Kidney International, Vol. 68 (2005), pp. 2189–2196

Effects of renin-angiotensin system blockade on renalangiotensin-(1-7) forming enzymes and receptors

CARLOS M. FERRARIO, JEWELL JESSUP, PATRICIA E. GALLAGHER, DAVID B. AVERILL,K. BRIDGET BROSNIHAN, E. ANN TALLANT, RONALD D. SMITH, and MARK C. CHAPPELL

Hypertension and Vascular Disease Center, Wake Forest University School of Medicine, Winston-Salem, North Carolina

Effects of renin-angiotensin system blockade on renalangiotensin-(1-7) forming enzymes and receptors.

Background. Angiotensin-converting enzyme (ACE)2, a ho-mologue of ACE, which is insensitive to ACE inhibitors andforms angiotensin-(1-7) [Ang-(1-7)] from angiotensin II (AngII) with high efficiency was investigated in response to chronicblockade with lisinopril, losartan, and both drugs combined.

Methods. Thirty-six adult Lewis rats were assigned to receivethese medications in their drinking water for 2 weeks while theirarterial pressure, water intake, and urine volume were recordedthroughout the study. Measures of renal excretory variables in-cluded assessing excretion rates of angiotensin I (Ang I), Ang IIand Ang-(1-7) while blood collected at the completion of thestudy was used for measures of plasma angiotensin concentra-tions. Samples from renal cortex were assayed for renin, an-giotensinogen (Aogen), neprilysin, angiotensin types 1 and 2(AT1 and AT2) and mas receptor mRNAs by semiquantitativereverse transcriptase (RT) real-time polymerase chain reaction(PCR). ACE2 activity was determined as the rate of Ang IIconversion into Ang-(1-7).

Results. Comparable blood pressure reductions were ob-tained in rats medicated with either lisinopril or losartan,whereas both drugs produced a greater decrease in arterial pres-sure. Polyuria was recorded in all three forms of treatment as-sociated with reduced osmolality but no changes in creatinineexcretion. Lisinopril augmented plasma levels and urinary ex-cretion rates of Ang I and Ang-(1-7), while plasma Ang II wasreduced with no effect on urinary Ang II. Losartan producedsimilar changes in plasma and urinary Ang-(1-7) but increasedplasma Ang II without changing urinary Ang II excretion. Com-bination therapy mimicked the effects obtained with lisinoprilon plasma and urinary Ang I and Ang-(1-7) levels. Renal cortexAogen mRNA increased in rats medicated with either lisino-pril or the combination, whereas all three treatments produceda robust increase in renal renin mRNA. In contrast, ACE,ACE2, neprilysin, AT1, and mas receptor mRNAs remainedunchanged with all three treatments. Renal cortex ACE2 activ-ity was significantly augmented in rats medicated with lisinoprilor losartan but not changed in those given the combination.

Key words: lisinopril, losartan, angiotensin-converting enzyme 2,angiotensin-(1-7), hypertension, renal function.

Received for publication March 9, 2005and in revised form May 20, 2005Accepted for publication June 28, 2005

C© 2005 by the International Society of Nephrology

Conclusion. Our data revealed a role for ACE2 in Ang-(1-7)formation from Ang II in the kidney of normotensive rats asprimarily reflected by the increased ACE2 activity measured inrenal membranes from the kidney of rats given either lisino-pril or losartan. The data further indicate that increased levelsof Ang-(1-7) in the urine of animals after ACE inhibition orAT1 receptor blockade reflect an intrarenal formation of theheptapeptide.

As the newest homologue of angiotensin-convertingenzyme (ACE), ACE2 has emerged as a novel site fortherapeutic intervention within the cascade of the renin-angiotensin system (RAS) since the enzyme has a sub-strate preference for hydrolyzing angiotensin II (Ang II)into the vasodilator and antiproliferative peptide,angiotensin-(1-7) [Ang-(1–7)] [1]. As reviewed elsewhere[2], previous studies from our laboratory [3–7] showedthat ACE2 functions as a component of the counterregu-latory mechanism through which Ang-(1-7) opposes theeffects of Ang II on blood pressure and cellular growth, aswell as exerting important antihypertensive actions. Theimportance of ACE2 in cardiovascular regulation becameapparent in studies showing that ACE2 mRNA and pro-tein were reduced in the kidney of experimental modelsof hypertension while in ACE2 knockout mice reducedleft ventricular contractility and left ventricular dilationcould be rescued by crossing these animals with the ACEknockout mouse [4].

