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provided by Elsevier - Publisher Connector http://www.kidney-international.org original article & 2011 International Society of Nephrology

Amelioration of cisplatin nephrotoxicity by genetic or pharmacologic blockade of prostaglandin synthesis Zhanjun Jia1,2, Ningning Wang1,2, Toshinori Aoyagi1, Haiping Wang1, Haiying Liu1 and Tianxin Yang1

1Department of Internal Medicine, University of Utah, Salt Lake City, Utah, USA

Nephrotoxicity is a common complication of cisplatin Cis-diamminedichloroplatinum (cisplatin), an inorganic chemotherapy that limits its clinical use. Here, we determined molecule, is one of the simplest and most effective whether arachidonic acid metabolism has a role in the chemotherapeutic agents and is widely used for treatment pathogenesis of cisplatin nephrotoxicity in mice. Three days of various types of solid tumors, including testicular, ovarian, following cisplatin injection, wild-type mice displayed renal head, neck, and uterine cervical carcinoma and nonsmall cell functional and structural abnormalities consistent with lung carcinoma.1–4 Unfortunately, its clinical use is limited by nephrotoxicity accompanied by elevated circulating and severe side effects in normal tissues. Nephrotoxicity is a renal levels of TNF-a and renal levels of IL-1b,subunitsof frequent adverse effect with about 25–35% patients displaying NADPH oxidase, thiobarbituric acid-reactive substances, and decline of renal function after a single dose of cisplatin.5 PGE2. These indices of kidney injury, inflammation, oxidative Despite intensive investigation, the mechanisms underlying stress, and arachidonate metabolism were all diminished in cisplatin-induced nephrotoxicity are not fully understood. microsomal prostaglandin E synthase-1 (mPGES-1) null mice; After administration, cisplatin is taken up in renal a phenotype recapitulated by treatment of wild-type mice tubular cells at high concentrations, leading to tubular with the COX-2 inhibitor celecoxib. Following cisplatin injury and cell death. The signaling mechanisms res- administration, there was paralleled induction of COX-2 and ponsible for cisplatin-induced cytotoxicity appear to be mPGES-1 in renal parenchymal cells. Interestingly, mPGES-1 multifactorial, involving inflammation, oxidative stress, and null mice were not protected from acute kidney injury caused caspase.4 by ischemia–reperfusion or endotoxin. Hence, our results Prostaglandin E2 (PGE2), a major product of arachidonic suggest the activation of COX-2/mPGES-1 pathway in renal acid (AA) metabolism, has an established role in mediating 6 parenchymal cells may selectively mediate cisplatin-induced pain and inflammatory responses. The biosynthesis of PGE2 renal injury. This may offer a novel therapeutic target for requires three sequential steps of the cyclooxygenase (COX) management of the adverse effect of cisplatin chemotherapy. pathway: the release of AA from membrane glyceropho- Kidney International (2011) 79, 77–88; doi:10.1038/ki.2010.331; spholipids by phospholipase A2, conversion of AA to the published online 15 September 2010 unstable intermediate PGH2 by COX-1 or COX-2, and finally KEYWORDS: cisplatin; cyclooxygenase-2; cytokines; microsomal isomerization of PGH2 to PGE2 by prostaglandin E synthase prostaglandin E synthase-1; nephrotoxicity (PGES).7,8 To date, at least three major forms of PGES have been cloned and characterized: membrane-associated PGES (mPGES)-1, mPGES-2, and cytosolic PGES.8 mPGES-1 is a 16-kDa membrane-bound protein encoded by a 2.0-kb transcript. Similar to COX-2, mPGES-1 expression in inflammatory cells is highly inducible in macrophages in vitro9,10 and in the spleen and lung in vivo11–13 in response to proinflammatory stimuli. Moreover, mPGES-1-deficient mice exhibit blunted pain and inflammatory responses.9,14 mPGES-1 has also be implicated to have a key role in a 15 Correspondence: Tianxin Yang, Department of Internal Medicine, University number of chronic inflammatory diseases. Owing to the of Utah and VA Medical Center, 30 N 1900 E, Room 4R312, Salt Lake City, cardiovascular consequences of COX-2 inhibitors, mPGES-1 Utah 84132, USA. E-mail: [email protected] has been considered as a promising target for the next 2These authors contributed equally to this work. generation of analgesic drugs. Here, we describe a novel role Received 6 September 2009; revised 11 June 2010; accepted 29 June of mPGES-1 and COX-2 in the pathogenesis of cisplatin 2010; published online 15 September 2010 nephrotoxicity.

Kidney International (2011) 79, 77–88 77 original article Z Jia et al.: mPGES-1 and cisplatin nephrotoxicity

RESULTS were subjected to cisplatin treatment and the severity of renal mPGES-1 deletion attenuates cisplatin-induced renal dysfunction was reflected by blood urine nitrogen (BUN) and dysfunction and tubular damage plasma creatinine (Cr). At baseline, the values of BUN and To investigate the role of mPGES-1 in cisplatin nephro- plasma Cr were similar between the genotypes. Cisplatin toxicity, mPGES-1 wild-type (WT) and knockout (KO) mice treatment remarkably elevated BUN and plasma Cr in WT mice. These increases were attenuated by 80% for plasma Control Cr and 38% for BUN in the KO mice (Figure 1). Grossly, the Cisplatin degree of kidney swelling secondary to cisplatin-induced P <0.01 P <0.01 renal injury appeared similar between the genotypes 200 (Figure 2a) as confirmed by the similar increase in kidney wet weight (WT: 0.283±0.027 vs 0.162±0.013 g, Po0.05; 150 P<0.01 KO: 0.279±0.023 vs 0.171±0.015 g, Po0.05). However, 100 following cisplatin treatment, the WT kidney became pale and ischemic contrasting to the normal appearance of the KO

BUN (mg/dl) 50 kidney (Figure 2a). Microscopically, at baseline, there was no 0 obvious difference in the renal morphology between the WT KO genotypes. While, following cisplatin treatment, WT mice displayed severe renal pathological changes characterized 2.5 P <0.01 P <0.01 by distortion of the overall renal morphology, dilation 2 of renal tubules, and appearance of protein cast, most of 1.5 which were significantly attenuated in the KO mice (Figure 2b). The difference in the renal injury between the geno- 1 P<0.05 types was further reflected by the semiquantitative histolo- 0.5 gical damage score (Figure 2c). Acute renal failure is often Plasma Cr (mg/dl) 0 associated to fluid retention, leading to a fall in hematocrit. WT KO We found that hematocrit significantly decreased in WT Figure 1 | Assessment of renal function after cisplatin treatment. mice after cisplatin treatment and this decrease was less in Blood urine nitrogen (BUN) (a) and plasma creatinine (Cr) (b)in the KO mice (Figure 3a). Interestingly, the body weight membrane-associated prostaglandin E synthase-1 (mPGES-1) þ / þ loss, which reflects the general or gastrointestinal toxicity and / mice at 72 h following treatment with vehicle or cisplatin. Wild-type (WT)/vehicle: N ¼ 6; knockout (KO)/vehicle: N ¼ 6; WT/ of cisplatin, was not different between the genotypes cisplatin: N ¼ 16; KO/cisplatin: N ¼ 16. Data are mean±s.e. (Figure 3b).

