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Effects of renin-angiotensin system blockade on renal angiotensin-(1-7) forming enzymes and receptors
Kidney International, Vol. 68 (2005), pp. 2189–2196
Effects of renin-angiotensin system blockade on renal angiotensin-(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 renal angiotensin-(1-7) forming enzymes and receptors. Background. Angiotensin-converting enzyme (ACE)2, a homologue of ACE, which is insensitive to ACE inhibitors and forms angiotensin-(1-7) [Ang-(1-7)] from angiotensin II (Ang II) with high efficiency was investigated in response to chronic blockade with lisinopril, losartan, and both drugs combined. Methods. Thirty-six adult Lewis rats were assigned to receive these medications in their drinking water for 2 weeks while their arterial pressure, water intake, and urine volume were recorded throughout the study. Measures of renal excretory variables included assessing excretion rates of angiotensin I (Ang I), Ang II and Ang-(1-7) while blood collected at the completion of the study was used for measures of plasma angiotensin concentrations. Samples from renal cortex were assayed for renin, angiotensinogen (Aogen), neprilysin, angiotensin types 1 and 2 (AT 1 and AT 2 ) and mas receptor mRNAs by semiquantitative reverse transcriptase (RT) real-time polymerase chain reaction (PCR). ACE2 activity was determined as the rate of Ang II conversion into Ang-(1-7). Results. Comparable blood pressure reductions were obtained in rats medicated with either lisinopril or losartan, whereas both drugs produced a greater decrease in arterial pressure. Polyuria was recorded in all three forms of treatment associated with reduced osmolality but no changes in creatinine excretion. Lisinopril augmented plasma levels and urinary excretion rates of Ang I and Ang-(1-7), while plasma Ang II was reduced with no effect on urinary Ang II. Losartan produced similar changes in plasma and urinary Ang-(1-7) but increased plasma Ang II without changing urinary Ang II excretion. Combination therapy mimicked the effects obtained with lisinopril on plasma and urinary Ang I and Ang-(1-7) levels. Renal cortex Aogen mRNA increased in rats medicated with either lisinopril or the combination, whereas all three treatments produced a robust increase in renal renin mRNA. In contrast, ACE, ACE2, neprilysin, AT 1 , and mas receptor mRNAs remained unchanged with all three treatments. Renal cortex ACE2 activity was significantly augmented in rats medicated with lisinopril or 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, 2005 and in revised form May 20, 2005 Accepted 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 as primarily reflected by the increased ACE2 activity measured in renal membranes from the kidney of rats given either lisinopril or losartan. The data further indicate that increased levels of Ang-(1-7) in the urine of animals after ACE inhibition or AT 1 receptor blockade reflect an intrarenal formation of the heptapeptide.
As the newest homologue of angiotensin-converting enzyme (ACE), ACE2 has emerged as a novel site for therapeutic intervention within the cascade of the reninangiotensin system (RAS) since the enzyme has a substrate 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] showed that ACE2 functions as a component of the counterregulatory mechanism through which Ang-(1-7) opposes the effects of Ang II on blood pressure and cellular growth, as well as exerting important antihypertensive actions. The importance of ACE2 in cardiovascular regulation became apparent in studies showing that ACE2 mRNA and protein were reduced in the kidney of experimental models of hypertension while in ACE2 knockout mice reduced left ventricular contractility and left ventricular dilation could be rescued by crossing these animals with the ACE knockout mouse [4]. The abundance of ACE2 in the heart led us to investigate its role in cardiac function, particularly since previous experiments showed that Ang-(1-7), solely localized in rat cardiac myocytes [8], exerted important cardioprotective actions in terms of reversal of cardiac hypertrophy and cardiac dysfunction [7, 9]. Furthermore, both inhibition of Ang II synthesis as well as blockade of Ang II receptors increased cardiac ACE2 mRNA as well as ACE2 activity, determined as the rate of Ang II conversion into Ang-(1-7) in isolated cardiac membranes from the same animals [6]. To assess the effect of interruption of the pressor and proliferative arm of the RAS on the genes, proteins, and 2189
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receptors constituting the axis that opposes the activity of Ang II, we measured the effects of chronic administration of lisinopril, losartan, or both agents on the components of the RAS within the kidney of Lewis normotensive rats. The resultant effect of these treatments on the enzymes that participate in the renal formation of Ang-(1-7) from either Ang I (neprilysin) or Ang II (ACE2) are a focus of these studies since previous experiments indicated that ACE inhibition and angiotensin type 1 (AT 1 ) receptor blockade augmented cardiac and vascular ACE2 gene expression or activity [6, 7, 10]. Data on the effect of these treatments on cardiac ACE2 mRNA and ACE2 activity are reported elsewhere [6]. METHODS Animals Experiments were conducted in male Lewis rats (270 to 280 g) (Charles River Laboratories, Wilmington, MA, USA) housed in individual cages (12-hour light/dark cycle) with ad libitum access to rat chow and tap water using procedures approved by our Institutional Animal Care and Use Committee. Experimental protocol Thirty-six normotensive rats (age range 8 to 10 weeks) were randomly assigned to drink either tap water (vehicle) (N = 12) or tap-water to which lisinopril (10 mg/ kg/day) (N = 8), losartan (10 mg/kg/day) (N = 8), or both drugs [same doses (N = 8)] was added to their drinking water for 12 consecutive days. Rats were housed individually in metabolic cages for 24-hour collection of urine both before and throughout the study. During the treatment period, tail-cuff systolic blood pressure was determined for 3 days before and three times weekly after initiation of the treatments. Two weeks after completion of the treatment regimens, rats were anesthetized with halothane (1.5%) (Halocarbon Laboratories, River Edge, NJ, USA) for direct measurements of arterial pressure and heart rate using a catheter-tip pressure transducer inserted into a carotid artery (Model SPR-671) (Millar Instrument Co., Houston, TX, USA). A 5 mL sample of arterial blood was obtained for biochemical measurements. Cardiopulmonary excision was done in deeply anesthetized rats and the kidneys were removed for semiquantitative determinations of RAS gene expression. Biochemistry Blood was collected into chilled tubes containing a mixture of peptidase inhibitors: 25 mmol/L ethylenediaminetetraacetic acid (EDTA), 0.44 mmol/L 1,20-orthophenanthrolene monohydrate (Sigma Chemical Co., St. Louis, MO, USA), 1 mmol/L sodium para-chloromercuribenzoate, 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 centrifugation at 3000 rpm for 20 minutes, and aliquots of either plasma or serum were stored at −80◦ C until use. Frozen tissues were rapidly weighed and homogenized for later assay as described elsewhere [7]. Plasma and urinary concentrations of angiotensin peptides were measured by radioimmunoassay as