Anti-inﬂammatory Effects of v-3 Polyunsaturated Fatty ... · damage is increased by hypertension.1 High blood pressure (BP) can be reduced by certain dietary modi ﬁcations, such

ORIGINAL ARTICLE

Anti-inflammatory Effects of v-3 Polyunsaturated FattyAcids and Soluble Epoxide Hydrolase Inhibitors in

Angiotensin-II–Dependent Hypertension

Arzu Ulu, MSc,* Todd R. Harris, PhD,* Christophe Morisseau, PhD,* Christina Miyabe, BS,*Hiromi Inoue, DVM,† Gertrud Schuster, PhD,‡ Hua Dong, PhD,* Ana-Maria Iosif, PhD,§

Jun-Yan Liu, PhD,* Robert H. Weiss, MD,†¶ Nipavan Chiamvimonvat, MD,k¶ John D. Imig, PhD,**and Bruce D. Hammock, PhD*

Abstract: The mechanisms underlying the anti-inflammatory andantihypertensive effects of long-chain v-3 polyunsaturated fatty acids(v-3 PUFAs) are still unclear. The epoxides of an v-6 fatty acid,arachidonic acid epoxyeicosatrienoic acids also exhibit antihyperten-sive and anti-inflammatory effects. Thus, we hypothesized that themajor v-3 PUFAs, including eicosapentaenoic acid (EPA) and doco-sahexaenoic acid (DHA), may lower the blood pressure and attenuaterenal markers of inflammation through their epoxide metabolites.Here, we supplemented mice with an v-3 rich diet for 3 weeks ina murine model of angiotensin-II–dependent hypertension. Also,because EPA and DHA epoxides are metabolized by soluble epoxidehydrolase (sEH), we tested the combination of an sEH inhibitor andthe v-3 rich diet. Our results show that v-3 rich diet in combinationwith the sEH inhibitor lowered Ang-II, increased the blood pressure,further increased the renal levels of EPA and DHA epoxides, reduced

renal markers of inflammation (ie, prostaglandins and MCP-1),downregulated an epithelial sodium channel, and upregulated angioten-sin-converting enzyme-2 message and significantly modulated cyclo-oxygenase and lipoxygenase metabolic pathways. Overall, our findingssuggest that epoxides of the v-3 PUFAs contribute to lowering systolicblood pressure and attenuating inflammation in part by reduced pros-taglandins and MCP-1 and by upregulation of angiotensin-convertingenzyme-2 in angiotensin-II–dependent hypertension.

Key Words: v-3 polyunsaturated fatty acids, EPA, DHA, solubleepoxide hydrolase inhibitors, angiotensin-II–dependent hypertension

(J Cardiovasc Pharmacol� 2013;62:285–297)

INTRODUCTIONCardiovascular disease (CVD) is one of the main causes of

death worldwide, and thus is the subject of numerous studies toexplore potential new treatment strategies. Cardiovascular risk ofstroke, coronary artery disease, and hypertensive end-organdamage is increased by hypertension.1 High blood pressure (BP)can be reduced by certain dietary modifications, such as anincreased consumption of long-chain v-3 polyunsaturated fattyacids (v-3 PUFAs) found in fatty fish or fish oil.2 The AmericanHeart Association recommends that healthy adults consumea serving of fish at least 2 times a week and patients withcoronary heart disease take a supplement of 1 g of eicosapentae-noic acid (EPA) (20:5) and docosahexaenoic acid (DHA), 22:6every day.3 In addition to hypertension, the long-chain v-3 PU-FAs have the potential to protect against many CVDs,4,5 espe-cially coronary heart disease and atrial fibrillation. These fattyacids have been shown to exert anti-inflammatory,6–8 antiathero-sclerotic,9 and antiarrhythmic effects.2,10–12 There is a generalfeeling that consumption of v-3 lipids reduces cardiovascularrisk; however, some of the individual studies lack power to drawsolid conclusions, whereas some others have failed to showefficacy.13–17 The mechanisms of action of v-PUFAs are notwell understood.

In the v-6 fatty acid series, metabolites of arachidonicacid (ARA) are well known to influence inflammation andCVD.18–20 Of particular interest, the epoxides of ARA [epox-yeicosatrienoic acids (EETs)] have anti-inflammatory and anti-hypertensive properties similar to the ones observed with thelong-chain v-3 PUFAs.21–23 Moreover, in vitro DHA epoxides

Received for publication August 13, 2012; accepted April 24, 2013.From the *Department of Entomology and UC Davis Comprehensive Cancer

Center, University of California, Davis, CA; †Division of Nephrology;‡Department of Nutrition; kDivision of Cardiovascular Medicine, Depart-ment of Internal Medicine; §Division of Biostatistics, Department of PublicHealth Sciences, School of Medicine, University of California, Davis,USA; ¶US Department of Veterans’ Affairs Medical Center, Sacramento,CA; and **Department of Pharmacology and Toxicology, Medical Collegeof Wisconsin, Milwaukee, WI.

Supported by NIEHS grant R01 ES002710 and NIEHS Superfund ResearchProgram grant P42 ES004699; NIH grants 5UO1CA86402 (Early DetectionResearch Network), 1R01CA135401-01A1, and 1R01DK082690-01A1; andthe Medical Service of the US Department of Veterans’ Affairs (all to R.H.W.). Analytical work was partially supported by the NIH and NIDDK grantU24 DK097154. Studies and analyses performed by J. D. Imig were sup-ported by NIH grants HL59699 and DK38226. Analyses performed by A.-M.Iosif were supported by the National Center for Advancing TranslationalSciences, NIH, through grant UL1 TR000002. T. R. Harris is supported byNIH T32 training grant in basic and translational cardiovascular science (T32HL86350). B. D. Hammock is a George and Judy Marcus senior fellow of theAmerican Asthma Foundation, and was supported by NIEHS grant R01ES002710 and NIEHS Superfund Research Program grant P42 ES004699.

The University of California, Davis, has filed patents in the area of solubleepoxide hydrolase inhibitors for the treatment of diseases.

Supplemental digital content is available for this article. Direct URL citationsappear in the printed text and are provided in the HTML and PDFversions of this article on the journal’s Web site (www.jcvp.org).

Reprints: Bruce D. Hammock, PhD, One Shields Avenue, Department ofEntomology, University of California, Davis CA 95616 (e-mail:[email protected])

Copyright © 2013 by Lippincott Williams & Wilkins

J Cardiovasc Pharmacol� � Volume 62, Number 3, September 2013 www.jcvp.org | 285

[epoxydocosapentaenoic acids (EpDPEs)], similar to EETs,activate a large-conductance Ca2+-activated K+ channel24,25

that lead to vasodilatation ex vivo.26 Recently, regioisomericmixtures of EPA epoxides [epoxyeicosatetraenoic acids(EpETEs)] and EpDPEs were shown to reduce inflammatorypain, in which EpDPEs were demonstrated to be more effectivein reducing the nociceptive pain than the EpETEs and EETs.27

Interestingly, the regioisomers of DHA epoxides show unequaleffects on inflammatory pain. The bioactive v-3 PUFAs com-pete with ARA for metabolism by cytochrome P450 enzymes(CYP), specifically CYP2C and 2J, which largely generate theEETs.28,29 Based on these observations, we hypothesized thatEPA and DHA lower arterial BP and attenuate renal markers ofinflammation in vivo at least in part through their epoxidemetabolites in angiotensin-II–dependent hypertension. Toaddress this hypothesis, we conducted an experiment thatincluded controls and Ang-II–infused animals with and withoutan v-3 rich diet. We predicted that hypertensive animals trea-ted with an v-3 rich diet will have lower BP as compared totheir Ang-II–infused counterparts. Like the EETs, the EPA andDHA epoxides are very good substrates for the soluble epoxidehydrolase (sEH).27 Such hydrolytic metabolism has beenshown to reduce the bioactivity of EETs,30,31 and it should alsoreduce the bioactivity of these CYP-generated epoxy fattyacids (EpFAs) that are derived from EPA and DHA. Conse-quently, we tested the additional hypothesis that the presenceof an sEH inhibitor (sEHI) will further increase the effective-ness of the parent compounds by increasing the tissue levels ofthe epoxide metabolites, EpDPEs and EpETEs. To addressthese additional hypotheses, we also included Ang-II–infusedanimals treated with the combination of v-3 rich diet and sEHI(at low and high doses) and those treated only with the sEHI tocontrol for the potential antihypertensive effect of the sEHI.We expected to observe lower BP in Ang-II–infused animalstreated with the combination of the v-3 rich diet and sEHI ascompared to Ang-II–infused animals treated only with the v-3rich diet. To this end, we supplemented animals with an v-3rich diet in the presence of a potent sEHI in a murine model ofangiotensin-II–dependent hypertension.

Metabolic profiling was used to directly quantify themetabolites of the v-3 and v-6 PUFAs to gain insights intothe mechanism of action of the treatment with v-3 rich dietand the combination of the v-3 rich diet and sEHI. Weobserved that the v-3 rich diet, sEHI, and the combinationlower systolic blood pressure (SBP) in Ang-II–dependenthypertension. The findings of this study are consistent withour hypothesis that CYP metabolites of v-3 PUFAs that arestabilized by sEH inhibition have a role in lowering Ang-IIdependent increase in BP and in significant modulation of theCOX and LOX metabolic pathways in the ARA cascade.

METHODS

Animals and TreatmentsAll animal studies were approved by the University of

California Davis Animal Use and Care Committee and wereperformed in accordance with the National Institutes of HealthGuide for the care and use of laboratory animals. We avoided

using the C57/BL6 strain, which is poorly responsive to Ang-IIcompared to some other strains of mice.32 Instead, we usedSwiss Webster mice, in which the oxylipin profiles and thepharmacokinetics of the sEHIs have been well characterized,33,34

and who do not develop renal damage induced by angiotensin-II.This allowed examining the effects of the v-3 PUFAs solely onchanges in the BP and in renal eicosanoids. Eight week old maleSwiss Webster mice (Charles River Laboratories, Wilmington,MA) were acclimated to new housing conditions for 1 week andwere kept under a 12 hour light–dark cycle with free access towater and food for the duration of the experiment. Baseline BPswere established for each group of mice based on the average BPtaken for 3 days before treatment. Hypertension was inducedby infusion of Ang-II at a constant rate (20 ng/min or1 mg21$kg21$d21) for 14 days using subcutaneously implantedosmotic minipumps (Model 1002-Alzet, Cupertino, CA).

Mice were fed either a purified control diet (5% cornoil) or an v-3 rich diet consisting of the 2 major long-chainv-3 fatty acids, EPA (0.75%) and DHA (0.75%) at 90%purity (Larodan Fine Chemicals, Sweden). In the control diet,v-3 fatty acids replaced corn oil to retain constant dietary fat.The detailed composition and preparation of the diets aregiven in Supplemental Digital Content 1 (see Table S1A,http://links.lww.com/JCVP/A121). Based on the fatty acidanalysis of each diet (see Section A, Supplemental DigitalContent 1, http://links.lww.com/JCVP/A121, which de-scribes the method for fatty acid analysis), the total percent-age of the v-3 PUFAs was 0.6% and 23% for corn oil andv-3 rich diet, respectively (Table S1B, which presents thefatty acid composition of the v-3 rich diet). Animals wererandomly divided into 6 experimental groups: Animals in thecontrol group underwent sham surgery and the vehicle treat-ment, drinking water with 1% PEG400. All groups receivingAng-II are indicated by A in the group designation. A sec-ond group received Ang-II and the vehicle treatment (groupA). The other groups receiving Ang-II were randomly as-signed to receive the v-3 rich diet (group A-v3), sEHI (TPPUor UC1770,35 1-trifluoromethoxyphenyl-3-(1-propionylpiperi-din-4-yl) urea) alone (group A-TH), or the combination ofthe v-3 rich diet and sEHI at a low (group A-v3-TL) and highdose (group A-v3-TH). All groups receiving the sEHI TPPUare indicated by T. The 2 sEHI doses were 0.2 and 0.6 mg/kg,for TL (low dose TPPU) and TH (high dose TPPU), respec-tively. Additionally, we included a group treated only withAng-II (group A0) and a group treated with Ang-II andthe low dose sEHI (0.2 mg/kg) (group A0-TL) to test theeffect of sEHI alone on the BP in mice on a standard rodentchow diet (see Table S1B, Supplemental Digital Content 1,http://links.lww.com/JCVP/A121, which shows the fatty acidcomposition of the standard diet).

The v-3 rich diet was supplemented 1 week before theinduction of hypertension by Ang-II. The potent sEHI, TPPUwas administered in the drinking water with the cosolvent 1%PEG400 (polyethylene glycol) on the same day Ang-II infu-sion started. Fresh water containing TPPU was readministeredto the animals every 4 days. After administration of the dietfor 3 weeks, the animals were euthanized and tissues wereharvested and immediately frozen in liquid nitrogen to bestored at 2808C until analysis.

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Measurement of BPSBP was measured using a noninvasive tail-cuff blood

pressure system (Kent Scientific Corporation, Torrington, CT),as previously described.36–38 Animals were acclimated to thetail cuffs and the restraining procedure for 15 minutes for 3consecutive days, and then the actual measurements were per-formed every day at the same time by the same qualifiedoperator. The operator was blinded by placing cards in frontof the cages to block the treatment information. The cages withthose blocking cards were numbered and the data were savedwith those assigned numbers, which allowed blinded data anal-ysis as well. A standard method was followed for each animalto analyze the BP data. The first 3 values (acclimation values),highest and lowest values, and those that are associated withanimal movement were discarded, and the remaining readingswere averaged to determine the SBP (see Section B, Supple-mental Digital Content 1, http://links.lww.com/JCVP/A121,which describes the measurement of SBP in detail).

