Hypertension

Evidence That Links Loss of -2 With Increased Asymmetric Dimethylarginine Novel Explanation of Cardiovascular Side Effects Associated With Anti-Inflammatory Drugs

Blerina Ahmetaj-Shala, PhD*; Nicholas S. Kirkby, PhD*; Rebecca Knowles, MD; Malak Al’Yamani, MSc; Sarah Mazi, MRes; Zhen Wang, PhD; Arthur T. Tucker, PhD; Louise Mackenzie, PhD; Paul C. J. Armstrong, PhD; Rolf M. Nüsing, PhD; James A. P. Tomlinson, MD, PhD; Timothy D. Warner, PhD; James Leiper, PhD†; Jane A. Mitchell, PhD†

Background—Cardiovascular side effects associated with cyclooxygenase-2 inhibitor drugs dominate clinical concern. Cyclooxygenase-2 is expressed in the renal medulla where inhibition causes fluid retention and increased blood pressure. Downloaded from However, the mechanisms linking cyclooxygenase-2 inhibition and cardiovascular events are unknown and no biomarkers have been identified. Methods and Results—Transcriptome analysis of wild-type and cyclooxygenase-2–/– mouse tissues revealed 1 altered in the heart and aorta, but >1000 altered in the renal medulla, including those regulating the endogenous nitric oxide synthase inhibitors asymmetrical dimethylarginine (ADMA) and monomethyl-l-arginine. Cyclo-oxygenase-2–/– mice http://circ.ahajournals.org/ had increased plasma levels of ADMA and monomethyl-l-arginine and reduced endothelial nitric oxide responses. These genes and methylarginines were not similarly altered in mice lacking prostacyclin receptors. Wild-type mice or human volunteers taking cyclooxygenase-2 inhibitors also showed increased plasma ADMA. Endothelial nitric oxide is cardio- protective, reducing thrombosis and atherosclerosis. Consequently, increased ADMA is associated with cardiovascular disease. Thus, our study identifies ADMA as a biomarker and mechanistic bridge between renal cyclooxygenase-2 inhibition and systemic vascular dysfunction with nonsteroidal anti-inflammatory drug usage. Conclusions—We identify the endogenous endothelial nitric oxide synthase inhibitor ADMA as a biomarker and mechanistic by guest on December 4, 2017 bridge between renal cyclooxygenase-2 inhibition and systemic vascular dysfunction. (Circulation. 2015;131:633-642. DOI: 10.1161/CIRCULATIONAHA.114.011591.) Key Words: endothelium ◼ kidney ◼ nitric oxide ◼ pharmacology ◼ prostaglandins

yclooxygenase (COX) catalyzes the conversion of ara- Clinical Perspective on p 642 chidonic acid to prostaglandin H , which is then further C 2 metabolized by downstream synthase to a range of of this class of drugs inhibit both isoforms of COX1 and, . COX is expressed in 2 isoforms, constitutive as a consequence, are associated with gastrointestinal side COX-1 and inducible COX-2. Nonsteroidal anti-inflamma- effects.2 NSAID-induced gastrointestinal side effects are rel- tory drugs (NSAIDs) are widely used to treat inflammation atively common and can be serious, limiting the use of these and pain and show potential to prevent . COX-2 is drugs in some patients and proving fatal in others.3 COX-2 the therapeutic target of NSAIDs, although most members selective inhibitors such as Vioxx () and Celebrex

Received June 10, 2014; accepted November 21, 2014. From the Cardiothoracic Pharmacology, Vascular Biology, National Heart and Lung Institute, Imperial College, London, United Kingdom (B.A.-S., N.S.K., M.Al'Y., S.M., J.A.M.); The William Harvey Research Institute, Barts and the London School of Medicine and Dentistry, Queen Mary, University of London, London, United Kingdom (R.K., A.T.T., P.C.J.A., T.D.W.); King Fahad Cardiac Center of King Saud University, Riyadh, Saudi Arabia (M.Al'Y., S.M.,); MRC Clinical Sciences, Imperial College London, Nitric Oxide Signalling Group, Hammersmith Hospital, DuCane Road, London, United Kingdom (Z.W., J.A.P.T., J.L.); School of Life and Medical Sciences, University of Hertfordshire, College Lane, Hatfield, United Kingdom (L.M.); and Institute of Clinical Pharmacology, Johann Wolfgang Goethe-University, Theodor Stern Kai 7, Frankfurt, Germany (R.M.N.). *Drs Ahmetaj-Shala and Kirkby contributed equally. †Drs Leiper and Mitchell contributed equally. The online-only Data Supplement is available with this article at http://circ.ahajournals.org/lookup/suppl/doi:10.1161/CIRCULATIONAHA. 114.011591/-/DC1. Correspondence to Jane A. Mitchell, PhD, National Heart and Lung Institute, Imperial College, Dovehouse St, London SW36LY, UK. E-mail [email protected] © 2014 The Authors. Circulation is published on behalf of the American Heart Association, Inc., by Wolters Kluwer. This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution, and reproduction in any medium, provided that the original work is properly cited. Circulation is available at http://circ.ahajournals.org DOI: 10.1161/CIRCULATIONAHA.114.011591 633 634 Circulation February 17, 2015

() were originally developed to target COX-2 at Methods the site of inflammation while sparing COX-1 in the gut Experimental Animals and so reducing the gastrointestinal side effects typical of –/– –/– 11 4 Male or female COX-1 and COX-2 mice, back-crossed for >7 NSAIDs. However, now, cardiovascular side effects associ- generations onto a C57Bl/6J background (Harlan, UK) were used at ated with COX-2 inhibition dominate concern over NSAID 10 to 12 weeks of age. For transcriptomic studies, littermate COX- usage5–7 and have stopped the development of new drugs in 2+/+ mice from COX-2–/– colonies were used. For other studies, wild- this class. The cardiovascular side effects caused by the inhi- type mice maintained separately were used as controls. Animals 11 bition of COX-2 are mainly myocardial infarctions,8,9 which were genotyped before use. Mice lacking prostacyclin receptors (IP–/–), supplied by Dr Rolf Nüsing, were on a C57BL/6 background implies increased thrombosis and atherosclerosis and have as described.28 Animal studies were conducted in accordance with been considered to reflect local loss of the cardioprotective Animals (Scientific Procedures) Act 1986, which is a UK act after hormone prostacyclin by blood vessels. However, COX-2 is local ethical review. only sparsely detected in blood vessels, where, by contrast, COX-1 is readily detected. COX-2 is upregulated at sites of Pharmacological COX-2 Inhibition and Blood vascular inflammation, including atherosclerotic plaques, Pressure Measurement although, even at these areas, COX-1 drives prostacyclin (Pfizer, USA; 100 mg·kg–1·d–1) in drinking water was release.10 Although COX-2 is largely absent from blood ves- administered for 4 days to wild-type mice. Mean carotid arterial sels,11–13 there are key areas in the body, including the kid- blood pressure was measured by using radiotelemetry. In brief, a 1.4F 14–17 Millar MikroTip pressure catheter was inserted in the right common Downloaded from ney, where it is expressed constitutively. COX-2 in the carotid artery of spontaneously breathing mice anesthetized with kidney regulates natriuresis, diuresis, and vasodilation.16–18 inhaled isoflurane (Abbott, USA). Animals were allowed to recover NSAIDs, including COX-2 selective drugs, therefore have from the anesthetic, and measurements were made in conscious, unre- well-documented effects on the kidney and increase blood strained animals 21 days later. Blood pressure traces were recorded pressure by ≈1 to 2 mm Hg in normotensive individuals and continuously with the use of the PowerLab and Chart 5 software (AD Instruments Ltd, UK). Blood was obtained by tail nick in conscious by up to 14 mm Hg in patients with hypertension.19 In addi- http://circ.ahajournals.org/ animals into 3.2% citrate and plasma separated by centrifugation. tion, they are known to dysregulate glomerular filtration and 20–23 salt/water homeostasis. Given the well-established link Mouse Tissue Collection and Processing between impaired renal function and cardiovascular events, Mice were euthanized by CO2 narcosis. Blood collected from the including myocardial infarctions, it is logical to expect that inferior vena cava into heparin (10 U/mL final; Leo Laboratories, these changes in renal function may contribute to the car- UK) or clotting-tubes (Sarstdet, UK) was centrifuged for serum/ diovascular side effects associated with NSAID usage.24 plasma. Urine was collected by amniocentesis. Serum urea was However, the precise mechanism by which COX-2 regulates measured by a commercial veterinary diagnostics service (IDEXX

by guest on December 4, 2017 Laboratories, UK). renal function and the relevance of COX-2 in the kidney in comparison with other cardiovascular tissues are not known, and the debate continues regarding the relative contribution Microarray Analysis of vascular prostacyclin versus renal effects to cardiovas- Tissues were snap frozen and homogenized, and total RNA was 25 extracted by using a silica column–based kit (Nucleospin RNA II; cular side effects of NSAIDs. Consequently, there are no Machery-Nagal, UK). Samples were converted to cDNA, frag- biomarkers of NSAID-induced cardiovascular risk and no mented, labeled, and hybridized to MouseRef-8v3 BeadChip arrays avenues to protect those that might be more susceptible than (Illumina, UK). Data were quantile normalized and analyzed by the general population. using the linear models for microarray data modified t test method To address this issue, we have performed a transcriptomic and Benjamini-Hochberg false discovery rate correction by using GeneSpring GX 12.1 software (Agilent, USA). Differential expressed analysis of the 4 key cardiovascular tissues, kidney, heart, genes with a corrected P value of q<0.05 were considered statistically aorta, and blood, from wild-type and COX-2–deficient mice. significant. Focused pathway analysis was performed by examina- Pathway analysis revealed alterations in the kidney of genes tion of genes altered >1.3-fold (q<0.05) featuring in the following associated with nitric oxide (NO) biology, particularly those human (http://www.geneontology.org) pathways: (1) regulation of systemic arterial blood pressure (GO:0003073); regulating the synthesis and of the endogenous (2) regulation of blood vessel size (GO:0050880); (3) NO biosyn- NO synthase (NOS) inhibitors asymmetrical dimethylargi- thetic process (GO:0006809); (4) prostaglandin biosynthetic pro- nine (ADMA) and monomethyl-l-arginine (l-NMMA). We cess (GO:0001516); (5) regulation of angiotensin levels in blood went on to validate increased ADMA and l-NMMA levels (GO:0002002); (6) endothelin maturation (GO:0034959); (7) epi- in plasma of mice lacking COX-2 and in wild-type mice or nephrine biosynthetic process (GO:0042418); and (8) protein-argi- nine N-methyltransferase activity (GO:0090627). healthy human volunteers taking COX-2 selective inhibitors. Our work fits well with and helps to explain recent observa- tions showing that endothelial NO synthase (eNOS) responses Real-Time Quantitaive Polymerase Chain Reaction are reduced in vessels from COX-2–deficient mice.26 Because RNA was converted to cDNA using the iScript cDNA synthesis kit (BioRad, CA, USA). Quantitative polymerase chain reaction was per- NO release from endothelial cells performs the same protec- formed using a 7500 Fast Real-time polymerase chain reaction system tive role that prostacyclin performs on platelets and blood (Applied Biosystem). Ddah1 (5′-CACAGAAGGCCCTCAAGATCA-3′, vessels, and because it synergizes powerfully with prostacy- 5′-TCTCATAGACCTTTGCGCTTTC-3′), Ddah2 (5′-CCTGGTGCCA clin in platelets,27 our observations are the first to provide a CACCTTTCC-3′, 5′-AGGGTGACATCAGAGAGCTTCTG-3′) and Agxt2 (5′-GGCTTCCCCATGGCTGCAGTT-3′, 5′-CAATCACCTCA rational explanation for how the inhibition of COX-2 in the AGCACAGCAGATCC-3′). mRNA levels were d­etermined bus- kidney can remotely regulate vascular side effects throughout ing the iTAQ fast SybrGreen supermix with ROX (BioRad, the vasculature. CA, USA) expression assays and normalized to levels of Actb Ahmetaj-Shala et al Renal COX-2 and Regulation of ADMA and l-NMMA 635

