European Journal of Pharmacology 761 (2015) 199–205

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European Journal of Pharmacology journal homepage: www.elsevier.com/locate/ejphar

Pulmonary, gastrointestinal and urogenital pharmacology

Indomethacin induces endoplasmic reticulum stress, but not apoptosis, in the rat kidney Arumugam Suriyam Nagappan, Joe Varghese, Jithu V. James, Molly Jacob n Department of Biochemistry, Christian Medical College, Vellore 632002, India

art ic l e i nf o

a b s t r a c t

Article history: Received 26 November 2014 Received in revised form 20 April 2015 Accepted 21 April 2015 Available online 7 May 2015

Non-steroidal anti-inflammatory drugs (NSAIDs) are commonly used in clinical practice. However, their use is often associated with adverse effects in the gastrointestinal tract and kidney. Our earlier work with indomethacin, a prototype NSAID, has shown that it induced oxidative stress in the kidney in rats, an event that has been postulated to contribute to pathogenesis of its adverse effects in this organ. Endoplasmic reticulum (ER) stress responses have been shown to occur in response to oxidative stress. We investigated whether this occurred in the rat kidney, in response to indomethacin. For this, Wistar rats were orally gavaged with indomethacin (20 mg/kg). Markers of ER stress were studied in the kidneys 1, 12 and 24 h later. GRP78, p-PERK and nuclear sXBP-1, all markers of ER stress, were found to be increased in the rat kidney at 12 h, in response to indomethacin; levels of these markers fell by 24 h. The effects seen at 12 h were attenuated by pre-treatment with zinc, a known anti-oxidant, which has earlier been shown to ameliorate indomethacin-induced oxidative stress. Activation of an ER stress response was not associated with induction of apoptosis, as measured by markers of apoptosis such as release of cytochrome c from mitochondria into the cytosol, activation of caspases 3 and 9, cleavage of poly-ADP ribose polymerase and the presence of DNA laddering. We conclude that indomethacininduced oxidative stress activated ER stress, but did not lead to apoptosis in the rat kidney. & 2015 Elsevier B.V. All rights reserved.

Keywords: ER stress Indomethacin Kidney Zinc Apoptosis

1. Introduction Non-steroidal anti-inflammatory drugs (NSAIDs) often cause adverse effects in the gastrointestinal tract and kidneys (Epstein, 2002; Gabriel et al., 1991). Indomethacin, a prototype NSAID, has been shown to induce oxidative stress and mitochondrial dysfunction in the kidney (Basivireddy et al., 2004; Varghese et al., 2009). Reactive oxygen species (ROS) are known to break protein disulfide bonds, resulting in accumulation of unfolded or misfolded proteins in cells (Inagi, 2009). Accumulation of these unfolded proteins in the endoplasmic reticulum activates a repair mechanism known as endoplasmic reticulum stress (ER stress) response or unfolded protein response (UPR) (Kitamura, 2008; Malhotra and Kaufman, 2007; Yoshida, 2007). The ER stress response broadly

Abbreviations: ATF4, activating transcription factor-4; ATF6, activating transcription factor-6; CHOP, CCAAT/enhancer binding protein-homologous protein; cyt c, cytochrome c; eIF2α, eukaryotic translation initiation factor-2α; ER stress, endoplasmic reticulum stress; GRP 78, glucose-regulated protein 78; HO-1, heme oxygenase‐1; IRE1, inositol-requiring enzyme 1; Nrf2, nuclear factor E2 related factor 2; NSAID, non-steroidal anti-inflammatory drug; PARP, poly-ADP ribose polymerase; PERK, double-stranded RNA-activated protein kinase-like ER kinase; ROS, reactive oxygen species; sXBP, spliced form of X-box-binding protein n Corresponding author. Tel.: þ 91 416 2284267; fax: þ 91 416 2262788. E-mail addresses: [email protected] (A.S. Nagappan), [email protected] (J. Varghese), [email protected] (M. Jacob). http://dx.doi.org/10.1016/j.ejphar.2015.04.044 0014-2999/& 2015 Elsevier B.V. All rights reserved.