Quantification of Oxylipins and TPPU inthe Kidney

At the end of the Ang-II infusion, renal oxylipins andTPPU levels were determined using a solid phase extraction(SPE) method followed by a liquid chromatography electrosprayionization tandem mass spectrometry (LC/MS/MS) technique aspreviously described (see Section C and Table S2, Supplemen-tal Digital Content 1, http://links.lww.com/JCVP/A121, whichdetails oxylipin extraction and lists the analytes quantified alongwith their ionization conditions for LC/MS/MS analysis).39,40

Measurement of Proinflammatory Cytokinesin the Kidney

The concentration of common proinflammatory cyto-kines, IL-1b, IL-6, tumor necrosis factor-a, and monocyte che-motactic protein-1 (MCP-1) were determined using a mouseselective 4-plex kit (Bio-Rad, Hercules, CA) as describedbefore.41 Briefly, 100 mg kidney tissue excised from the renalcortex was subjected to lysis using a Bio-Rad cell lysis kit.The assay was performed according to the instructions of themanufacturer. The obtained concentrations were normalized tothe protein concentration of the lysate, and the results werereported as pg/mg protein.

Real-time Quantitative Reverse TranscriptionPolymerase Chain Reaction

Fold change in the mRNA expression of selectedgenes was determined using quantitative reverse transcriptionpolymerase chain reaction. Relative quantification ofthese data was processed by DD-Ct (cycle threshold)method (see Section D, Supplemental Digital Content 1,http://links.lww.com/JCVP/A121, which details the RT-PCRmethod).

Statistical AnalysesAll variables were summarized as mean 6 SEM. The

effects of Ang-II, v-3 rich diet, and TPPU on BP over thecourse of the study were analyzed using mixed-effects modelsfor longitudinal data. This approach allowed the use of all

available data for each animal, whereas accounting for thecorrelated nature of the data because of the repeated measure-ments on the same individual. The model was validated withresidual diagnostics.42 In secondary analyses, we examinedcumulative measures by calculating the area under the BP–time curve with respect to the control group, both for thewhole period of the study and for the past 3 days of measure-ments (days 7, 9, and 11). The remaining data (oxylipins,cytokines, gene expression, etc) were analyzed for the effectsof treatment (v-3 rich diet, TPPU, or the combination) ondependent measures. In cases where normality was met,random-effects 1-way ANOVA (analysis of variance) wasperformed, followed by pairwise comparisons (corrected formultiple comparisons using a layered Bonferroni correc-tion43). If the normality test failed, a Kruskal–Wallis 1-wayANOVA on the ranks was conducted, followed by Dunn’spost hoc tests. A threshold of P , 0.05 was used in the abovetests. Correlation between the variables was tested using Pear-son’s correlation, and the significance of the correlation wastested using a 2-tailed t test. All statistical tests were con-ducted in SPSS (IBM SPSS Statistics, version 19) exceptfor analyses of residuals, which were conducted in SAS Insti-tute, I, 2002–2010 (SAS/STAT Version 9.3, Cary, NC).

RESULTS

v-3 PUFAs Do Not Alter Food Intake orWeight Gain in Mice

There were no significant differences detected in foodintake (mice on the purified control diet consumed 4–5 g/d andmice on the v-3 rich diet consumed 3–4 g/d) and in the bodyweight gain among the treatment groups during the interven-tion period. Weight gain in all treatment groups was as follows:Control: 26 1 g, A: 1.46 0.9 g, A-v3: 1.1 + 0.5 g, A-v3-TL:1.6 6 0.2 g, A-v3-TH: 2 6 0.5 g, and A-TH: 1.3 6 0.4 g.Thus, our results fail to support that the v-3 PUFAs signifi-cantly alter the food intake or weight gain in mice during thecourse of this study.

v-3 PUFAs Exerted Antihypertensive Effects inAng-II-induced Hypertensive Animals

The time course of changes in BP across all the groups isshown in Figure 1. We examined the overall effect of Ang-IIand v3/TPPU treatments on the SBP data in 2 different ways(mixed-effects models with “group” or “Ang-II, v-3 rich diet,and TPPU” as between-subjects factor), which generated mutu-ally supporting results. The preliminary analysis of residualssuggested that the treated groups had similar values early onday 1 followed by a sharp increase and then a plateau in theAng-II treated group (see Section E and Table S3, Supple-mental Digital Content 1, http://links.lww.com/JCVP/A121,which summarizes the residuals diagnostics of the mixed-effects model). Therefore, the mixed-effects model included“group” (control, A, A-v3, A-v3-TL, A-v3-TH, and A-TH)as between-subjects factor and time (days 3–11) as repeatedmeasures. This analysis revealed only a significant main effectof treatment group (F = 15.75, P , 0.01). Pairwise groupcomparisons using LSD (least significant difference) tests

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revealed that Ang-II infusion increased SBP significantly com-pared to all 4 treatment groups and controls (all P , 0.05).Also, the SBP in group A-TH was significantly lower com-pared to group A-v3 (P = 0.03). Furthermore, SBP wasdecreased in A-v3-TH treated–animals as compared to A-v3(P = 0.002); whereas, in the A-v3-TL, this decrease missedstatistical significance (P = 0.07). The SBP in all treatmentgroups except for A-v3-TH (P = 0.106) differed significantlyfrom that of the controls (P , 0.05). To examine the separatedeffects of treatments on SBP, we analyzed the SBP data usingmixed-effects model with Ang-II, v3-rich diet and TPPU asthe 3 between-subjects factors (see Section E, SupplementalDigital Content 1, http://links.lww.com/JCVP/A121). Theresults of this analysis generated qualitatively supporting re-sults as compared to the mixed-effects model with “group” asbetween-subjects factor. To further explore the relationshipbetween A-v3 and A-v3-TL groups (and provide furtherconfirmation of the other group differences), we conductedsupplementary ANOVAs of the SBP data expressed asarea under the curve (AUC) for all days and for days 7–11(see Figure S1-Panel A, Supplemental Digital Content 1,http://links.lww.com/JCVP/A121) and a focused analysis forSBP data at day 11 (when the group differences were hypoth-esized to be maximal; see Section F, Supplemental Digital

Content 1, http://links.lww.com/JCVP/A121). Overall, theseadditional analyses generated results that were all in agree-ment with the results of the overall mixed-effects model: theAUC for groups A-v3-TL and A-v3-TH was lower than forA-v3 in both all days and in days 7–11 (though statisticalsignificance was reached only for the A-v3-TH group). Sim-ilarly, at day 11, SBP was lower in A-v3-TL and A-v3-THgroups compared to A-v3 (though in this case, only the for-mer difference reached statistical significance).

We observed that SBP values after 1 week were higherthan the earlier days in the experiment in group A-v3 (Fig. 1,solid reverse triangles), suggesting that the v-3 rich diet losessome of its effectiveness, with time in lowering BP. Therefore,we performed a post hoc analysis of these data using a pairedt test (2-tailed), which revealed a statistically significant differ-ence in SBP in the early versus late time points (after 1 week)(P , 0.05) in group A-v3.

The Effect of Diet on Arterial PressureThere was a large reduction in the SBP with sEHI alone

(A-TH) (Fig. 1, open squares). We tested the hypothesis thatsome of this reduction might be caused by the trace amounts ofa-linolenic acid (ALA) in the corn oil diet acting in concert withthe sEHI TPPU. We switched mice to a standard diet (2018Teklad Global 18% Protein Rodent Diet, Harlan Laboratories,Hayward, CA) that was previously used as a control diet toaccompany an v-3 rich diet. The standard diet had higheramounts of ALA than the corn oil diet used in our study44

(see Table S1B). We anticipated that this difference in theamount of ALA might reflect itself on changes in SBP and offeran explanation for the large reduction in SBP in animals treatedwith sEHI alone (group A-TH). We predicted that animals onthe diet with higher amount of ALA will have lower BP ascompared to those on the diet with lower amount of ALA.The mice were held on the standard diet for a week beforebeginning the experiment. After a 3-day acclimation period,baseline BP of these mice increased to 113 6 2 mm Hg,which was slightly higher than the baseline BP of animalsthat were on either the corn oil or the v-3 rich diet (107 64 mm Hg). On the standard diet, the BP increased from 11362 mm Hg to 1576 3 mm Hg (excluding baseline) in group A0,a significant change of 44 mm Hg (see Figure S2A, Supple-mental Digital Content 1, http://links.lww.com/JCVP/A121,which demonstrates time course of SBP in mice on a standarddiet). In contrast to this increase in SBP to 157 6 3 mm Hgon the standard diet in group A0, SBP increased only to122 6 4 mm Hg on the corn oil diet as in group A. TheSBP resulted in an average of 140 6 2 mm Hg in groupA0-TL, indicating that TPPU lowered but did not reduceSBP back to baseline at 0.2 mg/kg dose (Figure S2A). Thisreduction in SBP corresponds to a percentage change frombaseline of 23 6 2% when compared to a percentage changefrom baseline of 7 6 1% in group A-v3-TL (Fig. 1, opentriangles). This compares to a reduction to 107 6 2 mm Hgon the corn oil diet in group A-TH (Fig. 1, open squares).Also, because SBP reached a plateau after day 3 in groups A0and A0-TL, we calculated the AUCs from the SBP recordedon days 3–9 (AUC t(3–9)). Similar to the overall results, ingroup A0-TL, the AUCt(3–9) was significantly lower than

FIGURE 1. Antihypertensive effect of v-3 PUFAs on angiotensin-II–dependent hypertension. SBP was recorded with a non-invasive tail-cuff system. The x axis represents the time afterAng-II delivering osmotic minipumps were implanted. Adminis-tration of the v-3 rich diet started 1 week before the Ang-IIinfusion. TPPU was administered in the drinking water starting atthe same time as Ang-II infusion. Results are given as percentagechange from baseline taken before treatment for each group. Aindicates angiotensin treatment, v3 indicates v3-rich diet, andTL and TH refer to TPPU treatment at low and high doses,respectively. The chemical structure of TPPU is shown. Data aremean 6 SEM. Error bars were only shown unidirectional. Con-trols, n = 5; group A, n = 8; group A-v3, n = 8; group A-v3-TL,n = 7; group A-v3-TH, n = 8; and group A-TH, n = 8.



group A0 (see Figure S2B, Supplemental Digital Content 1,http://links.lww.com/JCVP/A121, which shows the AUC t (3–9)

in mice on a standard diet).

Effects of Treatments on the Plasma Levels of Ang-IIand Kidney

We measured the plasma levels of Ang-II to test whetherthe antihypertensive effect of the v-3 rich diet is directly asso-ciated with decreased plasma levels of Ang-II. Indeed, theplasma levels of Ang-II were reduced in group A-v3-TLas compared to group A (P , 0.05) (see Figure S3, Supple-mental Digital Content 1, http://links.lww.com/JCVP/A121,which shows plasma levels of Ang-II across all the groups).Inadequate amount of plasma was available from groups A-v3and A-TH, and thus, Ang-II levels could not be determined.

Examination of the general histology of the kidney tissueand serum kidney markers such as creatinine and urinary albuminshowed no statistically significant differences across the groups asexpected (see Figure S4A and S4B, Sections G and H, Sup-plemental Digital Content 1, http://links.lww.com/JCVP/A121,which show glomerular injury scores, renal function parameters,and results/discussion from these analyses).

Possible Mechanisms of Action of v-3PUFAs—Insights Gained FromMetabolic Profiling

To understand the possible mechanisms by which v-3PUFAs affect BP and renal inflammation, we measured thelevels of key oxidized metabolites of unsaturated fatty acidsin the kidney. Overall, the v-3 rich diet increased the renallevels of the EPA and DHA metabolites, specifically the epox-ide metabolites (Fig. 2A and see Tables S4 and S5, Supple-mental Digital Content 1, http://links.lww.com/JCVP/A121,which shows the renal levels of the epoxide and diol metabo-lites of ARA, linoleic acid (LA), EPA, and DHA). The epox-ide, diol metabolites and their corresponding epoxide-to-diolratios are shown in Figure 2A, Panels A–C for EPA, andPanels D–F for DHA. The EPA epoxides, EpETEs, decreasedto undetectable levels (except for 8, 9-EpETE) in group A ascompared to the controls. However, the renal levels of EpETEregioisomers increased in group A-v3 (Fig. 2A, panel A).Although epoxide metabolites of DHA (EpDPEs) were moreabundant than the EpETEs in the kidney (Fig. 2A, Panels Aand D), supplementation of the v-3 rich diet with TPPUresulted in a larger increase in EpETEs than the EpDPEs.Compared with group A-v3, the renal levels of EpDPEsand EpETEs further increased in groups A-v3-TL and A-v3-TH (P , 0.05); however, this increase was not dose-dependentfor the 2 doses tested. In parallel, the renal levels of eachregioisomer of the DHA epoxides significantly increased ingroup A-v3 (P , 0.05, Table S4). In contrast to v-3 fatty acidseries, the tissue levels of the epoxide metabolites of the v-6fatty acids decreased in group A-v3. The epoxide, diol metab-olites, and their corresponding epoxide-to-diol ratios are shownin Figure 2B, Panels A–C for ARA, and Panels D–F for LA.The availability of ARA and LA epoxides, EETs and epox-yoctadecenoic acid (EpOMEs), respectively, decreased in allgroups treated with Ang-II when compared to the controls(Fig. 2B, Panels A and D). In general, the tissue levels of EETs

tend to be lower in groups A-v3, A-v3-TL, and A-v3-TH ascompared to A-TH. As for EpOMEs, group A-v3 showedlower tissue levels than A-TH animals. In contrast to the EETs,tissue levels of EpOMEs increased in groups A-v3-TL andA-v3-TH when compared to group A-TH.

In mice fed a standard diet, the renal levels of the sum ofDHA and EPA epoxides were lower than those in mice feda corn oil diet; however, EETs and EpOMEs were comparablefor each diet (see Table S6, Supplemental Digital Content 1,http://links.lww.com/JCVP/A121, which shows the renal levelsof the epoxide and diol metabolites of ARA, LA, EPA and DHAin mice fed a standard diet). In addition, the renal levels ofEpDPEs and EpETEs but not EETs and EpOMEs were higherin group A-v3-TL (Table S4) as compared to A0-TL (Table S6).

