different mechanisms of acute versus long‐term antihypertensive … · 2015. 5. 27. ·...

ORIGINAL ARTICLE

Different mechanisms of acute versus long-term antihypertensive effects ofsoluble epoxide hydrolase inhibition: Studies in Cyp1a1-Ren-2 transgenic

rats

Alexandra Sporkov�a,* �S�arka J�ıchov�a,*† Zuzana Huskov�a,* Libor Kopkan,* Akira Nishiyama,‡

Sung H Hwang,§ Bruce D Hammock,§ John D Imig,¶ Elzbieta Kompanowska-Jezierska,** JanuszSadowski,** Herbert J Kramer†† and Lud�ek �Cervenka*‡‡

*Center for Experimental Medicine, Institute for Clinical and Experimental Medicine, Prague, Czech Republic,†Department of Physiology, ‡Department of Pharmacology, Kagawa University, Kagawa, Japan, §Department ofEntomology, UCD Comprehensive Cancer Center, University of California, Davis, CA, USA, ¶Department of

Pharmacology and Toxicology, Medical College of Wisconsin, Milwaukee, WI, USA, **Department of Renal and BodyFluid Physiology, M. Mossakowski Medical Research Centre, Polish Academy of Science, Warsaw, Poland, ††Section ofNephrology, Medical Policlinic, Department of Medicine, University of Bonn, Bonn, Germany and ‡‡Pathophysiology,

2nd Faculty of Medicine, Charles University, Prague, Czech Republic

SUMMARY

Recent studies have shown that the long-term antihyper-tensive action of soluble epoxide hydrolase inhibition (sEH)in angiotensin-II (AngII)-dependent hypertension might bemediated by the suppression of intrarenal AngII levels. Totest this hypothesis, we examined the effects of acute(2 days) and chronic (14 days) sEH inhibition on bloodpressure (BP) in transgenic rats with inducible AngII-dependent hypertension. AngII-dependent malignant hyper-tension was induced by 10 days’ dietary administration ofindole-3-carbinol (I3C), a natural xenobiotic that activatesthe mouse renin gene in Cyp1a1-Ren-2 transgenic rats. BPwas monitored by radiotelemetry. Acute and chronic sEHinhibition was achieved using cis-4-(4-(3-adamantan-1-yl-ure-ido)cyclohexyloxy) benzoic acid, given at doses of 0.3, 3, 13,26, 60 and 130 mg/L in drinking water. At the end ofexperiments, renal concentrations of epoxyeicosatrienoicacids, their inactive metabolites dihydroxyeicosatrienoicacids and AngII were measured. Acute BP-lowering effectsof sEH inhibition in I3C-induced rats was associated with amarked increase in renal epoxyeicosatrienoic acids to di-hydroxyeicosatrienoic acids ratio and acute natriuresis.Chronic treatment with cis-4-(4-(3-adamantan-1-yl-ureido)cy-clohexyloxy) benzoic acid in I3C-induced rats eliciteddose-dependent persistent BP lowering associated with a

significant reduction of plasma and kidney AngIIlevels. Our findings show that the acute BP-lowering effectof sEH inhibition in I3C-induced Cyp1a1-Ren-2 transgenicrats is mediated by a substantial increase in intrarenal ep-oxyeicosatrienoic acids and their natriuretic action withoutaltering intrarenal renin–angiotensin system activity. Long-term antihypertensive action of cis-4-(4-(3-adamantan-1-yl-ureido)cyclohexyloxy) benzoic acid in I3C-induced Cyp1a1-Ren-2 transgenic rats is mediated mostly by suppression ofintrarenal AngII concentration.Key words: angiotensin-II, cytochrome P-450 epoxygenase,

eicosanoids, epoxyeicosatrienoic acids, hypertension, solubleepoxide hydrolase.

INTRODUCTION

An increasing body of evidence indicates that cytochrome P-450 (CYP)-dependent metabolites of arachidonic acid, includingepoxyeicosatrienoic acids (EET), play an important role in theregulation of cardiovascular and renal functions.1–4 Numerousindependent studies have shown that increasing bioavailabilityof EET in tissues by preventing their degradation to the bio-logically inactive dihydroxyeicosatrienoic acids (DHETE) usingsoluble epoxide hydrolase (sEH) inhibition has antihypertensiveeffects.5–15 Based on these studies, it has been proposed thatthe EET-mediated antihypertensive actions are related to theirimmediate vasodilatory effects in addition to their direct influ-ence on renal tubular transport of sodium.3,4,16

We recently found that the sEH inhibitor cis-4-(4-(3-adaman-tan-1-yl-ureido) cyclohexyloxy) benzoic acid (c-AUCB), induceda significant reduction in plasma angiotensin-II (AngII) levelsand normalization of kidney AngII concentration in a Ren-2transgenic rat model of angiotensin-II (AngII)-dependent

Correspondence: Lud�ek �Cervenka, Center for Experimental Medicine,Institute for Clinical and Experimental Medicine, 1958/9 V�ıde�nsk�a,CZ-140 00 Prague 4, Czech Republic. Email: [email protected] 24 July 2014; revision 26 August 2014; accepted 29 August

2014.© 2014 Wiley Publishing Asia Pty Ltd

Clinical and Experimental Pharmacology and Physiology (2014) 41, 1003–1013 doi: 10.1111/1440-1681.12310

hypertension (TGR).17 Though c-AUCB significantly increasedthe intrarenal availability of biologically active EET in TGR,detailed analyses of the results of that study made us proposethat the antihypertensive action of sEH inhibition in TGR ispredominantly mediated by suppression of the systemic and in-trarenal renin–angiotensin system (RAS).17 As the dose ofc-AUCB used in that study was in the upper range of the dos-age recommended (26 mg/L in drinking water),18 double thedose we usually utilize,9–12,14,15 we considered it important toexamine whether the effect of sEH inhibition on the RAS mightbe dose-dependent.Of note, the most common model of AngII-dependent