(5′-CCAGGGTGTGATGGTGGGAATG-3′, 5′-CGCACGATTTCCC inhibitor Celebrex (celecoxib; 200 mg twice a day; Pfizer USA), or TCTCAGCTG-3′). For Ptgs2 (probe ID: Mm00478374_m1), Prmt1 the nonselective COX-1/COX-2 inhibitor Naprosyn (; 500 (probe ID: Mm00480133_m1), Arg1 (probe ID: Mm00475988_ mg twice a day; Roche Switzerland) for 7 days. After overnight m1), Arg2 (probe ID: Mm00477592_m1), 18S rRNA (probe ID: fasting, on day 0, before the first dose, and day 7, 2 hours after Mm03928990_g1), and Gapdh (probe ID: Mm99999915_g1) gene the final dose, blood was collected into citrate Vacutainers (BD expression levels were determined using TaqMan expression assays. Diagnostics, UK) and separated by centrifugation. This study was Genes were quantified relative to housekeeping genes (Actb or approved by the St Thomas’s Hospital Research Ethics Committee Gapdh/18S) by comparative Ct methods. (Ref. 07/Q0702/24) and conducted according to the Declaration of Helsinki. All volunteers gave written informed consent before Liquid-Chromatography Tandem Mass- entering the study. Spectrometry Measurements Samples were analyzed as described previously29 with a Agilent Statistical Methods 6400 series triple quadruple liquid-chromatography tandem mass- Data are mean±standard error of the mean. Statistical significance spectrometry using mobile phase (0.1% formic acid, 1% acetoni- (taken as P<0.05) was determined by using the following tests: trile). The mass spectrum parameters for detection were as follows: Mann–Whitney (1 variable, 2 groups), Wilcoxon signed-rank (1 vari- ADMA, mass-to-charge ratio (m/z): 203.3 to 46.0, collision energy: able, 2 paired groups), Kruskal–Wallis with the Dunn post hoc test 12; symmetrical dimethylarginine (SDMA), m/z: 203.3 to 70.2, colli- (1 variable, 3 groups), or Quades test (2 variables). All tests were sion energy: 24; l-NMMA, m/z: 189.3 to 70.2, collision energy: 24; performed using GraphPad Prism 6 software, with the exception of d7-ADMA, m/z: 210.0 to 46.0, collision energy: 24. Concentrations the Quades test, which was performed using R. Significance was of individual amino acids, l-NMMA, ADMA, SDMA, or creatinine assumed where P<0.05. No further correction has been made for mul-

Downloaded from were calculated from standard curves generated by using authentic tiple testing (typically <5 end points per animal). The exception is standards. analysis of transcriptomic data where false discovery rate correction has been applied as described above. Vasomotor Responses Results Mice were euthanized by CO2 narcosis, and the thoracic aorta was cut into 2-mm aortic rings, mounted in organ baths of a Mulvany-Halpern http://circ.ahajournals.org/ myograph at a resting tension of 13 kPa in Krebs with or without Expression of COX-2 in the Kidney l-arginine (100 μmol/L; Sigma, UK) and contracted with of endothe- As others have shown,16 constitutive expression of Ptgs2 lin (ET)-1 (30 pmol/L to 30 nmol/L; Sigma, UK) or the thromboxane gene, which encodes the COX-2 protein, was seen in the mimetic (1 nmol/L to 1 μmol/L). After washing, tissues were medulla region of the kidney, with lower levels in the cor- contracted with an EC80 concentration of U46619 (30–100 nmol/L) followed by acetylcholine (1 nmol/L to 10 μmol/L) or sodium nitro- tex (Figure 1A), which others have shown is relatively sparse prusside (SNP; 1 pmol/L to 10 μmol/L). For ET-1 studies, vessels unless animals are salt depleted.16 As seen by others, the were preincubated with the NOS inhibitor NG-nitro-l-arginine methyl loss of COX-2, but not of COX-1, resulted in renal compro- ester (100 μmol/L; Sigma, UK) to exclude any effect of changes in 30 by guest on December 4, 2017 mise associated with increased serum urea and creatinine the NO/ADMA pathway. Force was recorded via a PowerLab/800 (Figure 1B and 1C). (AD Instruments Ltd., UK) and analyzed by using Chart 6.0 acquisi- tion system (AD Instruments Ltd, UK). Effect of COX-2 on the Transcriptome in Clinical Study Cardiovascular Tissues Sixteen healthy young male volunteers aged 20 to 35 who had In the blood, heart, and aorta, COX-1 activity drives pros- abstained from NSAID use for 14 days previously were recruited. tanoid production with little or no contribution from COX- Volunteers received standard doses of the COX-2 selective 2.11,14 Consistent with this, no genes were altered by COX-2

Figure 1. Renal COX-2 expression and effects on renal function. A, Expression of COX-2 in the aorta, renal cortex, and renal medulla of wild-type mice. Changes in levels of serum urea (B) and serum creatinine (C) in wild-type, COX-1–/–, and COX-2–/– mice. Data are mean±SEM for n=8 to 14 mice in each group. P values by Kruskal–Wallis with the Dunn post hoc test. COX indicates cyclooxygenase; and SEM, standard error of the mean. 636 Circulation February 17, 2015

deletion in the blood of COX-2–/– mice, and, in the heart and NOS enzymes. Using quantitative polymerase chain reaction, aorta, only 1 gene was altered/decreased, Rgl1, in both tissues we confirmed that in COX-2–/– renal medulla expression of (Table I in the online-only Data Supplement). By contrast, in Prmt1 (Figure 2C), which drives methylarginine synthesis,29 the renal medulla, deletion of COX-2 altered the expression was increased; Ddah2, which breaks down methylarginine, of 1018 genes by >1.5-fold (Figure 2A; Table I in the online- was nonsignificantly increased (Figure II in the online-only only Data Supplement). To explore the consequences of this Data Supplement), whereas Agxt2, which degrades methylar- finding, we applied focused pathway analysis to specifically ginines,29 tended to be reduced (Figure 2D). Using quantita- examine changes in (1) blood pressure control, (2) vascular tive polymerase chain reaction , we also observed reduced tone, and (3) vascular hormones and identified alterations in expression of Ddah1, which, like Agxt2, is responsible for the expression of a number of genes involved in angioten- methylarginine breakdown29 (Figure 2E). sin, ET, and NO (Figure 2B). Changes in angiotensin genes (Anpep, Agt, Mme) were predictive of reduced activity, so Effect of COX-2 on Plasma Methylarginines and they were unlikely to be implicated in the deleterious effects eNOS Responses in Mouse Aorta of COX-2 inhibitors. ET-1 and ET Plasma levels of ADMA and l-NMMA were increased in (Edn1, Endra, Ednrb) was increased (Figure 2B). However, COX-2–/–, but not COX-1–/– mice (Figure 3A and 3B). Plasma levels of ET-1 in the plasma of COX-2–/– mice were not altered SDMA levels were not changed in COX-2–/– mice (Table II in (Figure IA in the online-only Data Supplement). Moreover, the online-only Data Supplement). Arginases, like NOS, use

Downloaded from contractile responses in the aorta to ET-1 were not signifi- l-arginine as a substrate and can be altered in inflammatory con- cantly altered in COX-2–/– mice (Figure IB in the online-only ditions. However, in the renal medulla, Arg1 was not expressed Data Supplement). We therefore focused on a cluster of genes and Arg2 was not altered in COX-2–/– mice (Figure IIIA in the (Agxt2, Ddah2, Prmt1) related to the turnover of methylargi- online-only Data Supplement). In addition, in the aorta, Arg2 nines such as ADMA and l-NMMA, which are inhibitors of was not expressed in and Arg1 was not altered in COX-2–/– mice http://circ.ahajournals.org/ by guest on December 4, 2017

Figure 2. Transcriptome profiling in cardiovascular tissues from COX-2–/– mice. A, Number of genes altered in the transcriptome of blood, heart, aorta, and kidney (renal medulla) by >1.2- to >3-fold. B, Focused pathway analysis of genes altered >1.3-fold (q<0.05). qPCR validation of changes in expression of Prmt1 (C), Agxt2 (D), and Ddah1 (E) in the renal medulla of COX-2–/– mice. Data are from n=7 to 8 mice. Data in C through E are mean±SEM. P values by Mann–Whitney U test. COX indicates cyclooxygenase; qPCR, quantitative polymerase chain reaction; and SEM, standard error of the mean. Ahmetaj-Shala et al Renal COX-2 and Regulation of ADMA and l-NMMA 637

Figure 3. Effect of COX gene deletion ADMA and l-NMMA in plasma. Levels of ADMA (A) and l-NMMA (B) were increased in the plasma of COX- 2–/– mice, but not in COX-1–/– mice. Data are from n=4 and presented as mean±SEM. P values by Kruskal–Wallis with the Dunn post hoc test. ADMA indicates asymmetrical dimethylarginine; COX, cyclooxygenase; l-NMMA, monomethyl-l-arginine; and SEM, standard error of the mean.