WT KO

WT control WT cisplatin

Control

KO control KO cisplatin

P <0.01 4 3.5 3 Cisplatin 2.5 2 1.5 damage 1 0.5 Score of tubular 0 WT KO Figure 2 | Morphological analysis of cisplatin-induced renal injury in membrane-associated prostaglandin E synthase-1 (mPGES-1) þ / þ and / mice. (a) Gross kidney appearance. (b) Hematoxylin and eosin staining (magnification: 200 shown) of renal cortex. (c) Renal injury score in cisplatin-treated mPGES-1 þ / þ and / mice. Wild-type (WT)/vehicle: N ¼ 6; knockout (KO)/vehicle: N ¼ 6; WT/cisplatin: N ¼ 9; KO/cisplatin: N ¼ 10. N ¼ 9 per group. Data are mean±s.e.

78 Kidney International (2011) 79, 77–88 Z Jia et al.: mPGES-1 and cisplatin nephrotoxicity original article

mPGES-1 deletion attenuates cisplatin-induced process.16 Therefore, our emphasis was placed on analysis of renal expression of cytokines the activation of TNF-a in response to cisplatin treatment. Activation of proinflammatory cytokine release is well known Immunoblotting demonstrated a marked increase in the total 16 to contribute to cisplatin nephrotoxicity. In particular, protein abundance of renal TNF-a in cisplatin-treated WT tumor necrosis factor-a (TNF-a) is shown to have a central mice and this increase was significantly attenuated in the role in the activation of the cytokine response in this disease KO mice (Figure 4a). This result was confirmed at mRNA level using quantitative reverse transcriptase (qRT)-PCR Control P <0.01 (Figure 4b). Moreover, circulating TNF-a levels were Cisplatin measured by using enzyme-linked immunosorbent assay. 60 P<0.01 P<0.05 Consistent with the gene expression data, the increases in 55 plasma TNF-a concentrations were less in the KO mice than 50 in WT controls (Figure 4c). Renal interleukin (IL)-1b mRNA 45 Hct (%) expression exhibited similar patterns of changes as TNF-a 40 (Figure 4d). 35 WT KO mPGES-1 deletion attenuates cisplatin-induced ROS

24 h 48 h 72 h production 0 In addition to inflammation, oxidative stress is another

–1 important factor involved in the pathogenesis of cisplatin nephrotoxicity. Therefore, we evaluated the effect of mPGES- –2 1 deletion on cisplatin-induced oxidative stress. The level of –3 kidney thiobarbituric acid-reactive substances (TBARS), an index of reactive oxygen species (ROS) generation, signifi- –4 WT KO cantly increased in WT mice after cisplatin treatment

Body weight decrease –5

compared with day 0 (g) and this increase was completely abolished in the KO mice –6 (Figure 5a). The increased ROS generation in cisplatin- treated WT mice was associated with elevated renal expres- Figure 3 | Effects of cisplatin-treatment on hematocrit (Hct) and phox phox body weight. Changes in hematocrit (Hct) (a) and body weight (b) sion of p47 (Figure 5b) and gp91 (Figure 5c), the two in membrane-associated prostaglandin E synthase-1 (mPGES-1) major subunits of NADPH oxidase, but not NOX-1 (Figure þ / þ and / mice following treatment with vehicle or cisplatin. 5d) or NOX-3 (Figure 5e). The stimulation of the expression Hct was determined at 72 h following cisplatin treatment and of p47phox and gp91phox was concomitantly suppressed in the body weight was monitored daily. Wild-type (WT)/vehicle: N ¼ 6; knockout (KO)/vehicle: N ¼ 6; WT/cisplatin: N ¼ 9; KO/cisplatin: KO mice (Figure 5b and c). Cisplatin-induced oxidative stress N ¼ 10. Data are mean±s.e. in WT mice was also associated with downregulation of

Control Cisplatin Control Cisplatin 30 P <0.01 P < 0.01 TNF-α WT 25 α-Tubulin α 20

1.05±0.2 4.5±0.19* 15 10 P<0.01 TNF-α Renal TNF- mRNA/GAPDH 5 KO α-Tubulin 0 WT KO 1.01±0.15 1.15±0.2 P<0.01 P<0.01 8 P<0.01 P<0.01 600 7 500 6 400 5 300 P<0.01 4 3

200 Renal IL- β 2 mRNA/GAPDH 100 1

Plasma TNF- α (pg/ml) 0 0 WT KO WT KO Figure 4 | The levels of proinflammatory cytokines in membrane-associated prostaglandin E synthase-1 (mPGES-1) þ / þ and / mice following treatment with vehicle or cisplatin. (a) Immunoblotting of tumor necrosis factor-a (TNF-a) and a-tubulin in the kidney. The densitometric value of TNF-a protein was normalized by a-tubulin. The mean values are shown below the immunoblot. *Po0.05 vs control. (b) Quantitative reverse transcriptase (qRT)-PCR analysis of renal TNF-a.(c) Enzyme-linked immunosorbent assay analysis of circulating TNF-a.(d), qRT-PCR analysis of renal interleukin (IL)-1b. Wild-type (WT)/vehicle: N ¼ 6; knockout (KO)/vehicle: N ¼ 6; WT/cisplatin: N ¼ 9; KO/cisplatin: N ¼ 10. Data are mean±s.e.