documented elsewhere [7, 11]. Serum ACE activity was determined by incubation of the sample with radiolabeled 3 H-Hip-Gly-Gly (pH 8.0) for 1 hour at 37◦ C. The intra-assay variation was 3.9% and the interassay variation averaged 5.9%. ACE2 activity was quantified as the rate of exogenous 125 I-Ang II conversion to 125 I-Ang-(1-7) in kidney membranes isolated from the renal cortex using procedures adapted from our previous study in rat heart [6]. The tissue was weighed and homogenized in 10 mmol/L Hepes, pH 7.4, 120 mmol/L NaCl, and 10 lmol/L ZnCl 2 . The homogenate was subject to centrifugation at 30,000g for 20 minutes. The supernatant was removed, the membranes were resuspended in the above buffer containing 0.5% Triton X-100, and incubated overnight on ice at 4◦ C. Following centrifugation, the soluble portion was incubated with 125 I-Ang II (2 × 106 cpm) (2200 Curies/mmol) in the Hepes buffer containing 10 lmol/L of amastatin, bestatin, SCH39370 (neprilysin inhibitor), chymostatin, lisinopril, benzyl succinate, and Z-prolyl prolinal (ZPP) at 37◦ C from 10 to 120 minutes and the reaction terminated by addition of 1% phosphoric acid. 125 I-Ang II was iodinated by the Chloramine T method and purified by high-performance liquid chromatography (HPLC). The samples were subjected to centrifugation and the supernatants were filtered 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 Waters 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% for 15 minutes at a flow rate of 0.35 mL/min at ambient temperature. The solvent system consisted of 0.1% phosphoric acid (mobile phase A) and 80% acetonitrile/0.1% phosphoric acid (mobile phase B) [12]. The eluted radioactive peaks were identified using an in-line c detector (BioScan, Washington, DC, USA) and the data were acquired and analyzed with Shimadzu version 7.2.1 acquisition software. ACE2 activity was expressed as the amount of Ang-(1-7) generated per minute per mg protein that was inhibited by addition of 10 lmol/L of the ACE2 inhibitor, MLN-4760 [13]. RNA isolation and reverse transcriptase (RT)/real-time polymerase chain reaction (PCR) RNA was isolated from kidney tissue, using the Trizol reagent (Gibco Invitrogen, Carlsbad, CA, USA), as
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P < 0.05 110 100 90 *
80 *
mm Hg
70
*
60 50 40 30 20 10 0 Before
After
Vehicle
Before
After
Lisinopril
Before
After
Losartan
directed by the manufacturer. The RNA concentration and integrity were assessed using an Agilent 2100 Bioanalyzer with an RNA 6000 Nano LabChip (Agilent Technologies, Palo Alto, CA, USA). Approximately 1 lg of total RNA was reverse transcribed using AMV reverse transcriptase in a 20 lL reaction mixture containing deoxyribonucleotides, random hexamers and RNase inhibitor in RT buffer. Heating the RT reaction product at 95◦ C terminated the reaction. For real-time PCR, 2 lL of the resultant cDNA was added to TaqMan(TM ) Universal PCR Master Mix (Applied Biosystems, Foster City, CA, USA) with the appropriate gene-specific primer/probe set and amplification was performed on an ABI 7000 Sequence Detection System. The primer/probe sets were purchased from Applied Biosystems with the exception of ACE2 (forward primer 5 -CCCAGAGAACAGTGGACCAAAA-3 ; reverse primer 5 -GCTCCACCACACCAACGAT-3 ; and probe 5 -FAM-CTCCCGCTTCATCTCC-NFQ-3 ) and mas (forward primer 5 -TTCATAGCCATCCTCAGCTTCT TG-3 ; reverse primer 5 -GTTCTTCCGTATCTTCACC ACCAA-3 ; and probe 5 -FAM-TTCACTCCGCTCA TGTTAG-NFQ-3 ). The mixtures were heated at 50◦ C for 2 minutes, at 95◦ C for 10 minutes followed by 40 cycles at 95◦ C for 15 seconds and 60◦ C for 1 minute. All reactions were performed in triplicate and 18S ribosomal RNA, amplified using the TaqMan Ribosomal RNA Control Kit (Applied Biosystems), served as an internal control. The results were quantified as Ct values, where Ct is defined as the threshold cycle of PCR at which the amplified product is first detected, and expressed as relative gene expression (the ratio of target/18S rRNA control).
Before
After
Combination
Fig. 1. Group averages of tail-cuff systolic blood pressure determinations in conscious Lewis normotensive rats before and after the completion of 2 weeks of oral administration of vehicle, lisinopril, losartan, or both drugs in combination. Values are means ± SE. ∗ P < 0.05 compared to their respective value before initiation of the medications. P value within graph denotes statistical difference between rats medicated with losartan or combination therapy.
Statistical analysis All values are expressed as mean ± SEM. One-way analysis of variance (ANOVA) followed by either the Bonferroni or the two-tailed Student t tests was used for comparing the differences at a P value <0.05.
RESULTS Twelve-day administration of the drugs either alone or in combination produced significant decreases in systolic blood pressure, more pronounced in rats medicated with the combination of lisinopril-losartan (Fig. 1). Direct measurements of mean arterial pressure at the end of the study in the anesthetized rats confirmed the observations obtained in conscious animals. None of the treatments had an effect on heart rate. As demonstrated elsewhere [6, 14, 15], rats given lisinopril increased plasma Ang I and Ang-(1-7) levels by an average of 1486% and 170%, respectively, whereas plasma Ang II and ACE were reduced by an average of 56% and 93%, respectively, compared to vehicle-treated rats (Table 1). Losartan treatment was associated with increases in plasma Ang I comparable to those found in lisinopril-treated rats. In addition, losartan stimulated large increases in plasma Ang II but not changes in plasma Ang-(1-7) and ACE activity (Table 1). Combination therapy obtunded but did not eliminate the significant rise in plasma Ang I levels, reduced Ang II levels to values below those found in vehicle but not lisinopril-treated rats, and increased plasma Ang-(1-7) concentrations above those found in rats given losartan alone (Table 1). ACE activity in 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 P value < Angiotensin II fmol/mL P value < Angiotensin-(1-7) fmol/mL P value< Angiotensin-converting enzyme activity nmol/min/mL P value<
94 ± 26
1397 ± 171 0.001 24 ± 6 0.05 78 ± 11 0.05 3.8 ± 0.1
909 ± 89a 0.001 332 ± 50a 0.001 51 ± 9 NS 49.9 ± 4.0a
433 ± 104a,b 0.01 22 ± 6b 0.05 89 ± 7b 0.01 4.2 ± 0.3b
0.001
NS
0.001
54 ± 9 46 ± 10 57.9 ± 4.8
Data are means ± SE. P value denotes statistical difference versus rats medicated with the vehicle. a < P 0.05 compared to lisinopril; b P < 0.05 compared to losartan.
Table 2. Effects of drug treatments on renal function variables Variables Water intake mL P value < Urinary output mL P value < Urine osmolality mOsm/mL P value< Sodium excretion mEq/24 hours P value< Potassium excretion mEq/24 hours P value < Creatinine excretion mg/24 hours P value <
Vehicle
Lisinopril
Losartan
Combination
26.70 ± 1.06
35.66 ± 1.48 0.001 21.33 ± 0.94 0.001 928 ± 158 0.001 1.99 ± 0.69 NS 3.28 ± 0.45 NS 9.93 ± 1.77 NS
26.31 ± NS 12.96 ± 0.65a 0.01 1850 ± 110a 0.01 1.70 ± 0.07 NS 4.32 ± 0.19 NS 12.65 ± 0.80 NS
40.26 ± 2.01b 0.001 15.26 ± 1.16b 0.001 1424 ± 167b 0.001 1.63 ± 0.20 NS 3.21 ± 0.32 NS 10.44 ± 0.97 NS
9.22 ± 0.64 2431 ± 140 1.90 ± 0.21 3.68 ± 0.28 11.56 ± 1.17
1.13a
Data are means ± SE. P value denotes statistical difference versus rats medicated with the vehicle. a < P 0.05 compared to lisinopril; b P < 0.05 compared to losartan.
