific and Innovative Research (AcSIR), CSIR-HRDC Campus, Sector 19, Kamla Nehru Nagar, Ghaziabad, Uttar Pradesh 201 002, India Animal House Facility, CSIR-Centre for Cellular and Molecular Biology (CCMB), Hyderabad, 500 007, India d Centre for Lipid Research, CSIR-Indian Institute of Chemical Technology (IICT), Hyderabad, 500 007, India b c

A R T I C L E I N F O

A B S T R A C T

Keywords: Ferulic acid (FA) Lipopolysaccharide (LPS) Acute kidney injury (AKI) Sepsis Antioxidants NFκB pathway

Sepsis-induced acute kidney injury (AKI) is responsible for 70–80% mortality in intensive care patients due to elevated levels of endotoxin, Lipopolysaccharide (LPS) caused by gram-negative infections. Ferulic acid (FA), a phenolic phytochemical is known for its renal protection on various induced models of nephrotoxicity. However, the curative effect of FA in LPS-induced AKI is not well studied. This study aimed to investigate the effect of FA on LPS-induced AKI in mice model and to understand the protective mechanisms involved, to provide evidence for FA in the treatment of AKI. Balb/c mice were treated with FA at 50 mg/kg and 100 mg/kg dosages after LPS stimulation (10 mg/kg). At the end of the intervention, we determined the concentrations of serum creatinine and blood urea nitrogen, inflammatory cytokines and histopathological changes in animals. Also, the relative protein expression level of TLR4 mediated NF-κB signaling pathway were studied in kidney tissues. FA treated animals showed upregulation of antioxidant defenses and suppression of inflammatory events by inhibiting TLR4 mediated NFκB activation. However, LPS alone administered group, resulted in rapid renal damage with increased levels of blood urea nitrogen and modest increase in creatinine; decreased antioxidant defenses and release of inflammatory cytokines. The histopathological analysis also revealed the protective action of the FA against sepsis induced fibrosis and renal damage. Our findings demonstrated that FA exhibits marked protective effects on LPS-induced AKI in mice suggesting its chemopotential role for treating AKI in humans.

1. Introduction Sepsis is known to be one of the major cause in the development of acute kidney injury (AKI) [1,2] and accounts for nearly 26%–50% of all AKI in the developed nations, compared with 7% to 10% of primary kidney disease-associated AKI [3]. Patients with concomitant sepsis and AKI have an unacceptably high mortality rate of approximately 70% compared with 39% in patients with non-septic AKI [4,5].

Sepsis occurs as a result of complex host-pathogen interactions, leading to release of inflammatory mediators, as well as reactive oxygen and reactive nitrogen intermediates [6]. The inflammatory processes and oxidative stress have a major role in the pathophysiology of sepsisinduced AKI [7]. The reactive oxygen species (ROS) generated through LPS stimuli can affect the pathogenesis of sepsis by two mechanisms: (a) modulating the innate immune signaling cascade, and (b) causing pathologic damage to cells and organs [8,9]. The innate immune signaling

Abbreviations: AKI, acute kidney injury; ANOVA, analysis of variance; BCA, bicinchoninic acid; BSA, bovine serum albumin; BUN, blood urea nitrogen; CAT, catalase; CD4, cluster of differentiation 4; CDNB, 1-chloro-2, 4-dinitrobenzene; COX-2, cycloxygenase-2; DAPI, 4′,6-diamidino-2-phenylindole; DTNB, dithiobis-nitrobenzoic acid; ECL, enhanced chemiluminescence; FA, Ferulic acid; FITC, Fluorescein isothiocyanate; GR, glutathione reductase; GSH, reduced glutathione; GSSG, oxidised glutathione; GST, glutathione-S-transferase; HO-1, Heme oxygenase 1; IκB, inhibitor of kappa B; IL-6, Interleukin 6; iNOS, inducible nitric oxide synthase; LPS, lipopolysaccharide; MDA, malondialdehyde; MT, Masson’s Trichome; MTT, 3(4, 5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; NADH, β-nicotinamide adenine dinucleotide hydrate; NADPH, β-nicotinamide adenine dinucleotide 3-phosphate reduced form; NDH, NADH dehydrogenase; NFκB, nuclear Factor-κB; Nrf2, nuclear factor erythroid 2-related factor 2; PI, propidium iodide; RIPA, radioimmunoprecipitation assay; ROS, reactive oxygen species; RNS, reactive nitrogen species; SEM, standard error of the mean; SDH, succinate dehydrogenase; SOD, superoxide dismutase; TBARS, thiobarbituric acid reactive substances; TLR-4, toll-like receptor 4; TNF-α, tumor necrosis factor-α; Vit C, vitamin C ⁎ Corresponding author at: Pharmacology and Toxicology Division, CSIR-Indian Institute of Chemical Technology (IICT), Hyderabad, 500 007, India. E-mail address: [email protected] (R. Sistla). https://doi.org/10.1016/j.biopha.2018.01.169 Received 16 November 2017; Received in revised form 27 January 2018; Accepted 29 January 2018 0753-3322/ © 2018 Elsevier Masson SAS. All rights reserved.

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on Animals (CPCSEA) guidelines under the approval and scrutiny of Institutional Animal Ethics Committee (IAEC) of CSIR-Indian Institute of Chemical Technology (IICT), Hyderabad, India (Approval No: IICT/ 09/2017).