hypertension is the one obtained by chronic infusion of subpres-sor doses of AngII that lead to suppression of endogenousRAS.19–21 Another possibility is the two-kidney, one-clip(2K1C) Goldblatt hypertension, where blood pressure (BP) ele-vation is the consequence of surgically-induced unilateral renalartery stenosis.22 Thus, induction of AngII-dependent hyperten-sion in these models results either from application of exoge-nous AngII leading to unphysiological suppression of RAS orfrom a surgical intervention that yields a variable degree ofRAS activation.19–24 Furthermore, the exact onset and the sever-ity of hypertension in these models often vary and cannot beprecisely controlled. To avoid these limitations, we used aninbred transgenic rat line (strain name: TGR(Cyp1a1-Ren-2))that offers a unique possibility to precisely control the develop-ment of AngII-dependent hypertension.25 This transgenic ratmodel allows assessment of the pathophysiological mechanismsof AngII-dependent hypertension involving increased activity ofendogenous RAS. Furthermore, we and others have shown thatthe gene expression, the level of endogenously produced AngIIand consequently the degree of hypertension can be preciselycontrolled.11,12,15,26–30

Our aim was to discover if sEH inhibition would result indose-dependent inhibition of the circulating and intrarenal RASin hypertensive Cyp1a1-Ren-2 transgenic rats. Therefore, weevaluated the effects of acute (2 days) and chronic (14 days)c-AUCB treatment on BP, and AngII, EET and DHETE concen-trations as affected by doses ranging from 0.3 to the highest sug-gested dose of 130 mg/L in drinking water.18 Furthermore, toelucidate the mechanism(s) responsible for the effects of sEHinhibition on RAS activity, Ren-2 renin gene expression andactivities/concentrations of individual components of the RASwere assessed in untreated and c-AUCB-treated Cyp1a1-Ren-2transgenic rats; this was carried out under control conditions andafter induction of the renin gene. Finally, our recent data17 sug-gested that after high-dose c-AUCB treatment, the pattern ofchanges was similar as that observed in AngII-dependent hyper-tension treated with angiotensin-converting enzyme inhibitor(ACEi).19,20,31,32 Therefore, we also determined the effects ofACEi treatment in non-induced animals and in Cyp1a1-Ren-2transgenic rats after induction of the renin gene on the profilesof hypertension and EET and DHETE concentrations, andexpression and activities/concentrations of individual componentsof the RAS. This was carried out to gain a deeper insight intothe possible role of the interaction between CYP-dependentmetabolites and the RAS in the antihypertensive potency of sEHinhibition.

RESULTS

Series 1: Effects of c-AUCB and ACEi in non-induced orI3C-induced Cyp1a1-Ren-2 transgenic rats on arterial bloodpressure, body weight, urinary sodium excretion, plasmac-AUCB concentrations and Ren-2 renin gene expression

As shown in Fig. 1, basal values of mean arterial pressure(MAP) were not significantly different among experimentalgroups. In untreated animals, I3C-induction resulted in the riseof MAP from 102 � 3 mmHg (day �10) to 171 � 3 mmHg

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Fig. 1 Time-course of mean arterial pressure (measured by radioteleme-try) in indole-3-carbinol (I3C)-induced and non-induced Cyp1a1-Ren-2transgenic rats, and effects of cis-4-(4-(3-adamantan-1-yl-ureido) cyclo-hexyloxy) benzoic acid (c-AUCB) or with angiotensin-converting enzymeinhibitor (ACEi) treatment. *P < 0.05 versus baseline values. ( ),Non-induced untreated; ( ), non-induced + c-AUCB (130 mL); ( ),non-induced + ACEi; ( ), 13C-induced untreated; ( ), 13C-induced + c-AUCB (0.3 mg/L); ( ), 13C-induced + c-AUCB (3 mg/L);( ), 13C-induced + c-AUCB (13 mg/L); ( ), 13C-induced +c-AUCB (26 mg/L); ( ), 13C-induced + c-AUCB (60 mg/L); ( ),13C-induced + c-AUCB (130 mg/L); ( ), 13C-induced + ACEi.

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1004 A Sporkov�a et al.

(day 0; P < 0.05). Non-induced rats remained normotensivethroughout the experiment and the treatment with the highestdose of c-AUCB (130 mg/L) did not have any significanteffect on their MAP. However, the treatment with ACEi(trandolapril, 6 mg/L) resulted in a significant decrease inMAP as compared to untreated non-induced rats (78 � 3 vs105 � 3 mmHg, on day 14; Fig. 1a). As also shown inFig. 1a, treatment with 0.3 mg/L of c-AUCB did not affectthe course of hypertension in I3C-induced rats as compared tountreated I3C-induced rats (175 � 4 vs 177 � 3 mmHg mea-sured on day 14). Treatment with 3 mg/L of c-AUCB signifi-cantly decreased MAP in I3C-induced rats, with maximalBP-lowering effect observed 2 days after initiation of c-AUCBadministration (from 173 � 4 to 137 � 2 mmHg, P < 0.05).On day 7, MAP returned to levels observed in I3C-induceduntreated rats. Treatment with 13 mg/L of c-AUCB causedmarked decreases in MAP with a maximum BP-lowering effecton day 4 of treatment (from 174 � 4 to 114 � 2 mmHg,P < 0.05), followed by a gradual return to levels observed inuntreated I3C-induced rats (Fig. 1a).As shown in Fig. 1b, treatment with 26 mg/L c-AUCB elicited

a profound decrease in MAP to normotensive levels (from173 � 4 to 113 � 3 mmHg, P < 0.05), then MAP slowlyincreased, but at the end of experiments still remained signifi-cantly lower compared to untreated I3C-induced rats (148 � 3 vs177 � 3 mmHg, P < 0.05). Treatment with 60 mg/L of c-AUCBhad similar rapid BP-lowering effects; however, the MAPremained within the normotensive range until the end of theexperiment (118 � 3 mmHg on day 14). Treatment with 130mg/L c-AUCB and administration of ACEi caused similar pro-found decrease in MAP to normotensive levels, and at the end ofthe experiment it even dropped below those observed in untreatednon-induced rats (91 � 3 and 89 � 3 vs 105 � 3 mmHg,P < 0.05 in both cases; Fig. 1b).As shown in Supplemental Fig. 1a, untreated and c-AUCB-