amount not different than that found in rats given lisinopril (Table 1). Table 2 shows that polydipsia associated with hyposmolar polyuria and no differences in either urinary Na+ and K+ excretion accompanied the administration of lisinopril. Although water intake was not changed in rats given losartan, these animals displayed increased urinary output and reduced urinary osmolality. Urinary excretion of Na+ and K+ in losartan-treated rats was not changed when compared to vehicle-treated controls (Table 2). Combination therapy mimicked the effect observed in rats medicated with lisinopril. None of the treatments caused significant changes in urinary creatinine excretion (Table 2). Figure 2 illustrates the effect of the various treatments on urinary excretion rates of Ang I, Ang II, and Ang(1-7). The patterns of urinary excretion of angiotensin peptides in Lewis rats do not differ from those previously reported for normotensive Sprague-Dawley rats [16] or spontaneously hypertensive rats given a combined ACE and neprilysin inhibitor [11]. Administration of an ACE inhibitor or the Ang II receptor antagonist was associated with augmented excretion rates of Ang I and Ang-(1-7) but not Ang II. In addition, the average values of urinary Ang-(1-7) excretion determined on the last day of the treatment 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 observed with the various protocols were associated only in part with changes in the expression of renal RAS genes (Table 3). All three treatments increased kidney cortex renin mRNA while angiotensinogen mRNA increased only in rats medicated with lisinopril or combination therapy. In contrast, ACE, neprilysin, ACE2, AT 1 , and mas receptor mRNAs were not changed by the treatments. ACE2 activity increased in the renal cortex of rats medicated with either lisinopril or losartan while ACE2 activity remained unchanged in rats given the combination therapy (Fig. 3). The increased ACE2 activity associated with treatment with either lisinopril or losartan correlated significantly with 24-hour urinary excretion rates 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 of Ang 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 the comparative effect of inhibition of Ang II synthesis versus Ang II receptor blockade, alone or in combination, on
Ferrario et al: Renal angiotensin peptides
Urinary Ang I excretion 2000
fmol/mL/24 h
1500
Vehicle Lisinopril * Losartan Combination
*
** **
**
**
1000
500
W B k. W 1 k. 2
W B k. W 1 k. 2
W B k. W 1 k. 2
W B k. W 1 k. 2
0
Urinary Ang II excretion 2800
fmol/mL/24 h
2400 2000
Vehicle Lisinopril Losartan Combination
1600 1200 800 400
W B k. W 1 k. 2
W B k. W 1 k. 2
W B k. W 1 k. 2
W B k. W 1 k. 2
0
Urinary Ang-(1−7) excretion Vehicle ** Lisinopril * Losartan Combination
*
*
W B k. W 1 k. 2
W B k. W 1 k. 2
** **
W B k. W 1 k. 2
4500 4000 3500 3000 2500 2000 1500 1000 500 0
W B k. W 1 k. 2
fmol/mL/24 h
5000
Fig. 2. Effects of the various treatments on urinary excretion rates of angiotensin I (Ang I), angiotensin II (Ang II), and angiotensin-(1-7) [Ang-(1-7)]. B is baseline. Values are means ± SE. ∗ P < 0.05 compared to the corresponding baseline values; ∗∗ P < 0.05 compared to animals medicated with lisinopril.
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renal excretory variables and expression of the genes encoding the active peptides and receptors of the RAS in the kidney of normotensive Lewis rats. The primary question we addressed in these experiments was whether administration of either lisinopril or losartan would modulate the renal expression of RAS genes, especially those genes accounting for the production (neprilysin and ACE2) and metabolism (ACE) of the vasodilator peptide Ang-(1-7) [17–19]. In agreement with previous studies [6, 16, 20], these medications effectively reduced the contribution of Ang II to blood pressure regulation as reflected by the decrease in arterial pressure and associated changes in both plasma and urinary excretion of Ang I, Ang II, and Ang-(1-7). Hemodynamic and renal excretion changes in Lewis rats given the vehicle, lisinopril, losartan, or their combination are similar to those reported in both SpragueDawley rats [16] and essential hypertensive subjects medicated with either captopril [21] or the dual vasopeptidase inhibitor omapatrilat [20]. The antihypertensive effect obtained with each agent alone or in combination was essentially the same, although the blood pressure of rats given combination therapy was reduced further when compared to vehicle but not the rats medicated with lisinopril alone. Urinary excretion rates of Ang I and Ang-(1-7) but not Ang II were augmented by all three treatment modalities, a finding that is consistent with a role of Ang-(1-7) in mediating the polyuria found in the treated animals [11, 16, 20]. Within the renal cortex, gene expression of renin was markedly increased by all treatments, whereas angiotensinogen mRNA increased only in rats exposed to blockade of ACE with lisinopril or the combination of lisinopril/losartan. Although both monotherapy and combination therapy caused marked increases in the urinary excretion of Ang I and Ang-(1-7), ACE, ACE2, and neprilysin mRNAs [genes of the enzyme axis regulating Ang-(1-7) formation and metabolism] did not change compared to vehicle-treated animals. Similarly, the treatments had no effect on the expression of AT 1 and mas receptor mRNAs. On the other hand, ACE2 activity, measured as the rate of generated Ang-(1-7) from Ang II in membranes from the renal cortex was augmented in rats medicated with lisinopril and losartan. A direct correlation of ACE2 activity with urinary Ang I and Ang-(1-7) as well as plasma Ang I and Ang(1-7) in rats medicated with either lisinopril or losartan provided evidence of its role in modulating the formation of Ang-(1-7) from Ang II. On the other hand, these experiments underscore our previous conclusion that the role of ACE2 in the regulation of cardiovascular function is more complex than anticipated. As reported by Gembardt et al [22], the effects of blockade of the RAS on ACE2 protein and activity may be both tissueand species-specific. This interpretation agrees with our
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Table 3. Real-time polymerase chain reaction (PCR) measures of kidney renin-angiotensin system (RAS) genes Kidney mRNAs Renin P value < Angiotensinogen mRNA P value < Angiotensin-converting enzyme P value < Neprilysin P value < Angiotensin-converting enzyme 2 P value < Angiotensin type 1 receptor P value < Angiotensin type 2 receptor mas receptor P value <
Vehicle
Lisinopril
Losartan
Combination
1.04 ± 0.12
30.79 ± 3.92 0.001 1.69 ± 0.17 0.01 1.23 ± 0.12 NS 1.04 ± 0.09 NS 1.10 ± 0.09 NS 1.33 ± 0.09 NS Not detected 1.07 ± 0.06 NS
2.96 ± 0.33a 0.001 1.19 ± 0.15a N.S. 1.02 ± 0.13 N.S. 1.06 ± 0.16 N.S. 1.07 ± 0.16 N.S. 1.47 ± 0.22 N.S. Not detected 1.01 ± 0.11 NS
22.38 ± 2.64b 0.001 1.63 ± 0.08 0.007 1.04 ± 0.15 NS 1.14 ± 0.17 NS 0.99 ± 0.14 NS 1.02 ± 0.13 NS Not detected 0.95 ± 0.14 NS
1.08 ± 0.15 1.09 ± 0.17 1.03 ± 0.11 1.05 ± 0.12 1.03 ± 0.13 Not detected 1.04 ± 0.12
Data are means ± SE of values expressed as relative gene expression units. P value denotes statistical difference versus rats medicated with the vehicle. a < P 0.05 compared to lisinopril; b P < 0.05 compared to losartan.