cascade results in expression of inflammatory genes, such as cycloxygenase 2 (COX-2), inducible nitric oxide synthase (iNOS), cell adhesion molecules, inflammatory cytokines, and chemokines through activation of the toll like receptor-4 (TLR-4) mediated NF-κB pathway [10,11]. The later results from ROS mediated peroxidation of lipids, oxidation of proteins, damage to nucleic acids, and ultimately cell death and tissue injury [12]. Therefore, suitable agents with the potential to inhibit oxidative stress as well as inflammatory events could be beneficial in treating sepsis-induced AKI. A number of phytoconsituents have been reported to suppress the activation of NF-κB transcription factor both in vitro [13,14] and in vivo [15–18] in response to LPS stimuli suggesting their protective effects against endotoxin-induced oxidative damage in organs [18]. Ferulic acid (FA; 4-hydroxy-3-methoxycinnamic acid), a ubiquitous phytochemical phenolic derivative of cinnamic acid is present majorly in plant cell wall components of Chinese medicinal herbs such as Angelica sinensis, Cimicifuga heracleifolia and Lignsticum chuangxiong [19]. This phenolic compound has divergent properties such as antioxidant, antihyperlipidemic, antidiabetic, antimicrobial, anti-inflammatory, antiatherogenic, anticarcinogenic, neuroprotective, and anti-hypertensive [20,21]. FA exhibited anti-inflammatory effects by inhibiting inflammatory cytokines production and the release of ROS and RNS by suppressing iNOS and COX-2 via activation of the NF-κB pathway in LPS-stimulated RAW 264.7 macrophages [22,23]. FA prevented sepsis mediated oxidative damage by improving the antioxidant status and decreasing the DNA damage in animals induced by cecal ligation and puncture method [24]. Studies have also shown the renal protective effect of FA against nephrotoxicity induced by cisplatin and glycerol in animal models [25,26]. Earlier report suggested that FA exhibits a potent anti-inflammatory activity against acute as well as chronic inflammatory diseases [27,28] However, to the best of our knowledge, the therapeutic effect and underlying protective mechanisms of FA against LPS induced AKI is not reported. The present study investigated the protective effect and underlying mechanisms of FA on LPS induced AKI in mice model.

2.3. Experimental design Animals were randomly divided into five groups consisting of 8 animals in each group and were treated as follows: VC: mice were administered with a suspension of 2% gum acacia, as the vehicle for 2 doses of FA and a single intraperitoneal injection of saline. LPS Con: mice were administered with a single intraperitoneal injection of LPS (10 mg/kg) in saline. FA Con: mice were intraperitoneally administered with two doses of FA (100 mg/kg) one hour before and 2 h after a single intraperitoneal injection of saline. LPS + FA50: mice were administered intraperitoneally with FA (50 mg/kg) one hour before and 2 h after a single intraperitoneal injection of LPS (10 mg/kg) in saline. LPS + FA100: mice were administered intraperitoneally with FA (100 mg/kg) one hour before and 2 h after a single intraperitoneal injection of LPS (10 mg/kg in saline). The LPS-induced acute kidney injury model was established as reported previously [18]. The dose of FA administered was selected based on literature followed by our pilot study. After 18 h of LPS administration, blood samples were collected through retro-orbital plexus and the animals were euthanized by CO2 asphyxiation. Serum was collected aseptically and estimated for various biochemical parameters. Animals were sacrificed and kidneys were excised and washed with saline solution. Left kidney tissues were fixed in 10% buffered formalin for histopathological analysis and the right kidney tissue was stored at −80 °C for further biochemical and immunoblot analysis. 2.4. Measurement of blood urea nitrogen and creatinine in serum Kidney injury was assessed by analyzing serum blood urea nitrogen and creatinine by kit (Siemens, India) and auto analyzer (Siemens, Dimension Xpandplus, USA). For BUN estimation 90 μl of reagent 1 (4.69 mmol/L α-ketoglutarate, 0.34 mmol/L NADH and 6.8 U/ml Urease) was added to 3 μl of serum sample and the assay volume was made up to 370 μl with assay diluent. The change in absorbance at 340 nm due to the disappearance of NADH is directly proportional to the BUN concentration in the sample and is measured using a bichromatic (340, 383 nm) rate technique. For creatinine estimation, 74 μl of reagent 1 (125 mM lithium picrate) and 18 μl of reagent 2 (2.7 mM potassium ferricyanide in 2M NaOH) were added to 20 μl of serum sample and the assay volume was made up to 370 μl with assay diluent and the absorbance was measured at 37 °C using a bichromatic (510, 577 nm) rate technique.

2. Materials and methods 2.1. Chemicals and reagents Trans-Ferulic acid (99% purity), Lipopolysaccharide from Escherichia coli (055:B5), Bradford reagent, Catalase, β-nicotinamide adenine dinucleotide 3-phosphate reduced form (NADPH), β-nicotinamide adenine dinucleotide hydrate (NADH), reduced glutathione (GSH), oxidized glutathione (GSSG), 1-chloro-2,4-dinitrobenzene (CDNB), 5, 5-dithio-bis (2-nitrobenzoic acid) (DTNB), 2-thiobarbituric acid (TBA), ascorbic acid, Griess reagent, protease inhibitor cocktail, Bovine serum albumin (BSA), MTT [3- (4, 5-dimethylthiazol-2-yl)-2, 5diphenyltetrazolium bromide], Triton X 100, Fetal Calf Serum, ammonium acetate, Copper(II) sulfate pentahydrate, Propidium iodide were purchased from Sigma (Sigma-Aldrich Co., St. Louis, MO, USA). Acetone, isopropanol and xylene were purchased from Merck Life Science Pvt. Ltd. (Mumbai, India).

2.5. Assay of renal antioxidant status The kidney tissue homogenate was prepared as described [29] and the supernatant obtained was used for the estimation of various antioxidants, like reduced glutathione (GSH), glutathione reductase (GR), glutathione S- transferase (GST), catalase (CAT), superoxide dismutase (SOD) and nitrite levels. The pellet obtained was mixed with 10% trichloroacetic acid, re-suspended and centrifuged at 1800 g for 10 min and the supernatant was used for the estimation of vitamin C and thiobarbituric acid reactive substance (TBARS) contents. GSH levels, vitamin C content, GR, GST and catalase activities were determined as reported earlier by our group [29]. Superoxide dismutase activity was measured using the SOD assay kit by following the manufacturer’s instructions (Sigma-Aldrich). The total protein content of the kidney tissue homogenates was estimated using Bradford reagent and BSA as standard.