Kidney International (2011) 79, 77–88 79 original article Z Jia et al.: mPGES-1 and cisplatin nephrotoxicity

Control abCispatin 25 P<0.01 P<0.01 200 P <0.01 P<0.01 20 150 15 /GAPDH 100 10 P<0.01 phox TBARS 50 5 p47 0 0 (nmol per g protein) WT KO WT KO cd P<0.01 P<0.01 1.8 6 1.6 5 1.4 1.2 4 1 /GAPDH 3 0.8 0.6

phox 2 0.4 1 NOX1/GAPDH 0.2

gp91 0 0 WT KO WT KO ef 1.4 1.4 1.2 1.2 1 1 0.8 0.8 0.6 0.6 0.4 0.4 0.2 0.2 NOX3/GAPDH 0 SOD1/GAPDH 0 WT KO WT KO

ghP <0.01 P<0.05 P<0.05 2 1.2 P<0.05 1 1.5 P <0.05 0.8 0.6 1 0.4 0.5

0.2 SOD3/GAPDH SOD2/GAPDH 0 0 WT KO WT KO Figure 5 | Evaluation of oxidative stress after cisplatin treatment. Measurements of kidney thiobarbituric acid-reactive substances (TBARS) (a) and quantitative reverse transcriptase (qRT)-PCR analysis of renal expression of p47phox (b) and gp91phox (c), NOX1 (d), NOX3 (e), superoxide dismutase (SOD1) (f), SOD2 (g), and SOD3 (h) in membrane-associated prostaglandin E synthase-1 (mPGES-1) þ / þ and / mice following treatment with vehicle or cisplatin. Wild-type (WT)/vehicle: N ¼ 6; knockout (KO)/vehicle: N ¼ 6; WT/cisplatin: N ¼ 9; KO/ cisplatin: N ¼ 10. Data are mean±s.e. superoxide dismutase 2 (SOD2) and SOD3 with unchanged treatment induced a marked, widespread upregulation of SOD1 (Figure 5f–h), suggesting reduced antioxidant capacity. mPGES-1 immunoreactivity in the kidney (Figure 7a). This The downregulation of SOD2 and SOD3 was completely enhanced labeling was completely competed out with the prevented in the KO mice (Figure 5f–h). Interestingly, renal immune peptide, thus confirming specificity of the antibody. SOD3 expression was even upregulated in the KO mice after The stimulation of renal mPGES-1 expression was further cisplatin treatment (Figure 5h). confirmed by immunoblotting (Figure 7b) and qRT-PCR (Figure 7c). In contrast, renal expression of mPGES-2 Assessment of apoptotic pathway (Figure 7d) and cytosolic PGES (Figure 7e) was unaffected Apoptosis can be induced within a few hours of cisplatin by neither cisplatin treatment nor mPGES-1 deletion. We treatment in cultured renal proximal tubular cells and may measured kidney PGE2 content using enzyme-linked im- represent an early event in the pathogenesis of cisplatin munosorbent assay. At 72 h after cisplatin treatment, kidney 4 nephotoxicity. It has also been shown that the Bak/Bax PGE2 content significantly increased in WT mice. This activation is a key element of the apoptotic pathway induced increase was markedly blunted in the KO mice (Figure 7f). by cisplatin.17 We compared renal mRNA expression of Bak/bax as well as Bcl-2 between the genotypes after cisplatin Role of COX-2 in cisplatin nephrotoxicity treatment. To our surprise, neither the induction of Bax/Bak To investigate the role of COX-2 in cisplatin nephrotoxicity, nor bcl-2 expression was affected in the KO mice (Figure 6). the COX-2 inhibitor celecoxib was used. celecoxib treatment These results revealed no evidence for changes in the was started 72 h before cisplatin treatment. Cisplatin treat- apoptotic pathway in mPGES-1 KO mice. ment induced renal dysfunction (Figure 8a and b), renal histological abnormalities (Figure 8c and d), and indices Activation of renal mPGES-1 by cisplatin treatment of proinflammatory cytokines (Figure 9a–c) and oxidative At baseline, immunostaining detected a low level of stress (Figure 9d–f), all of which were significantly attenuated mPGES-1 immunoreactivity in the collecting duct. Cisplatin by celecoxib. Immunostaining detected a marked, widespread

80 Kidney International (2011) 79, 77–88 Z Jia et al.: mPGES-1 and cisplatin nephrotoxicity original article

Control Cisplatin WT and KO mice (Figure 11). We found no difference in BUN or plasma Cr between the genotypes after I/R injury. a 6 P<0.05 Unexpectedly, renal mRNA expression of mPGES-1 along 5 P<0.05 with mPGES-2 and cytosolic PGES was significantly 4 decreased after I/R injury. Renal COX-2 expression tended 3 to be higher in the I/R vs sham group but this did not reach a 2 Bak/GAPDH statistical significance. Subsequently, we examined endotox- 1 in-induced renal dysfunction in mPGES-1 KO mice. 0 Similarly, endotoxin-induced increases in BUN and plasma WT KO Cr were not different between the genotypes nor was plasma