ACE2 activity [Ang-(1−7), fmol/mg/min]
100
* *
80
60
40
20
0 Vehicle
Lisinopril
Losartan
finding that the combination of lisinopril/losartan abrogated the increase in renal cortex ACE2 activity found in animals medicated with either agent alone even though this combination treatment continued to be associated with increases in plasma and urinary excretion rates of Ang I and Ang-(1-7). Additional evidence for a tissuespecific response of both the ACE2 gene and ACE2 activity is furnished by the finding that cardiac ACE2 activity increased in the hearts of Lewis rats medicated with the combination of lisinopril and losartan [6]. Two distinct biochemical axis-pathways determine the formation of Ang II and Ang-(1-7) from Ang I. Within the first arm of the system ACE cleaves Ang II from Ang I, whereas in most conditions, neprilysin is principally involved in the formation of Ang-(1-7) from Ang I [23]. The demonstration that ACE2 converts Ang II into Ang-(17) connects downstream the two axis of the RAS system [1, 18]. ACE2 is widely localized in cardiovascular tis-
Combination
Fig. 3. Effects of the various treatments on angiotensin-converting enzyme (ACE)2 activity measures determined as the rate of angiotensin-(1-7) [Ang-(1-7)] formation from angiotensin II (Ang II) in renal membranes isolated from the kidney cortex. Values are means ± SE. ∗ P < 0.05 compared to the vehicle group.
sues mainly in the media and endothelium of large and small arterial vessels, cardiac myocytes, and the tubular epithelia of the kidneys [24]. In addition Leydig cells in the rat testis, Leydig and Sertoli cells in the human testis, and the uteroplacental complex showed positive staining for ACE2 [25, 26]. Neoexpression of ACE2 in glomerular and peritubular capillary endothelium was reported in biopsies obtained from patients with nephropathies of various origins and in transplanted kidneys [27]. Tikellis et al [28] first showed that the expression of the ACE2 mRNA was decreased in diabetic renal tubules by approximately 50% and was not influenced by treatment with ramipril. This observation agrees with our findings and those obtained by Lely et al [27] in human subjects. On the other hand, we now show that inhibition of ACE caused a 72% increase in ACE2 activity in the renal cortex. The direct assessment of the functional activity of the ACE2 gene provides a strong argument of a role for
Ferrario et al: Renal angiotensin peptides
this enzyme in modulating intrarenal generation of Ang(1-7). Paralleling the effects of lisinopril on renal ACE2 activity, we also found that blockade of AT 1 receptors is associated with a 43% increase in renal ACE2 activity. Thus, both suppression of Ang II formation or blockade of Ang II binding to AT 1 receptors increased ACE2 activity within the renal cortex. Because the increased ACE2 activity is associated with elevations in both plasma concentrations of Ang-(1-7) and urinary excretion rates of Ang-(1-7), the data further underscore a functional role of this novel ACE homologue in the formation of Ang-(17) from Ang II. The significant correlations among renal cortex ACE2 activity and plasma and urine Ang I and Ang-(1-7) reinforced this interpretation. Additionally, Li et al [29] recently reported that ACE2 is widely localized in the tubules, glomeruli, and endothelial cells of the rat kidney; however, they demonstrate an important role of ACE2 in the formation of Ang-(1-7) from Ang I in isolated proximal tubules. Previous studies using purified preparations of ACE2 have emphasized that while Ang I and Ang II share similar Km values for the enzyme, the catalytic rate is far higher for Ang II than Ang I [1, 18, 30]. In addition, studies of Ang-(1-7) formation from Ang II in human hearts confirmed the involvement of ACE2 in the production of Ang-(1-7) [31, 32]. Because our ACE2 assay directly measured the rate of conversion of Ang II to Ang-(1-7) in isolated membranes of the renal cortex from the treated animals our results clearly implicate ACE2 in the formation of intrarenal Ang-(1-7). Combination therapy abrogated the increased renal ACE2 activity obtained in rats medicated with either agent alone. A differential effect of Ang II blockade was previously reported by us in the heart of Lewis rats after coronary artery ligation [6]. In those experiments, both losartan and the combination of lisinopril and losartan were associated with increased ACE2 activity, while lisinopril therapy reduced cardiac ACE2 activity to values found in vehicle-treated rats [6]. Therefore, in both this and in our previous study [6], the same therapeutic agents given to the same rat strain at the same doses and for the same period of time resulted in differential effects on cardiac and renal ACE2 mRNAs and activity. The differential effects on cardiac and renal ACE2 may not be attributed to hemodynamic factors since the fall in blood pressure obtained by single or combined administration of lisinopril and losartan were not different between the groups within this study or a cross-comparison with the results reported previously [6]. These findings and those reported elsewhere [22] suggest a tissue-specific regulation of the ACE2 gene expression and protein in response to maneuvers that alter either the formation of Ang II or its activity. Uncoupling between gene expression and protein is a known phenomenon as gene expression is regulated at transcription, translation, or by posttranslational modification. Furthermore, Zhong et al [33]
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showed that all-trans retinoic acid up-regulated ACE2 gene expression in the heart and kidney of spontaneously hypertensive rats, a finding that emphasizes a role of transcriptional factors in modulation of ACE2 mRNA. Further studies will be required to determine whether the absence of changes in renal cortical mRNA compared to cardiac mRNA in response to blockade of Ang II implies a differential action of these agents on transcription factors. While our studies shed no light on the signaling mechanisms that are a stimulus for ACE2 gene expression and protein activity, in both astrocytes and myocytes in culture we have preliminary evidence for a role of Ang II in reducing ACE2 mRNA while the addition of Ang-(17) into the medium blocks the inhibitory action of Ang II [Gallagher, Tallant, and Ferrario, unpublished observations]. If the in vitro data can be interpolated to the current findings, we can surmise that the combination therapy producing a marked increase in plasma Ang-(17) and suppressing Ang II formation should prevent stimulation of ACE2 activity. On the other hand, additional factors may be at play since the reduction in ACE2 activity found in animals receiving the combination therapy was not associated with a decrease in urinary excretion rates of Ang I or Ang-(1-7). These data suggest that renal excretion of Ang-(1-7), although reflecting primarily intrarenal formation of the peptide, may be under the control of other Ang-(1-7) forming enzymes, as demonstrated by us previously [11, 20]. The observation that renal neprilysin mRNA was not changed by any of the treatments alone or in combination does not necessarily exclude this Ang-(1-7)–forming enzyme from participating in the increased Ang-(1-7) found in the urine of all treatment groups since neprilysin activity was not determined in these experiments.
CONCLUSION The lessons learned from this study are (1) renal ACE2 activity increases as a result of suppression of Ang II synthesis or activity, a finding that suggests a feed forward mechanism for the components of the RAS axis regulating the production of Ang-(1-7); (2) although our experiments confirmed that ACE2 is not sensitive to blockade of ACE, our data now show that ACE inhibition is associated with increased renal ACE2 activity; (3) the absence of changes in renal AT 1 and mas receptors with the various treatment protocols suggests that the enzymes rather than the receptors affect the regulation of the system in these conditions; and (4) the experiments reported here and those described elsewhere [34], collectively, implicate increased Ang-(1-7) as a factor contributing to the renoprotective actions of blockade of Ang II with either ACE inhibitors or Ang II receptor blockers.