2.2. Animal studies Healthy female Balb/c mice, weighing between 20–25 g were acclimatized to the laboratory conditions for about seven days before initiation of the study in the BIOSAFE, an animal quarantine facility of the institute (Registration No: 97/GO/RBi/S/1999/CPCSEA). All animals were given free access to the standard pellet diet and fresh drinking water ad libitum. They were housed under standard laboratory conditions with 12 h/12 h of light/dark cycle, 22 ± 3 °C temperature and 56 ± 2% relative humidity throughout the study period. All protocols involving experimental animals were performed as per Committee for the Purpose of Control and Supervision of Experiments 305

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haematoxylin-eosin (H&E) for pathological evaluation and with Masson’s trichrome (MT) for fibrosis. The renal morphology and pathology were evaluated by optical microscopy using Zeiss microscope (Axioplan 2 Imaging, AxioVision software). For MT staining, blue linear deposits were interpreted as positive areas for collagen staining. The semiquantitative evaluation was performed by measuring the area of positive staining for fibrosis in 3 different fields and the average was recorded. The level of expression of CD4 (cluster of differentiation 4) protein in inflammatory cells as well as the localization and expression of NF-κB (p65) was checked using immuno fluorescence assay as reported [31]. Briefly, the paraffin sections (5 μm) of kidney tissues were deparaffinized with xylene for one hour and fixed with acetone for 20 min followed by permeabilization with 0.5% (v/v) Triton X 100 in PBS for 10 min at room temperature. After blocking with 5% fetal calf serum (FCS) in PBS for 1 h, the sections were incubated with fluorescein isothiocyanate (FITC)-labeled anti-mouse CD4 antibody (1:100) (BD Biosciences, USA), NFκB (p65) (1:200) (Cell Signalling Technologies, USA) for 1 h at room temperature in dark in a humidified chamber. The slides were washed with PBS for two minutes. The slides treated with NFκB (p65) antibody were then incubated with anti-rabbit Cy3 conjugated secondary antibody (BD Biosciences, USA), for one hour at room temperature in dark in a humidified chamber and washed with PBS. To reduce auto fluorescence, the sections were treated with CuSO4 (10 mM) in ammonium acetate buffer (50 mM CH3COONH4, pH 5.5) for 30 min. The slides were washed with PBS and the CD4 antibody treated sections were counterstained with propidium iodide (PI) for 5 min to visualize the nuclei in red and mounted in vector shield (Vector laboratories). Whereas the sections treated with NFκB (p65) antibody were counterstained and mounted with DAPI (4′,6-diamidino2-phenylindole) to visualize the nuclei in blue. Confocal laser scanning immuno fluorescence microscopy (CLSM) was carried out using a Zeiss LSM 510 META confocal microscope. Image analysis was done using LSM510 META software (Carl Zeiss).

2.6. Estimation of renal lipid peroxidation and nitrites The extent of renal lipid peroxidation was determined upon reaction with thiobarbituric acid as described [30]. Briefly, the supernatant obtained from the TCA (10% trichloroacetic acid in distilled water) fraction was allowed to react with thiobarbituric acid (0.067% in distilled water). The amount of TBARS formed was measured at 532 nm as nmol of malondialdehyde equivalents (MDA eq)/g tissue. The nitrite levels in the kidney tissue homogenate were estimated by using Griess reagent (Sigma Aldrich). In brief, the sample was incubated with Griess reagent (1% sulphanilamide in 5% phosphoric acid and 0.1% N-(1napthyl) ethylenediamine hydrochloride in distilled water) and the absorbance of the resulting azo compound was measured at 540 nm. The amount of nitrites present in the sample was quantified using a standard curve plotted using sodium nitrite as standard. 2.7. Estimation of renal mitochondrial redox and enzyme activity The mitochondria rich fractions were isolated as reported [29]. The MTT reduction rate was used to assess the activity of the mitochondrial respiratory chain in isolated mitochondria as described [29] and the amount of formazan crystals was measured at 580 nm. Mitochondrial redox activity was expressed as percentage activity where vehicle control group was taken as 100%. The levels of mitochondrial NADH dehydrogenase and Succinate dehydrogenase were estimated as reported [29]. The activity of cytochrome c oxidase was measured using the cytochrome c oxidase assay kit as per manufacturer’s instruction (Sigma-Aldrich). 2.8. Estimation of inflammatory cytokines in kidney tissue Kidney tissue was homogenized in phosphate buffer saline (50 mM, pH 7.4) with 1% protease inhibitor cocktail to prepare 10% tissue homogenate. The homogenate was centrifuged at 5000 g for 20 min and the supernatants were used for the estimation of TNF-α and IL-6 levels by enzyme linked immunosorbent assay (ELISA) using respective kits (BD Opt EIA, BD Biosciences, San Diego, CA, USA) as per manufacturer’s instruction.

2.11. Statistical analyses All statistical analyses were performed using one way ANOVA with the Graph Pad Prism, version 5.0 software. Comparisons between groups were performed by applying post-hoc Dunnett's multiple comparison procedures with reference to LPS Control group. Results were expressed as Means ± Standard error of the mean (S.E.M) (n = 8). Statistical significance was considered at p < .05.

2.9. Western blot analysis Radioimmunoprecipitation (RIPA) lysis buffer and NE-PER nuclear and cytoplasmic extraction kit containing 1% Halt protease inhibitor cocktail (Pierce Biotechnology, Rockford, IL, USA) were used to isolate the total, nuclear and cytoplasmic fraction from the kidney tissues as described in manufacturer instructions. The protein concentration in each fraction was determined by using Bicinchoninic acid (BCA) protein assay kit (Pierce Biotechnology, Rockford, IL, USA) against BSA as standard. In total protein extract, expressions levels of NF-κB (p65), Heme oxygenase 1 (HO-1), Toll like receptor 4 (TLR-4), Cycloxygenase-2 (COX-2), inducible nitric oxide synthase (iNOS); in nuclear fractions, expression of NF-κB (p65), Nrf-2, and in cytoplasmic fraction, the expressions of IκBα, phospho-IκBα and Nrf-2 were estimated. All the antibodies were procured from Cell Signaling Technology and used at recommended dilutions. The blots were visualized using chemiluminescent detection reagents (Supersignal West Pico, Pierce Biotechnology, Rockford, IL, USA) and Vilber-Fusion-Western blotChemiluminescence Imaging system. The densitometry analysis of each blot was performed using Image J software, NIH, USA.