b 4 P<0.01 P<0.01 TNF-a level (Figure 12). 3.5 3 2.5 DISCUSSION 2 PGs have a pivotal role in mediating the inflammatory 1.5 response as highlighted by analgesic properties of nonster- Bax/GAPDH 1 0.5 oidal anti-inflammatory drugs. PGE2 is one of the major 0 proinflammatory PG derived from the coordinated action of WT KO COX-2 and mPGES-1. Similar to COX-2, mPGES-1 is c 1.6 expressed at low level under baseline and can be induced 1.4 by proinflammatory stimuli and suppressed by corticoster- 1.2 18 1 oids. Studies, mainly from disruption of mPGES-1 gene in 0.8 mice, indicate the key roles of mPGES-1-derived PGE2 in a 0.6 number of pathological conditions, including chronic Bcl-2/GAPDH 0.4 inflammation, fever, pain, anorexia, atherosclerosis, stroke, 0.2 0 tumorigenesis, rheumatoid arthritis, osteoarthritis, and WT KO myositis.15 So far, renal function of mPGES-1 has been Figure 6 | Assessment of apoptotic pathway after cisplatin examined under physiological conditions, including low and treatment. Quantitative reverse transcriptase (qRT)-PCR analysis high salt intake,19–21 water loading,22 and angiotensin II of renal Bak (a), Bax (b), and Bcl-2 (c) in membrane-associated infusion,23 and angiotensin-converting enzyme inhibition;24 prostaglandin E synthase-1 (mPGES-1) þ / þ and / mice following treatment with vehicle or cisplatin. Wild-type (WT)/ the majority of these studies support a physiological role of vehicle: N ¼ 6; knockout (KO)/vehicle: N ¼ 6; WT/cisplatin: N ¼ 9; mPGES-1 in regulation of sodium and water balance, and KO/cisplatin: N ¼ 10. Data are mean±s.e. blood pressure.25 However, little is known concerning the pathological role of mPGES-1 in renal disease. To our induction of COX-2 immunoreactivity in the kidney knowledge, this study is the first to demonstrate a role of (Figure 10a), a pattern analogous to that of mPGES-1. mPGES-1 in renal pathology in general and in the model of Consistent with this finding, qRT-PCR detected a sevenfold cisplatin nephrotoxicity in particular. The pathological role stimulation of renal COX-2 mRNA expression (Figure 10b). of mPGES-1 in cisplatin nephrotoxicity is demonstrated by Kidney PGE2 content was significantly increased by cisplatin comparing renal functional and structural abnormalities, and and was almost completely normalized by celecoxib cytokine expression between mPGES-1 WT and KO mice at (Figure 10c). To address the specificity of celecoxib, we 72 h following cisplatin treatment. A single intraperitoneal tested its effect on COX-1 activity in mice. A separate group injection of WT mice with cisplatin (20 mg/kg) led to severe treated with indomethacin was used a positive control. Ex renal failure (BUN and plasma Cr), renal tubular damage, vivo production of thromboxane B2 (TXB2) from the blood accompanied by marked induction of cytokine expression. was determined to reflect COX-1 activity from the platelets. In contrast, the KO mice were largely resistant to cisplatin- We found that TXB2 production was unaffected by celecoxib induced changes in renal function and structure, and treatment but was remarkably suppressed by indomethacin cytokine expression. treatment (vehicle: 13.76±1.38 ng/ml; celecoxib: 14.21± Inflammation is well recognized to have an important role 1.75 ng/ml, P40.05 vs vehicle; indomethacin: 1.25± in cisplatin nephrotoxicity. Renal, circulatory, and urinary 0.71 ng/ml, Po0.01 vs vehicle; N ¼ 5–7 per group). These levels of TNF-a, along with renal levels of other cytokines and results suggested that the celecoxib treatment at the current chemokines, including IL-1b, regulated upon activation dose and duration did not affect COX-1 activity. normal T cell expressed and secreted (RANTES), macro- phage-inflammatory protein-2 (MIP2), monocyte chemotac- Role of mPGES-1 in other models of acute kidney injury (AKI) tic protein-1 (MCP-1), and transforming growth factor-b1, are To understand whether the renoprotective effect of mPGES-1 upregulated by cisplatin administration.16,26 Importantly, deletion was specific to the cisplatin model, we compared inhibition of TNF-a by pharmacological inhibitors or gene renal ischemia–reperfusion (I/R) injury between mPGES-1 knockout ameliorate cisplatin nephrotoxicity; the increases in

Kidney International (2011) 79, 77–88 81 original article Z Jia et al.: mPGES-1 and cisplatin nephrotoxicity

Control Cisplatin mPGES-1 peptide 1.2 1 0.8 0.6 0.4 0.2

mPGES-2/GAPDH 0 WT KO

Control Cisplatin 1.6 1.4 1.2 mPGES-1 1 0.8 α-Tubulin 0.6 0.4

cPGES/GAPDH 0.2 1.02±0.12 1.71±0.15* 0 WT KO Control Cisplatin P <0.01 P<0.01 P<0.01 150 5 content

4 2 100 P<0.05 3 50

2 0 (pg per mg protein)

Kidney PGE WT KO 1 mPGES-1/GAPDH 0 WT KO Figure 7 | Stimulation of renal membrane-associated prostaglandin E synthase-1 (mPGES-1) expression by cisplatin. (a) Immunohistochemistry of mPGES-1 in the renal cortex of wild-type (WT) mice treated with vehicle or cisplatin. Specificity of the antibody was validated by the using the competing mPGES-1 peptide. Arrow denotes the collecting duct (CD). Shown are representative photomicrographs (hematoxylin and eosin staining; magnification: 200) from three independent experiments. (b) Immunoblotting analysis of renal mPGES-1 protein expression in WT mice treated with vehicle or cisplatin. Immunoblotting of a-tubulin serves as a loading control. The densitometric value of mPGES-1 protein was normalized by a-tubulin. The mean values are shown below the immunoblot. *Po0.05 vs control. (c) Quantitative reverse transcriptase (qRT)-PCR analysis of mPGES-1 in the kidneys of vehicle or cisplatin-treated mPGES-1 þ / þ and / mice. (d) qRT-PCR analysis of renal mPGES-2. (e) qRT-PCR analysis of renal cytosolic PGES (cPGES). (f) Kidney prostaglandin E2 (PGE2) content in the two genotypes treated with vehicle or cisplatin. PGE2 was determined by enzyme immunoassay and normalized by protein content. (b) N ¼ 4–5 per group; (c–f)WT/vehicle:N ¼ 6; knockout (KO)/ vehicle: N ¼ 6; WT/cisplatin: N ¼ 9; KO/cisplatin: N ¼ 10. Data are mean±s.e. cisplatin-induced increases in IL-b, transforming growth Abundant evidence suggests involvement of oxidative factor-b, MCP-1 RANTES, and MIP1 are also blunted.16 stress in the pathogenesis of cisplatin nephrotoxicity. In this Likewise, TNF-a is responsible for production of other regard, increased renal generation of ROS is demonstrated in cytokines and chemokines, leading to inflammatory injury in whole animal models and also in cultured renal tubular cells the kidney. Together, these findings establish a central role of after cisplatin administration. A large number of antioxidant TNF-a in the pathogenesis of cisplatin renal injury. As agents, including N-acetlcysteine, vitamin E, selenium, previously reported,16,26 we found remarkable increases of dimethylthiourea, lecithinized SOD (L-SOD), cannabidiol, circulating TNF-a and renal TNF-a mRNA and protein melatonin and apocynin, and many others, are consistently expression in WT mice following cisplatin administration. In reported to protect against cisplatin nephrotoxicity.28–36 contrast, these increases were significantly ameliorated in While, the sources of ROS generation during cisplatin mPGES-1 KO mice. It seems reasonable to speculate that nephrotoxicity are still not well defined. We found that suppressing renal TNF-a expression may represent a primary cisplatin treatment significantly increased ROS generation in mechanism for the renoprotective effect of mPGES-1 the kidney as reflected by increased lipid peroxidation, deletion. Similar to TNF-a, renal IL-1b mRNA expression accompanied by increased renal expression of NADPH in cisplatin-treated WT mice increased by sevenfold that was oxidase subunits p47phox and gp91phox and decreased renal almost completely blocked in mPGES-1 KO mice. Indeed, expression of SOD2 and SOD3. These findings suggest that TNF-a and IL-1b are often elevated in parallel, stimulate the activation of NADPH oxidase together with reduced production of each other, and act synergistically to stimulate antioxidant capacity may contribute to cisplatin-induced the production of other cytokines and chemokines.27 oxidative stress. In support of our findings, the NADPH