consists of two distinct but inter-related pathways: the “light” and “dark” pathways (Inagi, 2009; Kitamura, 2008; Yoshida, 2007). The light pathway involves cellular adaptation to stress by translational suppression, induction of ER chaperones (such as 150 kDa oxygen-regulated protein [ORP150], 78 kDa glucose-regulated protein [GRP78], GRP94, calreticulin) and ER-associated degradation [ERAD] to eliminate immature proteins or unfolded proteins by proteasomal degradation (Vembar and Brodsky, 2008; Yoshida, 2007). These responses collectively attempt to rescue the cell from damage induced by the stress. On the other hand, severe and prolonged stress that is likely to result in cellular damage that is beyond repair results in activation of a second pathway, called the “dark” pathway (Inagi, 2009; Kitamura, 2008; Yoshida, 2007). This results in induction of apoptosis and eventually cell death (Inagi, 2009; Kitamura, 2008; Yoshida, 2007). Accumulation of unfolded proteins in the ER triggers the release of GRP78 from trans-membrane ER proteins, such as inositol-requiring enzyme 1 (IRE1), double-stranded RNA-activated protein kinase-like ER kinase (PERK) and activating transcription factor-6 (ATF6) (Inagi, 2009). IRE1 is an endonuclease that catalyzes alternative splicing of mRNA for X-box-binding protein (XBP). This leads to a functionally active transcription factor, sXBP-1, that initiates transcription of ER stress response genes (Hahmann et al., 2011; Inagi, 2009). PERK inhibits protein translation by phosphorylation and resultant inactivation of eukaryotic initiation factor-2α (eIF2α) (Malhotra and Kaufman,

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2007). Both ATF-6 and ATF4 have been shown to be activated by ER stress and this, in turn, activates apoptosis (Kim et al., 2011; Kitamura, 2008; Carlisle et al., 2014). Indomethacin has been shown to activate ER stress responses and apoptosis in various cells in vitro (Suemasu et al., 2009; Tsutsumi et al., 2004; Okamura et al., 2008; Franceschelli et al., 2011). However, more recently, Matsumoto et al. (2013) have shown that apoptosis did not occur in HepG2 cells, in response to ER stress. It is not known whether ER stress and/or apoptosis occur in the kidney in vivo following indomethacin administration. Zinc, a transition metal that has powerful anti-oxidant effects (Powell, 2000), has been shown to ameliorate indomethacin-induced oxidative stress in the rat kidney (Varghese et al., 2009) and small intestine (Basivireddy et al., 2002; Sivalingam et al., 2011). However, it is not known whether pre-treatment with zinc affects a possible ER stress response, following indomethacin administration.

2. Materials and methods 2.1. Materials Indomethacin (1-[p-chlorobenzoyl]-5-methoxy-2-methylindole-3acetic acid), bovine serum albumin (BSA), TRI reagent, ammonium persulphate, N,N,N0 ,N0 -tetramethylethylenediamine (TEMED), 2-mercaptoethanol, sodium dodecyl sulfate (SDS), protease inhibitors (aprotinin, leupeptin and phenylmethylsulfonyl fluoride [PMSF]), phosphatase inhibitors (sodium orthovanadate and sodium fluoride), 7amino-4-trifluoromethylcoumarin (AFC), caspase-3 substrate (AcDEVD-AFC), caspase-9 substrate (Ac-LEHD-AFC) and primary antibody for β-actin were purchased from Sigma, St. Louis, USA. Agarose was purchased from Genei, Bangalore, India. Zinc sulfate was purchased from Sarabhai Merck Limited, Baroda, India. Cell strainer of 100 mM pore size was purchased from BD Biosciences, California, USA. ApoAlert caspase-3 fluorescent assay kits were from Clontech Laboratories, CA. ECL DualVue western blotting markers were purchased from GE Healthcare Bio-sciences Corp, Piscataway, USA. Primary antibodies for GRP78 (cat# SC 1051), XBP-1 (cat# SC 7160), phospho-PERK (cat# SC 32577) and CHOP (cat# SC 575) were purchased from Santa Cruz Biotechnology, Inc., CA, USA. Poly-ADP ribose polymerase (PARP) (cat# 9542) and cytochrome c (cat# 4272) rabbit polyclonal primary antibodies were purchased from Cell Signaling Technology, Inc., Danvers, MA, USA. Rabbit anti-goat IgG (HþL) peroxidase-conjugated (cat# 31402), goat anti-rabbit IgG (HþL) peroxidase-conjugated (cat# 31462), goat anti-mouse IgG (HþL) peroxidase-conjugated (cat# 31430) secondary antibodies and Super Signal West Dura extended duration substrate (cat# 34075) were purchased from Thermo Scientific, IL, USA. Polyvinylidene difluoride (PVDF) membrane (0.45 mm) was obtained from Millipore, Bangalore, India.