The abundance of ACE2 in the heart led us to investi-gate its role in cardiac function, particularly since previ-ous experiments showed that Ang-(1-7), solely localizedin rat cardiac myocytes [8], exerted important cardiopro-tective actions in terms of reversal of cardiac hypertrophyand cardiac dysfunction [7, 9]. Furthermore, both inhibi-tion of Ang II synthesis as well as blockade of Ang II re-ceptors increased cardiac ACE2 mRNA as well as ACE2activity, determined as the rate of Ang II conversion intoAng-(1-7) in isolated cardiac membranes from the sameanimals [6].

To assess the effect of interruption of the pressor andproliferative arm of the RAS on the genes, proteins, and

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receptors constituting the axis that opposes the activity ofAng II, we measured the effects of chronic administrationof lisinopril, losartan, or both agents on the componentsof the RAS within the kidney of Lewis normotensive rats.The resultant effect of these treatments on the enzymesthat participate in the renal formation of Ang-(1-7) fromeither Ang I (neprilysin) or Ang II (ACE2) are a focus ofthese studies since previous experiments indicated thatACE inhibition and angiotensin type 1 (AT1) receptorblockade augmented cardiac and vascular ACE2 geneexpression or activity [6, 7, 10]. Data on the effect of thesetreatments on cardiac ACE2 mRNA and ACE2 activityare reported elsewhere [6].

METHODS

Animals

Experiments were conducted in male Lewis rats (270to 280 g) (Charles River Laboratories, Wilmington, MA,USA) housed in individual cages (12-hour light/dark cy-cle) with ad libitum access to rat chow and tap waterusing procedures approved by our Institutional AnimalCare and Use Committee.

Experimental protocol

Thirty-six normotensive rats (age range 8 to 10 weeks)were randomly assigned to drink either tap water (ve-hicle) (N = 12) or tap-water to which lisinopril (10 mg/kg/day) (N = 8), losartan (10 mg/kg/day) (N = 8), or bothdrugs [same doses (N = 8)] was added to their drinkingwater for 12 consecutive days. Rats were housed individ-ually in metabolic cages for 24-hour collection of urineboth before and throughout the study.

During the treatment period, tail-cuff systolic bloodpressure was determined for 3 days before and threetimes weekly after initiation of the treatments. Two weeksafter completion of the treatment regimens, rats wereanesthetized with halothane (1.5%) (Halocarbon Labo-ratories, River Edge, NJ, USA) for direct measurementsof arterial pressure and heart rate using a catheter-tippressure transducer inserted into a carotid artery (ModelSPR-671) (Millar Instrument Co., Houston, TX, USA).A 5 mL sample of arterial blood was obtained for bio-chemical measurements. Cardiopulmonary excision wasdone in deeply anesthetized rats and the kidneys were re-moved for semiquantitative determinations of RAS geneexpression.

Biochemistry

Blood was collected into chilled tubes containing amixture of peptidase inhibitors: 25 mmol/L ethylenedi-aminetetraacetic acid (EDTA), 0.44 mmol/L 1,20-ortho-phenanthrolene monohydrate (Sigma Chemical Co., St.Louis, MO, USA), 1 mmol/L sodium para-chloromercuri-benzoate, and 3 lmol/L WFML (rat renin inhibitor

acetyl-His-Pro-Phe-Val-Statine-Leu-Phe) (ANASPEC).After 30 minutes on ice, blood cells were isolated by cen-trifugation at 3000 rpm for 20 minutes, and aliquots ofeither plasma or serum were stored at −80◦C until use.Frozen tissues were rapidly weighed and homogenizedfor later assay as described elsewhere [7]. Plasma andurinary concentrations of angiotensin peptides were mea-sured by radioimmunoassay as documented elsewhere[7, 11].