82 Kidney International (2011) 79, 77–88 Z Jia et al.: mPGES-1 and cisplatin nephrotoxicity original article

P<0.01 P<0.05 P<0.01 P<0.05 250 1.6 1.4 200 1.2 150 1 0.8 100 0.6

BUN (mg/dl) 0.4

50 Plasma Cr (mg/dl) 0.2 0 0 Control Cisplatin Cisplatin Control Cisplatin Cisplatin +celecoxib +celecoxib

Control Cisplatin Cisplatin+celecoxib P <0.05 4.5 4 3.5 3 2.5 2 1.5 1 0.5 Score of tubular damage 0 Cisplatin Cisplatin +celecoxib Figure 8 | Renal functional and structural damage in control mice and in mice treated with cisplatin alone or in combination with celecoxib. Celecoxib was started at 72 h before cisplatin treatment. Blood urine nitrogen (BUN) (a) and plasma creatinine (Cr) (b), and renal histology (c: hematoxylin and eosin staining; d: renal injury score) were evaluated at 72 h after cisplatin. In c, the magnification is 200. Control: N ¼ 6; cisplatin: N ¼ 12; cisplatin þ celecoxib: N ¼ 8 per group. Data are mean±s.e.

200 600 P<0.01 P<0.05 P<0.01 P<0.01 500 150 400 300 100

200 TBARS TNF- α in 50 100 plasma (pg/ml)

0 (nmol per g protein) 0 Control Cisplatin Cisplatin Control Cisplatin Cisplatin +celecoxib +celecoxib

45 P<0.01 P<0.01 6 P<0.01 P<0.05 40 35 5 30 4

25 /GAPDH 20 3

15 phox 2 10 1

TNF- α /GAPDH 5 0 gp91 0 Control Cisplatin Cisplatin Control Cisplatin Cisplatin +celecoxib +celecoxib

P<0.01 P<0.01 P<0.01 P<0.05 16 25 14 12 20 10 8 15 6 /GAPDH 10

4 phox 5 IL-1 β /GAPDH 2 0 p47 0 Control Cisplatin Cisplatin Control Cisplatin Cisplatin +celecoxib +celecoxib Figure 9 | The levels of circulating or renal proinflammatory cytokines and oxidative stress in control mice and in mice treated with cisplatin alone or in combination with celecoxib. (a) Enzyme-linked immunosorbent assay analysis of circulating tumor necrosis factor-a (TNF-a). (b) Quantitative reverse transcriptase (qRT)-PCR analysis of renal TNF-a.(c) qRT-PCR analysis of renal interleukin (IL)-1b. (d) Measurements of renal thiobarbituric acid-reactive substances (TBARS). (e) qRT-PCR analysis of renal gp91phox.(f) qRT-PCR analysis of renal p47phox. The gene expression was normalized by GAPDH. Control: N ¼ 6; cisplatin: N ¼ 12; cisplatin þ celecoxib: N ¼ 8 per group. Data are mean±s.e.

Kidney International (2011) 79, 77–88 83 original article Z Jia et al.: mPGES-1 and cisplatin nephrotoxicity

a Control Cisplatin

COX-2

bcP<0.01 200 10 P<0.01 P<0.01 9 8 150

7 content 6 2 5 100 4 3 50 2

1 (pg per mg protein) COX-2/GAPDH 0 PGE Kidney 0 Control Cisplatin Control Cisplatin celecoxib Figure 10 | Stimulation of renal cyclooxygenase 2 (COX-2) expression by cisplatin. (a) COX-2 immunoreactivity in the kidney of wild-type (WT) mice treated with vehicle or cisplatin. Shown are representative photomicrographs (hematoxylin and eosin staining; magnification: 200 shown) from three independent experiments. (b) Quantitative reverse transcriptase (qRT)-PCR analysis of renal COX-2 in vehicle or cisplatin-treated mice. (c) Enzyme-linked immunosorbent assay analysis of kidney prostaglandin E2 (PGE2) content (normalized by protein content). Control: N ¼ 6; cisplatin: N ¼ 12; cisplatin þ celecoxib: N ¼ 8 per group. Data are mean±s.e.

250 1.8 1.5 1.5 200 1.2 1.2 150 0.9 0.9 100 0.6 0.6 * BUN (mg/dl) 50 mRNA/ β -actin 0.3

0.3 Renal mPGES-1 Plasma Cr (mg/dl) 0 0 0 WT I/R KO I/R WT I/R KO I/R Sham I/R

1.2 1.4 2.5 1 1.2 2 0.8 1 0.8 1.5 0.6 0.6 1 0.4 * 0.4 * Renal cPGES mRNA/ β -actin mRNA/ β -actin

Renal COX-2 0.5 mRNA/ β -actin Renal mPGES-2 0.2 0.2 0 0 0 Sham I/R Sham I/R Sham I/R Figure 11 | Role of membrane-associated prostaglandin E synthase-1 (mPGES-1) in renal ischemia–reperfusion (I/R) injury in mice. mPGES-1 þ / þ and / mice were subjected to bilateral renal ischemia for 30 min by clamping renal artery, followed by reperfusion. Blood urine nitrogen (BUN) (a) and plasma creatinine (Cr) (b) were determined at 24 h after I/R injury. In a separate experiment, mPGES-1 þ / þ mice were sham operated or subjected to the I/R procedure. Renal mRNA expression of mPGES-1 (c), mPGES-2 (d), cytosolic PGES (cPGES) (e), and cyclooxygenase 2 (COX-2) (f) was determined at 24 h after I/R injury. N ¼ 5–7 per group. *Po0.05 vs sham. Data are mean±s.e. oxidase inhibitor apocynin ameliorates cisplatin-induced of the NADPH oxidase system in macrophages as well as in renal dysfunction and renal histological damage.35 We further other noninflammatory cells.23,37 observed that mPGES-1 deletion remarkably attenuated One of the most novel findings is the demonstration of the cisplatin-induced ROS generation and p47phox and gp91phox specific pathogenic role of mPGES-1 in cisplatin nephro- expression, and restored SOD2 and SOD3 levels. However, toxicity but not other types of AKI induced by I/R or these findings are at odds with our previous observation that lipopolysaccharide (LPS). The mechanism underlying the PGE2 directly attenuates angiotensin II-induced ROS activa- distinct responses of mPGES-1 KO mice to different insults is tion of NADPH oxidase in cultured vascular smooth muscle unclear but could be related to the differences in the cellular cells.23 We suspect that the antioxidant effect of mPGES-1 sources of cytokine production. Increased TNF-a production deletion during cisplatin nephrotoxicity may be secondary to in response to cisplatin treatment has been demonstrated in the suppressed inflammatory response. Indeed, proinflam- renal epithelial cells in vitro, raising the possibility that renal matory cytokines including TNF-a are well-known activators parenchymal cells may be a major source of TNF-a