Considering the high abundance and their likely genera-tion by the sEH, we examined the changes in the renal levels ofthe diols of EPA and DHA from EpETEs and EpDPEs,respectively [di-hydroxy-eicosatetraenoic acid (DiHETE) anddi-hydroxy-docosapentaenoic acid (DiHDPE), respectively](Fig. 2A, Panels B and E). The renal levels of DiHETE andDiHDPE decreased in group A but normalized in group A-v3.As expected, treatment with TPPU at either dose (groups A-v3-TL and A-v3-TH) resulted in a significant dose-dependentdecrease in the production of DiHETE and DiHDPE (P ,0.01). Each of the dihydroxy-fatty acid regioisomers of EPAand DHA changed similarly, and thus showed an identical pat-tern to the summed tissue levels of diols across all the groups.The tissue levels of the diol derivatives of the v-6 fatty acids,dihydroxyeicosatrienoic acids (DHETs), and dihydroxyoctade-cenoic acid (DiHOMEs) decreased in group A as compared tocontrols (Fig. 2B, Panels B and E). The tissue levels of DHETsbut not DiHOMEs decreased in group A-v3 as compared togroup A. The tissue levels of both diols decreased only slightlyin group A-v3-TH in comparison with the A-v3-TL (P. 0.05).

Contribution of sEH to the AntihypertensiveEffects of v-3 PUFAs

To test the hypothesis that the v-3 PUFAs act in partthrough their epoxides, we further examined the epoxide-to-diol ratio of the EPA and DHA metabolites across all thegroups. The trend of the change in epoxide-to-diol ratio forEPA and DHA followed an almost identical pattern to eachother across all the groups (Fig. 2A, Panels C and F). Althoughthe EpETE-to-DiHETE and EpDPE-to-DiHDPE ratios increasedonly slightly in group A-v3, the ratios increased significantly ingroups A-v3-TL and A-v3-TH with a larger increase of theEpDPE-to-DiHDPE ratio in the latter group (P , 0.01). In thev-6 fatty acid series, the renal EET:DHET ratio decreased in allgroups as compared to controls (Fig. 2B, panel C). As for the v-6linoleate, the EpOME:DiHOME ratio significantly increased ingroups A-v3-TL and A-v3-TH as compared to groups A, A-v3,and A-TH (Fig. 2B, panel F). To test if TPPU reached effectivelevels (above the IC50) for inhibition of sEH, we determined theconcentration of TPPU in the kidney: 7 6 0.8 mg/gtissue and12 6 0.8 mg/gtissue, and in the plasma: 610 6 130 nM and18006 445 nM in groups A-v3-TL and A-v3-TH, respectively.

We observed that the sum of EpDPEs (includes the 10,11-, 13, 14-, 16, 17-, and 19, 20-EpDPE) have an inverse



correlation with SBP (R = 20.36, P = 0.04), whereas the sumof EpETEs (includes the 8, 9-, 11, 12-, 14, 15- and 17, 18-EpETE) have a weaker correlation (R =20.29, P = 0.1), but ina similar direction as the EpDPEs. The EpDPE regioisomers,16, 17- and 19, 20-EpDPE showed an inverse correlationwith SBP (R = 20.35, P , 0.05). The tissue levels of sumEETs and EpOMEs did not correlate with SBP (R =20.02 andR = 20.04, respectively).

Inhibition of sEH Enhances theAnti-inflammatory Effects of v-3 PUFAs inthe Kidney

To assess the anti-inflammatory effects of the v-3 PU-FAs, we quantified the tissue levels of common proinflamma-tory and anti-inflammatory metabolites produced by the COX-2 and LOX (lipoxygenase) enzymes in the ARA cascade (Fig.3 and Table S7A and B, which show the renal levels of theCOX-2 and LOX metabolites of ARA and EPA, respectively).

First, we determined the tissue levels of ARA-derivedprostaglandins (PGs) in the kidney. While the tissue levels ofPGE2, PGD2, and 6-keto-PGF1a increased by approximately12, 10 and 7 fold; respectively, in group A, the 3 PGs decreasedin group A-v3 (P , 0.05, Fig. 3, Panels A–C). In comparison

with group A-v3, PGs decreased to control levels in groupsA-v3-TL and A-v3-TH. Also, the tissue levels of PGD2 furtherdecreased in group A-v3-TL as compared to group A-v3-TH(P , 0.01). Similarly, the tissue levels of thromboxane B2 andPGF2a increased in group A and decreased in groups A-v3,A-v3-TL, and A-v-TH to control levels (Table S7A).

Second, we examined the changes in the LOX-derivedARA metabolites hydroxy-eicosatetraenoic acids (HETEs).The tissue levels of 11-, 12-, and 15-HETEs increased by 1.5-to 6-fold in group A as compared to controls (Fig. 3, PanelsD–F; and Table S7A). In contrast to group A, the tissue levelsof these HETEs decreased in group A-v3, and furtherdecreased in groups A-v3-TL and A-v3-TH. The tissue lev-els of 20-HETE slightly increased in group A (P . 0.05), andfurther increased in groups A-v3-TL and A-v3-TH (P ,0.05), but not in groups A-v3 and A-TH (Table S7A).

We also quantified the COX-2 and LOX metabolitesthat are generated from EPA. Although the major LOXproducts of EPA, the HEPE (hydroxy-eicosapentaenoic acid)(consisting of the 5-, 8-, 12-, 15-HEPE regioisomers),decreased in group A as compared to controls, the tissuelevels of these metabolites tremendously increased in groupA-v3. The presence of TPPU resulted in lower tissue levels ofHEPE (except for 5- and 15-HEPE) in groups A-v3-TL and

FIGURE 2. Effects of the dietary v-3 PUFAs and TPPU on the epoxide and diol metabolites of EPA, DHA, ARA, and LA in Ang-IIdependent hypertension. The changes in the abundance of the epoxides, diols, and corresponding epoxide-to-diol ratios inresponse to the given treatments are shown. A, Renal levels of EPA and DHA metabolites are shown in separate Panels. Panel A,sum EpETE (includes the 8, 9-, 11, 12-, 14, 15-, and 17, 18-EpETE); panel B, sum DiHETE (includes the 8, 9-, 11, 12-, 14, 15-, and17, 18-DiHETE); panel C, sum EpETE:DiHETE ratio; panel D, sum EpDPE (includes the 10, 11-, 13, 14-, 16, 17-, and 19,20-EpDPE); panel E, sum DiHDPE (includes the 10, 11-, 13, 14-, 16, 17-, and 19, 20-DiHDPE); and panel F, sum EpDPE:DiHDPEratio. B, Renal levels of ARA and LA metabolites are shown in separate Panels. Panel A, sum EETs (includes the 8, 9-, 11, 12-, and 14,15-EET); panel B, sum DHETs (includes the 8, 9-, 11, 12-, and 14, 15-DHET); panel C, sum EET:DHET ratio; panel D, sum EpOMEs(includes the 9, 10-, and 12, 13-EpOME); panel E, sum DiHOMEs (includes the 9, 10- and 12, 13-DiHOME); and panel F, sumEpOME:DiHOME ratio. Results are given as percentage of controls (n = 5). Group A, n = 8; group A-v3, n = 8; group A-v3-TL, n = 7;group A-v3-TH, n = 14; and group A-TH, n = 8. Statistically significant differences were determined by 1-way ANOVA followed bypairwise comparisons. P, 0.05; *, compared with controls;F, compared with group A; ¥, compared with group A-v3; $, comparedwith group A-v3-TH; and #, compared with group A-TH. Data are mean 6 SEM. For primary data see Tables S4 and S5.



A-v3-TH as compared to group A-v3 (Table S7B). In addi-tion, other metabolites of ARA and LA were reduced by thecombination of v-3 PUFAs and TPPU (see Table S8, Supple-mental Digital Content 1, http://links.lww.com/JCVP/A121,which presents the renal levels of other enzymatic productsof ARA and LA). We did not determine the renal levels ofthe COX-2 or LOX metabolites of DHA, because the analyticalmethods for the quantification of those metabolites are still indevelopment.

Finally, we determined the concentrations of majorproinflammatory cytokines as markers of inflammation inthe kidney (Fig. 4 and see Table S9, Supplemental DigitalContent 1, http://links.lww.com/JCVP/A121, which showsthe renal levels of the major proinflammatory cytokinesacross all the groups). As expected, the tissue levels ofMCP-1 significantly increased in group A as compared tocontrols (P , 0.05). The tissue levels of MCP-1 decreasedsignificantly in group A-v3 as compared to group A, andfurther decreased in group A-v3-TL as compared to groupA-v3 (P , 0.01). The tissue levels of MCP-1 showed aninverse correlation with the renal levels of the sum EpETEand sum EpDPE (R = 20.47, P , 0.01).

The v-3 PUFAs Modulate Renal GeneExpression of the ARA Cascade Enzymes, anEpithelial Sodium Channel andAngiotensin-Converting Enzyme-2

To obtain a better mechanistic view, we examined therenal mRNA expression profiles of the major ARA cascade

enzymes, an epithelial sodium channel (ENaC) and angio-tensin-converting enzyme-2 that are closely related to BPregulation (Fig. 5). First, we examined the effects of the v-3PUFAs on the expression of genes encoding the enzymes inthe ARA cascade, Ephx2 (epoxide hydrolase 2), Ptgs-2 (pros-taglandin endoperoxide synthase 2), and Alox5 (arachidonate5-lipoxygenase). The renal mRNA expression of Ephx2increased 10% in group A (Fig. 5, panel A). In comparisonwith group A-v3, Ephx2 was downregulated in group A-v3-TL and A-v3-TH (P , 0.05). The downregulation of Ephx2exhibited an inverse correlation with the tissue levels of sumEpETEs and sum EpDPEs (R = 20.4, P , 0.01). The Ptgs-2message decreased in the renal cortex across all the groupswith a slight downregulation in groups A-v3-TL or A-v3-TH(P , 0.05) (Fig. 5, panel B). In contrast, Alox5 message didnot change across any of the groups (Fig. 5, panel C).

Next, we examined whether the v-3 PUFAs mayreduce arterial BP by altering the gene expression of anENaC, and thus regulate sodium balance in the kidney. Wedetermined the mRNA levels of the a-subunit of ENaC gene,namely Scnn1a (Fig. 5, panel D). In group A, Scnn1a mRNAexpression increased by ; 50% compared with controls. TheScnn1a message was not altered in group A-v3 as comparedto group A; however, it was significantly downregulated ingroup A-v3-TH (P , 0.05) and further downregulated ingroup A-v3-TL (P , 0.01). The expression profile of theScnn1a message showed a significant inverse correlation withthe tissue levels of sum EpETE and sum EpDPE (R = 20.4,P , 0.01).

FIGURE 3. Anti-inflammatory effects of the v-3PUFAs in the kidney in Ang-II dependent hyper-tension. Inflammatory status in the kidney, re-flected by the major PGs (panel A, PGE2; panel B,PGD2; and panel C, 6-keto-PGF1a) and reflectedby major LOX metabolites (panel D, 11-HETE;panel E, 12-HETE; and panel F, 15-HETE) areshown. Results are given as the percentage ofcontrols (n = 5). Group A, n = 8; group A-v3, n =8; group A-v3-TL, n = 7; group A-v3-TH, n = 13–14; and group A-TH, n = 7–8. Statistically signifi-cant differences were determined by 1-way AN-OVA followed by pairwise comparisons. P , 0.05;*, compared with controls; F, compared withgroup A; ¥, compared with group A-v3; $, com-pared with group A-v3-TH; and #, compared withgroup A-TH. Data are mean 6 SEM. For originaldata see Table S7A.



Last, we tested the hypothesis that upregulation of theAce-2 (angiotensin-I-converting enzyme-2) might be involvedin the mechanism of action of the v-3 PUFAs and sEH inhi-bition. The Ace-2 message was upregulated by 3-fold in

A-v3-TL and A-v3-TH as compared to the controls (P ,0.05, Fig. 5, panel E). The Ace-2 message inversely correlatedwith last day SBP (R = 20.3, P = 0.1), plasma levels ofAng-II (R = 20.5, P = 0.09), and the tissue levels of sumEpDPEs and sum EpETEs (R = 0.7, P , 0.01). Among theDHA epoxides, the tissue levels of 19, 20-EpDPE and 16,17-EpDPE showed a strong correlation (R = 0.6, P , 0.01),whereas 13, 14-EpDPE showed a moderate correlation (R =0.4, P , 0.01) with the upregulation of Ace-2.

DISCUSSIONOur major goal was to elucidate the biology and

mechanism of action of the v-3 PUFAs in a murine modelof Ang-II dependent hypertension. We found that the v-3PUFAs in combination with an sEH inhibitor contributeseffectively to the reduction of Ang-II dependent increase inBP and to the attenuation of the renal markers of inflamma-tion. Metabolic profiling methods that directly quantify theoxylipins that are derivatives of v-3 and v-6 PUFAs suggestthat the epoxides of the v-3 PUFAs are in part responsible forthe antihypertensive and anti-inflammatory effects of thesecompounds. In addition, our results suggest that sEH hasa significant role in the metabolism and function of the epox-ides of the v-3 PUFAs. Dietary v-3 PUFAs, when sEH wasinhibited, not only increased the bioactive epoxides, but alsomodulated both the COX and the LOX pathways in the ARAcascade in Ang-II–infused mice. The increase in EpDPEs onsupplementation with an v-3 rich diet and sEH inhibitor andtheir inverse correlation with the SBP suggest that these

FIGURE 5. Changes in the mRNAexpression of genes encoding themajor ARA cascade enzymes (Ephx2,Ptgs-2, and Alox5 genes) and genesencoding an ENaC, a-ENaC (Scnn1agene), and an angiotensin I-convert-ing enzyme-2 (Ace-2) in the kidney.Panel A, Ephx2; panel B, Ptgs-2; panelC, Alox5; panel D, Scnn1a; and panelE, Ace-2. Positive mRNA expressionvalues represent upregulation,whereas negative values representdownregulation of the gene of inter-est. Results are given as percent of thecontrols (n = 5). Group A, n = 8; groupA-v3, n = 8; group A-v3-TL, n = 7;group A-v3-TH, n = 14; and groupA-TH, n = 8. Statistically significantdifferences were determined by 1-wayANOVA followed by pairwise com-parisons. P , 0.05; *, compared withcontrols; F, compared with group A;¥, compared with group A-v3; $,compared with group A-v3-TH; #,compared with group A-TH. Data aremean 6 SEM.