Serum ACE activity was determined by incubation ofthe sample with radiolabeled 3H-Hip-Gly-Gly (pH 8.0)for 1 hour at 37◦C. The intra-assay variation was 3.9% andthe interassay variation averaged 5.9%. ACE2 activitywas quantified as the rate of exogenous 125I-Ang II con-version to 125I-Ang-(1-7) in kidney membranes isolatedfrom the renal cortex using procedures adapted from ourprevious study in rat heart [6]. The tissue was weighed andhomogenized in 10 mmol/L Hepes, pH 7.4, 120 mmol/LNaCl, and 10 lmol/L ZnCl2. The homogenate was subjectto centrifugation at 30,000g for 20 minutes. The super-natant was removed, the membranes were resuspendedin the above buffer containing 0.5% Triton X-100, andincubated overnight on ice at 4◦C. Following centrifuga-tion, the soluble portion was incubated with 125I-Ang II(2 × 106 cpm) (2200 Curies/mmol) in the Hepes buffercontaining 10 lmol/L of amastatin, bestatin, SCH39370(neprilysin inhibitor), chymostatin, lisinopril, benzyl suc-cinate, and Z-prolyl prolinal (ZPP) at 37◦C from 10 to120 minutes and the reaction terminated by addition of1% phosphoric acid. 125I-Ang II was iodinated by theChloramine T method and purified by high-performanceliquid chromatography (HPLC). The samples were sub-jected to centrifugation and the supernatants were fil-tered through a 0.22 l membrane prior to HPLC analysis.Ang-(1-7) was resolved from Ang II on a Shimadzu(Kyoto, Japan) reverse-phase HPLC equipped with a Wa-ters NovaPak C18 column (2.1 × 150 mm) (Milford, MA,USA). We employed a linear gradient from 10% to 25%mobile phase B for 20 minutes and isocratic at 25% for15 minutes at a flow rate of 0.35 mL/min at ambienttemperature. The solvent system consisted of 0.1% phos-phoric acid (mobile phase A) and 80% acetonitrile/0.1%phosphoric acid (mobile phase B) [12]. The eluted ra-dioactive peaks were identified using an in-line c detec-tor (BioScan, Washington, DC, USA) and the data wereacquired and analyzed with Shimadzu version 7.2.1 ac-quisition software. ACE2 activity was expressed as theamount of Ang-(1-7) generated per minute per mg pro-tein that was inhibited by addition of 10 lmol/L of theACE2 inhibitor, MLN-4760 [13].

RNA isolation and reverse transcriptase (RT)/real-timepolymerase chain reaction (PCR)

RNA was isolated from kidney tissue, using the Trizolreagent (Gibco Invitrogen, Carlsbad, CA, USA), as

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Fig. 1. Group averages of tail-cuff systolicblood pressure determinations in consciousLewis normotensive rats before and after thecompletion of 2 weeks of oral administrationof vehicle, lisinopril, losartan, or both drugsin combination. Values are means ± SE. ∗P <

0.05 compared to their respective value beforeinitiation of the medications. P value withingraph denotes statistical difference betweenrats medicated with losartan or combinationtherapy.