84 Kidney International (2011) 79, 77–88 Z Jia et al.: mPGES-1 and cisplatin nephrotoxicity original article

Control finding is surprising in light of the well-documented LPS 14 120 proinflammtory role of mPGES-1. One explanation is that P <0.01 100 like renal I/R injury, endotoxin-induced AKI may also be P<0.01 chiefly mediated by bone marrow-derived cells. Although this 80 notion has not been directly assessed, infiltration of 60 leukocytes has been demonstrated in the septic kidney.44,45 40 BUN (mg/dl) It is interesting to note that the degree of body weight 20 loss after cisplatin treatment was not different between the 0 genotypes, suggesting that mPGES-1 may not mediate the WT KO general or gastrointestinal toxicity of cisplatin. Moreover, we found no difference in the degree of kidney swelling 0.4 P <0.01 P <0.01 indicative of the response to acute kidney injury. This 0.35 0.3 finding raises a possibility that mPGES-1 may have a role in a 0.25 particular stage, for example, the late rather than early stage 0.2 of the injury process. Apoptosis may represent an early event 4 0.15 in the pathogenesis of cisplatin nephrotoxicity. In cultured 0.1 renal proximal tubular cells, apoptosis is induced over a few Plasma Cr (mg/dl) 46,47 0.05 hours after exposure to cisplatin, and is preceded and 17 0 mediated at least in part by the Bak/Bax activation. We WT KO found that neither the induction of Bax/Bak nor bcl-2 140 P <0.05 expression was affected in mPGES-1 KO mice. These results 120 suggest that mPGES-1 may act independently of the Bak/Bax P<0.01 100 pathway, although other apoptotic pathways may still be 80 involved. 60 COX-2 inhibitors produce adverse effects on renal function particularly during low salt intake in animals and 40 is occasionally associated with acute renal failures in humans, Plasma TNF- α (pg/ml) 20 highlighting the important role of this enzyme in renal 0 48,49 WT KO physiology. However, the role of COX-2 in renal Figure 12 | Analysis of renal function and plasma TNF-a after pathophysiology is poorly defined with conflicting reports 50 51 lipopolysaccharide (LPS) treatment. Blood urine nitrogen (BUN) on detrimental vs beneficial effects of COX-2 inhibitors in (a), plasma creatinine (Cr) (b), and plasma tumor necrosis factor-a renal I/R injury. We found that renal COX-2 expression was (TNF-a)(c) in membrane-associated prostaglandin E synthase-1 induced in parallel with mPGES-1 in renal parenchymal cells (mPGES-1) þ / þ and / mice at 24 h following an intraperitoneal injection of vehicle or lipopolysaccharide (LPS) following cisplatin administration. Importantly, the COX-2 (10 mg/kg). N ¼ 8 per group. Data are mean±s.e. inhibitor celecoxib ameliorated the cisplatin-induced renal dysfunction and structural damage, recapitulating the production in cisplatin nephrotoxicity.38,39 Zhang et al.40 phenotype of mPGES-1 KO mice. Moreover, cisplatin- elegantly employed bone marrow transplantation technique induced increases in renal PGE2 content and cytokine to demonstrate that the production of TNF-a by parench- expression were similarly suppressed by celecoxib. The similar ymal cells, rather than by bone marrow-derived infiltrating beneficial effects produced by the COX-2 inhibitor and immune cells, is responsible for cisplatin nephrotoxicity. On mPGES-1 deletion substantiate the important role of the contrary, increasing evidence supports a primary role of proinflammatory PGE2 in mediating cisplatin-induced renal bone marrow-derived cells in the pathogenesis of renal injury. However, our results do not agree with the study by 41–43 52 ischemic injury. A specific role of PGE2 in cisplatin- Greene et al. who found no beneficial effect of the COX-2 induced renal injury can also be reflected by the differential inhibitor SC236 on cisplatin nephrotoxicity in rats. The regulation of mPGES-1 and COX-2 in the two renal injury reason for the discrepancy is unclear but could be related to models. The parallel induction of mPGES-1 and COX-2 the differences in experimental protocols or animal species. following cisplatin treatment occurred predominantly in For example, the COX-2 inhibitors were administered at 72 h renal proximal tubular cells, coinciding with the location of (our study) vs 2 h (Greene’s study) before cisplatin treatment. TNF-a production. In contrast, renal expression of neither The efficacy of COX-2 inhibition as reflected by suppression mPGES-1 nor COX-2 was induced during I/R injury; renal of tissue PGE2 production was validated in our but not mPGES-1 expression was even downregulated. Likely, a Greene’s study. More perplexing is that another COX-2 specific coupling between PGE2 and TNF-a induction in inhibitor nimesulide produced a worsening effect on renal proximal tubular cells may underlie the pathophysiol- cisplatin-induced nephrotoxicity in spontaneously hyperten- ogy of cisplatin nephrotoxicity. Similarly, mPGES-1 KO mice sive rats.53 This phenomenon may highlight the antihyper- were not protected against endotoxin-induced AKI. This tensive and renal protective role of renal COX-2 in