(Figure IIIB in the online-only Data Supplement). In line with vessels via NO independently of the endothelium was not this finding, l-arginine was not significantly altered in COX- altered (Figure 5B), nor was their contractile response to 2–/– mice (Figure IIIC in the online-only Data Supplement), U46619 (Figure V in the online-only Data Supplement). resulting in ADMA:l-arginine (Figure IIID in the online-only The reduced eNOS responses in the aorta of COX-2–/– mice Data Supplement) and l-NMMA:l-arginine (Figure IIIE in were reversed in the presence of l-arginine (100 μmol/L; the online-only Data Supplement) ratios being increased. In Figure 5C). No effect of l-arginine (Figure 5D) was seen on agreement with these data, the pharmacological inhibition of response to SNP. These effects were not due to local changes

Downloaded from COX-2 in wild-type mice with parecoxib (100 mg/kg for 4 in aortic Nos3, Ddah1, Ddah2, or Agxt2 gene expression days) blocked diurnal blood pressure decline (Figure 4A) and (Figure VI in the online-only Data Supplement). plasma ADMA (Figure 4B) and l-NMMA levels (Figure 4C), as well. l-Arginine levels (Figure IVA in the online-only Data Effect of Loss of the Prostacyclin Receptor (IP) on Supplement) along with ADMA:l-arginine (Figure IVB in Renal Gene Expression and Plasma ADMA Levels

http://circ.ahajournals.org/ the online-only Data Supplement) and l-NMMA:l-arginine Prostacyclin, which acts via cell surface IP receptors, is (Figure IVC in the online-only Data Supplement) ratios were an important COX-derived mediator in the cardiovascular increased by parecoxib. system. To establish the relative role of prostacyclin, in Increased plasma levels of methylarginines are associated comparison with the myriad of other COX products, in our in other models with reduced eNOS activity in vessels ex study, we performed experiments using IP–/– mice. None of vivo31 and hypertension in vivo. Aorta from COX-2–/– mice our genes of interest (Prmt1, Agxt2, Ddah1, Ddah2, or Arg2) had a reduced eNOS response to acetylcholine (Figure 5A). were altered in the renal medulla from IP–/– mice (Figure VII However, their response to the NO-donor SNP that relaxes in the online-only Data Supplement). In line, the increase by guest on December 4, 2017 seen in ADMA levels associated with the loss of IP gene was, although statistically significant (Figure VIIIA in the online-only Data Supplement), minor in magnitude in com- parison with what we report in COX-2–/– mice (Figure VIIIB in the online-only Data Supplement). l-Arginine (Figure VIIIC in the online-only Data Supplement) and l-NMMA (Figure VIIID in the online-only Data Supplement) lev- els were decreased, whereas those of SDMA (Table II in the online-only Data Supplement) were not affected by the loss of IP receptors. These changes may reflect altered renal function in IP–/– mice, because creatinine levels were elevated in plasma (Figure VIIIE in the online-only Data Supplement).

Effect of Inhibition of COX-2 in Healthy Human Volunteers In direct corroboration of our findings in mice, the inhibition of COX-2 with standard anti-inflammatory doses of celecoxib (200 mg twice a day) for 7 days (n=8) increased ADMA levels in healthy human volunteers (Table). Naproxen (500 mg twice a day), which inhibits COX-2, but also inhibits COX-1, simi- larly increased ADMA levels in volunteers taking the drug for Figure 4. Effect of parecoxib on blood pressure and 7 days (n=8; Table). methylarginines. Effect of parecoxib on mean arterial blood pressure (A) and plasma ADMA (B) and l-NMMA (C) levels in mice. Data are mean±SEM for n=3 to 7. Data in A were analyzed Discussion by Quades 2-way ANOVA. Data in B and C were analyzed The notion that COX-2 is constitutively expressed in the by the Mann–Whitney U test. ADMA indicates asymmetrical dimethylarginine; ANOVA, analysis of variance; l-NMMA, kidney is not new and is, in fact, based on observations 32 monomethyl-l-arginine; and SEM, standard error of the mean. made as early as 1994, well before selective COX-2 638 Circulation February 17, 2015

Figure 5. Effect of COX-2 gene deletion of eNOS responses in aorta. Acetylcholine (ACh; A) or sodium nitroprusside (SNP; B) induced the relaxation of aorta from wild-type and COX-2–/– mice. Maximum relaxation responses of the aorta from wild-type and COX-2–/– mice induced by Ach (C) or SNP (D) in the absence or presence of l-arginine (100 μmol/L). Data are the mean±SEM for n=6 to 10. P values for A and C were by Quades 2-way ANOVA and for B and D were by Mann–Whitney U test. ANOVA indicates analysis of variance; COX, cyclooxygenase; and eNOS, endothelial nitric oxide synthase. Downloaded from

http://circ.ahajournals.org/ inhibitors were introduced to the clinic. Similarly, the idea as the heart and major blood vessels, conditional knockout that the inhibition of COX-2 in the kidney could explain mice where either endothelial26 or cardiomyocyte34 COX-2 the cardiovascular side effects of NSAIDs has been sug- is deleted have been used to implicate a role of COX-2 in gested.18–20 However, this view has been somewhat side- cardiovascular function in these structures; although, in the lined by the idea that vascular COX-2 drives prostacyclin heart, the findings with conditional knockout mice were throughout the systemic circulation,7 and, until our recent questioned and suggested to be confounded by the non- studies,14,33 the relative importance of the kidney in compar- specific effects of tamoxifen/cre toxicity.35 Nevertheless, to

by guest on December 4, 2017 ison with large vessels had not been specifically addressed. identify the gross effect of COX-2 at these sites in compari- Using a COX-2 luciferase knock-in mouse, we performed son with the renal medulla and to identify the novel mech- an unbiased systems-based analysis of COX-2 expression anisms of NSAIDs, we investigated the effects of COX-2 in an extensive panel of organs and tissues in the mouse gene deletion on the transcriptome. In line with constitu- and found that renal COX-2 was localized to an intense hot tive COX-2 gene activity that we recently reported,14 we spot in the center of the kidney, aligned to the inner region found that only 1 gene is altered in the heart and aorta of of the renal medulla. Here, we validated our earlier report COX-2–/– mice, whereas >600 genes were changed by >1.5- and used polymerase chain reaction to show that the COX-2 fold in the renal medulla by COX-2 deletion. Clearly, these (Ptgs2) gene is highly enriched in the medulla in compari- findings implicate a dominant role for constitutive COX-2 son with the renal cortex. This is in line with what is known in the kidney in comparison with the heart or aorta. This about COX-2 in the kidney, which under normal or high- may suggest that endothelial cells outside large vessels, salt conditions is predominantly expressed in the medulla potentially in the renal circulation, mediate the effects pre- region, particularly in the interstitial fibroblasts.14,16 viously reported.26 Because of the large number of genes Although COX-2 expression and activity are low relative altered in the renal medulla by COX-2 deletion, we chose to those of COX-1 in other cardiovascular structures, such to apply a focused gene pathway analysis of transcripts

Table. Plasma Methylarginines In Healthy Volunteers Taking Naproxen or Celecoxib

ADMA, μmol/L l-NMMA, μmol/L SDMA, μmol/L Creatinine (Fold Change) Control 0.81±0.044 0.30±0.024 0.44±0.063 1.00±0.11 Naproxen 1.01±0.049 0.31±0.020 0.58±0.049 1.51±0.05 (P<0.01) (P=0.84) (P=0.19) (P<0.01) Control 0.75±0.067 0.29±0.013 0.44±0.049 1.00±0.08 Celecoxib 0.90±0.050 0.33±0.025 0.51±0.045 1.55±0.08 (P=0.05) (P=0.31) (P=0.81) (P<0.01)

Methylarginine (ADMA, l-NMMA, and SDMA) and creatinine levels in the plasma of patients before (control) and after 7 days of standard anti-inflammatory dosing with naproxen or celecoxib (n=8). Data are represented as mean±SEM. P values by a Wilcoxon signed rank test in comparison with baseline values for each individual. ADMA indicates asymmetrical dimethylarginine; l-NMMA, monomethyl-l-arginine; SDMA, symmetrical dimethylarginine; and SEM, standard error of the mean. Ahmetaj-Shala et al Renal COX-2 and Regulation of ADMA and l-NMMA 639

relating to control of blood pressure, vascular tone, and vas- pressure31 similar in magnitude to that seen in the current cular hormones to extract leads for understanding how renal study. Further, we have demonstrated that plasma increases COX-2 can regulate systemic cardiovascular homeostasis. of ADMA and l-NMMA in Ddah1-deficient mice, in the Using this approach, we identified a number of groups of concentration range shown here in COX-2–/– mice, were genes, for example, related to ET and angiotensin signal- associated with vascular dysfunction manifesting as ing. Angiotensin genes were altered in a way that would not reduced acetylcholine-induced vasodilator responses in predict cardiovascular side effects and so were not pursued endothelium intact segments of aorta.31 In the aorta, ace- in this study. Changes in ET-1 genes may well be impor- tylcholine-induced vasodilation is mediated predominately tant in the effects of NSAIDs in some settings, but, in our by NO derived from eNOS, with no role for local prosta- model using healthy mice, the changes we found in the renal cyclin release. Thus, in this work, the loss of acetylcho- medulla did not ultimately affect plasma ET-1 levels or vas- line-induced vasodilation was attributed to the inhibition cular responses to ET-1 in the aorta ex vivo. We therefore of eNOS as a direct consequence of increased ADMA and focused our study around genes altered that regulate the l-NMMA.31 In the current study, we have taken a similar synthesis and metabolism of the endogenous eNOS inhibi- approach and found that aorta from COX-2–/– mice had a tors ADMA and l-NMMA. The kidney is a major site of reduced eNOS-dependent response to acetylcholine, but a methylarginine synthesis and clearance from the circula- normal response to the endothelial-independent NO donor tion and changes to these pathways have the potential to SNP. This observation is consistent with recent work from

Downloaded from systemically regulate vascular health. Specifically, Prmt1, others showing reduced eNOS activity in the aorta of COX- first identified because of its association with the TIS21 2–/– mice,26 although, in that study, a role for natural eNOS gene,36 methylates arginine residues in proteins leading to inhibitors was not addressed. ADMA and l-NMMA are the release of mature ADMA and l-NMMA after proteoly- competitive inhibitors of eNOS, and, as such, their effects sis, was increased by COX-2 deletion, whereas both Agxt237 are reversed when the natural substrate, l-arginine, is and Ddah131, enzymes that metabolize methylarginines, increased in the system.38 To establish the specific role of http://circ.ahajournals.org/ were decreased in COX-2–/– renal medulla. ADMA and l-NMMA in the loss of eNOS function in the To define the interaction between COX-2 and the methylar- aorta, in our study, we assessed the ability of l-arginine to ginine pathways and to validate biologically our gene expres- rescue the vascular dysfunction. In wild-type mouse aorta, sion data, we measured the levels of ADMA, SDMA, and l-arginine did not amplify acetylcholine-induced vasodila- l-NMMA. ADMA and l-NMMA are both competitive inhibi- tion, consistent with l-arginine availability not being rate tors of eNOS, whereas SDMA is biologically inactive and not limiting in healthy blood vessels.39 In contrast, l-arginine cleared by DDAH1. COX-1–/– mice exhibited normal renal, reversed the impairment in endothelial function present in by guest on December 4, 2017 plasma, and urinary levels of each methylarginine. In contrast, aorta from COX-2–/– mice; returning acetylcholine-depen- the plasma levels of ADMA and l-NMMA were increased, dent vasodilation to wild-type levels, without affecting whereas l-arginine was unchanged, in samples from COX-2–/– smooth muscle sensing of NO. It must be remembered that mice. These findings are in line with others showing increased this effect cannot be explained by any loss of COX-2 activ- methylarginines in models where the activity of PRMT1 is ity locally within the vessel, because, in this tissue, COX-1, elevated and AGXT2 is reduced.29 We found that SDMA is and not COX-2, is expressed.11,14 Rather, this is explained not significantly increased in COX-2–/– mouse plasma, impli- by changes in kidney function, leading to increased circu- cating an additional functional role for reduced DDAH1 in lating ADMA and l-NMMA, which then act in a endocrine this model. It should be noted that nonrenal factors may act manner on the endothelium in the systemic circulation. Our together with events in the kidney to regulate plasma ADMA findings that the reduced eNOS activity seen in vessels from in COX-2–/– mice. COX-2–/– mice could be prevented by exogenous l-arginine After demonstrating that COX-2 deletion increases clearly implicate a role for ADMA and l-NMMA and are ADMA and l-NMMA, we next sought to establish if in line with others showing that ADMA and l-NMMA spe- this could be recapitulated with a therapeutically rel- cifically and actively accumulate in endothelial cells and evant COX-2 selective drug in wild-type mice. We chose that this results in decreased eNOS activity ex vivo, which parecoxib, which is the prodrug of and a licensed is reversible by l-arginine.40 therapy for acute pain. It is highly COX-2 selective,1 and, Finally, to establish if the phenomenon demonstrated in because it is water soluble, it can be easily administered mice, ie, that loss/inhibition of COX-2 results in increased to mice in drinking water. Similar to our findings in COX- circulating levels of ADMA also occurs in humans, we per- 2–/– mice, pharmacological COX-2 inhibition by parecoxib formed a clinical study in which healthy male volunteers produced an increase in circulating levels of ADMA and were treated with standard doses of the selective COX-2 l-NMMA. This further supports our findings, confirming inhibitor celecoxib or naproxen, a dual COX-1/COX-2 that increased levels of endogenous NOS inhibitors is not inhibitor.1 At therapeutic doses, both of these drugs inhibit a developmental consequence of COX-2 deletion; rather, it COX-2 by ≈ 80%.1 Creatinine levels were increased to a is an acute biochemical consequence of the loss of COX-2 similar level by celecoxib and naproxen treatment high- activity. lighting the effect of COX-2 inhibition on renal function. In We have shown previously that the global heterozy- direct corroboration of our findings in mice, both celecoxib gous deletion of the Ddah1 gene results in increased lev- and naproxen increased ADMA levels. These observations els of plasma ADMA and l-NMMA and increased blood suggest that, in humans, after even short-term inhibition of 640 Circulation February 17, 2015