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ACKNOWLEDGMENTS The work described here was supported in part by grants HL-051952, HL-068258, HL-056973, HL-67363, HL-42631, and the American Heart Association, AHA-151521. Unrestricted grants from the Unifi Corporation (Greensboro, NC) and the Farley-Hudson Foundation (Jacksonville, 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 and Vascular Disease Center, Wake Forest University School of Medicine, Winston-Salem, NC 27157. E-mail: [email protected]
REFERENCES 1. RICE GI, THOMAS DA, GRANT PJ, et al: Evaluation of angiotensinconverting enzyme (ACE), its homologue ACE2 and neprilysin in angiotensin peptide metabolism. Biochem J 383:45–51, 2004 2. YAGIL Y, YAGIL C: Hypothesis: ACE2 modulates blood pressure in the mammalian organism. Hypertension 41:871–873, 2003 3. CHAPPELL MC, MODRALL JG, DIZ DI, FERRARIO CM: Novel aspects of the renal renin-angiotensin system: Angiotensin-(1-7), ACE2 and blood pressure regulation. Contrib Nephrol 143:77–89, 2004 4. CRACKOWER MA, SARAO R, OUDIT GY, et al: Angiotensinconverting enzyme 2 is an essential regulator of heart function. Nature 417:822–828, 2002 5. FERRARIO CM, CHAPPELL MC, TALLANT EA, et al: Counterregulatory actions of angiotensin-(1-7). Hypertension 30:535–541, 1997 6. FERRARIO CM, JESSUP JA, CHAPPELL MC, et al: Effect of angiotensin converting enzyme inhibition and angiotensin II receptor blockers on cardiac angiotensin converting enzyme 2. Circulation 111:2605– 2610, 2005 7. ISHIYAMA Y, GALLAGHER PE, AVERILL DB, et al: Upregulation of angiotensin-converting enzyme 2 after myocardial infarction by blockade of angiotensin II receptors. Hypertension 43:970–976, 2004 8. AVERILL DB, ISHIYAMA Y, CHAPPELL MC, FERRARIO CM: Cardiac angiotensin-(1-7) in ischemic cardiomyopathy. Circulation 108:2141–2146, 2003 9. LOOT AE, ROKS AJ, HENNING RH, et al: Angiotensin-(1-7) attenuates the development of heart failure after myocardial infarction in rats. Circulation 105:1548–1550, 2002 10. IGASE M, STRAWN WB, GALLAGHER PE, et al: Angiotensin ii AT1 receptors regulate ACE2 and angiotensin-(1-7) expression in aorta of spontaneously hypertensive rats. Am J Physiol Heart Circ Physiol 289:H1013–H1019, 2005 11. FERRARIO CM, AVERILL DB, BROSNIHAN KB, et al: Vasopeptidase inhibition and Ang-(1-7) in the spontaneously hypertensive rat. Kidney Int 62:1349–1357, 2002 12. ALLRED AJ, DIZ DI, FERRARIO CM, CHAPPELL MC: Pathways for angiotensin-(1-7) metabolism in pulmonary and renal tissues. Am J Physiol 279:F841–F850, 2000 13. DALES NA, GOULD AE, BROWN JA, et al: Substrate-based design of the first class of angiotensin-converting enzyme-related carboxypeptidase (ACE2) inhibitors. J Am Chem Soc 124:11852– 11853, 2002 14. IYER SN, CHAPPELL MC, AVERILL DB, et al: Vasodepressor actions of angiotensin-(1-7) unmasked during combined treatment with lisinopril and losartan. Hypertension 31:699–705, 1998 15. NAKAMURA S, AVERILL DB, CHAPPELL MC, et al: Angiotensin receptors contribute to blood pressure homeostasis in salt-depleted SHR. Am J Physiol Regul Integr Comp Physiol 284:R164–R173, 2003
16. YAMADA K, IYER SN, CHAPPELL MC, et al: Differential response of angiotensin peptides in the urine of hypertensive animals. Regul Pept 80:57–66, 1999 17. CHAPPELL MC, PIRRO NT, SYKES A, FERRARIO CM: Metabolism of angiotensin-(1-7) by angiotensin converting enzyme. Hypertension 31:362–367, 1998 18. VICKERS C, HALES P, KAUSHIK V, et al: Hydrolysis of biological peptides by human angiotensin-converting enzyme-related carboxypeptidase. J Biol Chem 277:14838–14843, 2002 19. WELCHES WR, BROSNIHAN KB, FERRARIO CM: A comparison of the properties, and enzymatic activity of three angiotensin processing enzymes: Angiotensin converting enzyme, prolyl endopeptidase and neutral endopeptidase 24.11. Life Sci 52:1461–1480, 1993 20. FERRARIO CM, SMITH RD, BROSNIHAN KB, et al: Effects of omapatrilat on the renin angiotensin system in salt sensitive hypertension. Am J Hyperten 15:557–564, 2002 21. LUQUE M, MARTIN P, MARTELL N, et al: Effects of captopril related to increased levels of prostacyclin and angiotensin-(1-7) in essential hypertension. J Hypertens 14:799–805, 1996 22. GEMBARDT F, STERNER-KOCK A, IMBODEN H, et al: Organ-specific distribution of ACE2 mRNA and correlating peptidase activity in rodents. Peptides 26:1270–1277, 2005 23. FERRARIO CM, IYER SN: Angiotensin-(1-7): A bioactive fragment of the renin-angiotensin system. Regul Pept 78:13–18, 1998 24. DANILCZYK U, ERIKSSON U, OUDIT GY, PENNINGER JM: Physiological roles of angiotensin-converting enzyme 2. Cell Mol Life Sci 61:2714– 2719, 2004 25. BROSNIHAN KB, NEVES LA, JOYNER J, et al: Enhanced renal immunocytochemical expression of ANG-(1–7) and ACE2 during pregnancy. Hypertension 42:749–753, 2003 26. DOUGLAS GC, O’BRYAN MK, HEDGER MP, et al: The novel angiotensin-converting enzyme (ACE) homolog, ACE2, is selectively expressed by adult Leydig cells of the testis. Endocrinology 145:4703–4711, 2004 27. LELY AT, HAMMING I, VAN GH, NAVIS GJ: Renal ACE2 expression in human kidney disease. J Pathol 204:587–593, 2004 28. TIKELLIS C, JOHNSTON CI, FORBES JM, et al: Characterization of renal angiotensin-converting enzyme 2 in diabetic nephropathy. Hypertension 41:392–397, 2003 29. LI N, ZIMPELMANN J, CHENG K, et al: The role of angiotensin converting enzyme 2 in the generation of angiotensin 1-7 by rat proximal tubules. Am J Physiol Renal Physiol 288:F353–F362, 2005 30. TURNER AJ: Exploring the structure and function of zinc metallopeptidases: Old enzymes and new discoveries. Biochem Soc Trans 31:723–727, 2003 31. ZISMAN LS, KELLER RS, WEAVER B, et al: Increased angiotensin(1-7)-forming activity in failing human heart ventricles: Evidence for upregulation of the angiotensin-converting enzyme Homologue ACE2. Circulation 108:1707–1712, 2003 32. ZISMAN LS, MEIXELL GE, BRISTOW MR, CANVER CC: Angiotensin(1-7) formation in the intact human heart: In vivo dependence on angiotensin II as substrate. Circulation 108:1679–1681, 2003 33. ZHONG JC, HUANG DY, YANG YM, et al: Upregulation of angiotensin-converting enzyme 2 by all-trans retinoic acid in spontaneously hypertensive rats. Hypertension 44:907–912, 2004 34. FERRARIO CM, AVERILL DB, BROSNIHAN KB, et al: Angiotensin(1-7): Its contributions to arterial pressure control mechanisms, in Handbook of Experimental Pharmacology:Angiotensin (Vol 1), edited byUnger T, Scholkens BA, Heidelberg, Germany, 2004, pp 478–518