directed by the manufacturer. The RNA concentrationand integrity were assessed using an Agilent 2100Bioanalyzer with an RNA 6000 Nano LabChip (AgilentTechnologies, Palo Alto, CA, USA). Approximately1 lg of total RNA was reverse transcribed using AMVreverse transcriptase in a 20 lL reaction mixturecontaining deoxyribonucleotides, random hexamersand RNase inhibitor in RT buffer. Heating the RTreaction product at 95◦C terminated the reaction. Forreal-time PCR, 2 lL of the resultant cDNA was addedto TaqMan(TM) Universal PCR Master Mix (AppliedBiosystems, Foster City, CA, USA) with the appropriategene-specific primer/probe set and amplification wasperformed on an ABI 7000 Sequence Detection System.The primer/probe sets were purchased from AppliedBiosystems with the exception of ACE2 (forward primer5′-CCCAGAGAACAGTGGACCAAAA-3′; reverseprimer 5′-GCTCCACCACACCAACGAT-3′; and probe5′-FAM-CTCCCGCTTCATCTCC-NFQ-3′) and mas(forward primer 5′-TTCATAGCCATCCTCAGCTTCTTG-3′; reverse primer 5′-GTTCTTCCGTATCTTCACCACCAA-3′; and probe 5′-FAM-TTCACTCCGCTCATGTTAG-NFQ-3′). The mixtures were heated at 50◦Cfor 2 minutes, at 95◦C for 10 minutes followed by40 cycles at 95◦C for 15 seconds and 60◦C for 1 minute.All reactions were performed in triplicate and 18Sribosomal RNA, amplified using the TaqMan RibosomalRNA Control Kit (Applied Biosystems), served asan internal control. The results were quantified as Ctvalues, where Ct is defined as the threshold cycle ofPCR at which the amplified product is first detected,and expressed as relative gene expression (the ratio oftarget/18S rRNA control).

Statistical analysis

All values are expressed as mean ± SEM. One-wayanalysis of variance (ANOVA) followed by either theBonferroni or the two-tailed Student t tests was used forcomparing the differences at a P value <0.05.

RESULTS

Twelve-day administration of the drugs either aloneor in combination produced significant decreases in sys-tolic blood pressure, more pronounced in rats medicatedwith the combination of lisinopril-losartan (Fig. 1). Directmeasurements of mean arterial pressure at the end of thestudy in the anesthetized rats confirmed the observationsobtained in conscious animals. None of the treatmentshad an effect on heart rate.

As demonstrated elsewhere [6, 14, 15], rats given lisino-pril increased plasma Ang I and Ang-(1-7) levels byan average of 1486% and 170%, respectively, whereasplasma Ang II and ACE were reduced by an average of56% and 93%, respectively, compared to vehicle-treatedrats (Table 1). Losartan treatment was associated withincreases in plasma Ang I comparable to those foundin lisinopril-treated rats. In addition, losartan stimulatedlarge increases in plasma Ang II but not changes in plasmaAng-(1-7) and ACE activity (Table 1). Combination ther-apy obtunded but did not eliminate the significant rise inplasma Ang I levels, reduced Ang II levels to values belowthose found in vehicle but not lisinopril-treated rats, andincreased plasma Ang-(1-7) concentrations above thosefound in rats given losartan alone (Table 1). ACE activityin rats given combination therapy was suppressed to an

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Table 1. Profile of plasma angiotensin peptides and angiotensin-converting enzyme (ACE) activity changes by treatment groups

Variables Vehicle Lisinopril Losartan Combination

Angiotensin I fmol/mL 94 ± 26 1397 ± 171 909 ± 89a 433 ± 104a,b

P value < 0.001 0.001 0.01Angiotensin II fmol/mL 54 ± 9 24 ± 6 332 ± 50a 22 ± 6b

P value < 0.05 0.001 0.05Angiotensin-(1-7) fmol/mL 46 ± 10 78 ± 11 51 ± 9 89 ± 7b

P value< 0.05 NS 0.01Angiotensin-converting enzyme 57.9 ± 4.8 3.8 ± 0.1 49.9 ± 4.0a 4.2 ± 0.3b

activity nmol/min/mLP value< 0.001 NS 0.001

Data are means ± SE. P value denotes statistical difference versus rats medicated with the vehicle.aP < 0.05 compared to lisinopril; bP < 0.05 compared to losartan.