COX-2, renal dysfunction, including the reduction in filtra- are potential biomarkers for the cardiovascular risk in patients tion drive, increases the levels of ADMA. It is important to taking traditional NSAIDs, and COX-2 selective inhibitors, note, however, that ADMA, l-NMMA, and SDMA are simi- as well. In addition, if endogenous inhibitors of NOS are larly cleared by the kidney. Thus, the finding that ADMA is mechanistically involved in the cardiovascular toxicity asso- increased without a concomitant increase in other methylar- ciated with COX-2 inhibitor use in humans, then there is the ginines suggests that some additional specific pathways may very clear potential for l-arginine to be used as a preventa- be altered by COX-2 inhibition. We recognize that the rela- tive treatment in at-risk patients.50 Moreover, the potential for tive change in ADMA levels in our human study was much genetic mutations in genes such as PRMT1, DDAH1, and lower than seen in either COX-2–/– mice or in wild-type mice AGXT2 to be associated with increased risk of cardiovascular treated with a COX-2 inhibitor. We do not suggest that, in side effects in patients taking NSAIDs should be considered. this small sample size with healthy volunteers, the increase would be sufficient to adversely affect cardiovascular func- Acknowledgments tion, simply that, it provides a proof-of-concept study and We thank Hime Gashaw, Ivana Vojnovic, Mathew Delahaye, Olga that, in a much larger cohort, such as the Framingham Boruc, and Maxim Freydin for technical assistance. study41 and in patients with inflammation or cardiovascu- lar disease, this small but significant increase could account Sources of Funding for the equally small but significant increase in cardio- This work was supported by the Wellcome Trust (0852551Z108/Z),

Downloaded from vascular events seen in patients taking anti-inflammatory the BHF (FS/12/53/29643), and MRC Intramural funding. drugs, including COX-2 inhibitors. Indeed, as reported in the Framingham study41 in large populations, even rela- Disclosures tively small changes in ADMA levels are associated with Dr Mitchell is a member of the scientific advisory board for Antibe significant increases in the risk of cardiovascular events, Therapeutics, and Drs Mitchell and Warner have been expert wit- and the plasma levels of ADMA and l-NMMA, which oth- nesses for cases associated with Pfizer products. The other authors http://circ.ahajournals.org/ report no conflicts. ers have shown specifically can accumulate in endothelial cells,40 are highly correlated with cardiovascular disease in a range of human clinical studies.42–46 This is relevant because References COX-2 selective drugs, including celecoxib and rofecoxib, 1. Warner TD, Giuliano F, Vojnovic I, Bukasa A, Mitchell JA, Vane JR. Nonsteroid drug selectivities for cyclo-oxygenase-1 rather than cyclo- increase the risk of cardiovascular events even in healthy oxygenase-2 are associated with human gastrointestinal toxicity: a full in individuals – that is to say, in those people without overt vitro analysis. Proc Natl Acad Sci U S A. 1999;96:7563–7568. evidence of cardiovascular disease, requiring medication, 2. Wallace JL, McKnight W, Reuter BK, Vergnolle N. NSAID-induced gas-

by guest on December 4, 2017 tric damage in rats: requirement for inhibition of both cyclooxygenase 1 or recent previous events, because these individuals were and 2. Gastroenterology. 2000;119:706–714. excluded from trials. Furthermore, although the efficacy tri- 3. Wallace JL. Prostaglandins, NSAIDs, and gastric mucosal protection: why als for COX-2 inhibitors were performed in patients with doesn’t the stomach digest itself? Physiol Rev. 2008;88:1547–1565. doi: arthritis,47 who are commonly in a category of increased 10.1152/physrev.00004.2008. 4. Mitchell JA, Warner TD. Cyclo-oxygenase-2: pharmacology, physiol- risk of cardiovascular disease owing to general inflamma- ogy, biochemistry and relevance to NSAID therapy. Br J Pharmacol. tion and age, the Adenomatous Polyp Prevention on Vioxx 1999;128:1121–1132. doi: 10.1038/sj.bjp.0702897. (APPROVE)48 (rofecoxib) and Adenoma Prevention With 5. Antman EM, Bennett JS, Daugherty A, Furberg C, Roberts H, Taubert Celecoxib (APC)49 (celecoxib) trials were conducted on vol- KA; American Heart Association. Use of nonsteroidal antiinflammatory drugs: an update for clinicians: a scientific statement from the American unteers without arthritis. In both the APPROVE and APC Heart Association. Circulation. 2007;115:1634–1642. doi: 10.1161/ trials, increased cardiovascular events were noted, which in CIRCULATIONAHA.106.181424. the case of rofecoxib lead to the withdrawal of the drug. 6. Warner TD, Mitchell JA. COX-2 selectivity alone does not define the car- –/– diovascular risks associated with non-steroidal anti-inflammatory drugs. The differences seen in ADMA in COX-2 mice and Lancet. 2008;371:270–273. doi: 10.1016/S0140-6736(08)60137-3. human volunteers taking NSAIDs for 1 week are probably 7. Funk CD, FitzGerald GA. COX-2 inhibitors and cardiovascu- explained by the duration and extent of loss of COX-2 activity. lar risk. J Cardiovasc Pharmacol. 2007;50:470–479. doi: 10.1097/ The relatively small increase in ADMA seen in humans versus FJC.0b013e318157f72d. 8. McGettigan P, Henry D. Cardiovascular risk with non-steroidal anti- mice administered inhibitors is less obviously explained, but inflammatory drugs: systematic review of population-based controlled it could be due to species differences and the fact that humans observational studies. PLoS Med. 2011;8:e1001098. doi: 10.1371/journal. are outbred, and so responses are likely to be more variable. In pmed.1001098. addition, mice treated with parecoxib were chronically instru- 9. Kohli P, Steg PG, Cannon CP, Smith SC Jr, Eagle KA, Ohman EM, Alberts MJ, Hoffman E, Guo J, Simon T, Sorbets E, Goto S, Bhatt DL; REACH mented, which may also influence responses. Registry Investigators. NSAID use and association with cardiovascular Methylarginines inhibit eNOS, reducing NO released outcomes in outpatients with stable atherothrombotic disease. Am J Med. across the entire vascular tree. NO performs functions similar 2014;127:53–60.e1. doi: 10.1016/j.amjmed.2013.08.017. 10. Wong E, Huang JQ, Tagari P, Riendeau D. Effects of COX-2 inhibitors on to prostacyclin in vessels and platelets. Moreover, NO and aortic prostacyclin production in cholesterol-fed rabbits. Atherosclerosis. prostacyclin synergize strongly to inhibit platelet activation.27 2001;157:393–402. Our data provide the first explanation for how the inhibition 11. Kirkby NS, Lundberg MH, Harrington LS, Leadbeater PD, Milne of COX-2 in the kidney can lead to reduced vascular function GL, Potter CM, Al-Yamani M, Adeyemi O, Warner TD, Mitchell JA. Cyclooxygenase-1, not cyclooxygenase-2, is responsible for physiological and firmly establish a COX-2/eNOS axis involving ADMA. production of prostacyclin in the cardiovascular system. Proc Natl Acad Our results also show that ADMA and other methylarginines Sci U S A. 2012;109:17597–17602. doi: 10.1073/pnas.1209192109. Ahmetaj-Shala et al Renal COX-2 and Regulation of ADMA and l-NMMA 641

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Clinical Perspective Nonsteroidal anti-inflammatory drugs are the most common over-the-counter medications worldwide and the mainstay ther- apy for those with chronic inflammatory conditions such as arthritis. Cyclooxygenase (COX)-2 is the therapeutic target of nonsteroidal anti-inflammatory drugs, but these drugs also inhibit the constitutive form COX-1, potentially causing serious gastrointestinal side effects. COX-2 selective drugs, such as celecoxib and rofecoxib, were introduced to reduce gastrointes- tinal side effects. However, it is now clear that COX-2 selective anti-inflammatory drugs and older-style nonsteroidal anti- inflammatory drugs like and increase the risk of cardiovascular events. These side effects are relatively rare, but concern over them has meant that there has been a return to prescribing older style drugs, which generally carry a similar risk of heart attacks and strokes but with the added disadvantage of increased gastrointestinal side effects. Currently there is no accepted unifying mechanism to explain how COX-2 inhibitors cause cardiovascular side effects, and, conse-

Downloaded from quently, there are no biomarkers or rescue strategies to identify or treat those few individuals who might be at risk. Our study shows methylarginines, which block cardioprotective endothelial nitric oxide synthase in vessels, are elevated when COX-2 is inhibited. Methylarginines are elevated in a range of cardiovascular diseases and as such are considered risk factors. Our work suggests methylarginines may also be biomarkers of the risk associated with COX-2 inhibition. If these findings are validated in a larger patient group, a simple test for methylarginines could be used to identify those individuals most at risk for cardiovascular events while taking COX-2 inhibitors. http://circ.ahajournals.org/ by guest on December 4, 2017 Evidence That Links Loss of Cyclooxygenase-2 With Increased Asymmetric Dimethylarginine: Novel Explanation of Cardiovascular Side Effects Associated With Anti-Inflammatory Drugs Blerina Ahmetaj-Shala, Nicholas S. Kirkby, Rebecca Knowles, Malak Al'Yamani, Sarah Mazi, Zhen Wang, Arthur T. Tucker, Louise Mackenzie, Paul C. J. Armstrong, Rolf M. Nüsing, James

Downloaded from A. P. Tomlinson, Timothy D. Warner, James Leiper and Jane A. Mitchell