3. Results 3.1. Ferulic acid improves biochemical indices of kidney injury in LPS challenged mice The preliminary indices of renal injury (BUN and creatinine) were estimated in serum samples of mice. The results indicated a significant (p < .001) increase in the serum BUN and modest increase in creatinine levels in LPS control group of mice when compared with the vehicle control group after 18 h of LPS administration (Table 1). Treatment with FA at 50 mg/kg and 100 mg/kg showed significant (p < .01) decrease in the serum BUN levels when compared with the LPS alone treated group of mice. 3.2. Ferulic acid ameliorates oxidative stress in LPS challenged mice We examined the levels of various endogenous enzymatic and nonenzymatic antioxidants in renal tissues. The renal content of GSH and its associated antioxidant enzyme activities of GST and GR were significantly (p < .01: GSH and GST; p < .001: GR) decreased in LPS alone administered mice when compared with the vehicle control group of mice (Table 1). However, treatment with FA at 50 and 100 mg/kg in

2.10. Histopathological and immunofluorescence analysis The kidney tissue was fixed in 10% buffered formalin, processed, embedded in paraffin and sectioned to approximately 5μm thickness using a microtome (Leica, Bensheim, Germany) and stained with 306

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Table 1 Effect of Ferulic acid on LPS induced kidney injury markers and renal antioxidant status. Parameters estimated

VC

FA Con

LPS Con

LPS + FA50

LPS + FA100

BUN Creatinine GSH levels GST activity GR activity Catalase activity SOD activity Vit C levels TBARS Nitrites level

22.80 ± 2.43 0.247 ± 0.02 58.18 ± 2.43 9.44 ± 0.66 5.64 ± 0.19 775.06 ± 30.19 100 ± 0.1.31 72.56 ± 4.71 5.68 ± 0.1.09 39.82 ± 2.35

26.0 ± 1.0 0.25 ± 0.034 55.17 ± 4.76 8.35 ± 0.34 5.41 ± 0.15 728.06 ± 27.36 97.82 ± 3.17 69.50 ± 1.81 6.27 ± 1.35 36.47 ± 8.55

96.43 ± 5.64### 0.45 ± 0.042### 38.47 ± 3.005## 6.09 ± 0.58## 2.59 ± 0.78### 579.70 ± 25.47# 84.96 ± 2.97## 43.69 ± 3.58## 12.91 ± 0.91### 117.1 ± 21.22###

68.29 ± 3.96* 0.32 ± 0.038 51.68 ± 2.11* 8.62 ± 0.43** 4.38 ± 0.4** 738.30 ± 42.52** 95.99 ± 1.23* 61.11 ± 5.33** 7.08 ± 1.09** 70.79 ± 9.01*

56.63 ± 12.68** 0.31 ± 0.035 53.71 ± 3.001** 9.16 ± 0.69*** 4.3 ± 0.15** 780.0 ± 45.48** 97.21 ± 0.16** 68.72 ± 3.2*** 6.61 ± 0.70*** 43.24 ± 7.1***

Where, #P < .05, ##P < .01 and ###P < .001 compared with vehicle control group. *P < .05, **P < .01 and ***P < .001 compared with LPS control group. All data were expressed as means ± S.E.M (n = 8). FA, Ferulic acid; VC, vehicle control mice; LPS Con, LPS control mice; FA Con, Ferulic acid control mice; LPS + FA 50, LPS + Ferulic acid (50 mg/kg) treated mice; LPS + FA 100, LPS + Ferulic acid (100 mg/kg) treated mice. BUN, Blood Urea Nitrogen (mg/dL); creatinine (mg/dL); GSH, reduced glutathione (mg/g of tissue); GST, glutathione S-transferase (nmoles of CDNB conjugated/min/ml); GR, glutathione reductase (U/ml protein); CAT, catalase (U/mg protein); SOD, superoxide dismutase (% of control), Vit C, vitamin C (mg/g of tissue); TBARS, thiobarbituric acid reactive substances (nmoles of MDA eq./g of tissue); Nitrites, (μmol/g of tissue); CDNB, 1-chloro-2, 4-dinitrobenzene.

3.5. Ferulic acid restores LPS-induced decline in mitochondrial redox and respiratory enzyme activity

LPS administered group significantly restored the levels of renal GSH content (p < .05 for 50 mg/kg and p < .01 for 100 mg/kg) and the activities of GST (p < .01 for 50 mg/kg and p < .001 for 100 mg/kg) and GR (p < .01 for both 50 and 100 mg/kg) when compared with LPS alone treated mice. Renal content of CAT, SOD and vitamin C were also significantly (p < .01: SOD; p < .05 CAT; p < .01: vitamin C) decreased in the LPS alone group when compared with the vehicle control group (Table 1). The treatment with FA at both the doses significantly restored the activities of CAT (p < .01 for both 50 and 100 mg/kg), SOD (p < .05 for 50 mg/kg and p < .01 for 100 mg/kg) and vitamin C (p < .01 for 50 and p < .001 for 100 mg/kg) when compared with the LPS alone treated group of mice. No significant changes (p > .05) were observed between FA control group and vehicle control group of mice.

We evaluated the effect of LPS stimuli on mitochondrial respiratory enzyme complexes and redox activity in kidney tissues. As shown in Fig. 2, the mitochondrial respiratory enzyme activities were significantly reduced such as NADH dehydrogenase (p < .05), succinate dehydrogenase (p < .001), and cytochrome c oxidase (p < .001) in LPS control group when compared with the vehicle control group of mice. FA treatment at both the doses significantly (p < .05 at 50 mg/ kg and p < .01 at 100 mg/kg) restored the levels of renal NADH dehydrogenase, succinate dehydrogenase and cytochrome c oxidase. Also, mitochondrial redox activity was measured by MTT reduction assay where the LPS control group showed significantly (p < .01) reduced redox activity as compared to vehicle control group. The FA treated group at both the doses significantly (p < .05 at 50 mg/kg and p < .01 at 100 mg/kg) increased mitochondrial redox activity. Furthermore, mitochondrial GSH and TBARS levels were significantly (p < .001) reduced in LPS control group when compared to vehicle control group, whereas FA (100 mg/kg) treated group significantly (p < .05 for GSH and p < .01 for TBARS) restored the mitochondrial GSH levels and reduced the TBARS levels as compared to LPS alone treated group.