Table 2. Effects of drug treatments on renal function variables

Variables Vehicle Lisinopril Losartan Combination

Water intake mL 26.70 ± 1.06 35.66 ± 1.48 26.31 ± 1.13a 40.26 ± 2.01b

P value < 0.001 NS 0.001Urinary output mL 9.22 ± 0.64 21.33 ± 0.94 12.96 ± 0.65a 15.26 ± 1.16b

P value < 0.001 0.01 0.001Urine osmolality mOsm/mL 2431 ± 140 928 ± 158 1850 ± 110a 1424 ± 167b

P value< 0.001 0.01 0.001Sodium excretion mEq/24 hours 1.90 ± 0.21 1.99 ± 0.69 1.70 ± 0.07 1.63 ± 0.20

P value< NS NS NSPotassium excretion mEq/24 hours 3.68 ± 0.28 3.28 ± 0.45 4.32 ± 0.19 3.21 ± 0.32

P value < NS NS NSCreatinine excretion mg/24 hours 11.56 ± 1.17 9.93 ± 1.77 12.65 ± 0.80 10.44 ± 0.97

P value < NS NS NS

Data are means ± SE. P value denotes statistical difference versus rats medicated with the vehicle.aP < 0.05 compared to lisinopril; bP < 0.05 compared to losartan.

amount not different than that found in rats given lisino-pril (Table 1).

Table 2 shows that polydipsia associated with hyposmo-lar polyuria and no differences in either urinary Na+ andK+ excretion accompanied the administration of lisino-pril. Although water intake was not changed in rats givenlosartan, these animals displayed increased urinary out-put and reduced urinary osmolality. Urinary excretionof Na+ and K+ in losartan-treated rats was not changedwhen compared to vehicle-treated controls (Table 2).Combination therapy mimicked the effect observed inrats medicated with lisinopril. None of the treatmentscaused significant changes in urinary creatinine excretion(Table 2).

Figure 2 illustrates the effect of the various treatmentson urinary excretion rates of Ang I, Ang II, and Ang-(1-7). The patterns of urinary excretion of angiotensinpeptides in Lewis rats do not differ from those previouslyreported for normotensive Sprague-Dawley rats [16] orspontaneously hypertensive rats given a combined ACEand neprilysin inhibitor [11]. Administration of an ACEinhibitor or the Ang II receptor antagonist was associatedwith augmented excretion rates of Ang I and Ang-(1-7)but not Ang II. In addition, the average values of urinaryAng-(1-7) excretion determined on the last day of thetreatment period correlated with plasma concentrations

of Ang I (r = 0.59, P < 0.001) and Ang-(1-7) (r = 0.55,P < 0.001).

Hemodynamic and renal excretory changes observedwith the various protocols were associated only in partwith changes in the expression of renal RAS genes(Table 3). All three treatments increased kidney cortexrenin mRNA while angiotensinogen mRNA increasedonly in rats medicated with lisinopril or combinationtherapy. In contrast, ACE, neprilysin, ACE2, AT1, andmas receptor mRNAs were not changed by the treat-ments. ACE2 activity increased in the renal cortex of ratsmedicated with either lisinopril or losartan while ACE2activity remained unchanged in rats given the combina-tion therapy (Fig. 3). The increased ACE2 activity asso-ciated with treatment with either lisinopril or losartancorrelated significantly with 24-hour urinary excretionrates of Ang I (r = 0.57, P < 0.001) and Ang-(1–7) (r =0.49, P < 0.02) as well as both plasma concentrations ofAng I (r = 0.68, P < 0.001) and Ang-(1–7) (r = 0.54,P < 0.01).

DISCUSSION

In the current study, we document for the first time thecomparative effect of inhibition of Ang II synthesis ver-sus Ang II receptor blockade, alone or in combination, on

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Fig. 2. Effects of the various treatments on urinary excretion rates ofangiotensin I (Ang I), angiotensin II (Ang II), and angiotensin-(1-7)[Ang-(1-7)]. B is baseline. Values are means ± SE. ∗P < 0.05 comparedto the corresponding baseline values; ∗∗P < 0.05 compared to animalsmedicated with lisinopril.

renal excretory variables and expression of the genes en-coding the active peptides and receptors of the RAS in thekidney of normotensive Lewis rats. The primary questionwe addressed in these experiments was whether adminis-tration of either lisinopril or losartan would modulate therenal expression of RAS genes, especially those genes ac-counting for the production (neprilysin and ACE2) andmetabolism (ACE) of the vasodilator peptide Ang-(1-7)[17–19]. In agreement with previous studies [6, 16, 20],these medications effectively reduced the contributionof Ang II to blood pressure regulation as reflected by thedecrease in arterial pressure and associated changes inboth plasma and urinary excretion of Ang I, Ang II, andAng-(1-7).