Effects of renin-angiotensin system blockade on renal angiotensin-(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 renal angiotensin-(1-7) forming enzymes and receptors. Background. Angiotensin-converting enzyme (ACE)2, a homologue of ACE, which is insensitive to ACE inhibitors and forms angiotensin-(1-7) [Ang-(1-7)] from angiotensin II (Ang II) with high efficiency was investigated in response to chronic blockade with lisinopril, losartan, and both drugs combined. Methods. Thirty-six adult Lewis rats were assigned to receive these medications in their drinking water for 2 weeks while their arterial pressure, water intake, and urine volume were recorded throughout the study. Measures of renal excretory variables included assessing excretion rates of angiotensin I (Ang I), Ang II and Ang-(1-7) while blood collected at the completion of the study was used for measures of plasma angiotensin concentrations. Samples from renal cortex were assayed for renin, angiotensinogen (Aogen), neprilysin, angiotensin types 1 and 2 (AT 1 and AT 2 ) and mas receptor mRNAs by semiquantitative reverse transcriptase (RT) real-time polymerase chain reaction (PCR). ACE2 activity was determined as the rate of Ang II conversion into Ang-(1-7). Results. Comparable blood pressure reductions were obtained in rats medicated with either lisinopril or losartan, whereas both drugs produced a greater decrease in arterial pressure. Polyuria was recorded in all three forms of treatment associated with reduced osmolality but no changes in creatinine excretion. Lisinopril augmented plasma levels and urinary excretion rates of Ang I and Ang-(1-7), while plasma Ang II was reduced with no effect on urinary Ang II. Losartan produced similar changes in plasma and urinary Ang-(1-7) but increased plasma Ang II without changing urinary Ang II excretion. Combination therapy mimicked the effects obtained with lisinopril on plasma and urinary Ang I and Ang-(1-7) levels. Renal cortex Aogen mRNA increased in rats medicated with either lisinopril or the combination, whereas all three treatments produced a robust increase in renal renin mRNA. In contrast, ACE, ACE2, neprilysin, AT 1 , and mas receptor mRNAs remained unchanged with all three treatments. Renal cortex ACE2 activity was significantly augmented in rats medicated with lisinopril or 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, 2005 and in revised form May 20, 2005 Accepted 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 as primarily reflected by the increased ACE2 activity measured in renal membranes from the kidney of rats given either lisinopril or losartan. The data further indicate that increased levels of Ang-(1-7) in the urine of animals after ACE inhibition or AT 1 receptor blockade reflect an intrarenal formation of the heptapeptide.
As the newest homologue of angiotensin-converting enzyme (ACE), ACE2 has emerged as a novel site for therapeutic intervention within the cascade of the reninangiotensin system (RAS) since the enzyme has a substrate 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] showed that ACE2 functions as a component of the counterregulatory mechanism through which Ang-(1-7) opposes the effects of Ang II on blood pressure and cellular growth, as well as exerting important antihypertensive actions. The importance of ACE2 in cardiovascular regulation became apparent in studies showing that ACE2 mRNA and protein were reduced in the kidney of experimental models of hypertension while in ACE2 knockout mice reduced left ventricular contractility and left ventricular dilation could be rescued by crossing these animals with the ACE knockout mouse [4]. The abundance of ACE2 in the heart led us to investigate its role in cardiac function, particularly since previous experiments showed that Ang-(1-7), solely localized in rat cardiac myocytes [8], exerted important cardioprotective actions in terms of reversal of cardiac hypertrophy and cardiac dysfunction [7, 9]. Furthermore, both inhibition of Ang II synthesis as well as blockade of Ang II receptors increased cardiac ACE2 mRNA as well as ACE2 activity, determined as the rate of Ang II conversion into Ang-(1-7) in isolated cardiac membranes from the same animals [6]. To assess the effect of interruption of the pressor and proliferative arm of the RAS on the genes, proteins, and 2189
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receptors constituting the axis that opposes the activity of Ang II, we measured the effects of chronic administration of lisinopril, losartan, or both agents on the components of the RAS within the kidney of Lewis normotensive rats. The resultant effect of these treatments on the enzymes that participate in the renal formation of Ang-(1-7) from either Ang I (neprilysin) or Ang II (ACE2) are a focus of these studies since previous experiments indicated that ACE inhibition and angiotensin type 1 (AT 1 ) receptor blockade augmented cardiac and vascular ACE2 gene expression or activity [6, 7, 10]. Data on the effect of these treatments on cardiac ACE2 mRNA and ACE2 activity are reported elsewhere [6]. METHODS Animals Experiments were conducted in male Lewis rats (270 to 280 g) (Charles River Laboratories, Wilmington, MA, USA) housed in individual cages (12-hour light/dark cycle) with ad libitum access to rat chow and tap water using procedures approved by our Institutional Animal Care and Use Committee. Experimental protocol Thirty-six normotensive rats (age range 8 to 10 weeks) were randomly assigned to drink either tap water (vehicle) (N = 12) or tap-water to which lisinopril (10 mg/ kg/day) (N = 8), losartan (10 mg/kg/day) (N = 8), or both drugs [same doses (N = 8)] was added to their drinking water for 12 consecutive days. Rats were housed individually in metabolic cages for 24-hour collection of urine both before and throughout the study. During the treatment period, tail-cuff systolic blood pressure was determined for 3 days before and three times weekly after initiation of the treatments. Two weeks after completion of the treatment regimens, rats were anesthetized with halothane (1.5%) (Halocarbon Laboratories, River Edge, NJ, USA) for direct measurements of arterial pressure and heart rate using a catheter-tip pressure transducer inserted into a carotid artery (Model SPR-671) (Millar Instrument Co., Houston, TX, USA). A 5 mL sample of arterial blood was obtained for biochemical measurements. Cardiopulmonary excision was done in deeply anesthetized rats and the kidneys were removed for semiquantitative determinations of RAS gene expression. Biochemistry Blood was collected into chilled tubes containing a mixture of peptidase inhibitors: 25 mmol/L ethylenediaminetetraacetic acid (EDTA), 0.44 mmol/L 1,20-orthophenanthrolene monohydrate (Sigma Chemical Co., St. Louis, MO, USA), 1 mmol/L sodium para-chloromercuribenzoate, 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 centrifugation at 3000 rpm for 20 minutes, and aliquots of either plasma or serum were stored at −80◦ C until use. Frozen tissues were rapidly weighed and homogenized for later assay as described elsewhere [7]. Plasma and urinary concentrations of angiotensin peptides were measured by radioimmunoassay as documented elsewhere [7, 11]. Serum ACE activity was determined by incubation of the sample with radiolabeled 3 H-Hip-Gly-Gly (pH 8.0) for 1 hour at 37◦ C. The intra-assay variation was 3.9% and the interassay variation averaged 5.9%. ACE2 activity was quantified as the rate of exogenous 125 I-Ang II conversion to 125 I-Ang-(1-7) in kidney membranes isolated from the renal cortex using procedures adapted from our previous study