3.3. Ferulic acid enhances nuclear translocation of Nrf2 and increases HO1 expression in kidney tissues The treatment with FA substantially improved the antioxidant levels in sepsis induced renal tissues. Further, to determine the influence of FA treatment on the Nrf2 signaling pathway, we assessed the nuclear translocation of Nrf2 and the expression of HO-1 in kidney tissues by western blotting. We observed a moderate increase (p < .05) in nuclear accumulation of Nrf2 protein in kidneys of LPS alone treated mice when compared with the vehicle control mice (Fig. 1). Treatment with FA at both the doses further intensified (p < .01 for 50 mg/kg and p < .001 for 100 mg/kg) the nuclear accumulation of Nrf2 when compared with the LPS alone treated mice. Furthermore, the amount of HO-1 expression in the total protein extract of kidney tissues from LPS induced mice treated with FA at 50 and 100 mg/kg were significantly (p < .05 for 50 mg/kg and p < .01 for 100 mg/kg) increased in comparison to LPS alone treated mice.

3.6. Ferulic acid attenuates LPS-induced renal inflammation We estimated the levels of inflammatory cytokines in renal tissues. The concentrations of pro-inflammatory markers such as TNF-α and IL6 in renal tissues significantly (p < .001 for TNF-α and IL-6) increased after 18 h of LPS stimulation when compared to vehicle control as shown in Fig. 3. FA treatment at 50 mg/kg significantly (p < .05 for TNF-α; p < .01 for IL-6) and 100 mg/kg (p < .01 for TNF-α; p < .001 for IL-6) attenuated the elevated levels of TNF-α and IL6 as compared to LPS alone treated mice.

3.4. Ferulic acid attenuates LPS-induced renal lipid peroxidation and nitrative stress

3.7. Ferulic acid ameliorates histopathological changes and fibrosis induced by LPS

We determined the levels of nitrites and lipid peroxidation products in renal tissues of mice. LPS alone treated group showed significantly (p < .001) increased renal TBARS and nitrite levels in comparison with vehicle control group (Table 1). FA treated groups at both the doses (50 and 100 mg/kg) significantly (p < .01and p < .001 for 50 mg/kg and 100 mg/kg) attenuated the levels of renal TBARS levels and nitrite levels (p < .05 for 50 mg/kg and p < .001 for 100 mg/kg) in comparison with LPS alone administered mice.

Histopathological change is a direct indication of renal injury. To investigate the protective effects of FA on LPS-induced AKI, the histological changes of kidney tissues were examined. The H and E staining showed normal glomerular and tubular structure in vehicle control and FA control groups (Fig. 4A and C). However, in the LPS alone treated group (Fig. 4B), severe degeneration of cystic type in tubular regions of the kidney as well as dilatation of tubules with pushing of the basement layer of epithelium (appeared very thin indicated with the Red arrow) 307

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Fig. 1. Effect of FA on nuclear and cytoplasmic Nrf-2 and the total HO-1 expression. Immunoblot analyses showing (A) nuclear translocation of Nrf2, (B) HO-1 in kidneys. Immunoblots were representative of three independent experiments. Lamin B was used as internal control for nuclear fraction and β-actin was used as internal control for cytoplasmic and total protein fractions. Bar diagram showing densitometric analysis for relative expression of (C) nuclear Nrf2/cytoplasmic Nrf2 ratio, (D) Total HO-1 proteins. Values are the means ± SEM (n = 3). LPS and FA were administered to animals through intraperitoneal delivery. Where VC, group of animals treated with vehicle (2% gum acacia suspension) for 2 doses of FA and a single intraperitoneal injection of saline; LPS Con, group of animals treated with 2% gum acacia suspension for 2 doses of FA and a single dose of LPS (10 mg/kg in normal saline); LPS + FA50, group of animals treated with FA (50 mg/kg) one hour before and 2 h after a single dose of LPS (10 mg/kg in saline); LPS + FA100, group of animals treated with FA (100 mg/kg) one hour before and 2 h after a single dose of LPS (10 mg/kg in saline). FA, Ferulic acid; VC, Vehicle control; LPS Con, LPS Control; Nrf2, nuclear factor erythroid 2-related factor 2; HO-1, heme oxygenase-1 #p < .05 vs. vehicle control group, *p < .05, **p < .01 and ***p < .001 vs. LPS control group.

alone treated mice (Fig. 6F). Mice treated with LPS + FA50 (Fig. 6L) showed less expression of the CD4 protein in mononuclear cells infiltrated near renal pelvis compared with LPS alone treated animals. Further, mice treated with LPS + FA100 (Fig. 6O) showed expression of the CD4 protein in very few mononuclear cells infiltrated near renal pelvis region.

was observed. Tissue sections from groups treated with LPS + FA50 (Fig. 4D) showed moderate tubular degeneration and less number of tubules involved when compared with LPS alone treated animals. Further LPS + FA100 treated group (Fig. 4 E) showed mild tubular degeneration when compared with LPS alone treated animals. We further studied the protective effect of FA on LPS induced renal fibrosis by Masson’s trichrome staining. The vehicle control and FA control groups (Fig. 5A and C) showed normal renal pelvis surrounded by normal connective tissue. However, there was a marked proliferation of fibrous tissue in renal pelvis region of LPS alone treated mice showing bluish linear collagen deposition (Fig. 5B). Further, tissue sections from LPS + FA50 treated animals (Fig. 5D) showed mild proliferation of fibrous tissue in renal pelvis region and groups treated with LPS + FA100 (Fig. 5E) showed normal renal pelvis surrounded by normal connective tissue.