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hypertension.54,55 In contrast, salicylate reduces cisplatin Cisplatin treatment of animals nephrotoxicity in mice via inhibition of TNF-a,39 an effect Cisplatin was freshly prepared in saline at 1 mg/ml and injected similar to that of celecoxib in this study. The mechanism by intraperitoneally into mPGES-1 WT and KO mice at a dose of which salicylate protects against cisplatin nephrotoxicity 20 mg/kg; control group was administered with an equal volume of remains unclear and may involve inhibition of nuclear factor- saline. After 72 h, the animals were killed. To test the effect of the COX-2 inhibitor celecoxib on cisplatin nephrotoxicity, celecoxib was kB or COX-2 or both. The definitive role of COX-2 in cisplatin administered to mPGES-1 WT mice via daily gavage at 50 mg/kg/ nephrotoxicity needs to be tested by using COX-2 KO mice. day, starting at 72 h before cisplatin injection. Clinical trials demonstrate that regular use of nonsteroidal anti-inflammatory drugs reduces the risk of developing Effect of celecoxib on COX-1 activity 56 colorectal cancer by up to 40–50%. A large body of Thromboxane A2 (TXA2) is rapidly hydrolyzed nonenzymatically to experimental evidence has established a critical role of COX-2 form TXB2, which is then quickly metabolized (t1/2 ¼ 5–7min). The in tumorigenesis with the proven antitumorigenic activity of ex vivo production of TXB2 in blood samples has been used as an 57 64 COX-2 inhibitors. Among COX-2-derived products, PGE2 index of COX-1 activity. To address the specificity of celecoxib, we has received particular attention because of its direct role in examined its effect on COX-1 activity in comparison with the malignant progression of most solid tumors, including indomethacin. Briefly, mPGES-1 WT mice were administered with colon, breast, lung, head neck, uterus, and stomach vehicle, celecoxib (50 mg/kg/day), or indomethacin (2 mg/kg/day) via daily gavage for 5 days. The blood samples were taken via carcinomas.58 Increasing evidence suggests involvement of venipuncture and incubated at room temperature for 45 min, and mPGES-1 in tumor promotion.13,59–61 More importantly, then centrifuged at 2000 g at 4 1C for 5 min. The serum TXB2 level mPGES-1 deletion results in marked suppression of intestinal was measured by an enzyme immunoassay kit (Cayman Chemicals, 62 carcinogenesis in mice. In light of the antitumorigenic Ann Arbor, MI, USA). activity of COX-2 and mPGES-1 inhibitors together with their additional beneficial effects on cisplatin nephrotoxicity, Generation of mouse models with I/R and endotoxin-induced we suspect that the combination of either COX-2 or mPGES-1 renal injury inhibitor with the cisplatin may not only enhance the mPGES-1 WT and KO mice were subjected to renal I/R injury as 65 chemotherapeutic potential in solid tumors but may also previously described. Briefly, under general anesthesia, the left and minimize nephrotoxic effect of cisplatin. In support of this right renal arteries were located, isolated, and occluded for 30 min notion, administration of CS-706, a selective COX-2 using microaneurysm clips to produce ischemia. After removing the clips, verifying reperfusion, and closing the incision in two layers, inhibitor, in combination with cisplatin completely regressed anesthesia was terminated and 1 ml of 37 1C saline was injected into the tumors in a mouse model of colorectal adenocarcinoma, the abdomen to supplement fluid loss. A sham-operated group contrasting with the modest effect of a single treatment with underwent identical surgical procedures except that microaneurysm 63 either drug. clamps were not applied. In separate experiments, mPGES-1 WT In summary, this study examined the role of COX-2/ and KO mice were treated with a single intraperitoneal injection mPGES-1 pathway in a mouse model of cisplatin nephro- of vehicle or LPS at 10 mg/kg (Escherichia coli 0127:B8 (Sigma, toxicity. Following cisplatin administration, the expression of St Louis, MO, USA); dissolved in saline). At 24 h after I/R or LPS COX-2 and mPGES-1 was induced in parallel in renal injection, blood and kidney samples were harvested for evaluation of parenchymal cells. mPGES-1 KO mice were resistant to renal function and renal gene expression. cisplatin-induced renal dysfunction and structural damage, Immunohistochemistry accompanied with suppressed cytokine expression and Kidneys from vehicle or cisplatin-treated mice were fixed with 4% oxidative stress. The phenotype was recapitulated with the paraformaldehyde and embedded in paraffin. Kidney sections COX-2 inhibitor celecoxib. Overall, intervention of COX-2/ (4-mm thickness) were incubated in 3% H2O2 for 10 min at room mPGES-1 pathway in cisplatin-treated patients may offer a temperature to block endogenous peroxidase activity. The slides novel approach for management of the renal toxicity with were boiled in antigen retrieval solution (1 mmol/l Tris-HCl, added values of enhanced chemotherapeutic potential. 0.1 mmol/l EDTA, pH ¼ 8.0) for 15 min at high power in a microwave oven. The sections were incubated overnight at 4 1C MATERIALS AND METHODS with primary antibodies at appropriate dilutions (rabbit anti- Animals mPGES-1 and rabbit anti-COX-2 antibodies, Ann Arbor Cayman mPGES-1 null mice were originally generated in DBA/1lacJ Chemicals). After washing with phosphate-buffered saline, the background by Trebino et al.9 Owingtobreedingdifficulty,the/ secondary antibody was applied and the signals visualized using the mice were crossed with C57/BL6 129/SV mice and have been ABC kit (Santa Cruz Biotechnology, CA, USA). maintained in a mixed DBA/1lacJ C57/BL6 129/SV background for more than 20 intercrosses. The / mice were bred using Immunoblotting homozygous females to homozygous males. Nonlittermate WT mice The whole kidney was lysed and protein concentration was on the same genetic background were used as controls. Male mice determined by Coomassie reagent. Protein (60 mg) from whole (3–4 months old) were used for all experiments. All protocols kidney lysates were denatured in boiling water for 10 min, separated employing mice were conducted in accordance with the principles by SDS-polyacrylamide gel electrophoresis, and transferred onto and guidance of the University of Utah Institutional Animal Care nitrocellulose membranes. The blots were blocked overnight with and Use Committee. 5% nonfat dry milk in Tris-buffered saline (TBS), followed by

86 Kidney International (2011) 79, 77–88 Z Jia et al.: mPGES-1 and cisplatin nephrotoxicity original article

incubation for 1 h with rabbit anti-mPGES-1 at a dilution of 1:1000. Enzyme immunoassay After being washed with TBS, blots were incubated with a goat The whole kidney was homogenized in phosphate-buffered saline anti-horseradish peroxidase-conjugated secondary antibody (1:1000 and then centrifuged for 5 min at 10,000 r.p.m. The supernatant was dilution) and visualized with ECL kits (Amersham, Piscataway, NJ diluted 1:50 with enzyme immunoassay buffer. Concentrations of USA). PGE2 were determined by enzyme immunoassay (Cayman Chemi- cals) and normalized by protein concentrations. The plasma TNF-a qRT-PCR level was determined by using another enzyme immunoassay kit Total RNA isolation and reverse transcription were performed as (catalog number: 559732, BD OptEIA, BD Bioscience, San Jose, CA, previously described.19 Oligonucleotides were designed using USA) according to the manufacturer’s instructions. Primer3 software (available at http://frodo.wi.mit.edu/primer3/) and the sequences are shown in Table 1. qPCR amplification was Measurement of TBARS performed using the SYBR Green Master Mix (Applied Biosystems, The measurement of TBARS in the mouse kidney was based on the Warrington, UK) and the Prism 7500 Real-Time PCR Detection formation of malondialdehyde by using a commercially available System (Applied Biosystems, Foster City, CA, USA). Cycling TBARS Assay kit (catalog number: 10009055, Cayman Chemical) conditions were 95 1C for 10 min, followed by 40 repeats of 95 1C according to the manufacturer’s instructions. for 15 s, and 60 1C for 1 min. Statistical analysis Renal function and histology The data are reported as means±s.e. Statistical significance was Plasma Cr and BUN were determined to reflect renal function. For assessed by unpaired, two-tailed Student’s t-tests for single histology, kidneys were fixed with 4% paraformaldehyde and stained comparisons or analysis of variance for multiple comparisons. with hematoxylin and eosin. Tissues damage was indicated by Differences were considered to be significant when the probability tubular lysis, dilation, disruption, and cast formation. The degree of value was o0.05. tissue damage was scored based on the percentage of damaged tubules as previously described: 0, no damage; 1, o25%; 2, 25–50%; DISCLOSURE 66 3, 50–75%; 4, 475%. All the authors declared no competing interests.