standardized in our previous studies and has been shown to consistently produce oxidative stress in the kidney (Basivireddy et al., 2004; Varghese et al., 2009). To study the effects of zinc, zinc sulfate (ZnSO4  7H2O) at a dose of 50 mg/kg body weight (containing 11.4 mg of elemental zinc/kg body weight) (Joseph et al., 1999; Varghese et al., 2009) was administered by oral gavage, 2 h before the dose of indomethacin. Animals were killed by cervical dislocation under halothane anesthesia, at 1, 12 or 24 h after the indomethacin dose. The kidneys were removed, snap-frozen and stored at  70 1C till further use. 2.4. Isolation of cytosolic and nuclear proteins Cytosolic and nuclear protein extracts were prepared from whole kidney lysates, according to a previously described method (Hershfield et al., 2006). Purity of cytoplasmic and nuclear extracts were assessed by the absence of lactate dehydrogenase (LDH) activity in nuclear extracts and by showing the absence of DNA in cytosolic extracts, as assessed by agarose gel electrophoresis. 2.5. Isolation of rat kidney mitochondria Preparation of mitochondrial protein extracts was carried out from whole kidney lysates as described previously (Meimaridou et al., 2006). Purity of the mitochondrial preparation was confirmed by showing enrichment of succinate dehydrogenase (SDH) activity in the preparation. 2.6. Preparation of renal tissue lysates Tissue lysates were prepared as described (Kiroycheva et al., 2000). In brief, the frozen kidney was homogenized in ice-cold homogenizing buffer containing 50 mM Tris pH 7.5, 20 mM NaCl, 1 mM EDTA, 0.5% NP-40, phosphatase inhibitors (100 mM NaF, 1 mM Na3VO4) and protease inhibitors cocktails. The homogenates were centrifuged at 10,000g for 10 min. Resultant supernatants were used to determine levels of GRP78, phospho-PERK and CHOP by western blot analysis. 2.7. Isolation of DNA from rat kidney tissue DNA was isolated from snap-frozen rat kidney tissue using TRI reagent, according to the manufacturer's instructions. 2.8. Estimation of protein The protein content of the cytosolic, nuclear and mitochondrial preparations and tissue lysates were determined, as described previously (Lowry et al., 1951).

2.2. Animals 2.9. Assessment of markers of apoptosis Male Wistar albino rats (Rattus norvegicus) weighing 200–220 g were used for the study. All animals were maintained under conditions of controlled light (12 h light–dark cycles) and temperature (25oC 73oC). They had access to standard rodent chow and water ad libitum. All experiments performed on the animals were approved by the institutional animal ethics committee and Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA), Government of India. Six animals were used in each experimental group. 2.3. Dosing Rats were administered indomethacin by oral gavage at a dose of 20 mg/kg in 5% sodium bicarbonate. This dose has been

2.9.1. DNA laddering DNA that was isolated was electrophoresed at 80 V in a 1% agarose gel that contained 0.05% ethidium bromide, using Trisacetate-EDTA (TAE) buffer (containing 40 mM Tris-acetate and 1 mM EDTA, pH 8.3) to look for DNA laddering, which was taken as evidence of DNA fragmentation. The separated bands were visualized and documented, using an AlphaEase FC gel documentation system (Alpha Innotech Corporation, CA.). 2.9.2. Measurements of caspase activities Activities of caspases-3 and -9 were determined as described previously (Yang et al., 2003), with some modifications. In brief, the kidney tissue samples were homogenized in lysis buffer