and ACEi-treated non-induced rats showed a significant body-weight (BW) gain throughout the experiment, with final increasesby +57 � 3, +56 � 3 and +54 � 4 g, respectively (P < 0.05 vsinitial BW). In I3C-induced rats, the development of hypertensionwas associated with a profound loss of BW (from 320 � 8 to207 � 7 g, P < 0.05). In I3C-induced rats, neither the treatmentwith the highest dose of c-AUCB (130 mg/L) nor that with ACEifully prevented the BW losses (Fig. S1b).As shown in Fig. 2, the basal daily sodium excretion did

not significantly differ among the groups, and remained unal-tered in untreated non-induced as well as I3C-induced rats.The treatment with 130 mg/L of c-AUCB in non-induced ratsas well as the treatment with the lowest dose of c-AUCB(0.3 mg/L) in I3C-induced rats did not significantly alter dailysodium excretion (Fig. 2a). In contrast, the treatment with eachdose of c-AUCB (from 3 mg/L to 130 mg/L) and ACEi elic-ited marked increase in daily sodium excretion in I3C-inducedrats when measured on the second day of treatment (Fig. 2a,b). In addition, the treatment with ACEi caused a substantialincrease in daily sodium excretion in non-induced rats on thesecond day of treatment (Fig. 2a). However, already on day 8,sodium excretion returned to the levels observed in untreatednon-induced rats, and remained at those levels until the end ofthe experiment.

As shown in Fig. 3a, plasma concentrations of c-AUCB ingroups that were not treated with this sEH inhibitor were atzero level, whereas in c-AUCB-treated groups they increasedin a dose-dependent manner. Starting with the dose 3 mg/L, c-AUCB plasma concentration was already above the value ofIC50 for the specific sEH inhibition on the second day of treat-ment. This concentration was observed to effectively inhibitsEH activity in vitro as well as in vivo.18 Of special interest isthe finding that treatment with the highest dose of c-AUCB(130 mg/L) resulted in maximum plasma concentrations onday 7 of administration, and the levels were approximately 20-fold higher than the IC50 value required for the specific sEHinhibition.18

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Fig. 2 Effects of cis-4-(4-(3-adamantan-1-yl-ureido) cyclohexyloxy) ben-zoic acid (c-AUCB) or angiotensin-converting enzyme inhibitor (ACEi)treatment on daily sodium excretion (UNaV) in indole-3-carbinol (I3C)-induced and non-induced Cyp1a1-Ren-2 transgenic rats. *P < 0.05 versusbaseline values. ( ), Non-induced untreated; ( ), non-induced + c-AUCB (130 mL); ( ), non-induced + ACEi; ( ), 13C-induceduntreated; ( ), 13C-induced + c-AUCB (0.3 mg/L); ( ), 13C-induced + c-AUCB (3 mg/L); ( ), 13C-induced + c-AUCB (13 mg/L);( ), 13C-induced + c-AUCB (26 mg/L); ( ), 13C-induced + c-AUCB (60 mg/L); ( ), 13C-induced + c-AUCB (130 mg/L); ( ),13C-induced + ACEi.


Dose-dependent effects of c-AUCB 1005

As shown in Fig. 3b, the liver expression of Ren-2 reningene in the non-induced rats was virtually undetectable. Treat-ment with 130 mg/L of c-AUCB and ACEi did not changeliver Ren-2 renin gene expression in non-induced rats. I3Cadministration resulted in marked liver Ren-2 renin geneexpression, and it was substantially potentiated by effectivesEH and ACE inhibition.

Series 2: Effects of c-AUCB and ACEi in non-induced orI3C-induced Cyp1a1-Ren-2 transgenic rats on EET, DHETE,plasma and kidney tissue AngII concentrations, plasma reninactivity, and plasma and kidney tissue ACE activity

Figures 4a and 5a summarize the availability of biologicallyactive epoxygenase products in kidney tissue expressed as theratio of EET-to-DHETE. The ratios in untreated I3C-induced ratswere significantly lower than in untreated non-induced rats. Thetreatment with 130 mg/L of c-AUCB did not significantly changethe ratios in non-induced rats. As shown in Figs 4a and 5a, acute(2 days) as well as chronic (14 days) treatment with c-AUCBbeginning with the dose of 3 mg/L caused significant increase inthe EET-to-DHETE ratio. In addition, the increase in EET-to-DHETE ratios were similar in I3C-induced rats treated with eachof the other doses of c-AUCB (from 13 to 130 mg/L). However,the increases in the EET-to-DHETE ratio were greater in acutely-than in chronically-treated I3C-induced rats for all doses ofc-AUCB. The treatment with ACEi did not significantly changethe ratios of EET-to-DHETE in I3C-induced rats (Figs 4a,5a).As shown in Figs 4b and 5b, plasma AngII levels were signifi-

cantly higher in untreated I3C-induced rats than in untreatednon-induced rats. Neither acute (2 days) nor chronic (14 days)treatment with 130 mg/L of c-AUCB significantly changedplasma AngII levels in non-induced rats. Figure 4b shows thatacute treatment with 0.3, 3 or 13 mg/L of c-AUCB did not alterplasma AngII levels in I3C-induced as compared to untreatedI3C-induced rats. In addition, acute administration of 26, 60 and130 mg/L of c-AUCB in I3C-induced rats resulted in significantdecrease in plasma AngII levels as compared to untreated I3C-induced rats (106 � 6, 77 � 9 and 74 � 8 vs 148 � 9 fmol/mL, different at P < 0.05 in each case), but they remained signif-icantly higher than observed in untreated non-induced rats. Acutetreatment with ACEi normalized plasma AngII levels to valuesobserved in untreated non-induced rats (31 � 3 vs 24 � 5 fmol/mL). Acute treatment with ACEi did not alter plasma AngII lev-els in non-induced rats (Fig. 4b).Figure 5b shows that similarly to acute treatment, chronic