FIGURE 4. Anti-inflammatory effects of the v-3 PUFAs onMCP-1 in the kidney. The renal levels of a proinflammatorycytokine, MCP-1, were determined in the kidney at the end ofthe experiment. Results are given as percentage of the controls(n = 5). Group A, n = 8; group A-v3, n = 8; group A-v3-TL, n =7; group A-v3-TH, n = 13–14; and group A-TH, n = 7–8.Statistically significant differences were determined by 1-wayANOVA followed by pairwise comparisons. P , 0.05; *,compared with controls; F, compared with group A; ¥,compared with group A-v3; $, compared with group A-v3-TH; and #, compared with group A-TH. Data are mean6 SEM.See Table S9 for original data and other cytokines.



epoxides contribute to the reduction of SBP and attenuation ofrenal inflammation in Ang-II–dependent hypertension. Further-more, it seems that upregulation of Ace-2 and Scnn-1a mightcontribute to the mechanism of action of the v-3 PUFAs andsEH inhibition.

Antihypertensive Effects of the v-3 PUFAsAnimals receiving Ang-II had significantly higher SBP

compared to the rest of the groups suggesting that treatmentwith the v-3 rich diet, TPPU or combination of both wereeffective in lowering BP in this model. The antihypertensiveeffects of the v-3 PUFAs have been demonstrated in sponta-neously hypertensive rats and in humans.12,45,46 Consistentwith these previous reports, the v-3 PUFAs reduced SBP inAng-II dependent hypertension. The v-3 PUFAs were aseffective as TPPU at 0.6 mg/kg in reducing Ang-II inducedhypertension for the first several days of exposure (Fig. 1);however, this positive effect decreased with time to where the2 doses of sEHI with or without the v-3 rich diet were betterthan the v-3 rich diet alone. We do not have a full explanationfor the decreased antihypertensive effect observed with thev-3 rich diet on days 7, 9, and 11 of the study (Figure S1).However, coadministration of the v-3 rich diet with an sEHIresulted in a return to near normotensive BP. The results ofthe mixed-effects model with Ang-II, v-3 rich diet and TPPUas between-subjects factor supported these findings that maineffects of each of these factors are statistically significant, andthere is an interaction between the v-3 rich diet and TPPU inreducing SBP after day 1 in Ang-II–dependent hypertension.

Furthermore, our hypothesis that by the end of thetreatment period, the combination of the v-3 rich diet andTPPU would have a stronger effect on SBP compared tothe v-3 rich diet alone was supported by the results of theANOVA of SBP at day 11, which showed significantly lowerSBP with A-v3-TL, and a trend for lower SBP with A-v3-TH(P = 0.08). However, it is likely that the SBP decrease inA-v3-TH also represents a real effect rather than an artifact,because the mixed-effects model for the SBP data and AUCanalysis for days 7 through 11 (Figure S1, panel A) allshowed significantly lower SBP with A-v3-TH comparedto the v-3 rich diet alone. Because there were no detectabledifferences in the SBP between groups A-v3-TL and A-v3-TH (see Figure S1, panel B), the 2 doses of TPPU studiedhere did not result in a dose-dependent effect on the BP.Furthermore, the sEHI in combination with the v-3 rich dietclearly reduced biochemical indicators of inflammation in thekidney better than either treatment alone.

Contribution of Diet to Arterial PressureMice are known to concentrate v-3 PUFAs in a very

short time even on a low-fat diet,47 and ALA was shown toprevent v-3 PUFA deficiency–induced hypertension in the off-spring that are on a 10% ALA rich diet during the prenatalperiod and 24 weeks until after the weaning.48 In our study, weused a control diet supplemented with corn oil compared toa diet rich in v-3 PUFAs. The sEHI alone resulted in a signif-icant reduction of SBP in animals on the corn oil diet (Fig. 1).This could be in part due to the sEHI stabilizing trace levels ofepoxides from v-3 PUFAs derived from ALA present in the

corn oil (Table S1B). Of course, sEHIs are well known tostabilize epoxides of v-6 PUFAs, notably the antihypertensiveEETs. Therefore, we hypothesized that the v-3 PUFAs in thecorn oil diet that are comprised mostly of ALA (Table S1B)might have contributed to the normalized BP in group A-TH.To this end, we used a second control diet from Harlan thatwas previously used as a standard diet to compare with an v-3rich diet. When we put mice on the standard diet for 7 days,Ang-II infusion increased BP as expected (Figure S2A). A lowdose of sEHI of 0.2 mg/kg still reduced BP, which is consistentwith previous studies.37,49,50 Our data lacked the power todetermine whether this reduction was different from the highdose of sEHI alone on our standard diet without v-3 lipids.

Even though the amount of ALA was lower in the cornoil diet as compared to the standard diet, we observed higherrenal levels of the DHA and EPA epoxides in mice fed a cornoil diet as compared to mice fed a standard diet. In contrast tothe epoxides of v-3 PUFAs, the ARA and LA epoxides did notdiffer in mice on these 2 different diets. The differences in theSBP and renal DHA and EPA epoxides emphasize the contri-bution of the diet to the changes in BP. We do not have a fullexplanation of why the amount of ALA did not reflect itself intissue levels in our experiment. Of course, ALA conversioninto DHA is low, and small changes in levels of DHA epoxideslikely are overshadowed by endogenous EETs, which havestrong antihypertensive effects. However, the higher tissue lev-els of EPA and DHA epoxides in animals fed a corn oil diet areconsistent with the lower SBP observed in this group. Wedo not know whether there are dietary sources other than theALA that might explain the increase in tissue levels of DHAand EPA in mice fed the corn oil diet as compared to micefed the standard diet (see Supplemental Digital Content 1,http://links.lww.com/JCVP/A121, footnote in Table S1Bexplaining the differences in formulation and analysis of thefatty acid composition between the corn oil and standard diet).

Based on these results, it seems that mainly the increasein the EPA and DHA epoxides and not the ALA content ofthe diet are major contributors to the normalized BP in groupA-TH. However, because of the significant increase in thetissue levels of the epoxides of v-3 PUFAs and lower SBP ingroup A-v3 as compared to group A, we cannot attribute theobserved antihypertensive effects to sEH inhibition alone ingroups that received the combined treatment. Moreover, thedata showing that BP is only normalized in A-v3-TH and notin A-TH suggest that v-3 PUFAs (in addition to TPPU) alsocontribute to the reduction in BP in Ang-II dependent hyper-tension. Also, because it is hard to remove all v-3 lipids fromthe diet, we cannot say that the action of the sEHI occurred inthe absence of v-3 lipids (ie, in group A-TH). However, wecan say that the sEH inhibitor was effective even at the lowdose in reducing BP to a certain extent even when the amountof v-3 lipids in the diet is low.

Overall, the decrease in the effectiveness of the v-3PUFAs at days 7 through 11 (Fig. 1), and the data showingthat TPPU alone does not reduce BP back to control levels atthe low dose (Figure S2A) suggest that both the v-3 rich dietand TPPU were needed to maintain a stable reduction in BPas in A-v3-TL and A-v3-TH throughout the experiment. Insupport of this argument, ACE-2 message was downregulated



and Scnn1a message was upregulated in groups A-v3-TL andA-v3-TH as compared to A-v3 and A-TH (Fig. 5). Also, theanti-inflammatory effects (PGD2, 6-keto-PGF1a, 15-HETE,and MCP-1) observed with group A-v3-TL as compared togroups A-v3 and A-TH are consistent with the combinedeffects of the v3-PUFAs and TPPU.

This observation is further supported by the mixed-effects model with 3 between-subjects factor. As summarizedin Table S3, both the v-3 rich diet and the sEHI treatmentscontribute to reduced BP as indicated by the arbitrary unitsderived from the model. This model additionally indicatesthat sEHI treatment contributes more to reduced BP aloneor in combination with the v-3 PUFAs than the v-3 alone(P = 0.004).

Potential Role of Epoxide Metabolites in theAction of v-3 PUFAs

The overall increase in the EPA and DHA metabolitessuggests that 1 week pretreatment and the following 2-weekintervention with the v-3 rich diet allowed enough time forEPA and DHA to be incorporated into the membranes and tobe accumulated in the tissues. The inverse correlation ofEpDPEs with BP is consistent with the hypothesis thatEpDPEs contribute to lower BP in mice on the v-3 rich diet.Moreover, this correlation was stronger for EpDPEs than forEpETEs. Given the high abundance of EpDPEs in the kidney,these results suggest that DHA epoxides contribute to thereduction of SBP more than the EpETEs. Collectively, theseresults support the notion that DHA has antihypertensiveeffects as has been previously shown in spontaneously hyper-tensive rats.46,51 DHA has further been shown to activatelarge-conductance Ca2+-activated K+ channels on vascularsmooth muscle cells25 and cause vasodilatation.24 These anti-hypertensive effects of DHA have been shown to be depen-dent on CYP epoxygenase activity. Although our resultssuggest that DHA is more important than EPA in the kidneyin Ang-II–dependent hypertension, recent recommendationsin the literature propose consumption of both EPA and DHAfor maximum benefit from the v-3 PUFAs in cardiovasculardiseases.3 In addition, information on the total fatty acid com-position of the renal tissue would be helpful to understand thecontribution of the major parent fatty acids (ARA, ALA,DHA, and EPA) to the biosynthesis of the EPA and DHAepoxide metabolites.

We provide evidence that sEH inhibition increases theepoxide-to-diol ratio of the EPA and DHA epoxides in vivo.Assessment of the epoxide-to-diol ratio also allowed us toelucidate the contribution of the sEH to the metabolism ofepoxides that are derived from EPA and DHA. We show thatTPPU increases the epoxides of both EPA and DHA andreduces their corresponding diols, which results in an increasedepoxide-to-diol ratio. These observations are in line withprevious reports suggesting a key role for sEH in themetabolism and function of DHA and EPA epoxides, whichare excellent substrates for sEH.27,28 However, the effects ofTPPU on the tissue levels of DHA and EPA epoxides were notdose-dependent at the doses selected in our study. Our findingsagree with studies demonstrating that EpDPEs are more

abundant than the EpETEs.27,29 The ratio of EpDPEs toDiHDPEs provides a better indication of sEH inhibition (targetengagement) than the corresponding EET:DHET ratio.Accordingly, tissue levels of TPPU were at least 1000 timesabove its IC50 (2 nM or 2.50 1024 mg/gtissue) determinedagainst the recombinant mouse sEH using fluorescenta-cyanocarbonate as substrate. This suggests near-completeinhibition of sEH by TPPU in the kidneys. There are possibleexplanations for the unchanged EET:DHET ratio. The higherabundance of EPA and DHA, which competes with ARAmetabolism, might have affected the availability of the precur-sor fatty acid reservoir. Of course, further metabolism andexcretion of the diols and alternate routes of epoxide metabo-lism including relative incorporation into phospholipids couldhave an impact.52 These results are consistent with our hypoth-esis that EpDPE regioisomers have a role in the mechanism ofaction of DHA in reducing Ang-II induced hypertension.Among the DHA epoxides, we found 19, 20-EpDPE to bethe most abundant DHA epoxide in the kidney. However, itis currently not possible to make assumptions based on its highabundance whether this would be the most effective regioiso-meric DHA epoxide in reducing BP and inflammation. The 3regioisomeric epoxides of DHA, 13, 14-, 16, 17-, and 19,20-EpDPE have been shown to have excellent antihyperalge-sic activities in a rat inflammatory pain model.27 Among these3 regioisomeric DHA epoxides, 19, 20-EpDPE has beenshown to be the most abundant regioisomer in the centralnervous system of these rats; however, a less abundant regioi-someric epoxide, 13, 14-EpDPE is found to be more effica-cious than the 19, 20-EpDPE in this model. While studiesexamining the vascular reactivity and channel activity inresponse to DHA epoxides support the vasodilator effects ofDHA epoxides,24,26,53 the bioactivity and efficacy of differentregioisomeric epoxides of DHA in hypertension needs to beexplored in future studies.

Inhibition of sEH Increases the Efficacy of theAnti-inflammatory Effects of the v-3 PUFAs inthe Kidney

We examined the changes in renal eicosanoid levelsacross the treatment groups, because these metabolites areknown to effectively contribute to the long-term regulation ofBP in the kidney. As expected, we found that the v-3 PUFAswere more effective in attenuating renal markers of inflamma-tion when sEH was inhibited at either dose than when admin-istered alone. This increased effectiveness might be because ofan increase in the epoxides when sEH was inhibited. Of the 2doses, the anti-inflammatory effects of the v-3 PUFAs weremost evident in the presence of the lower dose TPPU. This is anadvantageous effect, because intake of drugs or drug-like com-pounds at high doses is usually associated with a higher risk ofadverse effects in comparison with intake at low doses. Eventhe higher dose of TPPU (0.6 mg/kg) used in this study, canstill be considered a low dose when compared to many drugson the market. For example, NSAIDs are usually taken at highdoses, which would be equivalent to over 1 or more mg/kgdose in mice. In group A-v3-TL, we observed enhanced tissuelevels of the epoxides (ie, EpETEs and EpDPEs), reduced renal



PGs (primarily PGE2 and PGD2), reduced LOX products(mainly HETEs), and reduced MCP-1 levels in comparisonwith group A-v3 (Figs. 2–4). Among the LOX products, theproinflammatory and the powerful vasoconstrictor, 20-HETE,followed a different pattern than the other HETE regioisomersacross the groups, which might be because this metabolite isproduced by a CYP rather than an LOX enzyme.