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Time after indomethacin administration 1h

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Mar +ve Con Indo Zn+I Zn

Mar Con Indo Zn+I Zn +ve

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100 kDa

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100 kDa GRP78 (78 kDa)

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GRP78 (78 kDa)

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GRP78 (78 kDa)

75 kDa

β-actin (42 kDa)

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β-actin (42 kDa)

GRP78 protein levels normalized to β-actin

1 0.8 0.6 0.4 0.2 0 Control Indo

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p-PERK protein levels normalized with β-Actin

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β-actin (42 kDa)

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1.2 1 0.8 0.6 0.4 0.2 0 Control Indo Zn+I 1h

Zn

Control Indo Zn+I 12 h

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Time after indomethacin administration

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75 kDa

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XBP -1 protein levels (Nuc/cytosol ratio)

Zn

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0.3 0.25

#

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Time after indomethacin administration Fig. 1. Effect of indomethacin, with or without zinc pre-treatment, on ER stress response proteins in the rat kidney at 1, 12 and 24 h after indomethacin administration. Representative images of western blots and densitometric quantification of blots for (A) GRP78, (B) phospho-PERK and (C) XBP-1(s) are shown. Each bar represents mean7S.D. (n¼6). * and # indicate Po0.05, when compared with corresponding control and indomethacin-treated groups respectively. The Mann–Whitney test was used for pairwise comparisons, following analysis by the Kruskal–Wallis test. “Mar” and “þve” lanes indicate protein molecular weight marker and HeLa whole cell lysate used as a positive control for GRP78, respectively.

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12 h

1h Con

CHOP (30 kDa)

Indo Zn+I

Zn

Mar

Con

Indo Zn+I

24 h Zn

Con

Mar

Indo

Zn+I

Zn

+ve

Mar

35 kDa

35 kDa

35 kDa

25 kDa

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β-actin (42 kDa)

Fig. 2. Effect of indomethacin, with or without zinc pretreatment, on CHOP levels in the rat kidney at 1, 12 and 24 h after indomethacin administration. Representative images of western blots for CHOP are shown. “Mar” and “þ ve” lanes indicate protein molecular weight marker and RAW264.7 þ LPS/PMA cell lysate used as a positive control for CHOP, respectively.

(containing 10 mM HEPES/KOH (pH7.2), 2 mM EDTA, 0.1% CHAPS, 5 mM dithiothreitol, 1 mM PMSF, 10 μg/ml aprotinin, and 20 μg /ml leupeptin). The lysate was centrifuged at 25,000g for 10 min at 41C. Fifty ml of the supernatant (containing  200 mg protein) was added to 50 ml of 2X reaction buffer (40 mM HEPES, pH 7.4, 0.2% CHAPS, 10 mM dithiothreitol and 4 mM EDTA). Fluorogenic substrate (Ac-DEVD-AFC for caspase-3 or Ac-LEHD-AFC for caspase-9) was added (50 mM final concentration) and the reaction mixture was incubated for 3 h at 37 1C. Fluorescence intensity (excitation at 400 nm and emission at 505 nm) was measured at 0, 1, 2 and 3 h time points, using a Varian Cary Eclipse fluorescence spectrophotometer. A standard curve was constructed using free AFC and results were expressed as amount of AFC released from the substrate per h per mg of protein.

attenuated at 24 h. Protein levels of CHOP, a marker of the dark pathway of ER stress, were not increased in response to indomethacin administration at any of the time points studied (Fig. 2). 3.2. Effect of zinc on indomethacin-induced ER stress responses in the kidney Indomethacin-induced increases in GRP78 protein levels and nuclear translocation of sXBP-1 at 12 h were significantly attenuated by zinc pre-treatment (Fig. 1A and C). Zinc also tended to reduce drug-induced elevations in phospho-PERK at 12 and 24 h, but the effects were not statistically significant (Fig. 1B). CHOP was unaffected by pre-treatment with zinc (Fig. 2). Treatment with zinc alone did not produce any changes in the parameters studied (Figs. 1 and 2).