Circulation. 2015;131:633-642; originally published online December 9, 2014; doi: 10.1161/CIRCULATIONAHA.114.011591 Circulation is published by the American Heart Association, 7272 Greenville Avenue, Dallas, TX 75231 http://circ.ahajournals.org/ Copyright © 2014 American Heart Association, Inc. All rights reserved. Print ISSN: 0009-7322. Online ISSN: 1524-4539

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SUPPLEMENTARY MATERIAL p=0.99 (a) ) 6 p=0.38 ml / pg

( 4

2

Plasma [ET-1] Plasma 0

Wild-type COX-1-/- COX-2-/-

(b) 140 COX-2+/+ 120 p=0.65 COX-2-/- 100 80 60 Force 40 20 (% of KPSS response) KPSS of (% 0

-11 -10 -9 -8 -7

[ET-1] (log M)

Supplementary Fig. 1: Effect of COX-1 and COX-2 gene deletion on (a) circulating endothelin-1 (ET-1) and (b) contractile effect of ET-1 on aorta. Aorta were incubated in Krebs’ buffer containing 100 µM L- NGmonomethyl-L-arginine to inhibit eNOS activity. Data is the mean ± S.E.M for (a) n=8-9 mice and (b) n=4 vessels from 2 mice. Data was analysed using (a) Kruskil-Wallis with Dunn’s post-hoc test and (b) by Quades two-way ANOVA. p=0.10 2.0

1.5

1.0

(fold-change) 0.5 Ddah2 mRNA Ddah2

0.0

COX-2+/+ COX-2-/-

Supplementary Fig 2: Effect of COX-2 gene deletion on Ddah2 expression in the renal medulla. The data is the mean mean ± S.E.M for n=7. Data was analysed using a Mann-Whitney U-test. (a) Renal Medulla (b) Aorta p=0.13 1.5 p=0.19 4

3 1.0

2 0.5 Arg2 mRNA Arg1 mRNA 1 (aorta; fold-change) (aorta;

(renal medulla; fold-change) medulla; (renal 0.0 0

+/+ -/- COX-2 COX-2 COX-2+/+ COX-2-/- (c) (d) p=0.63 0.8

M) 20

µ p=0.03 ( 15 0.6

10 0.4

5 0.2

0 ADMA : L-arginine ratio 0.0 Plasma [L-arginine] Plasma

COX-2+/+ COX-2-/- COX-2+/+ COX-2-/-

(e) p=0.03 0.20

0.15

0.10

0.05

0.00 L-arginine : L-NMMA ratio L-NMMA : L-arginine

COX-2+/+ COX-2-/-

Supplementary Fig. 3: Effect of COX-2 gene deletion on the expression of (a) Arg2 in the renal medulla, (b) Arg1 in the aorta and plasma levels of (c) L-arginine, (d) ratio of ADMA: L-arginine and (e) L-NMMA:L- arginine in the plasma. The data is the mean ± S.E.M for (a) and (b) n=7-8, (c-e) n=4. Data was analysed Mann-Whitney U-test. (a) (b)

M) 250 p=0.70 µ 0.015

( p=0.009p<0.01 200

150 0.010

100 0.005 50

0 ADMA : L-arginine ratio 0.000 Plasma [L-arginine] Plasma

Vehicle Parecoxib Vehicle Parecoxib (c)

0.015 p=0.006p<0.01

0.010

0.005

0.000 L-NMMA : L-arginine ratio L-arginine : L-NMMA

Vehicle Parecoxib

Supplementary Fig. 4: Effect of parecoxib (100 mg/kg/day) compared to vehicle (water) on plasma levels of (a) L-arginine and (b) ADMA:L-arginine and (c) L-NMMA:L-arginine ratios in plasma of wild type mice. The data is the mean ± S.E.M n=3-7. Data was anlaysed using a Mann-Whitney U-test. 20 COX-2+/+ p=0.12 COX-2-/- 15

(mN) 10

Force 5

0

-9 -8 -7 -6

[U46619] (log M)

Supplementary Fig. 5: Effect of COX-2 gene deletion on the contractile responses of U46619 in aorta. The data the mean ± S.E.M for n=6-10. Data was analysed using a Quades two-way ANOVA. (a) (b) p=0.13 2.0 3 p=0.09

1.5 2 1.0 1 (fold-change) (fold-change)

Nos3 mRNA Nos3 0.5 Ddah1 mRNA Ddah1

0.0 0

COX-2+/+ COX-2-/- COX-2+/+ COX-2-/-

(c) p=0.71 (d) 2.0 2.0 p=0.78

1.5 1.5

1.0 1.0 (fold-change) (fold-change)

0.5 Agxt2 mRNA 0.5 Ddah2 mRNA Ddah2

0.0 0.0

COX-2+/+ COX-2-/- COX-2+/+ COX-2-/-

Supplementary Fig. 6: Effect of COX-2 gene deletion on aortic expression of (a) Nos3, (b) Ddah1, (c) Ddah2 and (d) Agxt2. The data is the mean ± S.E.M for n=5-7 mice. Data was analysed using a Mann- Whitney U-test (a) (b) p=0.32 p=0.61 1.5 2.5

2.0 1.0 1.5

1.0 0.5 (fold-change) (fold-change) Prmt1 mRNA Prmt1 Agxt2 mRNA 0.5

0.0 0.0

+/+ -/- IP IP IP+/+ IP-/-

(c) (d) p=0.75 p=0.32 2.0 2.5

2.0 1.5 1.5 1.0 1.0 (fold-change) (fold-change)

0.5 mRNA Ddah2 Ddah1 mRNA Ddah1 0.5

0.0 0.0

IP+/+ IP-/- IP+/+ IP-/-

(e) p=0.49 1.5

1.0

0.5 (fold-change) Arg2 mRNA

0.0

IP+/+ IP-/-

Supplementary Fig. 7: Effect of IP gene deletion on the expression of (a) Prmt1, (b) Agxt2, (c) Ddah1, (d) Ddah2 and (e) Arg2 in the renal medulla. The data is the mean ± S.E.M n=8. Data was analysed using a Mann-Whitney U-test. (a) (b) p=0.03 10 p=0.0002p<0.01 20 M) µ 8 ( 15 6 10 4 p=0.0002p<0.01 5 2 Plasma [ADMA] Plasma (fold-change vs. control) vs. (fold-change Plasma [ADMA] Plasma 0 0

IP+/+ IP-/- IP+/+ IP-/- COX-2+/+ COX-2-/- (c) (d) p<0.01 M) 20 0.4 p=0.002 M) µ µ

( p=0.03 ( 15 0.3

10 0.2

5 0.1

0

Plasma [L-NMMA] Plasma 0.0 Plasma [L-arginine] Plasma

+/+ -/- IP IP IP+/+ IP-/-

(e)