3.9. Ferulic acid inhibits TLR-4 mediated nuclear translocation of NF-κB (p65) and IκBα degradation in renal tissue To investigate the anti-inflammatory mechanism of FA, TLR4 mediated NF-κB signaling pathway was studied. As shown in Fig. 7, there was a significant (p < .001) increase in the TLR-4 expression in the renal tissues of LPS-induced mice in comparison to the vehicle control mice. FA treated group showed significant (P < .01) downregulation of TLR-4 at both the doses. On the other hand, there was a marked (p < .001) degradation and phosphorylation of IκBα in the cytoplasmic fraction and a significant (p < .001) increase in the accumulation of nuclear NF-κB (p65) in LPS-induced mice in comparison to vehicle control mice. However, FA treated groups at both the doses markedly prevented the LPS-induced IκBα phosphorylation, IκBα degradation and subsequent nuclear translocation of NF-κB (p65) when compared with LPS alone treated group. Furthermore, immunostaining for NF-κB (p65) further confirmed its expression and localization in kidney sections Fig. 8. The vehicle

3.8. Ferulic acid reduces infiltration of inflammatory cells in renal tissue The involvement of inflammatory CD4 + cells in LPS induced AKI was studied by immunostaining the renal tissues with fluorescein isothiocyanate (FITC)-labeled anti-mouse CD4 antibody. The results revealed there was no infiltration of mononuclear cells near renal pelvis region in the vehicle control and FA control groups (Fig. 6C and I). However, over expression of the CD4 protein was observed in more number of mononuclear cells infiltrated near renal pelvis region of LPS 308

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Fig. 2. Effect of FA on LPS-induced impairment of mitochondrial respiratory enzymes, redox activity and oxidative stress. Treatment with FA restored LPS-induced decline in mitochondrial (A) NADH dehydrogenase (B) Succinate dehydrogenase, (C) Cytochrome c oxidase, (D) Redox (MTT reduction), (E) GSH content (F) TBARS in kidney tissues. The data were expressed as the means ± SEM of 8 animals. LPS and FA were administered to animals through intraperitoneal delivery. Where VC, group of animals treated with vehicle (2% gum acacia suspension) for 2 doses of FA and a single intraperitoneal injection of saline; LPS Con, group of animals treated with 2% gum acacia suspension for 2 doses of FA and a single dose of LPS (10 mg/kg in normal saline); FA Con, group of animals treated with FA (100 mg/kg) for 2 doses of FA and a single intraperitoneal injection of saline; LPS + FA50, group of animals treated with FA (50 mg/kg) one hour before and 2 h after a single dose of LPS (10 mg/kg in saline); LPS + FA100, group of animals treated with FA (100 mg/kg) one hour before and 2 h after a single dose of LPS (10 mg/kg in saline). FA, Ferulic acid; VC, Vehicle control; LPS Con, LPS Control; FA Con, Ferulic acid control; GSH, reduced glutathione; TBARS, thiobarbituric acid reactive substances; MTT, [3- (4, 5dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide]#p < .05, ##p < .01 and ###p < .001 vs. vehicle control group, *p < .05, **p < .01 vs. LPS control group.

Fig. 3. Effect of FA on LPS-induced renal inflammatory mediators. (A) Tumor necrosis factor- α (TNF-α) and (B) Interleukin-6 (IL-6) Values are the means ± SEM (n = 8). Where VC, group of animals treated with vehicle (2% gum acacia suspension) for 2 doses of FA and a single intraperitoneal injection of saline; LPS Con, group of animals treated with 2% gum acacia suspension for 2 doses of FA and a single dose of LPS (10 mg/kg in normal saline); FA Con, group of animals treated with FA (100 mg/kg) for 2 doses of FA and a single intraperitoneal injection of saline; LPS + FA50, group of animals treated with FA (50 mg/kg) one hour before and 2 h after a single dose of LPS (10 mg/kg in saline); LPS + FA100, group of animals treated with FA (100 mg/kg) one hour before and 2 h after a single dose of LPS (10 mg/kg in saline). FA, Ferulic acid; VC, Vehicle control; LPS Con, LPS Control; FA Con, Ferulic acid control. ###p < .001 vs. vehicle control group, *p < .05, **p < .01 and ***p < .01 vs. LPS control group.

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Fig. 4. Representative photomicrographs and light microscopic examination (H and E staining) of kidney tissues treated with LPS and/or FA (magnification × 200). Kidney tissue sections from Vehicle control (A) and FA Control (C) showing normal glomerulus and tubular regions. Kidney tissue sections from mice treated with LPS alone (B) showing severe degenerative (cystic type) changes in tubular regions of kidney [black arrow] as well as dilatation of tubules with pushing of the basement layer of epithelium [Red arrow]. Mice treated with LPS + FA50 (D) showing moderate tubular degenerative changes [black arrow] and less number of tubules involved. Mice treated with LPS + FA100 (E) showing normal glomerular and tubular structure with mild tubular degenerative changes [black arrow]. Scale bars: 50 μm. Where FA, Ferulic acid.

3.10. FA reduced iNOS and COX-2 expression induced by LPS

control mice (Fig. 8C) showed no nuclear localization of NF-κB (p65) in the tubular epithelial cells. However, the LPS alone treated group (Fig. 8F) showed an increased nuclear localization of NF-κB (p65) in the tubular epithelial cells when compared to vehicle control mice. On the other hand, LPS + FA50 and LPS + FA100 treated groups (Fig. 8I and L) showed mild nuclear translocation of NF-κB (p65) as compared to LPS control mice as indicated by arrows in tubules.

We also studied the effect of FA on the expression of iNOS and COX2 of NF-κB signaling pathway under LPS stimuli. As shown in Fig. 9, there was a significant (p < .001) overexpression of iNOS and COX-2 in LPS control group as compared to vehicle control. However, FA (100 mg/kg) treated group showed significant (p < .001) downregulation of iNOS and COX-2 further supporting the anti inflammatory mechanism of FA on LPS induced AKI.

Fig. 5. Representative photomicrographs and light microscopic examination Masson’s Trichome stained sections of kidney tissues (magnification × 200). The fibrous tissue stained blue color [black arrow]. The kidney tissue sections from Vehicle control group (A) and FA Control (C) showing normal renal pelvis. Kidney tissue sections from mice treated with LPS alone (B) showing the proliferation of fibrous tissue (Fibrosis) near renal pelvis region. Mice treated with FA at 50 mg/kg and LPS (D) showed mild proliferation of bluish fibrous tissue near renal pelvis region where as mice treated with FA at 100 mg/kg followed by LPS (E) showed apparently normal renal tissue. All the photomicrographs were captured at 50 μm scale bars. The bar diagram showing average fibrotic area of kidney tissues (F). The results were expressed as means ± S.E.M of 3 animals in each group. Where FA, Ferulic acid ###p < .001 vs. vehicle control group and ***p < .001 vs. LPS control group.