2.9.3. Western blot analyses of cytochrome c (cyt c) and poly ADP ribose polymerase (PARP) Cytochrome c (cyt c) levels in mitochondrial and cytosolic extracts, and PARP levels in the nuclear extract were determined by western blot analyses, as described previously (Luo et al., 2008; Meimaridou et al., 2006). The proteins of interests were detected by an enhanced chemiluminescence system, using a Super Signal West Dura Extended Duration Substrate kit (Thermo Scientific, USA), according to the manufacturer's instructions.

Release of cytochrome c from the mitochondria into the cytosol, PARP cleavage, fragmentation of DNA and activation of caspases 3 and 9, all markers of apoptosis, did not occur to any significant extent in the rat kidney in response to indomethacin (Fig. 3A–E). This indicates that apoptosis was not induced in response to indomethacin-induced activation of ER stress responses in the kidney.

2.10. Western blot analyses of markers of ER stress

3.4. Effect of zinc on parameters of apoptosis in the kidney

Protein levels of GRP78, phospho-PERK, CHOP (in renal tissue lysates) and X-box binding protein-1 (spliced) (sXBP-1) (in cytosolic and nuclear fractions) were determined by western blot analysis, as described previously (Chiang et al., 2011; Kato et al., 2013). The proteins of interests were detected as described in Section 2.9.3.

Pre-treatment with zinc or treatment with zinc alone did not affect any of the markers of apoptosis studied (Fig. 3A–E).

2.11. Statistical analysis The Statistical Package for the Social Scientist (SPSS), version 16, was used for statistical analysis. Data were analyzed by the Kruskal–Wallis test. This was followed by pairwise comparisons within each time period studied, using the Mann–Whitney test. A P-value o 0.05 was taken to indicate statistical significance in all cases.

3. Results 3.1. Effect of indomethacin on ER stress response in the kidney Levels of GRP78 were found to be elevated at 12 h and phosphorylation of PERK was found to occur at 12 h and 24 h after indomethacin administration (Fig. 1A and B). Levels of sXBP-1 protein in the nucleus were elevated at 12 h after the drug (Fig. 1C); the protein was not detectable at 1 h and 24 h. These results indicate that indomethacin activated ER stress responses in the rat kidney. The effects were maximal at 12 h and tended to be

3.3. Effect of indomethacin on parameters of apoptosis in the kidney

4. Discussion Our earlier work has shown that indomethacin induced oxidative stress and renal damage in the rat kidney (Basivireddy et al., 2004; Varghese et al., 2009; Nagappan et al, 2014). Oxidative stress is known to activate ER stress by inhibiting protein disulfide bond formation and protein folding (Inagi, 2009). We therefore investigated whether indomethacin-induced oxidative stress in the rat kidney led to ER stress responses. We found that it did so. This effect was most prominent at 12 h following indomethacin administration, at which time point, GRP78, phosphorylated PERK and nuclear sXBP-1 levels were found to be significantly increased. By 24 h, the ER stress response was attenuated, with only phospho-PERK levels remaining significantly elevated. GRP94, oxygen-regulated protein of 150 kDa (ORP150) and calreticulin are other markers of ER stress; they are resident ER chaperones that are induced along with GRP78 during ER stress. It would be expected that the levels of these proteins would also increase, along with GRP78, in this setting. This would provide additional evidence for ER stress induction. However, we were not able to study indomethacin-induced changes in these additional markers. Induction of ER stress, with consequent activation of CHOP, has been shown to mediate indomethacin-induced apoptosis in gastric mucosal cells and renal carcinoma cells in vitro (Ou et al., 2007;

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Western blot analysis of cytochrome c in cytosolic and mitochondrial fractions Time after indomethacin administration 1h Con Indo

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cytochrome c release (cyto/mito ratio)

0.6 0.5 0.4 0.3 0.2 0.1 0 Control Indo

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(Caspase-3 activity) nM of AFC release/h/mg protein