Overall, these results are consistent with our hypothesesthat the epoxide metabolites of v-3 PUFAs can be stabilized bysEH inhibition and EpDPEs and EpETEs contribute to theobserved anti-inflammatory effects of their corresponding par-ent fatty acids in Ang-II–dependent hypertension. Moreover,the inverse correlation between the tissue levels of MCP-1 andthat of epoxides offers a promising clue to the mechanism ofaction for the anti-inflammatory effects of the v-3 PUFAs inthe kidney in Ang-II dependent hypertension. Based on thedata on the largely inflammatory cytokines MCP-1, IL-1b,and IL-6, the lower dose of TPPU at 0.2 mg/kg with the v-3PUFAs has an additive effect in reducing these indicators ofrenal inflammation (Fig. 4). This is possibly because TPPUalone significantly reduced the tissue levels of the proinflam-matory cytokines MCP-1, tumor necrosis factor-a, and IL-1b(P , 0.05) as compared to group A (Table S9). Not only thetissue levels of MCP-1 but also the diol metabolites of EPA,DHA, and ARA decreased after TPPU treatment in our study(Fig. 2A and B, Panels B and E). This finding is consistent witha recent study reporting that the diol metabolites that arederived from ARA, the DHETs, promote MCP-1 mediated–monocyte chemotaxis, and suggesting that sEH inhibitors actin part by inhibiting MCP-1 induced–monocyte chemotaxisthrough a decrease in DHET levels.54 It was also intriguingthat the v-3 rich diet showed a further decrease in the tissuelevels of the LOX metabolites when sEH was inhibited. Themetabolites in the LOX pathway altered by v-3 PUFAs arelargely proinflammatory,55 and thus the observed decrease isindeed a favorable anti-inflammatory effect.

DHA has been shown to alter the major PGs and LOXmetabolites by competing with ARA.29 While we observed thatthe tissue levels of EETs and EpOMEs (Fig. 2B) decreased, thetissue levels of EpETEs and EpDPEs (Fig. 2A) increased ingroups treated with the v-3 rich diet as compared to the con-trols. Such differences in the relative abundance of epoxides ofthe v-3 and v-6 fatty acid series are suggestive of selectiveformation of epoxides of v-3 PUFAs among all 4 parent fattyacids (ARA, LA, and EPA/DHA). Thus, our results on theP450 branch of the ARA cascade are in good agreement withprevious studies7,28,29 suggesting that v-3 PUFAs exert theiranti-inflammatory effects, in part, by competitively inhibitingthe conversion of ARA to its respective metabolites in theCOX and LOX branches.

Effects of the v-3 PUFAs on mRNA Expressionof Ephx2, Ptgs-2, Alox5, Scnn1a, and Ace-2

The gene expression data provided additional insightsinto the biological effects of the v-3 PUFAs. Overall, theeffects of the v-3 PUFAs on gene expression were reversedby TPPU, which may be interpreted as a control of homeostasisand vascular tone in this model. Although the gene expression

data correlated generally with changes in oxylipin expressionfor Ephx2, Ptgs-2, and Scnn1a, the mRNA expression of Alox5was unaltered despite the reduced LOX metabolites. In groupsA-v3-TL and group A-v3-TH, the downregulation of Ephx2and Ptgs-2 was consistent with the reduced levels of diols fromEPA/DHA and reduced levels of PGs, respectively. Moreover,assuming that changes in the Ephx2 message are closely asso-ciated with the activity of sEH, reduced sEH expression shouldresult in reduced metabolism and thus increased levels ofEpETE and EpDPE. Indeed, we determined higher levels ofEpFAs in both groups A-v3-TL and A-v3-TH. Interestingly,Ephx2 was downregulated by the v-3 PUFAs only in thepresence of TPPU, although TPPU alone did not alter themRNA expression of the Ephx2 message. These data suggestthat the v-3 epoxides are involved in the downregulation ofEphx2message. A previous report has demonstrated a decreasein Ephx2 expression in the liver on dietary supplementationwith v-3 PUFAs.9 However, our study has examined theexpression of Ephx2 in the kidney not in the liver, and ina different disease model. The downregulation of the Ephx2message by the v-3 PUFAs in the presence of an sEHI is likelya desirable effect, because sEH has been shown to be involvedin the development and maintenance of hypertension.40

The gene expression profile of the Scnn1a providesanother potential mechanism underlying the v-3 PUFAs actionin BP regulation. ENaC is a constitutively active membrane-bound sodium channel, located at the apical membrane of theepithelial cells in the kidney. ENaC is a major contributor tothe regulation of BP, because it constitutes the ultimate step ofrenal sodium handling.56 The EETs inhibit ENaC and elicitnatriuresis by regulating tubular sodium transport and inhibitsodium reabsorption by altering the pressure–natriuresis mech-anism in the kidney.57–59 Because the changes in the transcrip-tion or translation of ENaC are closely associated with thenumber of channels and aldosterone-induced ENaC expressionis specific to the a-subunit of the ENaC,60 we looked at thea-subunit of ENaC, Scnn1a mRNA expression in the kidney.We found that the v-3 PUFAs downregulated the Scnn1amessage in the presence of TPPU, and this downregulationshowed an inverse correlation with the renal EpETE andEpDPE levels. These results suggest that the epoxides of thev-3 PUFAs, very much like the EETs, contribute to sodiumhandling in the kidney, which may be responsible, in part, fortheir antihypertensive effects.

The upregulation of Ace-2 and its inverse correlation withSBP suggest that the combination of the v-3 rich diet andTPPU modulates the renin–angiotensin–aldosterone system(RAAS). ACE-2 is considered as the counter regulatory com-ponent of the RAAS, because ACE-2 inactivates both Ang-Iand Ang-II by its exopeptidase action, which results in theformation of Ang 1, 7. The Ang 1, 7 opposes the vasoconstric-tor actions of Ang-II and induces vasorelaxation.61 The v-3PUFAs reduce ACE-2 activity62–64; and ACE-2 mRNA hasbeen reported to be downregulated in the aorta of the sponta-neously hypertensive rats fed a diet rich in ALA.65 Last, thestrong correlation between the upregulation of Ace-2 and thetissue levels of the EpDPEs and EpETEs and plasma levels ofAng-II suggests that these epoxides might modulate Ace-2 andthus contribute to the regulation of BP.



CONCLUSIONWe provided evidence that the long-chain v-3 PUFAs,

sEH inhibition, and coadministration of the v-3 PUFAs withsEHIs lower arterial BP and attenuate markers of renal inflam-mation in a murine model of angiotensin-II dependent hyper-tension. Our results also suggest that the v-3 PUFAs, incombination with an sEHI exhibit improved anti-inflamma-tory effects and modulate the ARA cascade by blocking boththe COX and the LOX pathways. Results on the anti-inflam-matory and antihypertensive effects of the v-3 PUFAs andsEH inhibition are consistent with decreased tissue levels ofPGs, LOX products, and MCP-1, increased tissue levels ofepoxides derived from EPA and DHA, the downregulation ofthe Scnn1a and upregulation of Ace-2 message in the kidney.Even though the v-3 rich diet and sEHI lead to changes in thetissue levels of the EPA and DHA metabolites which is re-flected in the inflammatory state of the kidney, we wereunable to show the clear reflection of these changes on SBP.

Our study forms a basis for understanding the biologyand underlying mechanisms of the long-chain v-3 PUFAs inAng-II dependent hypertension. We provide evidence for theanti-inflammatory effects of v-3 PUFAs and their modulationof the COX and LOX pathways in the ARA cascade. Fur-thermore, our data suggest that the epoxide metabolites of v-3PUFAs are among the mediators of the antihypertensive andanti-inflammatory effects of v-3 PUFAs in Ang-II dependenthypertension. The combination of v-3 supplementation andTPPU treatment presents an exciting alternative to currentanti-inflammatory and antihypertensive drugs on the market.The modulation of key enzymes and metabolites in the COX,LOX, and P450 pathways and the accompanying reduction inSBP and proinflammatory cytokines are exciting and haveimplications for not only hypertensive renal injury but alsoatherosclerosis and vascular remodeling. Future studies arewarranted to identify mediators of the anti-inflammatoryand antihypertensive effects of EPA and DHA independentlyfrom each other in angiotensin-II–dependent hypertension.

ACKNOWLEDGMENTSThe authors thank Dr Alan Buckpitt and Dr Kent

Pinkerton for their valuable assistance with renal metabolicprofiling and survival surgeries.

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1

SUPPLEMENTAL DIGITAL CONTENT 1

Anti-inflammatory Effects of Omega-3 Polyunsaturated Fatty Acids

and Soluble Epoxide Hydrolase Inhibitors in Angiotensin-II

Dependent Hypertension

Arzu Ulu, MSc*; Todd R Harris, PhD*; Christophe Morisseau, PhD*; Christina Miyabe, BS*; Hiromi Inoue, DVM#;

Gertrud Schuster, PhD§, Hua Dong, PhD*; Ana-Maria Iosif, PhD£, Jun-Yan Liu, PhD*; Robert H Weiss, MD# ¥;

Nipavan Chiamvimonvat, MDɸ ¥; John D Imig, PhD†; Bruce D Hammock, PhD*

*Department of Entomology and UC Davis Comprehensive Cancer Center, University of California, Davis;

#Division of Nephrology, ɸ Division of Cardiovascular Medicine, Department of Internal Medicine, §Department of

Nutrition, £Division of Biostatistics, Department of Public Health Sciences, School of Medicine, University of

California, Davis, USA; ¥U.S. Department of Veterans’ Affairs Medical Center, Sacramento, California, USA;

†Medical College of Wisconsin, Milwaukee, WI, USA.

Corresponding author:

Dr Bruce D. Hammock

E-mail: [email protected]

Telephone: 530-752-7519

Fax: 530-752-1537

Short title: Fish oil and sEH inhibitors in hypertension

mailto:[email protected]

2

INDEX Sections: A) Fatty acid analysis of the diets………………………………………………………… 3

Table S1A and Table S1B

B) Measurement of blood pressure………………………………………………………. 5

C) Oxylipin extraction and analysis………………………………………………………. 5

Table S2A and Table S2B

D) qRT-PCR gene expression assays…………………………………………..………….. 8

E) Analysis of the SBP data using mixed-effects model with three between subjects

factors………………………............................................................................................ 8

Mixed-effects models with residuals diagnostics……………………………………….. 9

Table S3

F) Examination of the effects of each treatment on blood pressure at day11……….……... 10

Figure S1, Figure S2A and Figure S2B

G) Assessment of renal function and histology ……………………………………............. 14

1. Methods

2. Results

Figure S3, Figure S4A and Figure S4B

H) Measurement of plasma creatinine……………………………………………………. 18

Tables:

Table S4……………………………………………………………………………….. 19

Table S5……………………………………………………………………………….. 20

Table S6…………………………………………………………………………………21

Table S7A……………………………………………………………………………… 22

Table S7B……………………………………………………………………………… 23

Table S8…………………………………………………………………………………24

3

Table S9…………………………………………………………………………………25

References ……………………………………………………………………………... 26

4

A) Fatty acid analysis of the diets

To quantify the fatty acid composition, lipids of the powered diets were extracted from

weighed aliquots, mixed with internal standards, transesterified and analyzed using gas

chromatography with mass spectral detection. Specifically, ~50 mg aliquot of diets were

weighed into a pre-tarred 2mL safe lock tubes, lipids were extracted with 400 μL isopropanol,

followed by 500 μL cyclohexane and phase separation with 550 μL 0.1 M ammonium acetate.

Samples were centrifuged at 3500rpm for 5min at room temperature. A 10 μL extract aliquot was

transferred to a 2 mL glass amber vial containing 3 μM SS-15:1n5-triacylglceride (NuChek Prep;

Elisian, MN) derivatization standard in 1:9 toluene/methanol (v/v). These samples were enriched

with 100μL 0.5M anhydrous sodium methoxide (Sigma-Aldrich; St. Louis, MO), incubated 1hr

at 60oC, enriched with 100μL 3N methanolic hydrochloric acid (Sigma-Aldrich; St. Louis, MO),

and incubated 30 min at 60oC. Reaction mixtures were neutralized by addition of 400μL of

0.25M KHCO3:0.5M K2CO3, and fatty acid methyl esters were extracted by vortexing with

400μL hexane, with phase separation being assisted by 5min centrifugation at 3500 rpm. A

100μL aliquot of the hexane extract was spiked with 10μL hexane containing 44μM C23:0 as an

internal standard. Analyte residues in 1μL sample aliquots were separated on a 30m x 0.25mm,

0.25μm DB-225 ms column and detected on an Agilent 5973 mass selective detector with

selected ion monitoring responses calibrated against a 7 point calibration curve used to quantify

the results. All reported results are corrected for surrogate loss.

TABLE S1A. Composition of mouse diets administered in the present study.

Components

Corn oil diet -3 rich diet*

(% weight)

Cornstarch 40 40

Casein 20 20

Dextrose 15 15

Sucrose 10 10

Cellulose 5 5

Mineral mix 3.5 3.5

Vitamin mix 1 1

Cysteine 0.3 0.3

Choline bitartrate 0.2 0.2

Corn oil (Mazola) 5 3.5

EPA - 0.75

DHA - 0.75

*Preparation: Briefly, dextrose, mineral mixture, vitamin mixture, cysteine and choline

bitartrate were combined and mixed for 30 min, then cornstarch, casein, sucrose and cellulose

were added to this mixture. The oil ingredients those are corn oil, 90% pure EPA and 90% pure

DHA (Larodan Fine Chemicals, Sweden) were weighed together in a glass beaker and added to

the dry ingredients of the diet. The diet lacking these omega-3 fatty acids (corn oil diet) was

prepared using the same ingredients except for the EPA and DHA, which was replaced by the

corn oil. Animals were started on corresponding diets one week before the induction of

hypertension. Daily food consumption was recorded.