administration of 0.3, 3 and 13 mg/L of c-AUCB did not signifi-cantly change plasma AngII levels in I3C-induced rats. In con-trast to acute treatment, chronic administration of 26, 60 and130 mg/L of c-AUCB decreased plasma AngII levels to valuesobserved in untreated non-induced rats. As with acute treatment,chronic treatment with ACEi in I3C-induced rats also decreasedplasma AngII levels to values observed in untreated non-inducedrats. Plasma AngII levels in I3C-induced rats decapitated onday 2 were higher compared to the values on day 14; inuntreated I3C-induced rats, the respective values were 148 � 9and 87 � 8 fmol/mL (different at P < 0.05). Chronic treatmentwith ACEi did not significantly change plasma AngII levels innon-induced rats (Fig. 5b).As shown in Figs 4c and 5c, kidney AngII concentrations were

substantially higher in untreated I3C-induced than in untreatednon-induced rats. Acute treatment with any dose of c-AUCB didnot affect kidney AngII concentrations in I3C-induced rats. Incontrast, acute treatment with ACEi significantly decreased kid-ney AngII concentrations as compared to untreated I3C-inducedrats (197 � 19 vs 514 � 34 fmol/g of tissue, P < 0.05), but theystill remained significantly higher compared to values observed in

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Fig. 3 Plasma (a) cis-4-(4-(3-adamantan-1-yl-ureido) cyclohexyloxy)benzoic acid (c-AUCB) concentrations ( ), 13C-induced + c-AUCB(0.3 mg/L); ( ), 13C-induced + c-AUCB (3 mg/L); ( ), 13C-induced + c-AUCB (13 mg/L); ( ), 13Cinduced + c-AUCB (26mg/L); ( ), 13C-induced + c-AUCB (60 mg/L); ( ), non-induced + c-AUCB (130 mg/L); ( ), 13C-induced + c-AUCB(130 mg/L) and (b) liver Ren-2 renin gene expression inindole-3-carbinol (I3C)-induced and non-induced Cyp1a1-Ren-2transgenic rats, and effects c-AUCB or angiotensin-convertingenzyme inhibitor (ACEi). ( ), Non-induced untreated; ( ), non-induced + c-AUCB (130 mL); ( ), non-induced + ACEi; ( ),13C-induced untreated; ( ), 13C-induced + c-AUCB (0.3 mg/L);( ), 13C-induced + c-AUCB (3 mg/L); ( ), 13C-induced + c-AUCB (13 mg/L); ( ), 13C-induced + c-AUCB (26 mg/L); ( ),13C-induced + c-AUCB (60 mg/L); ( ), 13C-induced + c-AUCB (130 mg/L); ( ), 13C-induced + ACEi; *P < 0.05 versusunmarked values, #P < 0.05 versus all the other values.



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Fig. 5 Effects of chronic (14 days) treatment with cis-4-(4-(3-adaman-tan-1-yl-ureido) cyclohexyloxy) benzoic acid (c-AUCB) or angiotensin-converting enzyme inhibitor (ACEi) on (a) the kidney tissue epoxyei-cosatrienoic acids (EET)-to-dihydroxyeicosatrienoic acids (DHETE)ratio, (b) plasma angiotensin-II (AngII) levels and (c) kidney AngIIlevels in indole-3-carbinol (I3C)-induced and non-induced Cyp1a1-Ren-2 transgenic rats. *P < 0.05 versus unmarked values, #P < 0.05 versusall the other values. ( ), Non-induced untreated; ( ), non-induced +c-AUCB (130 mL); ( ), non-induced + ACEi; ( ), 13C-induceduntreated; ( ), 13C-induced + c-AUCB (0.3 mg/L); ( ), 13C-induced +c-AUCB (3 mg/L); ( ), 13C-induced + c-AUCB (13 mg/L); ( ), 13C-induced + c-AUCB (26 mg/L); ( ), 13C-induced + c-AUCB (60 mg/L);( ), 13C-induced + c-AUCB (130 mg/L); ( ), 13C-induced + ACEi.

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Fig. 4 Effects of acute (2 days) treatment with cis-4-(4-(3-adamantan-1-yl-ureido) cyclohexyloxy) benzoic acid (c-AUCB) or angiotensin-con-verting enzyme inhibitor (ACEi) on (a) kidney tissue epoxyeicosatrie-noic acids (EET)-to-dihydroxyeicosatrienoic acids (DHETE) ratio, (b)plasma angiotensin-II (AngII) levels and (c) kidney AngII levels inindole-3-carbinol (I3C)-induced and non-induced Cyp1a1-Ren-2 trans-genic rats. *P < 0.05 versus unmarked values, #P < 0.05 versus all theother values. ( ), Non-induced untreated; ( ), non-induced + c-AUCB(130 mL); ( ), non-induced + ACEi; ( ), 13C-induced untreated; ( ),13C-induced + c-AUCB (0.3 mg/L); ( ), 13C-induced + c-AUCB(3 mg/L); ( ), 13C-induced + c-AUCB (13 mg/L); ( ), 13C-induced + c-AUCB (26 mg/L); ( ), 13C-induced + c-AUCB (60 mg/L);( ), 13C-induced + c-AUCB (130 mg/L); ( ), 13C-induced + ACEi.



non-induced rats (197 � 19 vs 74 � 7 fmol/g of tissue,P < 0.05). Acute treatment with ACEi did not alter kidney AngIIconcentrations in non-induced rats (Fig. 4c).In contrast to the results of acute treatment, chronic treatment

with 26 mg/L c-AUCB (Fig. 5c) markedly decreased kidneyAngII concentrations in I3C-induced rats compared to untreatedI3C-induced rats (117 � 9 vs 443 � 32 fmol/g of tissue,P < 0.05). Chronic administration of 60 mg/L of c-AUCB inI3C-induced rats decreased kidney AngII concentrations to valuesobserved in non-induced rats. Furthermore, treatment with130 mg/L c-AUCB and ACEi elicited significant decrease inkidney AngII concentrations even below the values observedin untreated non-induced rats (39 � 4 and 37 � 4 vs80 � 4 fmol/g of tissue, P < 0.05 in each case). Finally, chronictreatment with ACEi resulted in a significant decrease in kidneyAngII concentrations in non-induced rats (Fig. 5c).As shown in Figs 6a and 7a, plasma renin activities were