Cleaved PARP / uncleaved PARP ratio (normalized to β-actin)

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5 4 3 2 1 0

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(Caspase-9 activity) nM of AFC release/h/mg protein

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7 6 5 4 3 2 1 0 1h

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Time after indomethacin administration

Fig. 3. Effect of indomethacin, with or without zinc pretreatment, on markers of apoptosis in the rat kidney at 1, 12 and 24 h after indomethacin administration. Representative images of western blots and densitometric quantification of blots for (A) cytochrome c levels in cytosolic and mitochondrial fractions and (B) intact and cleaved PARP in nuclear fractions are shown. (C) Agarose gel electrophoresis of DNA in nuclear fractions and (D and E) levels of caspase-3 and -9 activities, analyzed by fluorescence spectrophotometry, are also shown. Each bar represents mean 7 S.D. (n¼ 6 in all cases). “Mar” indicates a protein molecular weight marker.

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Tsutsumi et al., 2004). However, in our study, we did not find evidence to suggest that this occurred in the kidney, following administration of indomethacin. A direct “topical” effect of indomethacin has been suggested to contribute to drug-induced ER stress and apoptosis in gastric mucosal and small intestinal epithelial cells in vitro and in vivo (Maity et al., 2009; Omatsu et al., 2009). Indomethacin is protein-bound in blood (Hvidberg et al., 1972). Hence, the kidney is not exposed to the direct topical effects of indomethacin. It is possible that this may partly account for the fact that, though the drug induced ER stress in the kidney, it did not induce apoptosis. These observations are similar to those reported by other authors, who have also shown that activation of ER stress was not accompanied by apoptosis. These studies were done in HepG2 cells and renal proximal tubular cells in vitro and showed that autophagy occurred in response to ER stress (Matsumoto et al, 2013; Kawakami et al, 2009). Thus, it appears that apoptosis is not an inevitable consequence of an ER stress response. It is possible that indomethacin-induced renal damage that we have reported earlier (Nagappan et al, 2014) may be mediated via autophagic events. However, we were not able to measure markers of autophagy in the kidney in the present study to confirm this. This is a limitation of the study. Activation of PERK, an ER stress transducer protein, is known to cause activation of both nuclear factor erythroid-derived-2-related factor-2 (Nrf2) and activating transcription factor 4 (ATF4) (Cullinan and Diehl, 2006; Kim et al., 2011). Nrf2 is a transcription factor that induces the expression of a number of anti-oxidant enzymes and molecules, including heme oxygenase-1 (HO-1). HO1 is thought to play an important part in the adaptive response triggered by ER stress (Kim et al., 2011; Lee et al., 2007; Liu et al., 2005) and is known to inhibit apoptosis via reduction of CHOP expression (Kim et al., 2011; Lee et al., 2007; Liu et al., 2005). In addition, HO-1 has been shown to inhibit caspase activation (Inguaggiato et al., 2001; Suzuki et al., 1998; Xu and El-Deiry, 2000). Our earlier work has shown that indomethacin activated Nrf2 and induced HO-1 in the kidney (Nagappan et al., 2014). Therefore, activation of HO-1 may play a role in suppressing activation of CHOP and preventing apoptosis in this setting. However, additional studies, using genetic and pharmacological models of inhibition, will be required to confirm this. Pre-treatment of the animals with zinc prevented the increase in GRP-78 and nuclear sXBP1 levels, induced by indomethacin at 12 h after the drug. Zinc is a potent anti-oxidant, and has been shown to ameliorate indomethacin-induced renal effects (Varghese et al., 2009). The results of the present study suggest that ER stress in this setting was triggered by indomethacininduced oxidative stress, which was ameliorated by pre-treatment with zinc, an effect that is probably due to its anti-oxidant property. The anti-oxidant effects of zinc are known to be mediated by different mechanisms (Powell, 2000). Long-term effects are known to be mediated by induction of metallothionein (MT), a low molecular weight protein that can act as a sink for reactive oxidant species (Powell, 2000). However, our earlier work has shown that zinc did not induce MT in the kidney within 24 h (Varghese et al., 2009). The short-term effects of zinc may be attributed to its ability to stabilize sulphydryl bonds in protein, thus aiding protein folding (Powell, 2000). Zinc also displaces redox active metals, such as iron and copper, from their intracellular binding sites (Powell, 2000). This inhibits formation of free radicals induced by these redox-active metals. It is possible that these mechanisms are involved in the protective effects of zinc in the setting of the present study. It has been reported that zinc, at high concentrations, can be toxic to cells and can, by itself, induce apoptosis (Bozym et al., 2010), hepatotoxicity and nephrotoxicity in rats (Faddah et al.,