M) 80 p=0.0006p<0.01 µ ( 60

40

20

0 Plasma [creatinine] Plasma

IP+/+ IP-/-

Supplementary Fig. 8: (a) Effect of IP gene deletion on plasma levels of ADMA. (b) Comparison of the effect of IP gene deletion versus COX-2 gene deletion on relative (fold) change in ADMA. (c) Effect of IP gene deletion on plasma levels of (c) L-arginine, (d) L-NMMA and (e) creatinine. The data is the mean ± S.E.M for n=8. Data was analysed using Mann-Whitney U-test Fold- Fold- Fold- Gene$Symbol$ Gene$Symbol$ Gene$Symbol$ Change$ Change$ Change$ Igfbp2' 3.2' Nupr1' 1.8' Spon2' 1.7' Upk2' 2.8' ' Arhgef4' 1.8' ' Spnb3' 1.7' ' Psca' 2.5' ' Stab1' 1.8' ' Sppl3' 1.7' ' Nrip3' 2.5' ' Ccdc3' 1.8' ' Sepw1' 1.7' ' Neurl' 2.4' ' Lgals3' 1.8' ' Pea15' 1.7' ' Rgs4' 2.4' ' Gpr123' 1.8' ' Kdelr3' 1.7' ' Egr1' 2.4' ' Jun' 1.8' ' Susd4' 1.7' ' Lypd2' 2.4' ' Col6a1' 1.8' ' Ctsk' 1.7' ' Npy' 2.3' ' Spon1' 1.8' ' Ptprb' 1.7' ' Lyz' 2.2' ' Slc24a3' 1.8' ' Rasl11b' 1.7' ' Spnb1' 2.2' ' Ophn1' 1.8' ' Ssbp4' 1.7' ' Cacna2d1' 2.1' ' Gprc5a' 1.8' ' Samd14' 1.7' ' Nbl1' 2.1' ' Elf3' 1.8' ' Abi3' 1.7' ' Loc100047934' 2.1' ' F13a1' 1.8' ' Fst' 1.7' ' Aoc3' 2.1' ' Serpinf1' 1.8' ' Tnrc6c' 1.7' ' Fbn1' 2.1' ' Asah3l' 1.8' ' Itgb4' 1.7' ' Lynx1' 2.1' ' Cntn1' 1.8' ' Cfp' 1.7' ' Ndn' 2.1' ' Tmem118' 1.8' ' D12ertd553e' 1.7' ' Aatk' 2.1' ' Megf9' 1.8' ' S100a13' 1.7' ' Purb' 2.0' ' Ptprs' 1.8' ' Txnip' 1.7' ' Zeb2' 2.0' ' Scara3' 1.8' ' Mmp17' 1.7' ' Upk1b' 2.0' ' Serpina3n' 1.8' ' Anxa2' 1.7' ' Lcn2' 2.0' ' D0h4s114' 1.8' ' Bok' 1.7' ' Sprr1a' 2.0' ' Cyp1b1' 1.8' ' Serinc2' 1.7' ' Klf5' 2.0' ' Gja1' 1.8' ' Aqp2' 1.7' ' Satb1' 2.0' ' Rhoj' 1.8' ' Loc100045019' 1.7' ' Ankrd1' 1.9' ' Vwf' 1.8' ' Cdkn1c' 1.7' ' Bc025575' 1.9' ' Loc100046044' 1.8' ' Stat3' 1.7' ' Cldn5' 1.9' ' Loc100043671' 1.8' ' Axin2' 1.7' ' Lyzs' 1.9' ' Wnt7b' 1.8' ' Pof1b' 1.7' ' Chst1' 1.9' ' Slc44a2' 1.8' ' Laptm5' 1.7' ' Flrt3' 1.9' ' P2ry6' 1.8' ' F2r' 1.7' ' Loc100047200' 1.9' ' Bcl9l' 1.8' ' Endod1' 1.7' ' Krt18' 1.9' ' Klf6' 1.8' ' Atf5' 1.7' ' Fxyd6' 1.9' ' Fscn1' 1.8' ' C030011o14rik' 1.7' ' Rab15' 1.9' ' Cldn4' 1.8' ' D16ertd472e' 1.7' ' Grit' 1.9' ' E430002d04rik' 1.8' ' Hist1h2af' 1.7' ' Tmem119' 1.9' ' Dusp7' 1.7' ' Stxbp1' 1.7' ' Col16a1' 1.9' ' Upk3a' 1.7' ' Ckb' 1.7' ' Krt14' 1.9' ' Cygb' 1.7' ' 2310022b05rik' 1.7' ' Nav1' 1.9' ' Il18r1' 1.7' ' Tmem110' 1.7' ' Loc100048721' 1.9' ' Hist1h2an' 1.7' ' Ap3m2' 1.7' ' Mmp14' 1.9' ' Gnaz' 1.7' ' Gjb6' 1.7' ' Scarf2' 1.9' ' Rgs5' 1.7' ' Ankrd47' 1.7' ' 9130213b05rik' 1.9' ' Pip4k2a' 1.7' ' Col1a2' 1.7' ' Ccdc80' 1.9' ' Ppp1r9b' 1.7' ' Des' 1.7' ' Ddit4' 1.8' ' Zfp52' 1.7' ' H2^aa' 1.7' ' ' ' ' Fold- Fold- Fold- Gene$Symbol$ Gene$Symbol$ Gene$Symbol$ Change$ Change$ Change$ Gp38' 1.7' Fxyd4' 1.6' Myst4' 1.6' Ppp1r3c' 1.7' ' D6wsu176e' 1.6' ' Gata2' 1.6' ' Emid2' 1.7' ' Slc1a3' 1.6' ' Scx' 1.6' ' Crip2' 1.6' ' Kpnb1' 1.6' ' Sidt1' 1.6' ' Notch1' 1.6' ' Ahi1' 1.6' ' A230050p20rik' 1.6' ' Ier3' 1.6' ' Edg2' 1.6' ' Npnt' 1.6' ' Fcho1' 1.6' ' 4930572j05rik' 1.6' ' Traf7' 1.6' ' Slco4a1' 1.6' ' Cugbp2' 1.6' ' Spire2' 1.6' ' Lrrc26' 1.6' ' Loc100045343' 1.6' ' Esam1' 1.6' ' Nnmt' 1.6' ' Tcof1' 1.6' ' Magee1' 1.6' ' Tiam1' 1.6' ' Loc676420' 1.6' ' Pkm2' 1.6' ' Actb' 1.6' ' Phc2' 1.6' ' Xlr4a' 1.6' ' Prkcbp1' 1.6' ' Ccdc85b' 1.6' ' Loc100042777' 1.6' ' Dkk3' 1.6' ' Adora1' 1.6' ' Ppap2b' 1.6' ' Myo9b' 1.6' ' Grasp' 1.6' ' Tspan17' 1.6' ' Dusp26' 1.6' ' Pdlim1' 1.6' ' Itga3' 1.6' ' Slc2a6' 1.6' ' Gja5' 1.6' ' Serf2' 1.6' ' Pitpnm2' 1.6' ' Sh3pxd2b' 1.6' ' Csf1' 1.6' ' 2310010m24rik' 1.6' ' Rcan2' 1.6' ' Sh3bgrl3' 1.6' ' Adamts2' 1.6' ' Sepn1' 1.6' ' Pip4k2b' 1.6' ' Pitx2' 1.6' ' Prelp' 1.6' ' Psmb10' 1.6' ' Pfkl' 1.6' ' Dusp3' 1.6' ' Hn1' 1.6' ' Bace1' 1.6' ' Capn13' 1.6' ' Bc021381' 1.5' ' Csnk1e' 1.6' ' Mmp2' 1.6' ' Col18a1' 1.5' ' Ccnd2' 1.6' ' Irf2bp1' 1.6' ' Btg1' 1.5' ' Chrnb1' 1.6' ' Sema3f' 1.6' ' Notch4' 1.5' ' Gstm2' 1.6' ' Hcn2' 1.6' ' Rapgefl1' 1.5' ' Efemp2' 1.6' ' Rgs10' 1.6' ' Fmnl3' 1.5' ' Dag1' 1.6' ' 9130404d14rik' 1.6' ' Cldn23' 1.5' ' Cav1' 1.6' ' Csrp1' 1.6' ' Upk1a' 1.5' ' Myd116' 1.6' ' Pik3r1' 1.6' ' Dusp8' 1.5' ' Pdgfra' 1.6' ' Pls3' 1.6' ' Phactr1' 1.5' ' Scnn1b' 1.6' ' Mbc2' 1.6' ' Mospd3' 1.5' ' Slc38a2' 1.6' ' Dab2ip' 1.6' ' Foxj2' 1.5' ' Rbms3' 1.6' ' Meis1' 1.6' ' Smarcd2' 1.5' ' Zfp46' 1.6' ' Axl' 1.6' ' Arrdc2' 1.5' ' Pdlim7' 1.6' ' Edn1' 1.6' ' Rem1' 1.5' ' Cxcl4' 1.6' ' Mef2c' 1.6' ' Slc9a1' 1.5' ' Kctd10' 1.6' ' Pold1' 1.6' ' Pdxp' 1.5' ' Wfdc2' 1.6' ' Ehd4' 1.6' ' Pacs1' 1.5' ' St6gal1' 1.6' ' Tmem23' 1.6' ' Mmd' 1.5' ' Cbr2' 1.6' ' Mmrn2' 1.6' ' Olfml3' 1.5' ' Bat2' 1.6' ' Bc063749' 1.6' ' Akap8l' 1.5' ' Timp2' 1.6' ' Mcl1' 1.6' ' Hoxd4' 1.5' ' Sort1' 1.6' ' Bc057552' 1.6' ' Pygo2' 1.5' ' Ccnd1' 1.6' ' Large' 1.6' ' Mfap2' 1.5' ' Smpdl3b' 1.6' ' Rassf5' 1.6' ' 8430408g22rik' 1.5' ' ' ' ' Fold- Fold- Fold- Gene$Symbol$ Gene$Symbol$ Gene$Symbol$ Change$ Change$ Change$ Vamp2' 1.5' Slco2a1' ^1.5' Fads2' ^1.5' Cox4i2' 1.5' ' Gpr177' ^1.5' ' Aadacl1' ^1.5' ' Ahdc1' 1.5' ' Syap1' ^1.5' ' Strap' ^1.5' ' Ptk7' 1.5' ' Dock1' ^1.5' ' Gm2a' ^1.5' ' Arid2' 1.5' ' Mto1' ^1.5' ' Rheb' ^1.5' ' Ptplad2' 1.5' ' Tmem174' ^1.5' ' 1810049h13rik' ^1.5' ' Tubb2b' 1.5' ' Commd1' ^1.5' ' Loc100047261' ^1.5' ' Fpr2' 1.5' ' 1110031b06rik' ^1.5' ' Tnfrsf21' ^1.5' ' Socs3' 1.5' ' Ppp1ca' ^1.5' ' Surf1' ^1.5' ' Hmha1' 1.5' ' Ppapdc1' ^1.5' ' Ppm1g' ^1.5' ' Zfhx3' 1.5' ' Tpk1' ^1.5' ' Eg434858' ^1.5' ' Cdc42ep5' 1.5' ' Acox2' ^1.5' ' Glb1l2' ^1.5' ' Tcf4' 1.5' ' Spg21' ^1.5' ' Hspe1' ^1.5' ' Atox1' 1.5' ' Cul3' ^1.5' ' 4930403o06rik' ^1.5' ' Vat1' 1.5' ' Pycrl' ^1.5' ' Mgst1' ^1.5' ' Cpne2' 1.5' ' Atpif1' ^1.5' ' Dnajb9' ^1.5' ' Mmp23' 1.5' ' Nrbf2' ^1.5' ' Snx6' ^1.5' ' Dpt' 1.5' ' Pdss1' ^1.5' ' Ttc8' ^1.5' ' Tkt' 1.5' ' Mcat' ^1.5' ' Pdcl' ^1.5' ' Fxyd5' 1.5' ' 2410022l05rik' ^1.5' ' Med21' ^1.5' ' Scamp5' 1.5' ' Fcgr2b' ^1.5' ' Rpl31' ^1.5' ' Atp6v0a1' 1.5' ' Creld2' ^1.5' ' Slc6a20b' ^1.5' ' Ednrb' 1.5' ' Eif4g2' ^1.5' ' Fgf9' ^1.5' ' S100a6' 1.5' ' Hax1' ^1.5' ' Adfp' ^1.5' ' Hexa' 1.5' ' Rps27a' ^1.5' ' Slc41a2' ^1.5' ' 2300002d11rik' 1.5' ' D10bwg1364e' ^1.5' ' Fga' ^1.5' ' Zfp259' 1.5' ' Acad10' ^1.5' ' Slc9a8' ^1.5' ' Smtn' 1.5' ' 1500032l24rik' ^1.5' ' Gng10' ^1.5' ' Aip' 1.5' ' Sdf2' ^1.5' ' 5730469m10rik' ^1.5' ' Stx12' 1.5' ' Loc100048331' ^1.5' ' Loc677528' ^1.5' ' Spink8' 1.5' ' Ctsf' ^1.5' ' Tmem159' ^1.5' ' Loc100047619' 1.5' ' Xdh' ^1.5' ' Dcun1d4' ^1.5' ' Tubb6' 1.5' ' 2810439f02rik' ^1.5' ' Mboat5' ^1.5' ' Bmpr1b' 1.5' ' Letm1' ^1.5' ' Dnajb12' ^1.5' ' Nfatc4' 1.5' ' Slc5a9' ^1.5' ' Rab38' ^1.5' ' Loc641240' 1.5' ' Ak2' ^1.5' ' Rpl36al' ^1.5' ' Brd2' 1.5' ' Zc3h14' ^1.5' ' Cyp27a1' ^1.5' ' Slc4a2' 1.5' ' E030010a14rik' ^1.5' ' Iyd' ^1.5' ' Capn5' 1.5' ' Caml' ^1.5' ' Tm2d3' ^1.5' ' Rab31' 1.5' ' Hpn' ^1.5' ' Atp5s' ^1.5' ' Rgma' 1.5' ' Cd55' ^1.5' ' Sat2' ^1.5' ' Atpbd1b' 1.5' ' Zfp207' ^1.5' ' Uqcrc2' ^1.6' ' Gfod2' 1.5' ' Ccnc' ^1.5' ' Mrpl30' ^1.6' ' Efna5' 1.5' ' Sod1' ^1.5' ' Nsdhl' ^1.6' ' Krt7' 1.5' ' Errfi1' ^1.5' ' Nqo1' ^1.6' ' Flywch2' 1.5' ' Aqp7' ^1.5' ' Med10' ^1.6' ' Cdh1' 1.5' ' Akr1c14' ^1.5' ' Epdr1' ^1.6' ' ' ' ' Fold- Fold- Fold- Gene$Symbol$ Gene$Symbol$ Gene$Symbol$ Change$ Change$ Change$ Ubr1' ^1.6' Exoc4' ^1.6' Dusp28' ^1.6' Chchd4' ^1.6' ' Cpn1' ^1.6' ' Loc668492' ^1.6' ' Ndufa9' ^1.6' ' Tmem5' ^1.6' ' 1110038d17rik' ^1.6' ' Ogfrl1' ^1.6' ' Susd3' ^1.6' ' Metap1' ^1.6' ' Eno1' ^1.6' ' Msrb2' ^1.6' ' Mettl9' ^1.6' ' Rnaset2b' ^1.6' ' Psmd12' ^1.6' ' Tmem9b' ^1.6' ' Arf5' ^1.6' ' Insig2' ^1.6' ' Ubl5' ^1.6' ' Suclg1' ^1.6' ' Gstt2' ^1.6' ' Stx18' ^1.6' ' Wdr55' ^1.6' ' Dnajc15' ^1.6' ' Rcan1' ^1.6' ' Loc667370' ^1.6' ' Nsbp1' ^1.6' ' Arl3' ^1.6' ' Sri' ^1.6' ' Cobll1' ^1.6' ' Capns1' ^1.6' ' Tceal8' ^1.6' ' Prosc' ^1.6' ' Mrps16' ^1.6' ' 4933434e20rik' ^1.6' ' Rala' ^1.6' ' Akr1c12' ^1.6' ' Fundc1' ^1.6' ' Bxdc2' ^1.6' ' Tmem77' ^1.6' ' Cldn1' ^1.6' ' Eif2b1' ^1.6' ' Ranbp9' ^1.6' ' Pfkp' ^1.6' ' P2rx4' ^1.6' ' Zfyve20' ^1.6' ' Net1' ^1.6' ' Htatip2' ^1.6' ' Ddx1' ^1.6' ' L7rn6' ^1.6' ' Maea' ^1.6' ' Agps' ^1.6' ' Decr2' ^1.6' ' Kctd12' ^1.6' ' Calm2' ^1.6' ' Arpc1a' ^1.6' ' Iars2' ^1.6' ' Anpep' ^1.6' ' Tmem85' ^1.6' ' Sypl2' ^1.6' ' Serpina1f' ^1.6' ' Pdzd11' ^1.6' ' Vps29' ^1.6' ' Gchfr' ^1.6' ' Ostf1' ^1.6' ' Snrpd1' ^1.6' ' Ccdc47' ^1.6' ' Fmo4' ^1.6' ' Bc048355' ^1.6' ' Ostm1' ^1.6' ' Pcmt1' ^1.6' ' Napsa' ^1.6' ' 2010316f05rik' ^1.7' ' Hist1h4h' ^1.6' ' Htra1' ^1.6' ' Thoc4' ^1.7' ' Dynll2' ^1.6' ' Rab1b' ^1.6' ' Ndufa4' ^1.7' ' Dpy30' ^1.6' ' 2810008m24rik' ^1.6' ' Nxt2' ^1.7' ' 5033414d02rik' ^1.6' ' Atp5d' ^1.6' ' Nbr1' ^1.7' ' Dab2' ^1.6' ' Nsmaf' ^1.6' ' 