markedly higher in untreated I3C-induced rats than in untreatednon-induced rats. As expected, plasma renin activity in untreatedI3C-induced rats from the chronic treatment protocol (i.e.14 days) was noticeably higher as compared to untreated I3C-induced rats from the acute treatment protocol (2 days). Neitheracute nor chronic treatment with c-AUCB significantly changedplasma renin activities in non-induced or I3C-induced rats. Incontrast, acute as well as chronic treatment with ACEi elicitedsignificant increase in plasma renin activities in non-induced rats.Remarkably, chronic treatment with ACEi increased plasma reninactivity in non-induced rats to values that were observed inuntreated I3C-induced rats (Fig. 7a).As shown in Figs 6b,c and 7b, there were no significant differ-

ences in plasma and kidney ACE activities estimated as the ratioof AngII-to-AngI among non-induced and I3C-induced rats, andacute treatment with any dose of c-AUCB did not significantlyalter them (Fig. 6b,c). In contrast, acute treatment with ACEicaused significant decrease in plasma and kidney ACE activitiesin non-induced as well as in I3C-induced rats (Fig. 6b,c).Figure 7b and c show that chronic treatment with 0.3, 3 and

13 mg/L of c-AUCB did not change plasma and kidney ACEactivities in I3C-induced rats. In contrast, chronic treatment with26, 60 and 130 mg/L of c-AUCB profoundly decreased plasmaand kidney ACE activities in I3C-induced rats, bringing themclose to the levels observed in I3C-induced rats exposed tochronic ACEi treatment. Similar results regarding plasma and kid-ney ACE activities were obtained by direct measurement of ACEactivity.

DISCUSSION

The first major finding of the present study was that the acute(2 days’ treatment) BP-lowering effect of sEH inhibition in ourmodel of AngII-dependent hypertension was associated with asubstantial increase in renal bioavailability of EET (increasedEET-to-DHETE) and natriuresis, whereas intrarenal AngII con-centration remained unaltered. This is in accordance with the vastevidence indicating antihypertensive properties of EET are largelyrelated to their natriuretic potency.1–4,9–16 Indeed, EET have beenshown to inhibit sodium reabsorption in the proximal tubule byblocking the sodium-hydrogen exchanger38 and in the corticalcollecting duct by blocking the epithelial sodium channels.39 In

agreement with our previous studies,11,12,15 the I3C-inducedCyp1a1-Ren-2 transgenic rats showed reduced availability of bio-logically active epoxygenase metabolites. The data accord also

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Fig. 6 Effects of acute (2 days) treatment with cis-4-(4-(3-adamantan-1-yl-ureido) cyclohexyloxy) benzoic acid (c-AUCB) or angiotensin-convert-ing enzyme inhibitor (ACEi) on (a) plasma renin activities, (b) ratios ofplasma angiotensin-II (AngII) to AngI levels, and (c) ratios of kidney An-gII to AngI levels in indole-3-carbinol (I3C)-induced and non-inducedCyp1a1-Ren-2 transgenic rats. *P < 0.05 versus unmarked values,#P < 0.05 versus all the other values. ( ), Non-induced untreated; ( ),non-induced + c-AUCB (130 mL); ( ), non-induced + ACEi; ( ), 13C-induced untreated; ( ), 13C-induced + c-AUCB (0.3 mg/L); ( ), 13C-induced + c-AUCB (3 mg/L); ( ), 13C-induced + c-AUCB (13 mg/L);( ), 13C-induced + c-AUCB (26 mg/L); ( ), 13C-induced + c-AUCB(60 mg/L); ( ), 13C-induced + c-AUCB (130 mg/L); ( ), 13C-induced + ACEi.



with our recent finding that chronic inhibition of sEH normalizedintrarenal EET bioavailability and improved the pressure–natriuresis relationship in 2K1C Goldblatt hypertensive and

I3C-induced Cyp1a1-Ren-2 transgenic rats,9,11,12. This furthersupports the concept that net intrarenal deficiency of EET con-tributes to the impairment of the pressure–natriuresis mecha-nism.12,40 In accordance with the concept first proposed byGuyton et al.41 and validated by several other groups,38–45 theimpairment of this mechanism is the crucial factor responsible forthe development and maintenance of hypertension.Acute administration of c-AUCB at the dose of 3 mg/L signifi-

cantly increased intrarenal availability of biologically active EETand other epoxy fatty acids; starting at the dose 13 mg/L andhigher, even the intrarenal EET in I3C-induced Cyp1a1-Ren-2transgenic rats increased to values significantly higher than thosein non-induced rats. Of note, acute administration of c-AUCBat doses below 13 mg/L did not alter plasma AngII levels inI3C-induced Cyp1a1-Ren-2 transgenic rats. However, starting atthe dose 26 mg/L, these levels were reduced but still remaineddistinctly higher than in non-induced rats. Furthermore, acuteadministration of c-AUCB at any dose did not significantlychange kidney AngII in our hypertensive rats. Taken together,these findings strongly suggest that acute sEH inhibition does notalter intrarenal RAS activity, whereas acute ACEi treatmentresults in plasma AngII normalization and in striking suppressionof kidney AngII concentrations. Therefore, based on our earlierand present findings, we propose that the mechanism underlyingBP-lowering in response to acute sEH inhibition is related tomarkedly enhanced intrarenal availability of EET and their directinhibitory influence on renal tubular sodium transport, and is notcrucially dependent on the alterations in RAS activity.9–12,14