in rat heart [6]. The tissue was weighed and homogenized in 10 mmol/L Hepes, pH 7.4, 120 mmol/L NaCl, and 10 lmol/L ZnCl 2 . The homogenate was subject to centrifugation at 30,000g for 20 minutes. The supernatant was removed, the membranes were resuspended in the above buffer containing 0.5% Triton X-100, and incubated overnight on ice at 4◦ C. Following centrifugation, the soluble portion was incubated with 125 I-Ang II (2 × 106 cpm) (2200 Curies/mmol) in the Hepes buffer containing 10 lmol/L of amastatin, bestatin, SCH39370 (neprilysin inhibitor), chymostatin, lisinopril, benzyl succinate, and Z-prolyl prolinal (ZPP) at 37◦ C from 10 to 120 minutes and the reaction terminated by addition of 1% phosphoric acid. 125 I-Ang II was iodinated by the Chloramine T method and purified by high-performance liquid chromatography (HPLC). The samples were subjected to centrifugation and the supernatants were filtered 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 Waters 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% for 15 minutes at a flow rate of 0.35 mL/min at ambient temperature. The solvent system consisted of 0.1% phosphoric acid (mobile phase A) and 80% acetonitrile/0.1% phosphoric acid (mobile phase B) [12]. The eluted radioactive peaks were identified using an in-line c detector (BioScan, Washington, DC, USA) and the data were acquired and analyzed with Shimadzu version 7.2.1 acquisition software. ACE2 activity was expressed as the amount of Ang-(1-7) generated per minute per mg protein that was inhibited by addition of 10 lmol/L of the ACE2 inhibitor, MLN-4760 [13]. RNA isolation and reverse transcriptase (RT)/real-time polymerase chain reaction (PCR) RNA was isolated from kidney tissue, using the Trizol reagent (Gibco Invitrogen, Carlsbad, CA, USA), as
Ferrario et al: Renal angiotensin peptides
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P < 0.05 110 100 90 *
80 *
mm Hg
70
*
60 50 40 30 20 10 0 Before
After
Vehicle
Before
After
Lisinopril
Before
After
Losartan
directed by the manufacturer. The RNA concentration and integrity were assessed using an Agilent 2100 Bioanalyzer with an RNA 6000 Nano LabChip (Agilent Technologies, Palo Alto, CA, USA). Approximately 1 lg of total RNA was reverse transcribed using AMV reverse transcriptase in a 20 lL reaction mixture containing deoxyribonucleotides, random hexamers and RNase inhibitor in RT buffer. Heating the RT reaction product at 95◦ C terminated the reaction. For real-time PCR, 2 lL of the resultant cDNA was added to TaqMan(TM ) Universal PCR Master Mix (Applied Biosystems, Foster City, CA, USA) with the appropriate gene-specific primer/probe set and amplification was performed on an ABI 7000 Sequence Detection System. The primer/probe sets were purchased from Applied Biosystems with the exception of ACE2 (forward primer 5 -CCCAGAGAACAGTGGACCAAAA-3 ; reverse primer 5 -GCTCCACCACACCAACGAT-3 ; and probe 5 -FAM-CTCCCGCTTCATCTCC-NFQ-3 ) and mas (forward primer 5 -TTCATAGCCATCCTCAGCTTCT TG-3 ; reverse primer 5 -GTTCTTCCGTATCTTCACC ACCAA-3 ; and probe 5 -FAM-TTCACTCCGCTCA TGTTAG-NFQ-3 ). The mixtures were heated at 50◦ C for 2 minutes, at 95◦ C for 10 minutes followed by 40 cycles at 95◦ C for 15 seconds and 60◦ C for 1 minute. All reactions were performed in triplicate and 18S ribosomal RNA, amplified using the TaqMan Ribosomal RNA Control Kit (Applied Biosystems), served as an internal control. The results were quantified as Ct values, where Ct is defined as the threshold cycle of PCR at which the amplified product is first detected, and expressed as relative gene expression (the ratio of target/18S rRNA control).
Before
After
Combination
Fig. 1. Group averages of tail-cuff systolic blood pressure determinations in conscious Lewis normotensive rats before and after the completion of 2 weeks of oral administration of vehicle, lisinopril, losartan, or both drugs in combination. Values are means ± SE. ∗ P < 0.05 compared to their respective value before initiation of the medications. P value within graph denotes statistical difference between rats medicated with losartan or combination therapy.
Statistical analysis All values are expressed as mean ± SEM. One-way analysis of variance (ANOVA) followed by either the Bonferroni or the two-tailed Student t tests was used for comparing the differences at a P value <0.05.
RESULTS Twelve-day administration of the drugs either alone or in combination produced significant decreases in systolic blood pressure, more pronounced in rats medicated with the combination of lisinopril-losartan (Fig. 1). Direct measurements of mean arterial pressure at the end of the study in the anesthetized rats confirmed the observations obtained in conscious animals. None of the treatments had an effect on heart rate. As demonstrated elsewhere [6, 14, 15], rats given lisinopril increased plasma Ang I and Ang-(1-7) levels by an average of 1486% and 170%, respectively, whereas plasma Ang II and ACE were reduced by an average of 56% and 93%, respectively, compared to vehicle-treated rats (Table 1). Losartan treatment was associated with increases in plasma Ang I comparable to those found in lisinopril-treated rats. In addition, losartan stimulated large increases in plasma Ang II but not changes in plasma Ang-(1-7) and ACE activity (Table 1). Combination therapy obtunded but did not eliminate the significant rise in plasma Ang I levels, reduced Ang II levels to values below those found in vehicle but not lisinopril-treated rats, and increased plasma Ang-(1-7) concentrations above those found in rats given losartan alone (Table 1). ACE activity in 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 P value < Angiotensin II fmol/mL P value < Angiotensin-(1-7) fmol/mL P value< Angiotensin-converting enzyme activity nmol/min/mL P value<
94 ± 26
1397 ± 171 0.001 24 ± 6 0.05 78 ± 11 0.05 3.8 ± 0.1
909 ± 89a 0.001 332 ± 50a 0.001 51 ± 9 NS 49.9 ± 4.0a
433 ± 104a,b 0.01 22 ± 6b 0.05 89 ± 7b 0.01 4.2 ± 0.3b
0.001
NS
0.001
54 ± 9 46 ± 10 57.9 ± 4.8
Data are means ± SE. P value denotes statistical difference versus rats medicated with the vehicle. a < P 0.05 compared to lisinopril; b P < 0.05 compared to losartan.
Table 2. Effects of drug treatments on renal function variables Variables Water intake mL P value < Urinary output mL P value < Urine osmolality mOsm/mL P value< Sodium excretion mEq/24 hours P value< Potassium excretion mEq/24 hours P value < Creatinine excretion mg/24 hours P value <
Vehicle
Lisinopril
Losartan
Combination
26.70 ± 1.06
35.66 ± 1.48 0.001 21.33 ± 0.94 0.001 928 ± 158 0.001 1.99 ± 0.69 NS 3.28 ± 0.45 NS 9.93 ± 1.77 NS
26.31 ± NS 12.96 ± 0.65a 0.01 1850 ± 110a 0.01 1.70 ± 0.07 NS 4.32 ± 0.19 NS 12.65 ± 0.80 NS
40.26 ± 2.01b 0.001 15.26 ± 1.16b 0.001 1424 ± 167b 0.001 1.63 ± 0.20 NS 3.21 ± 0.32 NS 10.44 ± 0.97 NS
9.22 ± 0.64 2431 ± 140 1.90 ± 0.21 3.68 ± 0.28 11.56 ± 1.17
1.13a
Data are means ± SE. P value denotes statistical difference versus rats medicated with the vehicle. a < P 0.05 compared to lisinopril; b P < 0.05 compared to losartan.