ACKNOWLEDGMENTS This work was supported by the NIH Grant DK079162 and a grant from the American Heart Association (to TY). We thank John McNeish Table 1 | Sequences of oligonucleotides quantatitive PCR (Pfizer, Groton, CT, USA) for providing breeder pairs of mPGES-1 KO Gene Primer sequence Accession number mice, and Shannon J Odelberg and Ivor J Benjamin (Cardiology, University of Utah) for technical assistance. TY is an Established 0 0 GAPDH 5 -GTCTTCACTACCATGGAGAAGG-3 M32599 Investigator of the American Heart Association. 50-TCATGGATGACCTTGGCCAG-30 mPGES-1 50-AGCACACTGCTGGTCATCAA-30 BC024960 50-CTCCACATCTGGGTCACTCC-30 REFERENCES mPGES-2 50-GCTGGGGCTGTACCACAC-30 NM_133783 1. Wang D, Lippard SJ. Cellular processing of platinum anticancer drugs. 50-GATTCACCTCCACCACCTGA-30 Nat Rev Drug Discov 2005; 4: 307–320. cPGES 50-GGTAGAGACCGCCGGAGT-30 NM_019766 2. Cohen SM, Lippard SJ. Cisplatin: from DNA damage to cancer chemotherapy. Prog Nucleic Acid Res Mol Biol 2001; 67: 93–130. 50-TCGTACCACTTTGCAGAAGCA-30 0 0 3. Arany I, Safirstein RL. Cisplatin nephrotoxicity. Semin Nephrol 2003; 23: COX-2 5 -AGGACTCTGCTCACGAAGGA-3 NM_011198 460–464. 0 0 5 -TGACATGGATTGGAACAGCA-3 4. Pabla N, Dong Z. Cisplatin nephrotoxicity: mechanisms and 0 0 TNF-a 5 -TCCCCAAAGGGATGAGAAG-3 NM_013693 renoprotective strategies. Kidney Int 2008; 73: 994–1007. 50-CACTTGGTGGTTTGCTACGA-30 5. Luke DR, Vadiei K, Lopez-Berestein G. Role of vascular congestion in IL-1b 50-ACTGTGAAATGCCACCTTTTG-30 NM_008361 cisplatin-induced acute renal failure in the rat. Nephrol Dial Transplant 50-TGTTGATGTGCTGCTGTGAG-30 1992; 7:1–7. NOX1 50-CACTCCCTTTGCTTCCATCT-30 NM_172203 6. Breyer MD, Breyer RM. G protein-coupled prostanoid receptors and the 50-ATGTTGCTATCCCAGCCAGT-30 kidney. Annu Rev Physiol 2001; 63: 579–605. NOX-3 50-GGAGGAGGTCGCATCATT-30 NM_198958 7. Smith WL, Garavito RM, DeWitt DL. Prostaglandin endoperoxide H 50-CACGCATACAAGACCACAGG-30 synthases (cyclooxygenases)-1 and -2. J Biol Chem 1996; 271: p47phox 50-GTCGTGGAGAAGAGCGAGAG-30 NM_010876 33157–33160. 8. Murakami M, Kudo I. Prostaglandin E synthase: a novel drug 50-CGCTTTGATGGTTACATACGG-30 0 0 target for inflammation and cancer. Curr Pharm Des 2006; 12: gp91phox 5 -CCGTATTGTGGGAGACTGGA-3 NM_007807 943–954. 0 0 5 -CTTGAGAATGGAGGCAAAGG-3 9. Trebino CE, Stock JL, Gibbons CP et al. Impaired inflammatory and pain 0 0 SOD1 5 -AAGGCCGTGTGCGTGCTGAA-3 NM_921076 responses in mice lacking an inducible prostaglandin E synthase. 50-CAGGTCTCCAACATGCCTCT-30 Proc Natl Acad Sci USA 2003; 100: 9044–9049. SOD2 50-CGGCCTACGTGAACAATCTC-30 NM_013671 10. Uematsu S, Matsumoto M, Takeda K et al. Lipopolysaccharide-dependent 50-GATAGCCTCCAGCAACTCTCC-30 prostaglandin E(2) production is regulated by the glutathione-dependent SOD3 50-TTCTTGTTCTACGGCTTGCTAC-30 NM_011435 prostaglandin E(2) synthase gene induced by the Toll-like receptor 50-CTCCATCCAGATCTCCAGCACT-30 4/MyD88/NF-IL6 pathway. J Immunol 2002; 168: 5811–5816. bak 50-CGCTACGACACAGAGTTCCA-30 NM_007523 11. Boulet L, Ouellet M, Bateman KP et al. Deletion of microsomal 50-TCCATCTGGCGATGTAATGA-30 prostaglandin E2 (PGE2) synthase-1 reduces inducible and basal PGE2 production and alters the gastric prostanoid profile. J Biol Chem 2004; bax 50-TGCAGAGGATGATTGCTGAC-30 NM_007527 279: 23229–23237. 50-GATCAGCTCGGGCACTTTAG-30 0 0 12. Murakami M, Nakashima K, Kamei D et al. Cellular prostaglandin Bcl-2 5 -GAGCGTCAACAGGGAGATGT-3 NM_009741 E2 production by membrane-bound prostaglandin E synthase-2 via 0 0 5 -CTCACTTGTGGCCCAGGTAT-3 both cyclooxygenases-1 and -2. J Biol Chem 2003; 278: 37937–37947.

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