The second critically important finding of the present studywas that sustained antihypertensive action of chronic (14 days’treatment) sEH inhibition with c-AUCB is dose-dependent; itis associated with both normalization of intrarenal EET, and sig-nificant reduction of plasma and kidney AngII levels. This isin agreement with our recent findings where a high dose ofc-AUCB (26 mg/L) led to a significant reduction in plasma An-gII and normalization of kidney AngII concentrations in TGR,another rat model of AngII-dependent hypertension, whereaslow-dose treatment (3 mg/L), which still efficiently blocked sEH,did not alter circulating and tissue RAS.17,33 The c-AUCBinduced BP lowering persisted throughout the 2-week period oftreatment. Two features of this response were very remarkable.First, the BP lowering effect of c-AUCB was dose-dependent.Second, the degree of concurrent suppression of AngII concentra-tions was also dose-dependent and parallel to the BP change. Incontrast, in I3C-induced Cyp1a1-Ren-2 transgenic rats, chronicc-AUCB treatment did not alter intrarenal EET in a dose-depen-dent manner: the maximal effect was already observed at thedose of 13 mg/L. Admittedly, our assays of plasma c-AUCBconcentration showed that each dose was highly above the rangeof IC50 for the specific sEH inhibition.18 Furthermore, althoughchronic c-AUCB treatment increased intrarenal EET in our hyper-tensive rats to values observed in non-induced rats, the effect ofchronic treatment on the ratio of EET-to-DHETE was signifi-cantly smaller compared to that of acute c-AUCB treatment.Taken together, these data strongly suggest that sustained antihy-pertensive actions of sEH inhibition with c-AUCB were specifi-cally mediated by the suppression of circulating and especiallyintrarenal AngII levels rather than by normalization of the intrare-nal availability of biologically active EET. This conclusion fits

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(b)

(c)

Fig. 7 Effects of chronic (14 days) treatment with cis-4-(4-(3-adaman-tan-1-yl-ureido) cyclohexyloxy) benzoic acid (c-AUCB) or angiotensin-converting enzyme inhibitor (ACEi) on (a) plasma renin activities, (b)ratios of plasma angiotensin-II (AngII) to AngI levels and (c) ratios ofkidney AngII to AngI levels in indole-3-carbinol (I3C)-induced and non-induced Cyp1a1-Ren-2 transgenic rats. *P < 0.05 versus unmarked val-ues, #P < 0.05 versus all the other values. ( ), Non-induced untreated;( ), non-induced + c-AUCB (130 mL); ( ), non-induced + ACEi; ( ),13C-induced untreated; ( ), 13C-induced + c-AUCB (0.3 mg/L); ( ),13C-induced + c-AUCB (3 mg/L); ( ), 13C-induced + c-AUCB (13 mg/L);( ), 13C-induced + c-AUCB (26 mg/L); ( ), 13C-induced + c-AUCB(60 mg/L); ( ), 13C-induced + c-AUCB (130 mg/L); ( ), 13C-induced + ACEi.



well with recent data showing the crucial importance of anenhanced intrarenal AngII in the pathophysiology of AngII-dependent hypertension.20,27,31,32

What are the mechanisms responsible for the effects ofchronic sEH inhibition by c-AUCB on plasma and kidneyAngII levels in I3C-induced Cyp1a1-Ren-2 transgenic rats?

We found that chronic c-AUCB treatment caused dose-dependentsuppression of plasma as well as kidney AngII concentrations.It will be recalled that inhibition of RAS activity by pharmaco-logical blockade of AngII type 1 receptor leads to a marked ele-vation of circulating AngII levels. This is the consequence of theinterruption of the short-loop negative feedback wherein AngIItype 1 receptor activation suppresses renin secretion anddecreases plasma AngII levels.46,47 It is therefore expected thatthe blockade of the RAS activity by chronic c-AUCB treatmentmust be either at the renin or at the ACE level. In this context, afurther important finding of the present study is that chronic sEHinhibition by c-AUCB did not suppress the expression of theRen-2 renin gene in I3C-induced Cyp1a1-Ren-2 transgenic rats.In contrast, these animals showed a marked increase in theexpression of Ren-2 renin gene, suggesting that I3C-inducedCyp1a1-Ren-2 transgenic rats responded to suppression of AngIIconcentrations by a compensatory rise in renin gene expression.This is consistent with the concept of disruption of the short-loopnegative feedback of AngII on the renin gene expression andsecretion.46,47 In addition, our present results clearly show thatchronic sEH inhibition by c-AUCB did enhance plasma reninactivity in I3C-induced Cyp1a1-Ren-2 transgenic rats. These find-ings strongly suggest that the blockade of the RAS activity bychronic c-AUCB treatment did not occur at the renin level.In this regard, of special interest is our finding that I3C-induced

Cyp1a1-Ren-2 transgenic rats treated chronically with ACEi orwith high doses of c-AUCB (either treatment leading to sustainednormalization of BP) affected AngII concentrations, and plasmaand kidney ACE activity in the same way. This strongly suggeststhat c-AUCB, a new orally active sEH inhibitor, blocks the activ-ity of the RAS at the ACE level, an unexpected finding in a studythat was not designed to explore the ACEi-like action ofc-AUCB. In addition, a limitation of our present study was that itwas not designed to delineate the strict correlation betweendecreases in BP and BW in response to dose-dependent treatmentand changes in intrarenal AngII concentrations in I3C-inducedCyp1a1-Ren-2 transgenic rats. It is clear that additional studiesare required to address this issue more precisely.Furthermore, the evidence that chronic treatment with ACEi

and the highest dose of c-AUCB (130 mg/L) decreased kidneyAngII concentrations in I3C-induced Cyp1a1-Ren-2 transgenicrats even below the levels observed in non-induced rats furtheremphasizes the critical role of the intrarenal AngII content in thepathophysiology of hypertension in this model. The presentresults corroborate those of the previous studies showing thatsuppression of intrarenal RAS activity is the crucial mechanismunderlying effective long-term antihypertensive treatment inAngII-dependent models of hypertension.20,22,31,32

With regard to BW loss in I3C-induced Cyp1a1-Ren-2 trans-genic rats, previous studies have shown that malignant hyperten-sion in this model is characterized by augmented activity of the

RAS, rapid increase in BP and an intensive pressure diuresisassociated with profound loss of BW, identical to that of ourpresent study.25–29 In our present study, the decrease in BW inuntreated I3C-induced Cyp1a1-Ren-2 transgenic rats was not sim-ply the result of reduced food intake (and consequently loss inmuscle weight etc.), because our data from monitoring foodintake show that these animals reduced food intake by approxi-mately 7%, which was not significantly different from foodintake of either non-induced rats or I3C-induced and pharmacolog-ically treated rats. Taken together, our present data show that theloss in BW is a consequence of the accentuated pressure diuresisaccompanying the development of malignant hypertension.However, it is obvious that future studies should more complexlyaddress the mechanism(s) responsible for the BW loss afterinduction of the renin gene in Cyp1a1-Ren-2 transgenic rats.In conclusion, the present findings show that the acute BP-

lowering effects of sEH inhibition in I3C-induced Cyp1a1-Ren-2transgenic rats are mediated by a substantial increase of intrarenalbiologically active epoxygenase products and their natriureticaction. In contrast, the long-term antihypertensive action of sEHinhibition in this rat model is chiefly due to the suppression ofintrarenal AngII concentration that is mediated by the ACEi-likeaction of c-AUCB.