5

TABLE S1B. Percentage fatty acid composition of total fat for the corn oil, -3 rich and

standard diets.

Fatty Acids C: D Corn oil

diet (%) -3 rich

diet (%)

Standard diet*

(Harlan-Teklad)

(%)

Saturated fats

Palmitic C16:0 9.53 7.58 7.64

Magaric C17:0 0.04 0.03 0.00

Stearic C18:0 1.17 0.93 1.5

Arachidic C20:0 0.23 0.19 0.01

Behenic C22:0 0.06 0.05 0.03

Lignoceric C24:0 0.07 0.06 0.00

Monounsaturated fats

trans -7-hexa-deanoic C16:1n7t 0.02 0.02 N/A

Palmitoleic C16:1n7 0.06 0.07 0.07

cis-10-Heptadecenoic C17:1n7 0.02 0.02 N/A

Oleic C18:1n9 26.97 20.90 12.59

cis-Vaccenic C18:1n7 0.18 0.15 N/A

Gadoleic C20:1n9 0.08 0.07 0.17

n-6 polyunsaturated fats

Linoleic C18:2n6 60.92 46.35 31.35

Conjugated linoleic 9c,11t-CLA 0.01 0.01 N/A

Eicosadienoic C20:2n6 0.00 0.01 0.00

Arachidonic C20:4n6 0.00 0.22 0.00

Docosapentaenoic C22:5n6 0.00 0.07 0.00

n-3 polyunsaturated fats

α- linolenic C18:3n3 0.63 0.53 2.76

Eicosatetraenoic C20:4n3 0.00 0.06 N/A

Eicosapentaenoic C20:5n3 0.01 11.53 0.00

Docosapentaenoic C22:5n3 0.00 0.19 0.00

Docosahexaenoic C22:6n3 0.00 10.98 0.00

Σ saturated fatty acids 11.10 8.84 9.59

Σ monounsaturated fatty acids 27.33 21.22 12.84

Σ (n-6) polyunsaturated fatty acids 60.93 46.66 31.35

Σ (n-3) polyunsaturated fatty acids 0.64 23.28 2.76

Σ (n-6)/ Σ (n-3) 95.4 2 11.4

*2018 Teklad Global 18% Protein Rodent Diet, Harlan Laboratories, Hayward, CA. Fatty analysis

data are reprinted from Arendash et al.9 The fatty acid analysis of the corn oil and -3 rich diet, but

not the standard diet (reprinted from the analysis of Arendash et al) was performed by us. Of note,

while the corn oil diet was a purified diet and used in its powder form, the standard diet was a

commercially available regular rodent chow in pellet form, which might vary with changes in the

ingredients and processing by the manufacturer.

N/A: not available

B) Measurement of blood pressure

6

To avoid the investigator bias, we blinded the operator (A.U.) by placing cards that block the

treatment information associated with each cage during the blood pressure recordings. Also, each

cage and associated blocking card was given a number. Measurements were saved by the cage

number and the number identifying each animal (for example; Cage 1-mouse 1), which also

provided blinded data analysis. For each animal, we used the exact same method to analyze the

data. Briefly, we exclude the first 3 (acclimation), the highest and the lowest value among the 20

cycles and last we eliminate the readings associated with animal movement. Then, we average

the remaining blood pressure readings to obtain the final systolic blood pressure for each animal.

Animal movement results in inaccurate measurements, because the software recognizes the

movements as a reference point on the blood pressure curve.

C) Oxylipin extraction and analysis

Fifty mg of murine kidney was homogenized in 400 µL ice-cold methanol containing 0.1%

glacial acetic acid and 0.1% BHT and added with 10 µL anti-oxidant solution (0.2 mg/ mL BHT

(butyl hydroxytoluene) and EDTA in 1:1 ratio methanol and water) and 10 µL internal standard.

Then, tissue homogenates were centrifuged at 10,000 rpm at 4°C for 10 min. Collected

supernatants were extracted using SPE cartridges (Waters Oasis HLB C18 cartridge), then eluted

by ethyl acetate, and thoroughly dried using a vacuum centrifuge. The samples were then re-

dissolved in 50 µL additional standard solution (instrumental standard) in methanol and

quantified using an LC/MS/MS technique.1

TABLE S2A. HPLC solvent gradient to separate ARA, LA, EPA and DHA metabolites.

Gradienta

(min)

Aqueous

phase b

Organic

Phase c

0 65 35

0.25 65 35

1 55 45

3 45 55

8.5 34 66

12.5 28 72

15 18 82

16.5 5 95

18 5 95

18.1 65 35

21.5 65 35 a0.25 ml/min flow rate, b Distilled Water 99.9, Acetic Acid 0.1, volume %, c ACN 80, Methanol 20, Acetic Acid 0.1, volume %.

7

TABLE S2B. Retention time, selected reaction monitoring (SRM) and internal standard

information used to quantify all analytes.

Peak Name

Precursor ion

(m/z)

Product ion

(m/z)

Surrogate Internal

Standards

Retention time

(min)

6-keto-PGF1α 369.3 163.2 d4-6-keto-PGF1a 3.54

d4-6-keto-PGF1 α 373.3 167.1 Instrumental standard (IS)* 3.56

Resolvin E1 349.3 195 d4-PGE2 3.58

d4-TXB2 373.3 173.2 IS 4.28

TXB2 369.2 169.1 d4-TXB2 4.29

PGE3 349.3 269.2 d4-PGE2 4.37

PGD3 349.3 269.2 d4-PGE2 4.61

PGF2 α 353.2 309.2 d4-PGE2 4.75

d4-PGE2 355.2 275.3 IS 4.97

PGE2 351.2 271.3 d4-PGE2 4.99

PGD2 351.2 271.3 d4-PGE2 5.29

17,18-DiHETE 335.3 247.2 d11-14,15-DHET 8.34

14,15-DiHETE 335.3 207.2 d11-14,15-DHET 8.84

Instrumental Standard (IS) 339.2 214.3 None 8.86

11,12-DiHETE 335.2 167.1 d11-14,15-DHET 9.01

12,13-DiHOME 313.2 183.2 d11-14,15-DHET 9.18

8,9-DiHETE 335.2 127.1 d11-14,15-DHET 9.31

9,10-DiHOME 313.2 201.2 d11-14,15-DHET 9.54

d11-14,15-DHET 348.2 207.1 IS 10.0

19,20-DiHDPE 361.2 273.2 d11-14,15-DHET 10.12

14,15-DHET 337.2 207.1 d11-14,15-DHET 10.13

16,17-DiHDPE 361.2 233.2 d11-14,15-DHET 10.64

11,12-DHET 337.2 167.1 d11-14,15-DHET 10.79

13,14-DiHDPE 361.2 193.2 d11-14,15-DHET 10.86

10,11-DiHDPE 361.2 153.2 d11-14,15-DHET 11.17

8,9- DHET 337.2 127.1 d11-14,15-DHET 11.31

d6-20-HETE 325.2 281.2 d11-14,15-DHET 11.95

20-HETE 319.2 275.1 d6-20-HETE 12.03

15-HEPE 317.2 219.2 d8-12-HETE 12.05

8-HEPE 317.2 155.2 d8-12-HETE 12.32

12-HEPE 317.2 179.2 d8-12-HETE 12.51

5-HEPE 317.2 115.1 d8-12-HETE 12.92

d4-9-HODE 299.2 172.3 IS 13.13

13-HODE 295.2 195.2 d4-9-HODE 13.17

9-HODE 295.2 171.1 d4-9-HODE 13.24

15-HETE 319.2 219.2 d8-12-HETE 13.82

17(18)-EpETE 317.2 215.2 d11-11(12)-EET 13.99

11-HETE 319.2 167.2 d8-12-HETE 14.32

d8-12-HETE 327.2 184.2 IS 14.56

9-oxo-ODE 293.2 185.1 d4-9-HODE 14.57

15-oxo-ETE 317.2 113.1 d11-11(12)-EET 14.57

14(15)-EpETE 317.2 207.2 d11-11(12)-EET 14.66

8-HETE 319.2 155.2 d8-5-HETE 14.68

12-HETE 319.2 179.2 d8-12-HETE 14.75

11(12)-EpETE 317.2 167.2 d11-11(12)-EET 14.83

8(9)-EpETE 317.2 127.2 d11-11(12)-EET 14.98

8

9-HETE 319.2 167.2 d8-12-HETE 15.05

15(S)-HETrE 321.2 221.2 d8-12-HETE 15.12

d8-5-HETE 327.2 116.1 IS 15.13

12-oxo-ETE 317.2 153.1 d11-11(12)-EET 15.28

5-HETE 319.2 115.2 d8-5-HETE 15.31

19(20)-EpDPE 343.2 241.2 d11-11(12)-EET 15.98

12(13)-EpOME 295.2 195.2 d4-9(10)-EpOME 16.06

d4-9(10)-EpOME 299.2 172.2 IS 16.16

14(15)-EET 319.2 219.3 d11-11(12)-EET 16.24

9(10)-EpOME 295.2 171.1 d4-9(10)-EpOME 16.26

16(17)-EpDPE 343.2 233.2 d11-11(12)-EET 16.46

13(14)-EpDPE 343.2 193.2 d11-11(12)-EET 16.55

10(11)-EpDPE 343.2 153.2 d11-11(12)-EET 16.66

d11-11(12)-EET 330.2 167.2 IS 16.68

11(12)-EET 319.2 167.2 d11-11(12)-EET 16.81

8(9)-EET 319.2 167.2 d11-11(12)-EET 16.98

*1-Adamantan-1-yl-3-decyl-urea is used as an additional (instrumental) standard. IS was used to

quantify the surrogate internal standards and to calculate extraction recovery after SPE extraction of the

oxylipins from the kidney. All the analytes were quantified using the deuterated surrogate standards.

D) qRT-PCR gene expression assays Kidneys were perfused with cold PBS, and immediately snap frozen in the Trizol reagent

(Invitrogen, Carlsbad, CA) and then stored at -80°C until processing. Total RNA was extracted

from 10 µg of the renal cortex using a pure link Micro total RNA purification kit (Invitrogen,

Carlsbad, CA). Quantity and quality of the RNA samples were assayed by a spectrophotometer

and then 1 µg DNase treated total RNA was converted to cDNA using a high-capacity cDNA

reverse transcriptase kit (Applied Biosystems, Foster City, CA). In the resultant cDNA samples,

fold change in mRNA expression was determined using TaqMan gene expression assays. The

following TaqMan gene expression assays (Applied Biosystems, Foster City, CA) were

employed: Ephx2 (Mm00514706_m1), Ptgs-2 (prostaglandin endoperoxide synthase 2,

Mm00478374_m1), Alox5 (arachidonate 5-lipoxygenase, Mm01182747_m1*), Scnn1a

(epithelial sodium channel, Mm00803386_m1) and Ace-2 (angiotensinI converting enzyme-2,

Mm01159003_m1). Experiments were run on the same day in duplicate along with a house

keeping gene, mouse GAPDH (glyceraldehyde 3-phosphate dehydrogenase message). Fold

change in gene expression was normalized to the endogenous control, GAPDH, and to the

calibrator samples (controls).

E) Analysis of the SBP data using mixed-effects model with three between

subjects factors We performed additional statistical analyses of the SBP data to examine the individual

effects of the given treatments. To this end, we analyzed the SBP data using Ang-II, -3 rich

diet and TPPU as three between subjects factors and time (after day 1) as repeated measures in a

mixed-effects model. While Ang-II and -3 rich diet were analyzed at two levels as present and

absent (+/-), TPPU was analyzed at three levels as absent, low dose and high dose (- / low/ high).

We performed full factorial analysis of the fixed effects that are ‘Ang-II’, ‘-3 rich diet’, ‘TPPU’

and ‘time’, while including ‘subjects’ as random effects. This analysis revealed statistically

significant main effects of Ang-II (F= 51.5, P < 0.01), TPPU (F= 18.7, P < 0.01), -3 rich diet

9

(F= 10.8, P < 0.01) and an interaction for -3 rich diet and TPPU (F= 4.07, P = 0.045). The

findings obtained from the mixed effects model with three between subjects factors are

consistent with the mixed effects model analysis with ‘group’ as between subjects factor (see

Results). It is worth noting that there is a discrepancy in the interpretation of the results when

the data are analyzed with mixed-effects model with ‘group’ as one between subjects factor

versus with mixed-effects model with three between subjects factor. Statistical analysis using

three between subjects factors allowed examining individual effects of each treatment and

therefore provides evidence on how much each treatment (e.g. Ang-II, -3 rich diet, TPPU)

contributes to the changes in blood pressure. The mixed effects model with only ‘group’ as

between subjects factor allowed making group contrasts while relying on pairwise comparisons.

Mixed-effects models with residuals diagnostics:

We used residuals analysis of the SBP data which showed as expected that the treated groups

had indistinguishable values on day 1. This was followed by a sharp increase in SBP in the Ang-

II treated group and then a plateau. To capture these salient features of the data and separate out

the main effects of -3 rich diet and TPPU over the course of the study, we fitted a linear mixed-

effects core model allowing for a constant trajectory for the control group, a different value for

the Ang-II treated groups on day 1, and different effects for Ang-II, -3 rich diet, and TPPU

after day 1. To this core model we added and tested terms for an additional Ang-II effect after

the third day (to evaluate further changes in SBP after the first increase in SBP), for TPPU (to

evaluate the benefit of a higher dose versus a lower one) and for the interaction between TPPU

and -3 rich diet. None of these terms were significant, and they were not retained in the model.

Table S3 summarizes the results of the mixed-effects models with residuals diagnostics used to

analyze the blood pressure data. The results revealed that Ang-II infused mice experienced an

increase in blood pressure after day 1 (estimate = 20.14, SE = 4.05, p < 0.0001). After the first

day, mice receiving Ang-II and TPPU experienced a reduction in blood pressure of about 11

arbitrary units, which was statistically significant (p < 0.01), and those receiving the -3 rich diet

also experienced a 4.5 unit reduction in blood pressure (p = 0.24).