1300018j18rik' ^1.7' ' Als2cr2' ^1.6' ' Hagh' ^1.6' ' Top1' ^1.7' ' Rps12' ^1.6' ' Cyp4f14' ^1.6' ' Agpat2' ^1.7' ' Steap2' ^1.6' ' Pitpnc1' ^1.6' ' Rnf11' ^1.7' ' Slc25a33' ^1.6' ' Efr3a' ^1.6' ' Echs1' ^1.7' ' Slc35a1' ^1.6' ' 1700034h14rik' ^1.6' ' Tspo' ^1.7' ' Tmem131' ^1.6' ' Tbca' ^1.6' ' Entpd5' ^1.7' ' Hpd' ^1.6' ' Gtpbp4' ^1.6' ' Arl6ip5' ^1.7' ' Papss2' ^1.6' ' Ppp2cb' ^1.6' ' Afmid' ^1.7' ' Tomm7' ^1.6' ' Acn9' ^1.6' ' Loc100046457' ^1.7' ' Yif1a' ^1.6' ' 4931417g12rik' ^1.6' ' Rabggtb' ^1.7' ' Cyb5r4' ^1.6' ' Gjb2' ^1.6' ' Atp5j' ^1.7' ' Mrps31' ^1.6' ' Fbxo9' ^1.6' ' Nat2' ^1.7' ' Actn4' ^1.6' ' Slc34a3' ^1.6' ' Hsd17b2' ^1.7' ' Rab17' ^1.6' ' Bc026682' ^1.6' ' Hdhd2' ^1.7' ' Sypl' ^1.6' ' Hmox2' ^1.6' ' Pcbd1' ^1.7' ' Atp6v0e' ^1.6' ' A030007l17rik' ^1.6' ' 2510010f15rik' ^1.7' ' Mrpl40' ^1.6' ' Loc100047012' ^1.6' ' Fkbp2' ^1.7' ' ' ' ' Fold- Fold- Fold- Gene$Symbol$ Gene$Symbol$ Gene$Symbol$ Change$ Change$ Change$ Sec61b' ^1.7' Qdpr' ^1.7' 0610012d14rik' ^1.8' Tmem106a' ^1.7' ' Lpar3' ^1.7' ' Rps16' ^1.8' ' Car3' ^1.7' ' Car12' ^1.7' ' Eg632778' ^1.8' ' 1810022c23rik' ^1.7' ' Ncoa4' ^1.7' ' Tfam' ^1.8' ' Galnt1' ^1.7' ' Gnas' ^1.7' ' Sqstm1' ^1.8' ' Synj2bp' ^1.7' ' Trappc4' ^1.7' ' Proc' ^1.8' ' Bc040758' ^1.7' ' Ufsp2' ^1.7' ' Bud31' ^1.8' ' Lrrc8' ^1.7' ' 1500031l02rik' ^1.7' ' Socs2' ^1.8' ' Col4a5' ^1.7' ' Cideb' ^1.7' ' Echdc2' ^1.8' ' Rpl27' ^1.7' ' Gm1821' ^1.7' ' Eg622339' ^1.8' ' Dtymk' ^1.7' ' Tmem166' ^1.7' ' Ndufa12' ^1.8' ' 1110019n10rik' ^1.7' ' Ppp1cb' ^1.7' ' Kl' ^1.8' ' Ndufc1' ^1.7' ' Mcts1' ^1.7' ' Plscr1' ^1.8' ' Mlstd2' ^1.7' ' Mpst' ^1.7' ' Pgam1' ^1.8' ' Psen1' ^1.7' ' Clptm1' ^1.7' ' Fh1' ^1.8' ' Ly6c1' ^1.7' ' Gcdh' ^1.7' ' Rnf4' ^1.8' ' 2410129h14rik' ^1.7' ' Tmem116' ^1.7' ' Loc100048480' ^1.8' ' 2310045a20rik' ^1.7' ' Mdh1' ^1.7' ' Atp6v1g1' ^1.8' ' Hmgn2' ^1.7' ' Klk1b5' ^1.7' ' Ddx52' ^1.8' ' Fbxo3' ^1.7' ' Rbbp7' ^1.7' ' Coasy' ^1.8' ' Mrpl22' ^1.7' ' Slc22a18' ^1.7' ' Cox15' ^1.8' ' Zfp313' ^1.7' ' Crym' ^1.7' ' Jtb' ^1.8' ' Cnih' ^1.7' ' Ppp2r1a' ^1.8' ' Tmem9' ^1.8' ' Dcxr' ^1.7' ' Mrpl4' ^1.8' ' Rab18' ^1.8' ' Klhl9' ^1.7' ' Asl' ^1.8' ' Usp16' ^1.8' ' Acss2' ^1.7' ' Suclg2' ^1.8' ' Capn7' ^1.8' ' Leprel1' ^1.7' ' Xylb' ^1.8' ' Ntan1' ^1.8' ' 4631427c17rik' ^1.7' ' Nhsl1' ^1.8' ' Rps27l' ^1.8' ' Pcx' ^1.7' ' Hnrpf' ^1.8' ' Mertk' ^1.8' ' Fads3' ^1.7' ' Sdhb' ^1.8' ' Ube2k' ^1.8' ' Mrpl9' ^1.7' ' Loc654426' ^1.8' ' Rps13' ^1.8' ' Ndrl' ^1.7' ' Cyba' ^1.8' ' Gde1' ^1.8' ' Hpgd' ^1.7' ' Zfp810' ^1.8' ' Nfs1' ^1.8' ' Dnajc6' ^1.7' ' Hnrph1' ^1.8' ' Rpl9' ^1.8' ' Gstp1' ^1.7' ' Tmed10' ^1.8' ' Thnsl2' ^1.8' ' Ubb' ^1.7' ' Pcdh24' ^1.8' ' Ufc1' ^1.8' ' Btf3' ^1.7' ' G6pc' ^1.8' ' 2310005n03rik' ^1.8' ' Jak2' ^1.7' ' Tmem41a' ^1.8' ' Ap2a2' ^1.8' ' Ube2e3' ^1.7' ' Mrps22' ^1.8' ' Bc051227' ^1.8' ' Pvalb' ^1.7' ' Plscr2' ^1.8' ' Ccdc91' ^1.8' ' Tfg' ^1.7' ' Ces3' ^1.8' ' 2610204l23rik' ^1.8' ' Pcca' ^1.7' ' Sumo2' ^1.8' ' Nap1l1' ^1.8' ' Mpv17l' ^1.7' ' Gss' ^1.8' ' Acad8' ^1.8' ' Uchl3' ^1.7' ' D10ertd322e' ^1.8' ' Bc038156' ^1.9' ' Acsm5' ^1.7' ' Pdia3' ^1.8' ' Agt' ^1.9' ' Tnfaip8' ^1.7' ' Eef1e1' ^1.8' ' Efhd1' ^1.9' ' Serpina1d' ^1.7' ' Rars2' ^1.8' ' Paqr9' ^1.9' ' ' ' ' Fold- Fold- Fold- Gene$Symbol$ Gene$Symbol$ Gene$Symbol$ Change$ Change$ Change$ Acsl1' ^1.9' Dpep1' ^1.9' Sumo3' ^2.0' Cndp1' ^1.9' ' Slc22a2' ^1.9' ' Hsd17b11' ^2.0' ' Il13ra1' ^1.9' ' Hist1h2bm' ^1.9' ' Slc2a2' ^2.0' ' Sqle' ^1.9' ' Cox7a2l' ^1.9' ' Akr1a4' ^2.0' ' Sod3' ^1.9' ' Loc100043257' ^1.9' ' Cbs' ^2.0' ' Wwp1' ^1.9' ' Myo5a' ^1.9' ' Loc100046650' ^2.0' ' Scoc' ^1.9' ' Msn' ^1.9' ' Abcc2' ^2.0' ' Sult1d1' ^1.9' ' Rps4x' ^1.9' ' Tcp1' ^2.0' ' Adss' ^1.9' ' Car4' ^1.9' ' Dio1' ^2.1' ' Slc39a11' ^1.9' ' Nus1' ^1.9' ' Slc26a4' ^2.1' ' Cmbl' ^1.9' ' Pepd' ^1.9' ' Syngr2' ^2.1' ' Acaa2' ^1.9' ' Arl5a' ^1.9' ' Slc25a5' ^2.1' ' Hspd1' ^1.9' ' Gpr137b' ^1.9' ' Tfrc' ^2.1' ' Mrpl18' ^1.9' ' Slc6a13' ^1.9' ' Hip2' ^2.1' ' Tmco3' ^1.9' ' Pdzd3' ^2.0' ' Ctsh' ^2.1' ' Atp5l' ^1.9' ' Ndufb6' ^2.0' ' Tsc22d1' ^2.1' ' Nudt19' ^1.9' ' Hnrnpa2b1' ^2.0' ' Gatad1' ^2.1' ' Galm' ^1.9' ' Timm8b' ^2.0' ' Rab5a' ^2.1' ' Eif5a' ^1.9' ' Ccbl2' ^2.0' ' Cth' ^2.1' ' Gna11' ^1.9' ' Tmem126a' ^2.0' ' Rps2' ^2.1' ' Slc13a3' ^1.9' ' Car14' ^2.0' ' Aldh1l1' ^2.1' ' Tst' ^1.9' ' Pbld' ^2.0' ' Bpnt1' ^2.1' ' Gsto1' ^1.9' ' 2810410p22rik' ^2.0' ' Slc46a3' ^2.1' ' Scl0003251.1_2 ' Eif3eip' ^2.0' ' Zfp330' ^2.1' ' 1' ^1.9' Zmynd10' ^2.0' ' Mme' ^2.1' ' Znrf2' ^1.9' ' Rdh14' ^2.0' ' Jam4' ^2.1' ' Grpel1' ^1.9' ' Bola3' ^2.0' ' L2hgdh' ^2.1' ' Slc35a3' ^1.9' ' Mapk14' ^2.0' ' Tmem59' ^2.1' ' Il1rl1l' ^1.9' ' Aqp11' ^2.0' ' Frrs1' ^2.1' ' Pex7' ^1.9' ' Lonp2' ^2.0' ' 5730437n04rik' ^2.1' ' Ppt1' ^1.9' ' A530050d06rik' ^2.0' ' Slc7a9' ^2.1' ' Mrpl3' ^1.9' ' Gfm1' ^2.0' ' 1810063b05rik' ^2.1' ' As3mt' ^1.9' ' Ndufa6' ^2.0' ' Vil1' ^2.1' ' Pdia4' ^1.9' ' 0610010d20rik' ^2.0' ' Ebp' ^2.1' ' Nit2' ^1.9' ' Rpl24' ^2.0' ' Agxt2' ^2.1' ' Nudt4' ^1.9' ' 4833421e05rik' ^2.0' ' Dgat2' ^2.1' ' Morf4l1' ^1.9' ' Arsb' ^2.0' ' Apom' ^2.1' ' Eif3s8' ^1.9' ' Adh5' ^2.0' ' Sar1b' ^2.1' ' 9630015d15rik' ^1.9' ' Dhrs1' ^2.0' ' Loc667609' ^2.1' ' Ceacam2' ^1.9' ' Capn2' ^2.0' ' Sult1c2' ^2.1' ' Nox4' ^1.9' ' Acadm' ^2.0' ' Cyp2j11' ^2.1' ' Sap30' ^1.9' ' Slc13a2' ^2.0' ' Gsta2' ^2.1' ' Cml1' ^1.9' ' Snx7' ^2.0' ' Kdelr2' ^2.1' ' Atp5o' ^1.9' ' Loc100047353' ^2.0' ' Higd1c' ^2.1' ' Adhfe1' ^1.9' ' Tmem139' ^2.0' ' Hist1h2bj' ^2.1' ' Commd3' ^1.9' ' H47' ^2.0' ' Bc026585' ^2.1' ' Psmb7' ^1.9' ' Cyc1' ^2.0' ' Cyp2d26' ^2.1' ' ' ' ' Fold- Fold- Fold- Gene$Symbol$ Gene$Symbol$ Gene$Symbol$ Change$ Change$ Change$ 2310008m10rik' ^2.1' Gnmt' ^2.3' Ndufs2' ^2.6' Nola3' ^2.1' ' D630023f18rik' ^2.3' ' Pxmp2' ^2.6' ' Dpp4' ^2.2' ' Loc100045617' ^2.3' ' Slc19a1' ^2.6' ' Mtap7' ^2.2' ' Ldhd' ^2.3' ' Ai747699' ^2.6' ' Ahcyl1' ^2.2' ' Pdk3' ^2.3' ' Gstm5' ^2.6' ' Nt5e' ^2.2' ' Tinag' ^2.3' ' Ppcs' ^2.6' ' Spcs1' ^2.2' ' Eef2' ^2.3' ' Folr1' ^2.6' ' Etfa' ^2.2' ' Defb29' ^2.3' ' Clrn3' ^2.6' ' Loc100045567' ^2.2' ' Vdac3' ^2.4' ' Hist1h2bf' ^2.6' ' Sepp1' ^2.2' ' Abhd3' ^2.4' ' Bc026439' ^2.6' ' Bc055107' ^2.2' ' Acot3' ^2.4' ' Cldn2' ^2.6' ' Bc016495' ^2.2' ' Loc381629' ^2.4' ' Lrrk2' ^2.6' ' Acad9' ^2.2' ' Upb1' ^2.4' ' Loc100047046' ^2.7' ' 1110008f13rik' ^2.2' ' Cryz' ^2.4' ' D630042f21rik' ^2.7' ' Slc25a15' ^2.2' ' Loc268782' ^2.4' ' Csad' ^2.7' ' Pcbp2' ^2.2' ' Tspan3' ^2.4' ' Etfdh' ^2.7' ' Nudt12' ^2.2' ' Bc006662' ^2.4' ' Mrpl53' ^2.7' ' Ubie' ^2.2' ' Bhmt2' ^2.4' ' Nme2' ^2.7' ' Ottmusg000000 ' Tmem33' ^2.4' ' Chuk' ^2.7' ' 05148' ^2.2' Galnt11' ^2.4' ' Fgf1' ^2.7' ' Slc5a2' ^2.2' ' Ppa2' ^2.4' ' Hint1' ^2.7' ' Slc25a30' ^2.2' ' Calr' ^2.4' ' Dhrs4' ^2.7' ' Sfrs5' ^2.2' ' Hist1h2bn' ^2.5' ' Col4a3' ^2.8' ' Adi1' ^2.2' ' Hao3' ^2.5' ' Loc100047937' ^2.8' ' Loc677144' ^2.2' ' Loc100044204' ^2.5' ' Mpp1' ^2.8' ' Ela1' ^2.2' ' Calml4' ^2.5' ' Sephs2' ^2.8' ' B230339h12rik' ^2.2' ' H3f3a' ^2.5' ' Coq9' ^2.8' ' Sigirr' ^2.2' ' C1qbp' ^2.5' ' Ugt3a2' ^2.8' ' Supt4h2' ^2.2' ' Qprt' ^2.5' ' Rps6' ^2.9' ' Slc12a3' ^2.2' ' Mtch2' ^2.5' ' Kng2' ^2.9' ' Khk' ^2.2' ' Arl6' ^2.5' ' Loc333331' ^2.9' ' 2010311d03rik' ^2.2' ' Ai317395' ^2.5' ' Gm853' ^2.9' ' Dars' ^2.3' ' Slc7a7' ^2.5' ' Prdx3' ^2.9' ' Bc021608' ^2.3' ' Dnajc12' ^2.5' ' Fmo1' ^2.9' ' Smpdl3a' ^2.3' ' 2610528j11rik' ^2.5' ' Nckap1' ^2.9' ' Bc021785' ^2.3' ' Sucnr1' ^2.5' ' Ostb' ^2.9' ' H2afz' ^2.3' ' Cct6a' ^2.5' ' Glud1' ^2.9' ' Agtr1a' ^2.3' ' Igfbp3' ^2.5' ' Oxct1' ^3.0' ' Ak3l1' ^2.3' ' Pah' ^2.5' ' Pcbp1' ^3.0' ' Pdhb' ^2.3' ' Loc100048413' ^2.5' ' Mep1b' ^3.1' ' Ndfip1' ^2.3' ' Frap1' ^2.5' ' Cat' ^3.1' ' Slc25a36' ^2.3' ' Ddc' ^2.5' ' Rbpms2' ^3.2' ' Ndufc2' ^2.3' ' Klk1b4' ^2.5' ' Slc27a2' ^3.2' ' Nola2' ^2.3' ' Hist1h2bh' ^2.6' ' Plekhb2' ^3.2' ' F13b' ^2.3' ' Vps35' ^2.6' ' Slc6a19' ^3.2' ' Rpl7a' ^2.3' ' Eps8' ^2.6' ' Rgl1' ^3.2' ' Iah1' ^2.3' ' Loc433479' ^2.6' ' Slc22a9' ^3.2' ' ' ' ' Fold- Gene$Symbol$ Change$ Ivns1abp' ^3.2' Slc47a1' ^3.3' ' 5033411d12rik' ^3.3' ' Ugt3a1' ^3.3' ' 2500002l14rik' ^3.3' ' Rps7' ^3.4' ' Klk1b27' ^3.4' ' Lgmn' ^3.5' ' Slc17a3' ^3.6' ' Cyp4a31' ^3.6' ' Gpx1' ^3.6' ' Rnaset2' ^3.7' ' Tcn2' ^3.8' ' Cml3' ^3.9' ' Ehhadh' ^3.9' ' Slc16a4' ^4.0' ' Idh1' ^4.0' ' Fbp1' ^4.2' ' Scp2' ^4.2' ' Spp2' ^4.3' ' Slc22a13' ^4.3' ' Hrsp12' ^4.5' ' Atp5f1' ^5.0' ' Miox' ^5.0' ' Eg433923' ^5.1' ' Chpt1' ^5.3' ' Loc100046918' ^5.4' ' Tmem27' ^5.6' ' Cyp51' ^5.9' ' Cyp4b1' ^6.4' ' Ugt1a10' ^6.5' ' Hspa8' ^7.6' ' Dnase1' ^8.7' ' ! ' ADMA (µM) L-NMMA (µM) SDMA (µM) Creatinine (fold change) Wild-type 0.28 ± 0.070 ± 0.24 ± 1.00 ± 0.02 0.01 0.02 0.04 COX-1-/- 0.86 ± 0.085 ± 0.63 ± 0.89 ± 0.26 0.01 0.14 0.05 (p=0.16) (p=0.99) (p=0.09) (p=0.46) COX-2-/- 3.36 ± 0.38 ± 1.25 ± 1.53 ± 1.16* 0.11* 0.90 0.18* (p=0.007)(p<0.01) (p=0.02) (p>0.99) (p=0.01)