METHODS

The studies were carried out in accordance with guidelines andpractices established by the Committee for Animal Care and Useat the Institute of Clinical and Experimental Medicine (IKEM,Prague, Czech Republic; protocol #26-2010 issued by Ministryof Health of Czech Republic).

Animals and diets

Experiments were carried out in male Cyp1a1-Ren-2 transgenicrats generated by inserting the mouse Ren-2 renin gene, fusedto the cytochrome P-450 (Cyp1a1) promoter, into the genomeof the Fischer 344 rat. The Cyp1a1 promoter is not constitu-tively expressed in the liver; however, after exposure to variousnatural xenobiotics, such as indole-3-carbinol (I3C), which canbe easily given in the diet, the expression of the Cyp1a1 pro-moter is rapidly enhanced and results in a marked increase ofthe expression of the Ren-2 renin gene in the liver. Enhancedexpression of the Ren-2 renin gene increases AngII levels.25–30

All animals used in the present study were bred at the Centerfor Experimental Medicine of the Institute for Clinical andExperimental Medicine from stock animals supplied from theCenter for Cardiovascular Science, University of Edinburgh, UK(we acknowledge the generous gift from Professor Mullins). Alldiets used in the present study were produced by Albert Weber– Services to Experimental Medicine (SEMED, Prague 4, CzechRepublic). Rats were fed either a rat chow without I3C (non-induced groups) or a rat chow containing 0.3% I3C (I3C-induced groups). It has been shown that I3C is a dietary sup-plement that does not show any harmful biological effects intransgene-negative rats, but causes a very strong induction ofCyp1a1 through activation of the aryl hydrocarbon receptor,which is a basic helix-loop-helix-transcription factor that bindsto the Cyp1a1 promoter.25



Chemicals

Cis-4-(4-(3-adamantan-1-yl-ureido)cyclohexyloxy) benzoic acid(c-AUCB), a sEH inhibitor, was prepared freshly and given indrinking water as described previously.9–12 The following dosesof c-AUCB per 1 L of water were examined: 0.3, 3, 13, 26, 60and 130 mg. Trandolapril (Gopten; Abbot, Prague, CzechRepublic), an ACEi, was used at a dose of 6 mg/L drinkingwater. Our recent studies have shown that this high dose com-pletely blocks the development of the AngII-dependent form ofhypertension.33–35

Series 1: Measurements of BP (radiotelemetry), urinecollections in conscious animals, and measurement of plasmac-AUCB levels and Ren-2 renin gene expression

In accordance with the recommendation for BP measurement inexperimental animals, we used a radiotelemetry system for directBP measurements.36 Rats were anaesthetized with a combinationof tiletamine, zolazepam (Zoletil, 8 mg/kg; Virbac SA, CarrosCedex, France) and xylazine (Rometar, 4 mg/kg; Spofa, Prague,Czech Republic) intramuscularly, and TA11PA-C40 radiotelemet-ric probes (Data Sciences International, St. Paul, MN, USA) wereimplanted for direct BP measurements as described previ-ously.9,11,12,15 Rats were allowed 10 days to recover before basalBP was recorded. Basal BP was determined for 4 days, and theninduction of the renin gene was carried out until the end of theexperiment that lasted 24 days.Our previous studies have shown that after 10 days of

induction a stable, severe hypertension develops.11,12,26,27 Then,the treatment with either c-AUCB or ACEi was carried out for14 days. Untreated conscious non-induced, and I3C-inducedrats and non-induced rats treated with ACEi served as controls.The animals were housed individually in metabolic cages, BPwas continuously recorded and urine was collected for 24 hinto sterile tubes. The 24-h urine samples were collected beforethe start of pharmacological treatment (day �2), and then ondays 2, 8 and 12 of treatment. Urine volumes were determinedfor each urine collection, and the samples were centrifuged(600 g/5 min; 4°C) and preserved for analysis. Urinary concen-tration of sodium was assessed by flame photometry. Thewhole aforementioned procedure was recently described indetail.9,11,33–35 At the end of experiments, all rats were decapi-tated to collect blood for measurement of plasma c-AUCB asdescribed previously,10,11 and the livers were removed foranalysis of Ren-2 renin gene expression by s techniquedescribed in detail previously.37 Briefly, total RNA wasextracted from liver tissue using Trizol (Invitrogen, Carlsbad,CA, USA) according to the manufacturer’s directions. TotalRNA treated with DNase I (Fermentas, Thermoscientific, Wal-tham, MA, USA) was reverse transcribed and amplified usingOne Step SYBR PrimeScript RT–PCR Kit II (TAKARA BIO,Shiga, Japan) following the manufacturer’s recommendations,with total volume of 20 lL. All samples were analysed in trip-licates. The primer sequences for renin-2 were: forward: 50-GCCTCAGCAAGACTGATTCC-30, reverse: 50-ATATTCATGTAGTCTCTTCTCC-30, and for b-actin: forward: 50-TGACTGACTACCTCATGAAGA-30 and reverse: 50-CACGTCACACTTCATGATG-30. Polymerase chain reactions amplifications were

carried out using the ABI Prism 7300 sequence detection sys-tem (Applied Biosystems, Foster City, CA, USA) following thereaction parameters recommended by the manufacturer, using2 mg RNA per sample. b-Actin was used as an endogenouscontrol gene, and negative controls contained water instead ofcDNA. In all experiments, relative gene expression was calcu-lated by the Δ cycle threshold (Ct) method. Briefly, the resul-tant mRNA was normalized to a calibrator; in each case, thecalibrator chosen was the basal sample. Final results wereexpressed as the n-fold difference in gene expression relativeto b-actin mRNA and calibrator as follows: n-fold = 2 – (ΔCtsample/ΔCt basal), where ΔCt values of the sample and cali-brator were determined by subtracting the average Ct value ofthe transcript under investigation from the average Ct value ofthe b-actin mRNA gene for each sample.Only animals with stable BP records during the basal period

were used; these were randomly divided into the following exper-imental groups:

Group 1: Non-induced + untreated (n = 6)Group 2: Non-induced + c-AUCB (130 mg/L; n = 7)Group 3: Non-induced + ACEi (n = 7)Group 3: I3C-induced + untreated (n = 7)Group 4: I3C-induced + c-AUCB (0.3 mg/L; n = 7)Group 5: I3C-induced + c-AUCB (3 mg/L; n = 7)Group 6: I3C-induced + c-AUCB (13 mg/L; n = 8)Group 7: I3C-induced + c-AUCB (26 mg/L; n = 8)Group 8: I3C-induced + c-AUCB (60 mg/L; n = 7)Group 9: I3C-induced + c-AUCB (130 mg/L; n = 8)Group 10: I3C-induced + ACEi (n = 7)

Series 2: Effects of c-AUCB and ACEi in non-induced orI3C-induced Cyp1a1-Ren-2 transgenic rats on EET, DHETE,plasma and kidney tissue AngII concentrations, plasma reninactivity, and plasma and kidney tissue ACE activity

It is now generally recognized that plasma and tissue AngII con-centrations in anaesthetized animals are higher than thoseobtained from rats decapitated while conscious, and that normo-tensive animals show a greater increase in renin secretion inresponse to anaesthesia and surgery than AngII-induced hyperten-sive intrarenal renin-depleted animals.9–12,14,15,19,20,22,31 There-fore, in the present study we determined AngII levels in separategroups of conscious rats (as described in series 1) that weredecapitated either on day 2 (to evaluate acute effects) or onday 14 (to evaluate chronic effects) after introduction of pharma-cological treatment (n = 8 in each group). Plasma and whole kid-ney AngI and AngII concentrations were assessed byradioimmunoassay as described in detail in our previousstudies.26,27 In the present study, for measurements of plasmarenin activities, the commercially available RIA kit was used(Cisbio Bioassays, Paris, France); for measurements of plasmaand kidney ACE activities, the commercially available RIA kitwas also used (B€uhlmann Laboratories, Allschwil, Switzerland).In this protocol, one unit of ACE activity was defined as theamount of enzyme required to release 1 lmol of hippuric acidper minute per liter of sample at 37°C. In addition, in the presentstudy, ACE activities were also estimated as the ratio of AngII toAngI because we have recently found, in accordance with one



original study in AngII-infused hypertensive rats, that in this An-gII-dependent model of hypertension the ratio is a very reliableindex for assessment of circulating and especially tissue ACEactivity.19,26,27 The level of the arachidonic acid metabolites EETand DHETE were measured in the kidney cortex. Samples wereextracted, separated by reverse-phase, high performance liquidchromatography, and analysed by negative-mode electrosprayionization and tandem mass spectroscopy as described previ-ously.9–12 The procedure of blood sampling and the assays ofindividual components of RAS and EET and DHETE assays areroutinely used in our laboratory, and this standardized approachallows us to compare the results to those of our previous studiesevaluating the role of the RAS and CYP-dependent metabolitesin the pathophysiology of the AngII-dependent form ofhypertension.9–12,14,15,17,26,27

Statistical analysis

All values are expressed as means � SEM. GRAPH-PAD PRISM

software (Graph Pad Software, San Diego, CA, USA) was usedand, when appropriate, statistical analysis was carried out usingone-way analysis of variance (ANOVA). ANOVA for repeatedmeasurements, followed by Student–Newman–Keuls test, wascarried out for the analysis within groups (e.g. before and aftereither I3C or pharmacological treatment). Values exceeding the95% probability limits (P < 0.05) were considered statisticallysignificant.

ACKNOWLEDGEMENTS

This study was principally supported by grant No. NT/12171-5awarded by the Internal Grant Agency of the Ministry of Healthto ZH. AS was supported by a Marie Curie Fellowship from theEuropean Commission Program PEOPLE (IRG 247847). TheInstitute for Clinical and Experimental Medicine (IKEM) is arecipient of the project of the Ministry of Health of the CzechRepublic for the development of research organization 00023001(institutional support). SJ is supported the Grant Agency ofCharles University No. 266213. The Center for ExperimentalMedicine (IKEM) received financial support from the EuropeanCommission within the Operational Program Prague–Competi-tiveness; project “Rozvoj infraskruktury PEM” (#CZ.2.16/3.1.00/28025). JDI was supported by NIH grants HL59699 andDK38226. SHH was supported by a fellowship from the NIEHSSupported Basic Research Program. Partial support was providedby NIEHS Grant R01 ES02710, R01 ES013933 and P42ES013933, and by West Coast Center U24 DK097154 awardedto BDH. BDH is a George and Judy Marcus Senior Fellow ofthe American Asthma Foundation. BDH was funded by EicOsisto develop soluble epoxide hydrolase inhibitors for treatinginflammatory and neuropathic pain. The other authors declare nopotential conflict of interest.

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SUPPORTING INFORMATION

Additional Supporting Information may be found in the online version of this article:

Figure S1. Time-course of body weight in indole-3-carbinol (I3C)-induced and noninduced Cyp1a1-Ren-2 transgenic rats and effectsc-AUCB or ACEi treatment.



Figure legends for supplemental figure.

Supplemental figure 1. Time‐course of body weight in indole‐3‐carbinol (I3C)‐induced and

noninduced Cyp1a1‐Ren‐2 transgenic rats and effects c‐AUCB or ACEi treatment. *P<0.05

versus baseline values. # P<0.05 versus c‐AUCB‐ or ACEi‐treated I3C‐induced rats at the same

time point.

different mechanisms of acute versus long‐term antihypertensive … · 2015. 5. 27. ·...

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