TABLE S3. Summary (parameter estimates and standard errors) of the random-effects models

assessing the relationship between Ang-II, -3 rich diet, and TPPU and blood pressure.

Estimate* (SEM) P value

Model Term

Intercept (average blood pressure for control mice) 2.64 (3.29) 0 .43

Estimated difference between Ang-II treated mice and controls

on day 1

7.11 (7.79) 0.37

Ang-II effect after day 1 20.14 (4.05) < 0.0001

-3 rich diet effect after day 1 -4.51 (3.83) 0.24

TPPU effect after day 1 -11.25 (3.89) 0.004

*Arbitrary units.

F) Examination of the effects of each treatment on blood pressure at day11

10

In order to test our specific hypotheses regarding group differences in SBP a one way

ANOVA of SBP at day 11 was conducted, which revealed a significant effect of group (F = 4.65,

P = 0.002). Pairwise comparisons using Student-Newman Keuls post hoc tests showed that at

day 11 SBP was significantly lower in all treatment groups compared to Ang-II, but also in A-

3-TL compared to A-3 (P = 0.05), a trend for lower SBP in A-3-TH compared to A-3 (P =

0.08) and in A-TH compared to A-3 (P = 0.06).

11

Figure S1 Examination of the area under the systolic blood pressure (SBP) – time curve across

all the groups from days 1-11 and from days 7-11. Panel A: Area under the SBP-time curve corrected to the baseline readings with respect to

control group average trajectory was calculated by trapezoidal rule as described before.11

Differences in the AUC values among the groups were examined by one-way ANOVA. P <

0.05 was considered statistically significant. Data are mean ± SEM. Controls, n=5; group A,

n=8; group A-3, n=8; group A-3-TL, n=7; group A-3-TH, n=8; group A-TH, n=8. *

compared with controls, ɸ compared with group A. Panel B: Regression lines of the group

means of the AUCs in A-3-TH and A-3-TL relative to the group A-3. Slopes of each

regression line were compared to test if group A-3-TH and A-3-TL differ from each other. A

t-test revealed that neither slope was different than zero or from each other.

Baseline blood pressure values were established for each group of mice based on average

blood pressure taken for 3 days before treatment. Baseline blood pressures (mean ± SEM) were

as follows: Controls, 105 ± 4 mmHg; group A, 99 ± 2 mmHg; group A-3, 102 ± 2.5 mmHg;

group A-3-TL, 125 ± 8 mmHg, group A-3-TH, 104 ± 2 mmHg; group A-TH, 107 ± 2.5

mmHg.

A

AUC t (0-11)

Are

a u

nd

er

the c

urv

e

(% c

han

ge*d

ays)

0

100

200

300

400

500AUC t (7-11)

Are

a u

nd

er

the c

urv

e

(% c

han

ge*d

ays)

0

40

80

120

160

200

Control

Group A

Group A- 3

Group A- 3-TL

Group A- 3-TH

Group A-TH

Control

Group A

Group A- 3

Group A- 3-TL

Group A- 3-TH

Group A-TH

P = 0.06

P = 0.1

P = 0.055

Control

Group A

Group A- 3

Group A- 3-TL

Group A- 3-TH

Group A-TH

B

Are

a u

nd

er

the c

urv

e

t(0-1

1)

40

80

120

160

200A- 3-TH

A- 3-TL

group A- 3 group A- 3-T(H or L)

slope: -55

slope: -120

12

Figure S2A Time course of systolic blood pressure in mice that are on a standard diet.

A different set of mice (Swiss Webster, 8 weeks old) were fed a standard rodent chow

(2018 Teklad Global 18% Protein Rodent Diet, Harlan Laboratories, Hayward, CA) throughout

the whole experiment. Systolic blood pressure was measured using tail cuff blood pressure

system as described in the methods section. Briefly, baseline blood pressure readings were

collected following acclimation of the animals to the tail cuff blood pressure measurements.

Then, TPPU was administered in the drinking water at 0.2 mg/kg dose. Blood pressure was

recorded 1 and 4 days after the administration of TPPU. During this time TPPU did not alter

control blood pressure levels until after Ang-II was administered. Five days after the TPPU

administration, hypertension was induced by subcutaneous implantation of osmotic mini-pumps

delivering Ang-II at 1 mg/kg/day. During the time of Ang-II infusion, animals continued to

receive TPPU in the drinking water at 0.2 mg/kg.

The arrow heads indicate the day on which the treatment started (TPPU and Ang-II).

Group A’, Ang-II alone and group A’-TL, Ang-II + TPPU (0.2 mg/kg). Data are mean ± SEM.

Time (days)

-5 -4-2 0 2 4 6 8 10

Sys

toli

c b

loo

d p

ress

ure

a

s %

ch

an

ge

fro

m b

ase

lin

e

-10

0

10

20

30

40

50

60Control (n=3)

Group A' (n= 8)

Group A'-TL (n=8)

TPPU

(day -5)

Ang-II

(day 0)

*P < 0.05

13

Figure S2B Area under the systolic blood pressure (SBP) – time curve from days 3-9 from data

in Figure S2A.

Area under the SBP-time curve was calculated by trapezoidal rule as described before.11

The

area was calculated from blood pressure data obtained between days 3 and 9. Note that control

AUC lack data on day 5. Group A, Ang-II alone and group A-TL, Ang-II + TPPU (0.2 mg/kg).

Data are mean ± SEM.

While AUC values calculated using the SBP values recorded between days 3 and 9 differed

significantly between group A’ and group A’-TL, AUC values calculated from values of % change

from baseline did not differ between these two groups (P =0.07).

AU

C (

mm

Hg

*da

ys

)

0

200

400

600

800

1000

1200

AU

C (

% c

ha

ng

e*d

ays

)

0

50

100

150

200

250

300

Control Group A' Group A'-TL Control Group A' Group A'-TL

P < 0.01

P = 0.07

P = 0.04

14

G) Assessment of Renal Function and Histology

1. Methods

At the end of the experiment, renal injury was assessed by quantitative histology and by

measurements of the plasma levels of creatinine, Ang-II, and the urinary levels of albumin.

Multiple histological sections of each kidney were evaluated in a blinded fashion and scored by

the previously described method based on multiple criteria including cellularity and hypertrophy

of the glomerulus, mesangial expansion, interstitial inflammatory cell infiltration and arteriolar

thickening. 2, 3

Plasma creatinine was measured as described above. Urinary albumin, which is

considered as a biomarker for renal injury, was quantified using a commercially available kit

(Albuwell M, Exocell Inc, PA). For this measurement, mice were housed in metabolic cages (one

animal per cage) designed to collect urine free of food or feces. Urine was collected on dry ice

for the last 24 hours of the experiment. Samples were stored at -80C until analysis. Plasma

levels of Angiotensin-II were measured using an ELISA kit (Phoenix Pharmaceuticals,

Burlingame, CA) according to the manufacturer’s instructions.

2. Results

We determined the plasma creatinine and urinary albumin concentrations as markers of renal

function (P > 0.05). No significant differences were observed in the plasma creatinine levels

(controls: 0.25 ± 0.06 mg/dl, group A- 3-TL: < 0.2 mg/dl and group A-3-TH: 0.2 mg/dl) and

in the urinary albumin among the selected groups (controls: 15 ± 4.5 µg/day, group A- 3-TL:

20 ± 5 µg/day and group A- 3-TH: 10 ± 3 µg/day). Last, we measured plasma Ang-II levels

(Figure S3). Results showed an approximately 3 fold increase in plasma Ang-II levels when

animals were infused with Ang-II alone at 1 mg/kg/day. Interestingly, group A-3-TL had

significantly lower plasma levels of Ang-II compared with group A (P < 0.05, Kruskal Wallis

one-way ANOVA).

To substantiate these results, we examined the changes in renal tissue with given treatments

using glomerular injury scoring and general histology. When H & E stained sections were scored

blindly by the method described in Imig et al3, no significant differences were found among the

groups (Figure S4A). Accordingly, there was also no change in tubules or interstitial tissue

among the groups (Figure S4B). Overall, we did not observe any changes in the kidney that

might be driven by HTN. Since Swiss Webster mice are resistant to hypertension driven renal

damage4, 5

and particularly resistant to Ang II driven damage in the absence of salt2,3, 6-8

,

extensive histological changes in the kidney were not anticipated.

15

Figure S3 Plasma Angiotensin-II levels were decreased with the -3 rich diet and TPPU

treatment.

Blood was collected at the end of the Ang-II infusion. Plasma (200 µl) was used to

determine Ang-II levels. Inadequate amount of plasma was available from groups A-3 and A-

TH, and thus Ang-II levels could not be determined. No significant differences were observed

between sham operated controls (n=5) and group A (n=8). However, in groups A-3-TL (n=7),

plasma Ang-II levels were significantly lower than group A (P < 0.05). Data are mean ± SEM. *

compared with group A.

Pla

sm

a A

ng

-II

leve

ls

(fm

ol/

ml)

0

100

200

300

400

500

600

Control

Group A

Group A- 3-TL

Group A- 3-TH

*

16

Figure S4A No significant differences in glomerular injury scores were observed among any

groups following histological examination.

Kidneys were harvested and immediately fixed in 10% buffered formalin solution at the

end of Ang-II infusion. The 3-4 µm kidney sections stained with hematoxylin & eosin (H&E)

were prepared for light microscopic evaluation. Quantitative assessment was performed for

glomeruli in each H&E stained kidney section and scoring was completed at the Medical College

of Georgia (Dr John Imig). A score was given to each glomerulus by a blinded observer.

Glomerular injury was evaluated by criteria including 1) hypercellularity and hypertrophy of the

mesangial cells within the glomerulus, 2) apparent (open/ obvious) glomerular capillary loops

and mesangial expansion. Based on a scale of 0-4 (0 being the best and 4 being the worst).

Then, scores that are obtained from 250-400 glomeruli in the cortex from each kidney were

averaged for each slide and added to the appropriate treatment group. Data are mean ± standard

error. Sham operated controls, n=5; group A, n=5; group A-3, n=4; group A-3-TH, n=4; and

group A-TH, n=3. As expected, Swiss Webster mice are generally resistant to Ang-II driven

renal damage, particularly in the absence of salt.

Glo

meru

lar

inju

ry (

sco

re)

0.0

0.4

0.8

1.2

Control

Group A

Group A- 3

Group A- 3-TH

Group A-TH

17

Figure S4B No significant differences in renal injury were observed among any groups

following histological examination.

H & E stained kidney sections were examined for inflammation, degeneration of tubular

epithelial cells and presence of tubular casts, and arteriolar thickening. We found no significant

changes across the groups. Control (n=5); group A (n=5); group A-3 (n=4); group A-3-TH

(n=4); and group A-TH (n=3). Bar = 100 µm.

A. Control B. Group A

C. Group A-3 D. Group A-3-TH

E. Group A-TH

18

H) Measurement of plasma creatinine

Creatinine was purchased from Fisher Scientific (Pittsburgh, PA). The internal standard,

creatinine-d3 was purchased from C/D/N Isotopes Inc. (Pointe-Claire, Quebec, Canada). Thawed

plasma (30 µl) was mixed with 30 µL internal standard solution (1000 nM in methanol) on a

Vortex Mixer at 3,000 rpm for half min and then vortexed with 30 µl methanol at 5,000 rpm for

1 min. The mixed solution was centrifuged at 13,200 rpm for 5 min. The supernatant (25 µL)

was mixed with equal volume of 12-(3-cyclohexanyl-ureido)-dodecanoic acid (CUDA, 200 nM

in methanol) for analysis. Plasma creatinine was measured using an Agilent 1200 Series HPLC

(Agilent Technologies, Inc. Santa Clara, CA) coupled to an Applied Biosystems 4000 QTRAP

hybrid, triple-quadrupole MS instrument (Applied Biosystems, Foster City, CA). The instrument

was equipped with a linear ion trap and a Turbo V ion source and was operated in positive MRM

mode. Plasma creatinine was separated using a 100 mm X 4.6 mm Luna-C18 (2) 3 μm column

(Phenomenex, Torrance, CA) held at 40 ºC. Analytes were eluted by using the mixture of

water/acetonitrile/acetic acid (850/150/1, v/v) at a flow rate of 0.4 mL/min for 5 minutes. The

injection volume was 10 μL and the samples were kept at 10 ºC in the auto sampler. The mass

spectrometers were set with a positive electrospray mode with following parameters. CUR= 20

psi, GS1=25 psi, GS2=25 psi, IS=4500 V, CAD= HIGH, TEM=600oC, ihe=ON, DP=20 V. The

collision energies used for CAD (15 to 30 eV) varied according to molecular species and was

individually maximized for each compound. The parent and daughter ions for monitoring

creatinine, creatinine-d3, and CUDA are 114>86, 117.1>89.1, and 341.2>216.2, respectively.

19

TABLE S4. Renal epoxide metabolites of ARA, LA, EPA and DHA produced by P450s.