Wild type normal 0.41 ± 0.27 ± N/A N/A salt; vehicle 0.044 0.079 Wild type normal 0.86 ± 1.19 ± N/A N/A salt; parecoxib 0.13* 0.26* (p=0.005)(p<0.01) (p=0.02)

Wild type high 0.73 ± 0.78 ± N/A N/A salt; vehicle 0.14 0.17

Wild type high 1.12 ± 0.75 ± N/A N/A salt; parecoxib 0.10 0.07 (p=0.18) (p=1.00)

IP+/+ 2.93 ±! 0.30 ± 1.74 ± 1.0 ± 0.12 0.04 0.18 0.04 IP-/- 4.11 ± 0.11 ± 1.88 ± 1.85 ± 0.11* 0.02* 0.31 0.29* (p=0.0002)(p<0.01) (p=0.002)(p<0.01) (p=0.44) (p=0.0006)(p<0.01) !

Supplementary Table 2: Effect of gene deletion of COX inhibition on plasma methylarginines. Methylarginines (ADMA, L-NMMA and SDMA) and creatinine in the plasma of wild-type, COX-1-/-, COX-2-/- (p-values by Kruskal-Wallis with Dunn’s post-hoc test) and IP-/- mice and wild type mice treated with vehicle or parecoxib fed a normal or a high salt diet (p-values by Mann-Whitney U-test).