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Fig. 6. Confocal images of expression of CD4 protein in renal tissues by immunoflourescence assay (magnification × 400). Red panel demonstrates nuclei stained with PI and Green panel in the images demonstrate staining with CD4 (FITC labeled). Merged images demonstrate expression of CD4 protein in renal tissues indicated with white arrows. n = 3; Kidney tissue sections focusing from Vehicle control (A, B, C) and FA control (G, H, I) showing no expression of CD4 protein in renal tissue. Kidney tissue sections from mice treated with LPS alone (D, E, F) showing overexpression of CD4 protein in more number of mononuclear cells infiltrated near renal pelvis (Arrows in F). Mice treated with FA at 50 mg/kg and LPS (J, K, L) showing less expression of CD4 protein in mononuclear cells infiltrated near renal pelvis compared with LPS alone treated section (Arrows in L). Mice treated with FA at 100 mg/kg followed by LPS (M, N, O) showing expression of CD4 protein in very few mononuclear cells infiltrated near renal pelvis (Arrows in L). FA, Ferulic acid; VC, Vehicle control; LPS Con, LPS Control; CD4, cluster of differentiation 4. Scale bars: 100 μm.

4. Discussion

induced renal dysfunction (Table 1). Similarly, the effect of FA on the levels of BUN and creatinine were demonstrated in other models of kidney injury [25,26]. Lipopolysaccharide causes overproduction of ROS generation leading to decreased antioxidant defenses and lipid peroxidation of biological membranes with increased MDA production which aggravates tissue injury [6]. Our study indicated that FA treatment at both the doses increased the antioxidant levels and decreased the formation of lipid peroxides thereby minimizing the detrimental effects of ROS and RNS (Table 1). Our results were supported by previous findings [25,26]. The antioxidant potential of FA is attributed to its resonancestabilized phenoxy radical structure leading to quenching of free radicals [27]. Our results indicated the protective effect of FA is due to the enhanced activity of antioxidant enzymes, thereby counteracting the effect of LPS-induced free radicals. Nrf-2 is a key regulator of the cellular redox state in sepsis and acts as the main defense mechanism against oxidative stress in cells [34]. Moreover, the induction of antioxidant defense and phase II detoxifying enzymes has been positively associated with Nrf2 expression and its nuclear translocation [35]. Nrf2 is sequestered by the Kelch-like ECHassociated protein 1 (Keap1) in the cytosol and restricts its translocation

The present study showed that FA exhibited protective effects on LPS-induced acute kidney injury in mice. FA significantly reduced the levels of blood urea nitrogen, nitrites and inflammatory cytokines (TNFα and IL-6) and increased the levels of antioxidants (GSH, GR, GST, Catalase, SOD and Vitamin C). In addition, FA effectively attenuated the fibrosis and histological changes in renal tissue. Western blot analysis demonstrated that FA ameliorated LPS-induced acute kidney injury by enhancing antioxidant defenses and suppressing the inflammatory events through NFκB signaling pathway. Previous studies have shown the therapeutic effects of FA at both the doses (50 mg/kg and 100 mg/kg) against cardiotoxicity and epilepsy [32,33]. Furthermore, reports suggest that FA exhibited renal protective effects against cisplatin-induced and glycerol induced nephrotoxicities [25,26]. However, the therapeutic effect of FA against LPS induced acute kidney injury is not well studied. Hence, we studied the pharmacological activity and mechanisms of protection by FA (50 mg/kg and 100 mg/kg) against LPS induced acute kidney injury. Our findings indicated that FA treatment decreased the levels of serum blood urea nitrogen indicating its protection against sepsis 311

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Fig. 7. Effect of FA on LPS induced changes in NF-κB p65, TLR-4, p-IκBα and IκBα in LPS-induced AKI. Immunoblot analyses showing (A) nuclear NF-κB (p65) expression, (B) TLR4, phospho-IκBα and IκBα expressions in kidneys. Immunoblots were representative of three independent experiments. Lamin B was used as internal control for nuclear fraction and βactin was used as internal control for cytoplasmic and total protein fractions. Bar diagram showing densitometric analysis for relative expression of (C) nuclear NF-κB (p65)/Lamin B ratio, (D) phospho-IκBα/ IκBα ratio, (E) TLR-4/β-actin ratio. Values are the means ± SEM (n = 3). LPS and FA were administered to animals through intraperitoneal delivery. Where VC, group of animals treated with vehicle (2% gum acacia suspension) for 2 doses of FA and a single intraperitoneal injection of saline; LPS Con, group of animals treated with 2% gum acacia suspension for 2 doses of FA and a single dose of LPS (10 mg/kg in normal saline); LPS + FA50, group of animals treated with FA (50 mg/kg) one hour before and 2 h after a single dose of LPS (10 mg/kg in saline); LPS + FA100, group of animals treated with FA (100 mg/kg) one hour before and 2 h after a single dose of LPS (10 mg/kg in saline). FA, Ferulic acid; VC, Vehicle control; LPS Con, LPS Control; TLR, toll like receptor; NF-κB, nuclear factor kappa B; IκB, Inhibitor of kappa B ###p < .001 vs. vehicle control group, **p < .01 and ***p < .001 vs. LPS control group.

Nonetheless, the oxidative damage of mitochondria and the impairment of mitochondrial respiratory enzyme activities have been implicated in the pathogenesis of LPS induced AKI [42]. The mitochondrial ROS leads to decrease in the levels of antioxidants as well as the redox potential resulting in mitochondrial dysfunction [43]. However, our results indicated that FA treatment at both the doses improved the mitochondrial antioxidant status and the redox potential in LPS induced kidney damage (Fig. 2). An earlier study reported renal fibrosis in a swine model of endotoxemia-induced acute kidney injury [44]. We also observed induced proliferation of fibrous tissue in renal pelvis region of kidney tissues (Fig. 5) of LPS alone treated group. However, the treatment with FA significantly reduced the proliferation of fibrous tissue. Our findings were supported by previous studies demonstrating the protective effect of FA against renal and cardiac fibrosis [45,46]. Inflammation has a major role in the initiation and progression of LPS–induced renal damage [18]. LPS stimulation releases proinflammatory cytokines (TNF-α, IL-1β, IL-6, IL-18) and causes the infiltration of mononuclear cells leading to renal tissue damage [43]. It is well known that genes encoding for many pro-inflammatory cytokines are regulated by NF-κB transcription factors during sepsis induced renal damage [10,11,47]. Furthermore, a recent study demonstrated the role of CD4+ T-cells in LPS-induced acute kidney injury and other inflammatory disorders like atherosclerosis and abdominal aortic aneurysm [48–50]. Our results indicated that the FA treatment showed