Renal levels (ng/g tissue)

Epoxide

Metabolites Controls

(n=5)

Group A

(n=8) Group A-3

(n=8)

Group A-3-TL

(n=7)

Group A-3-TH

(n=14)

Group A-TH

(n=8)

AR

A 8,9-EET 19 ± 4.4 5.5 ± 0.9

* 3.6 ± 0.3

* 5.3 ± 1

* 5 ± 0.5

* 7.6 ± 0.6

*

11,12-EET 14 ± 0.5 3 ± 0.2* 1.8 ± 0.1

* ɸ 3 ± 0.5

* ¥ 2.7 ± 0.3

* ¥ #

3.8 ± 0.3* ¥

14,15-EET 4.5 ± 0.6 2 ± 0.2* 1.5 ± 0.3

* 1.2 ± 0.2

* ɸ #

1.7 ± 0.2* #

2.7 ± 0.2* ¥

LA

9,10-EpOME 11 ± 0.6 2.2 ± 0.2* 1.3 ± 0.1

* 8 ± 1

* ɸ ¥ # $ 5 ± 1

* ɸ ¥ # 2.8 ± 0.4

*

12, 13-EpOME 10 ± 0.7 2 ± 0.2* 1.3 ± 0.2

* 7 ± 1

* ɸ ¥ # 6 ± 1

* ɸ ¥ # 3 ± 0.3

*

EP

A

8,9-EpETE 1.2 ± 0.05 0.3 ± 0.1 1 ± 0.3 4 ± 1* ɸ ¥

# 2.9 ± 0.6

ɸ ¥ # 0.4 ± 0.2

11,12-EpETEa

0.5 ± 0.1 < 0.1 1 ± 0.1 3 ± 1 2.2 ± 0.5 < 0.1

14,15-EpETEa 1.4 ± 0.2 < 0.1 1.1 ± 0.04 1 ± 0.2 1 ± 0.1 < 0.1

17,18-EpETEa 2 ± 0.2 < 0.1 2.3 ± 0.2 9.5 ± 1 8 ± 1 0.9 ± 0.1

DH

A

10,11-EpDPE 6 ± 1 1.1 ± 0.1* 2.8 ± 0.2

* ɸ 4.5 ± 0.8

ɸ ¥ #

3.9 ± 0.4* ɸ #

1.5 ± 0.2*

13,14-EpDPE 3.5 ± 0.6 0.7 ± 0.1* 2 ± 0.1

* ɸ 3 ± 0.4

ɸ # 3 ± 0.4

ɸ ¥ # 1 ± 0.1

* ¥

16,17-EpDPE 4 ± 0.6 0.7 ± 0.1* 1.9 ± 0.1

* 4.7 ± 0.7

ɸ ¥ # 4.9 ± 0.7

ɸ ¥ # 1.9 ± 0.2

*

19,20-EpDPE 9 ± 1 8 ± 1 20 ± 1* ɸ

32 ± 3* ɸ ¥ #

37 ± 2* ɸ ¥ #

20 ± 1* ɸ

P < 0.05 *compared with controls, ɸ compared with group A,

¥ compared with group A-3,

$ compared with group A-3-TH,

#

compared with group A-TH. Data are mean ± standard error. a Post hoc tests were not performed for these analytes, because at least one group has samples below 0.1 ng/g tissue concentration.

20

TABLE S5. Diols produced by sEH from the ARA, LA, EPA and DHA epoxides in the kidney.


Diol metabolites Controls

(n=5)

Group A

(n=8) Group A-3

(n=8)

Group A-3-TL

(n=7)

Group A-3-TH

(n=14)

Group A-TH

(n=8)

AR

A 8,9-DHET 0.9 ± 0.1 0.4 ± 0.1

* 0.3 ± 0.1

* 0.5 ± 0.1

* 0.6 ± 0.1

* ¥ 0.7 ± 0.1

ɸ ¥

11,12-DHET 1.5 ± 0.4 0.9 ± 0.2* 0.6 ± 0.1

* 1 ± 0.2 0.9 ± 0.1

* 1.1 ± 0.1

14,15-DHET 3.6 ± 0.5 2.7 ± 0.2 1.8 ± 0.1* 2.4 ± 0.2

* 1.9 ± 0.1

* # 3 ± 0.2

¥

LA

9,10-DiHOME 6 ± 1 2 ± 0.2* 1 ± 0.2

* 2.5 ± 0.3

* 1.8 ± 0.3

* 3 ± 0.3

*

12, 13-DiHOME 19.5 ± 3.5 5.2 ± 0.5* 2.8 ± 0.3

* 8 ± 1

* ¥ 6 ± 1

* 5.6 ± 0.8

*

EP

A

8,9-DiHETE a 0.5 ± 0.1 < 0.1 0.6 ± 0.1 1.7 ± 0.2 1 ± 0.1 < 0.1

11,12-DiHETE a 0.3 ± 0.1 < 0.1 0.5 ± 0.1 0.7 ± 0.1 0.6 ± 0.

1 < 0.1

14,15-DiHETE 2 ± 0.6 0.2 ± 0.02* 2.3 ± 0.3

ɸ 4 ± 0.3

* ɸ ¥ # $ 2.7 ± 0.3

ɸ # 0.3 ± 0.02

* ¥

17,18-DiHETE 4.6 ± 0.7 0.7 ± 0.1* 8 ± 1

* ɸ 7 ± 0.8

ɸ # $ 4 ± 0.4

ɸ ¥ # 0.7 ± 0.1

* ¥

DH

A

10,11-DiHDPE 2 ± 0.3 0.6 ± 0.1* 1.6 ± 0.1

ɸ 2.3 ± 0.4

ɸ # 1.7 ± 0.2

ɸ # 0.7 ± 0.1

* ¥

13,14-DiHDPE 3.5 ± 0.5 1.3 ± 0.1 3.4 ± 0.2 10 ± 3 ɸ #

6 ± 2* #

1.3 ± 0.1

16,17-DiHDPE 28 ± 4 9.4 ± 1* 16 ± 1

* ɸ 16 ± 2

* ɸ $ 11 ± 1

* ¥ 10 ± 1

*

19,20-DiHDPE 69.5 ± 19 25 ± 3* 58 ± 3

ɸ 48.5 ± 6

* ɸ # $ 24 ± 2

* ¥ # 13 ± 1

* ɸ ¥




#


21

TABLE S6. Renal levels of the epoxide and diol metabolites of of the ARA, LA, EPA and DHA

in mice fed a standard diet (Harlan-Teklad).

Tissue levels (ng/g tissue)

Epoxide metabolitesa

Control Group A’ Group A’-TL

Sum EETs 38 ± 4 42 ± 3 37 ± 4

Sum EpOMEs 22 ± 2 15 ± 1 14 ± 3*

Sum EpETEs 3 ± 0.2 2 ± 0.1* 1.5 ± 0.2*

Sum EpDPEs 15 ± 2 20 ± 1 31 ± 5* ɸ

Diol metabolitesa

Sum DHETs 6 ± 0.4 7 ± 0.2 8 ± 0.4* ɸ

Sum DiHOMEs 37 ± 6 41 ± 2 46 ± 9 ɸ

Sum DiHETEs 6 ± 0.5 2 ± 0.1* 2 ± 0.1*

Sum DiHDPEs 89 ± 10 80 ± 2 72 ± 10

aSum of ARA metabolites includes the 8, 9-, 11, 12- and 14, 15-fatty acid regioisomers.

Sum of LA metabolites includes the 9, 10- and 12, 13- fatty acid regioisomers. Sum of EPA

metabolites include the 8, 9-, 11, 12-, 14, 15- and 17, 18-fatty acid regioisomers, and sum of

DHA metabolites includes the 10, 11-, 13, 14-, 16, 17- and 19, 20- regioisomers. Control (sham

operated animals), n=3, group A’ (Ang-II alone), n=6, and group A’-TL (Ang-II + TPPU 0.2

mg/kg), n=6. Statistically significant differences (P < 0.05) were determined by one-way

ANOVA followed by pairwise comparisons. Data are mean ± SEM. * compared with controls, ɸ

compared with Group A’.

22

TABLE S7A. Renal inflammatory status across treatment groups reflected by the tissue levels of major pro- and anti-inflammatory

ARA metabolites generated from COX-2 and LOX enzymes.


ARA

metabolites

Controls

(n=5)

Group A

(n=8) Group A-3

(n=8)

Group A-3-TL

(n=7)

Group A-3-TH

(n=14)

Group A-TH

(n=8)

AR

A

CO

X-2

TXB2 1.4 ± 0.1 3.9 ± 0.9* 1.5 ± 0.2

ɸ 1.2 ± 0.3

ɸ # 1.4 ± 0.2

ɸ # 6 ± 0.7

* ɸ ¥

PGF2α 5 ± 1.6 45.6 ±

6.4*

14 ± 1.45 ɸ

5.7 ± 1.5 ɸ #

9.2 ± 7 ɸ #

69.5 ± 9.5* ɸ ¥

AR

A

LO

X

5-HETE a

< 0.1 8.4 ± 1.6 7.8 ± 1 2.5 ± 0.3 5 ± 1 13.5 ± 2

8-HETE a

< 0.1 1.6 ± 0.2 1 ± 0.2 < 0.1 1 ± 0.1 2.8 ± 0.2

9-HETE a

< 0.1 35 ± 8 17 ± 2 6.8 ± 2 13 ± 4 50 ± 6

11-HETE 24 ± 5 36 ± 8 17 ± 2 ɸ

7 ± 2* ɸ #

13 ± 4 ɸ #

50 ± 6* ɸ ¥

12-HETE 34 ± 12 70 ± 13 45 ± 5 13 ± 4 ɸ #

22 ± 4 ɸ #

113 ± 40* ɸ ¥

15-HETE 6 ± 2 37 ± 7* 27 ± 3

* 5 ± 1

ɸ ¥ # $ 18 ± 4

ɸ # 57 ± 5.5

* ɸ ¥

15(S)-HETrE a < 0.1 6.4 ± 1.3 5 ± 0.6 2.5 ± 0.3 3.7 ± 0.5 8.9 ± 0.75

20-HETE 3.8 ± 0.3 5.3 ± 1.2 5 ± 1.2 12.4 ± 3* ɸ ¥ #

8.5 ± 1* 5.5 ± 0.9




#


23

TABLE S7B. COX-2 and LOX products of EPA in the kidney.


EPA

metabolites

Controls

(n=5)

Group A

(n=8) Group A-3

(n=8)

Group A-3-TL

(n=7)

Group A-3-TH

(n=14)

Group A-TH

(n=8)

CO

X-2

Resolvin E1 1 ± 0.4 < 0.1 < 0.1 < 0.1 < 0.1 < 0.1

PGE3 a

< 0.1 < 0.1 4.5 ± 0.9 2.2 ± 0.6 5 ± 2.5 < 0.1

PGD3 a

0.9 ± 0.3 0.4 ± 0.1 2 ± 0.8 < 0.1 1 ± 0.3 0.3 ± 0.1

LO

X

5-HEPE a

4 ± 0.2 < 0.1 10 ± 2 14 ±2 9.5 ± 0.6 < 0.1

8-HEPE a

1 ± 0.8 < 0.1 1.7 ± 0.3 1 ± 0.1 1.1 ± 0.1 < 0.1

12-HEPE 45 ± 7 4.6 ± 2 170 ± 16* ɸ

72.5 ± 15 ɸ ¥ #

63 ± 12.5 ɸ ¥ #

7 ± 5¥

15-HEPE 2 ± 0.4 0.4 ± 0.1 9.6 ± 0.7* ɸ

18 ± 2* ɸ ¥ # $

10 ± 2* ɸ #

0.9 ± 0.3¥




#


24

TABLE S8. Other enzymatic products derived from arachidonic and linoleic acid in the kidney.

Among those metabolites, 9-HODE and 13- HODE showed a similar trend to that of HETEs. Consequently, there was a

strong correlation (R = 0.9) between these two HODE metabolites and the 15-HETE, which have been shown to activate PPAR-

gamma.10

The LA oxidation products also changed similarly to 9- HODE and 13- HODE across all the groups.


Oxidation

products

Controls

(n=5)

Group A

(n=8) Group A-3

(n=8)

Group A-3-TL

(n=7) Group A-3-TH

(n=14)

Group A-TH

(n=8)

9-HODE 19 ± 4 86 ± 18* 44 ± 6

ɸ 14 ± 1

ɸ ¥ # 33 ± 7

ɸ # 76 ± 5

* ¥

13-HODE 32 ± 6 95 ± 16* 56 ± 7.5

ɸ 22 ± 1.4

ɸ ¥ # 41 ± 7

ɸ # 89 ± 9

* ¥

9-oxo-ODE 53 ± 16 4 ± 0.6* 2.5 ± 0.6

* 5.5 ± 0.6

* 4 ± 0.8

* 4 ± 0.5

*

12-oxo-ETE 14.3 ± 3 22 ± 2 13 ± 1 9.3 ± 1 ɸ #

8 ± 1 ɸ #

35.5 ± 10* ɸ ¥

15-oxo-ETE 1.5 ± 0.3 3.6 ± 0.4* 1.8 ± 0.3

ɸ 0.8 ± 0.1

ɸ ¥ # 1.1 ± 0.2

ɸ ¥ # 4 ± 0.5

* ¥




#

compared with group A-TH. Data are mean ± standard error.

25

TABLE S9. Tissue levels of the major pro-inflammatory cytokines in the kidney.

Renal levels (pg/mg protein)

Cytokines Controls

(n=5)

Group A

(n=8) Group A-3

(n=8)

Group A-3-TL

(n=7)

Group A-3-TH

(n=14)

Group A-TH

(n=8)

MCP-1 5.4 ± 0.9 66 ± 16* 29 ± 3* 5.7 ± 0.45

¥ # 18 ± 4

30 ± 3*

TNF- α 43 ± 6 40 ± 11 40 ± 16 41 ± 5 35 ± 4¥ 30 ± 4

¥

IL-1β 4.7 ± 0.8 4 ± 0.5 2.5 ± 0.65 < 0.1 1 ±0.4 2 ±0.4*

IL-6 0.3 ± 0.1 0.2 ± 0.02 0.1 ±0.03 < 0.05 0.4 ±0.1 0.2 ±0.02




#

compared with group A-TH. Data are mean ± standard error.

26

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oxylipin metabolome by liquid chromatography electrospray ionization tandem mass

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Pollock JS, Pollock DM, & Imig JD. Soluble epoxide hydrolase inhibition protects the

kidney from hypertension-induced damage. J Am Soc Nephrol. 2004;15(5):1244-1253.

3. Manhiani M, Quigley JE, Knight SF, Tasoobshirazi S, Moore T, Brands MW, Hammock

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8. Li J, Carroll MA, Chander PN, Falck JR, Sangras B, & Stier CT. Soluble epoxide

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9. Arendash GW, Jensen MT, Salem N, Jr., Hussein N, Cracchiolo J, Dickson A, Leighty R,

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C. Group A-3 C. Group A-3

E. Group A-TH

C. Group A-3

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