Hemodynamic and renal excretion changes in Lewisrats given the vehicle, lisinopril, losartan, or their com-bination are similar to those reported in both Sprague-Dawley rats [16] and essential hypertensive subjectsmedicated with either captopril [21] or the dual vasopep-tidase inhibitor omapatrilat [20]. The antihypertensive ef-fect obtained with each agent alone or in combinationwas essentially the same, although the blood pressureof rats given combination therapy was reduced furtherwhen compared to vehicle but not the rats medicatedwith lisinopril alone. Urinary excretion rates of Ang I andAng-(1-7) but not Ang II were augmented by all threetreatment modalities, a finding that is consistent with arole of Ang-(1-7) in mediating the polyuria found in thetreated animals [11, 16, 20].

Within the renal cortex, gene expression of reninwas markedly increased by all treatments, whereas an-giotensinogen mRNA increased only in rats exposedto blockade of ACE with lisinopril or the combinationof lisinopril/losartan. Although both monotherapy andcombination therapy caused marked increases in the uri-nary excretion of Ang I and Ang-(1-7), ACE, ACE2,and neprilysin mRNAs [genes of the enzyme axis reg-ulating Ang-(1-7) formation and metabolism] did notchange compared to vehicle-treated animals. Similarly,the treatments had no effect on the expression of AT1

and mas receptor mRNAs. On the other hand, ACE2activity, measured as the rate of generated Ang-(1-7)from Ang II in membranes from the renal cortex wasaugmented in rats medicated with lisinopril and losar-tan. A direct correlation of ACE2 activity with urinaryAng I and Ang-(1-7) as well as plasma Ang I and Ang-(1-7) in rats medicated with either lisinopril or losartanprovided evidence of its role in modulating the forma-tion of Ang-(1-7) from Ang II. On the other hand, theseexperiments underscore our previous conclusion thatthe role of ACE2 in the regulation of cardiovascularfunction is more complex than anticipated. As reportedby Gembardt et al [22], the effects of blockade of theRAS on ACE2 protein and activity may be both tissue-and species-specific. This interpretation agrees with our

2194 Ferrario et al: Renal angiotensin peptides

Table 3. Real-time polymerase chain reaction (PCR) measures of kidney renin-angiotensin system (RAS) genes

Kidney mRNAs Vehicle Lisinopril Losartan Combination

Renin 1.04 ± 0.12 30.79 ± 3.92 2.96 ± 0.33a 22.38 ± 2.64b

P value < 0.001 0.001 0.001Angiotensinogen mRNA 1.08 ± 0.15 1.69 ± 0.17 1.19 ± 0.15a 1.63 ± 0.08

P value < 0.01 N.S. 0.007Angiotensin-converting enzyme 1.09 ± 0.17 1.23 ± 0.12 1.02 ± 0.13 1.04 ± 0.15

P value < NS N.S. NSNeprilysin 1.03 ± 0.11 1.04 ± 0.09 1.06 ± 0.16 1.14 ± 0.17

P value < NS N.S. NSAngiotensin-converting enzyme 2 1.05 ± 0.12 1.10 ± 0.09 1.07 ± 0.16 0.99 ± 0.14

P value < NS N.S. NSAngiotensin type 1 receptor 1.03 ± 0.13 1.33 ± 0.09 1.47 ± 0.22 1.02 ± 0.13

P value < NS N.S. NSAngiotensin type 2 receptor Not detected Not detected Not detected Not detectedmas receptor 1.04 ± 0.12 1.07 ± 0.06 1.01 ± 0.11 0.95 ± 0.14

P value < NS NS NS

Data are means ± SE of values expressed as relative gene expression units. P value denotes statistical difference versus rats medicated with the vehicle.aP < 0.05 compared to lisinopril; bP < 0.05 compared to losartan.