to the nucleus. The interaction with different types of inducers (electrophilic and oxidative stress) releases Nrf2 from Keap1 and facilitates its translocation into the nucleus, resulting in the activation of phase II cytoprotective genes [36]. HO-1 plays its antioxidant role by converting heme into the powerful prooxidant biliverdin and finally a strong antioxidant bilirubin [37]. Several phytonutrients with antioxidant potential have been reported to induce HO-1 expression thereby imparting protection against oxidative stress [36]. Our study indicated that treatment with FA at both the doses increased nuclear translocation of Nrf2 as well as upregulated HO-1 expression (Fig. 1) which is also responsible for its protection against LPS induced oxidative renal damage. These findings were supported by similar effects of FA reported previously [38–40]. Further, the lipid peroxidation marker, TBARS and tissue levels of nitrites are other indicators of renal oxidative stress [34]. Excess NO production has been associated with sepsis-related organ failure [6] leading to the formation of toxic lipid peroxides causing cellular damage. Moreover, LPS alone increases NO synthesis via activation of iNOS [41]. Peroxynitrite radical formed by the interaction of NO with superoxide anion leads to nitrosative stress and damage of cellular proteins [41]. Previous reports demonstrated that FA could reduce the levels of nitrites and iNOS expression in various disease condition [22,23,28]. Similarly, our finding indicates that FA treatment reduced levels of nitrites and iNOS expression in renal tissues upon LPS stimulation (Table 1) and (Fig. 9). 312

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Fig. 8. Confocal images of NFκB (p65) localization in tubular epithelial cells by immunohistochemistry (magnification × 400). Blue panel demonstrates nucleus of tubular epithelial cells stained with DAPI and Red panel in the images demonstrate presence of NF-κB (Cy3 labelled). Merged images demonstrate nuclear localization of NF-κB and distribution in renal tubules with white arrows pinpoint variation in color intensity owing to nuclear localization of NF-κB. Kidney tissue sections focusing from Vehicle control (A, B, C) showing no nuclear localization of NF-κB (p65). Kidney tissue sections from mice treated with LPS alone (D, E, F) showing nuclear localization of NF-κB (p65) in the tubular epithelial cells (Arrows in F). Mice treated with FA at 50 mg/kg and LPS (G, H, I) showing less number of nuclei with NFκB (p65) localization in tubular epithelial cells (Arrows in I). Mice treated with FA at 100 mg/kg followed by LPS (J, K, L) showing NF-κB (p65) localization in very few nuclei of tubular epithelial cells (Arrows in L). FA, Ferulic acid; VC, Vehicle control; LPS Con, LPS Control; NF-κB, nuclear factor kappa B.

Fig. 9. Effect of FA acid on LPS induced downstream proteins of NFκB pathway. Immunoblot analyses showing expression of (A) COX-2 (B) iNOS in kidneys. Immunoblots were representative of three independent experiments. β-actin was used as internal control for total protein fractions. Bar diagram showing densitometric analysis for relative expression of (C) COX-2/β-actin ratio, (D) iNOS/β-actin. Values are the means ± SEM (n = 3). LPS and FA were administered to animals through intraperitoneal delivery. Where VC, group of animals treated with vehicle (2% gum acacia suspension) for 2 doses of FA and a single intraperitoneal injection of saline; LPS Con, group of animals treated with 2% gum acacia suspension for 2 doses of FA and a single dose of LPS (10 mg/kg in normal saline); LPS + FA50, group of animals treated with FA (50 mg/kg) one hour before and 2 h after a single dose of LPS (10 mg/kg in saline); LPS + FA100, group of animals treated with FA (100 mg/kg) one hour before and 2 h after a single dose of LPS (10 mg/ kg in saline). FA, Ferulic acid; VC, Vehicle control; LPS Con, LPS Control; COX-2, cycloxygenase-2; iNOS, inducible nitric oxide synthase ###p < .001 vs. vehicle control group, **p < .01 ***p < .001 vs. LPS control group.

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less number of mononuclear cells with mild expression of the CD4 protein, infiltrated near renal pelvis as compared to LPS alone treated group (Fig. 6). Moreover, LPS alone treated group showed higher levels of inflammatory cytokines. However, the FA treatment significantly inhibited the release of inflammatory cytokines (TNF-α and IL-6) (Fig. 3). Similarly various phytoconstituents are reported to have inhibitory effect on cytokine release in LPS induced AKI [51–53]. NF-κB activation is mediated through TLR-4 receptors upon LPS stimulation leading to severe pathological renal damage [54–56]. We have observed that FA treatment at both the doses downregulated the TLR4 expression and subsequently inhibited IκBα phosphorylation and IκBα degradation leading to inhibition of the NF-κB signalling pathway (Figs. 7 and 8). We also showed that FA treatment at both the doses resulted in minimal localization of NF-κB in the renal tubules as compared to LPS alone treated group. It is evident that, FA imparts protection against sepsis induced acute kidney injury through inhibition of the NF-κB signaling pathway.

[10]

[11] [12]

[13]

[14]

[15]

[16]

5. Conclusion To our knowledge, this is the first report demonstrating that FA has a promising pharmacological intervention in the prevention of LPS-induced renal damage with negligible toxic effects. The protective action of FA facilitates in downregulation of oxidative stress, and inflammatory events via upregulation of Nrf2/HO-1 proteins and inhibition of NF-κB signaling pathways. Overall, FA based therapy alone as well as in combination with other phytochemicals may provide a novel strategy in treating sepsis induced renal injury.

[17]

[18]

[19]

[20]

Conflicts of interest

[21]

The authors declare no competing financial interest.

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Acknowledgements

[23]

Authors are grateful to Director, CSIR-IICT, Hyderabad, India for providing necessary facilities and continuous support. Salma Mukhtar Mir thanks Department of Science and Technology (DST), New Delhi, India for financial support as INSPIRE Fellowship (IF 120825). The author, SMM thanks Dr. Vinoth Rajendran for his critical evaluation and proof reading of our manuscript.

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