2012; Yeh et al., 2011). However, we have found that zinc, when administered alone, did not affect any of the parameters studied, showing that zinc was not toxic at the doses used in this study. We have studied markers of indomethacin-induced ER stress in the kidney using whole tissue homogenates. It would have been useful to carry out similar studies in the different regions of the kidney, such as the cortex and medulla. Our earlier work has shown that indomethacin produced ultrastructural changes in renal proximal tubules; these changes were associated with evidence of oxidative stress in whole tissue homogenates (Basivireddy et al, 2004) and in renal brush border membranes (Basivireddy et al, 2005). We have also shown that expression of KIM-1 (kidney injury molecule–1), a marker for renal tubular damage, was increased in the kidney in response to indomethacin (Nagappan et al, 2014). These studies suggest that the renal tubules are likely sites affected by indomethacin, where it is possible that ER stress occurred secondary to oxidative stress. It is a limitation of the present study that we were not able to carry out confirmatory studies on this aspect. Such investigations may be warranted in the future, to pursue in greater depth the findings of these studies.

5. Conclusion In conclusion, we have shown that indomethacin caused activation of the ER stress response in the kidney. This effect, which is likely to be induced by indomethacin-induced oxidative stress, may contribute to renal damage seen in response to the drug. Zinc pre-treatment attenuated these changes induced by indomethacin.

Conflict of interest The authors of this paper declare they have no conflicts of interest.

Acknowledgments This study was funded by the Department of Science and Technology (DST), New Delhi, India (Grant no. 100/IFD/6451/ 2006-2007) and Fluid Research Grants (IRB Min no. 7995) from the Christian Medical College (CMC), Vellore, Tamil Nadu, India. Mr. Arumugam Suriyam Nagappan has been funded on research fellowships from the Department of Science and Technology (DST), New Delhi, India (Grant no. 100/IFD/6451/2006-2007) (period of 2007–2010) and the Indian Council of Medical Research (ICMR), New Delhi, India (File no. 45/25/2011/Bio/BMS for the period of 2012–2014) during the conduct of this study. References Basivireddy, J., Jacob, M., Pulimood, A.B., Balasubramanian, K.A., 2004. Indomethacin-induced renal damage: role of oxygen free radicals. Biochem. Pharmacol. 67, 587–599. http://dx.doi.org/10.1016/j.bcp.2003.09.023. Basivireddy, J., Jacob, M., Balasubramanian, K.A., 2005. Indomethacin induces free radical-mediated changes in renal brush border membranes. Archives of Toxicology 79, 441–450. Basivireddy, J., Vasudevan, A., Jacob, M., Balasubramanian, K.A., 2002. Indomethacin-induced mitochondrial dysfunction and oxidative stress in villus enterocytes. Biochem. Pharmacol. 64, 339–349. Bozym, R.A., Chimienti, F., Giblin, L.J., Gross, G.W., Korichneva, I., Li, Y., Libert, S., Maret, W., Parviz, M., Frederickson, C.J., Thompson, R.B., 2010. Free zinc ions outside a narrow concentration range are toxic to a variety of cells in vitro. Exp. Biol. Med. 235, 741–750. http://dx.doi.org/10.1258/ebm.2010.009258. Carlisle, R.E., Brimble, E., Werner, K.E., Cruz, G.L., Ask, K., Ingram, A.J., Dickhout, J.G., 2014. 4-Phenylbutyrate inhibits tunicamycin-induced acute kidney injury via

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