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finding that the combination of lisinopril/losartan abro-gated the increase in renal cortex ACE2 activity found inanimals medicated with either agent alone even thoughthis combination treatment continued to be associatedwith increases in plasma and urinary excretion rates ofAng I and Ang-(1-7). Additional evidence for a tissue-specific response of both the ACE2 gene and ACE2 activ-ity is furnished by the finding that cardiac ACE2 activityincreased in the hearts of Lewis rats medicated with thecombination of lisinopril and losartan [6].

Two distinct biochemical axis-pathways determine theformation of Ang II and Ang-(1-7) from Ang I. Withinthe first arm of the system ACE cleaves Ang II from AngI, whereas in most conditions, neprilysin is principally in-volved in the formation of Ang-(1-7) from Ang I [23]. Thedemonstration that ACE2 converts Ang II into Ang-(1-7) connects downstream the two axis of the RAS system[1, 18]. ACE2 is widely localized in cardiovascular tis-

sues mainly in the media and endothelium of large andsmall arterial vessels, cardiac myocytes, and the tubularepithelia of the kidneys [24]. In addition Leydig cells inthe rat testis, Leydig and Sertoli cells in the human testis,and the uteroplacental complex showed positive stainingfor ACE2 [25, 26]. Neoexpression of ACE2 in glomeru-lar and peritubular capillary endothelium was reportedin biopsies obtained from patients with nephropathies ofvarious origins and in transplanted kidneys [27]. Tikelliset al [28] first showed that the expression of the ACE2mRNA was decreased in diabetic renal tubules by ap-proximately 50% and was not influenced by treatmentwith ramipril. This observation agrees with our findingsand those obtained by Lely et al [27] in human subjects.On the other hand, we now show that inhibition of ACEcaused a 72% increase in ACE2 activity in the renal cor-tex. The direct assessment of the functional activity ofthe ACE2 gene provides a strong argument of a role for

Ferrario et al: Renal angiotensin peptides 2195

this enzyme in modulating intrarenal generation of Ang-(1-7). Paralleling the effects of lisinopril on renal ACE2activity, we also found that blockade of AT1 receptors isassociated with a 43% increase in renal ACE2 activity.Thus, both suppression of Ang II formation or blockadeof Ang II binding to AT1 receptors increased ACE2 activ-ity within the renal cortex. Because the increased ACE2activity is associated with elevations in both plasma con-centrations of Ang-(1-7) and urinary excretion rates ofAng-(1-7), the data further underscore a functional roleof this novel ACE homologue in the formation of Ang-(1-7) from Ang II. The significant correlations among renalcortex ACE2 activity and plasma and urine Ang I andAng-(1-7) reinforced this interpretation. Additionally, Liet al [29] recently reported that ACE2 is widely local-ized in the tubules, glomeruli, and endothelial cells of therat kidney; however, they demonstrate an important roleof ACE2 in the formation of Ang-(1-7) from Ang I inisolated proximal tubules. Previous studies using purifiedpreparations of ACE2 have emphasized that while Ang Iand Ang II share similar Km values for the enzyme, thecatalytic rate is far higher for Ang II than Ang I [1, 18, 30].In addition, studies of Ang-(1-7) formation from Ang IIin human hearts confirmed the involvement of ACE2 inthe production of Ang-(1-7) [31, 32]. Because our ACE2assay directly measured the rate of conversion of AngII to Ang-(1-7) in isolated membranes of the renal cor-tex from the treated animals our results clearly implicateACE2 in the formation of intrarenal Ang-(1-7).

Combination therapy abrogated the increased renalACE2 activity obtained in rats medicated with eitheragent alone. A differential effect of Ang II blockadewas previously reported by us in the heart of Lewis ratsafter coronary artery ligation [6]. In those experiments,both losartan and the combination of lisinopril and losar-tan were associated with increased ACE2 activity, whilelisinopril therapy reduced cardiac ACE2 activity to val-ues found in vehicle-treated rats [6]. Therefore, in boththis and in our previous study [6], the same therapeuticagents given to the same rat strain at the same doses andfor the same period of time resulted in differential effectson cardiac and renal ACE2 mRNAs and activity. The dif-ferential effects on cardiac and renal ACE2 may not beattributed to hemodynamic factors since the fall in bloodpressure obtained by single or combined administrationof lisinopril and losartan were not different between thegroups within this study or a cross-comparison with theresults reported previously [6]. These findings and thosereported elsewhere [22] suggest a tissue-specific regula-tion of the ACE2 gene expression and protein in responseto maneuvers that alter either the formation of AngII or its activity. Uncoupling between gene expressionand protein is a known phenomenon as gene expres-sion is regulated at transcription, translation, or by post-translational modification. Furthermore, Zhong et al [33]

showed that all-trans retinoic acid up-regulated ACE2gene expression in the heart and kidney of spontaneouslyhypertensive rats, a finding that emphasizes a role of tran-scriptional factors in modulation of ACE2 mRNA. Fur-ther studies will be required to determine whether theabsence of changes in renal cortical mRNA compared tocardiac mRNA in response to blockade of Ang II im-plies a differential action of these agents on transcriptionfactors.

While our studies shed no light on the signaling mech-anisms that are a stimulus for ACE2 gene expression andprotein activity, in both astrocytes and myocytes in cul-ture we have preliminary evidence for a role of Ang IIin reducing ACE2 mRNA while the addition of Ang-(1-7) into the medium blocks the inhibitory action of AngII [Gallagher, Tallant, and Ferrario, unpublished obser-vations]. If the in vitro data can be interpolated to thecurrent findings, we can surmise that the combinationtherapy producing a marked increase in plasma Ang-(1-7) and suppressing Ang II formation should prevent stim-ulation of ACE2 activity. On the other hand, additionalfactors may be at play since the reduction in ACE2 activ-ity found in animals receiving the combination therapywas not associated with a decrease in urinary excretionrates of Ang I or Ang-(1-7). These data suggest that re-nal excretion of Ang-(1-7), although reflecting primarilyintrarenal formation of the peptide, may be under thecontrol of other Ang-(1-7) forming enzymes, as demon-strated by us previously [11, 20]. The observation thatrenal neprilysin mRNA was not changed by any of thetreatments alone or in combination does not necessarilyexclude this Ang-(1-7)–forming enzyme from participat-ing in the increased Ang-(1-7) found in the urine of alltreatment groups since neprilysin activity was not deter-mined in these experiments.

CONCLUSION

The lessons learned from this study are (1) renal ACE2activity increases as a result of suppression of Ang II syn-thesis or activity, a finding that suggests a feed forwardmechanism for the components of the RAS axis regulat-ing the production of Ang-(1-7); (2) although our exper-iments confirmed that ACE2 is not sensitive to blockadeof ACE, our data now show that ACE inhibition is associ-ated with increased renal ACE2 activity; (3) the absenceof changes in renal AT1 and mas receptors with the vari-ous treatment protocols suggests that the enzymes ratherthan the receptors affect the regulation of the system inthese conditions; and (4) the experiments reported hereand those described elsewhere [34], collectively, impli-cate increased Ang-(1-7) as a factor contributing to therenoprotective actions of blockade of Ang II with eitherACE inhibitors or Ang II receptor blockers.

2196 Ferrario et al: Renal angiotensin peptides

ACKNOWLEDGMENTS

The work described here was supported in part by grants HL-051952,HL-068258, HL-056973, HL-67363, HL-42631, and the American HeartAssociation, AHA-151521. Unrestricted grants from the Unifi Corpo-ration (Greensboro, NC) and the Farley-Hudson Foundation (Jack-sonville, NC) are also gratefully acknowledged. Merck and Co., Inc.,is also gratefully acknowledged for the gift of losartan.

Reprint requests to Carlos M. Ferrario, M.D., The Hypertension andVascular Disease Center, Wake Forest University School of Medicine,Winston-Salem, NC 27157.E-mail: cferrari@wfubmc.edu

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