amount not different than that found in rats given lisinopril (Table 1). Table 2 shows that polydipsia associated with hyposmolar polyuria and no differences in either urinary Na+ and K+ excretion accompanied the administration of lisinopril. Although water intake was not changed in rats given losartan, these animals displayed increased urinary output and reduced urinary osmolality. Urinary excretion of Na+ and K+ in losartan-treated rats was not changed when compared to vehicle-treated controls (Table 2). Combination therapy mimicked the effect observed in rats medicated with lisinopril. None of the treatments caused significant changes in urinary creatinine excretion (Table 2). Figure 2 illustrates the effect of the various treatments on urinary excretion rates of Ang I, Ang II, and Ang(1-7). The patterns of urinary excretion of angiotensin peptides in Lewis rats do not differ from those previously reported for normotensive Sprague-Dawley rats [16] or spontaneously hypertensive rats given a combined ACE and neprilysin inhibitor [11]. Administration of an ACE inhibitor or the Ang II receptor antagonist was associated with augmented excretion rates of Ang I and Ang-(1-7) but not Ang II. In addition, the average values of urinary Ang-(1-7) excretion determined on the last day of the treatment 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 observed with the various protocols were associated only in part with changes in the expression of renal RAS genes (Table 3). All three treatments increased kidney cortex renin mRNA while angiotensinogen mRNA increased only in rats medicated with lisinopril or combination therapy. In contrast, ACE, neprilysin, ACE2, AT 1 , and mas receptor mRNAs were not changed by the treatments. ACE2 activity increased in the renal cortex of rats medicated with either lisinopril or losartan while ACE2 activity remained unchanged in rats given the combination therapy (Fig. 3). The increased ACE2 activity associated with treatment with either lisinopril or losartan correlated significantly with 24-hour urinary excretion rates 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 of Ang 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 the comparative effect of inhibition of Ang II synthesis versus Ang II receptor blockade, alone or in combination, on
Ferrario et al: Renal angiotensin peptides
Urinary Ang I excretion 2000
fmol/mL/24 h
1500
Vehicle Lisinopril * Losartan Combination
*
** **
**
**
1000
500
W B k. W 1 k. 2
W B k. W 1 k. 2
W B k. W 1 k. 2
W B k. W 1 k. 2
0
Urinary Ang II excretion 2800
fmol/mL/24 h
2400 2000
Vehicle Lisinopril Losartan Combination
1600 1200 800 400
W B k. W 1 k. 2
W B k. W 1 k. 2
W B k. W 1 k. 2
W B k. W 1 k. 2
0
Urinary Ang-(1−7) excretion Vehicle ** Lisinopril * Losartan Combination
*
*
W B k. W 1 k. 2
W B k. W 1 k. 2
** **
W B k. W 1 k. 2
4500 4000 3500 3000 2500 2000 1500 1000 500 0
W B k. W 1 k. 2
fmol/mL/24 h
5000
Fig. 2. Effects of the various treatments on urinary excretion rates of angiotensin I (Ang I), angiotensin II (Ang II), and angiotensin-(1-7) [Ang-(1-7)]. B is baseline. Values are means ± SE. ∗ P < 0.05 compared to the corresponding baseline values; ∗∗ P < 0.05 compared to animals medicated with lisinopril.
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renal excretory variables and expression of the genes encoding the active peptides and receptors of the RAS in the kidney of normotensive Lewis rats. The primary question we addressed in these experiments was whether administration of either lisinopril or losartan would modulate the renal expression of RAS genes, especially those genes accounting for the production (neprilysin and ACE2) and metabolism (ACE) of the vasodilator peptide Ang-(1-7) [17–19]. In agreement with previous studies [6, 16, 20], these medications effectively reduced the contribution of Ang II to blood pressure regulation as reflected by the decrease in arterial pressure and associated changes in both plasma and urinary excretion of Ang I, Ang II, and Ang-(1-7). Hemodynamic and renal excretion changes in Lewis rats given the vehicle, lisinopril, losartan, or their combination are similar to those reported in both SpragueDawley rats [16] and essential hypertensive subjects medicated with either captopril [21] or the dual vasopeptidase inhibitor omapatrilat [20]. The antihypertensive effect obtained with each agent alone or in combination was essentially the same, although the blood pressure of rats given combination therapy was reduced further when compared to vehicle but not the rats medicated with lisinopril alone. Urinary excretion rates of Ang I and Ang-(1-7) but not Ang II were augmented by all three treatment modalities, a finding that is consistent with a role of Ang-(1-7) in mediating the polyuria found in the treated animals [11, 16, 20]. Within the renal cortex, gene expression of renin was markedly increased by all treatments, whereas angiotensinogen mRNA increased only in rats exposed to blockade of ACE with lisinopril or the combination of lisinopril/losartan. Although both monotherapy and combination therapy caused marked increases in the urinary excretion of Ang I and Ang-(1-7), ACE, ACE2, and neprilysin mRNAs [genes of the enzyme axis regulating Ang-(1-7) formation and metabolism] did not change compared to vehicle-treated animals. Similarly, the treatments had no effect on the expression of AT 1 and mas receptor mRNAs. On the other hand, ACE2 activity, measured as the rate of generated Ang-(1-7) from Ang II in membranes from the renal cortex was augmented in rats medicated with lisinopril and losartan. A direct correlation of ACE2 activity with urinary Ang I and Ang-(1-7) as well as plasma Ang I and Ang(1-7) in rats medicated with either lisinopril or losartan provided evidence of its role in modulating the formation of Ang-(1-7) from Ang II. On the other hand, these experiments underscore our previous conclusion that the role of ACE2 in the regulation of cardiovascular function is more complex than anticipated. As reported by Gembardt et al [22], the effects of blockade of the RAS on ACE2 protein and activity may be both tissueand species-specific. This interpretation agrees with our
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Table 3. Real-time polymerase chain reaction (PCR) measures of kidney renin-angiotensin system (RAS) genes Kidney mRNAs Renin P value < Angiotensinogen mRNA P value < Angiotensin-converting enzyme P value < Neprilysin P value < Angiotensin-converting enzyme 2 P value < Angiotensin type 1 receptor P value < Angiotensin type 2 receptor mas receptor P value <
Vehicle
Lisinopril
Losartan
Combination
1.04 ± 0.12
30.79 ± 3.92 0.001 1.69 ± 0.17 0.01 1.23 ± 0.12 NS 1.04 ± 0.09 NS 1.10 ± 0.09 NS 1.33 ± 0.09 NS Not detected 1.07 ± 0.06 NS
2.96 ± 0.33a 0.001 1.19 ± 0.15a N.S. 1.02 ± 0.13 N.S. 1.06 ± 0.16 N.S. 1.07 ± 0.16 N.S. 1.47 ± 0.22 N.S. Not detected 1.01 ± 0.11 NS
22.38 ± 2.64b 0.001 1.63 ± 0.08 0.007 1.04 ± 0.15 NS 1.14 ± 0.17 NS 0.99 ± 0.14 NS 1.02 ± 0.13 NS Not detected 0.95 ± 0.14 NS
1.08 ± 0.15 1.09 ± 0.17 1.03 ± 0.11 1.05 ± 0.12 1.03 ± 0.13 Not detected 1.04 ± 0.12
Data are means ± SE of values expressed as relative gene expression units. P value denotes statistical difference versus rats medicated with the vehicle. a < P 0.05 compared to lisinopril; b P < 0.05 compared to losartan.
ACE2 activity [Ang-(1−7), fmol/mg/min]
100
* *
80
60
40
20
0 Vehicle
Lisinopril
Losartan
finding that the combination of lisinopril/losartan abrogated the increase in renal cortex ACE2 activity found in animals medicated with either agent alone even though this combination treatment continued to be associated with increases in plasma and urinary excretion rates of Ang I and Ang-(1-7). Additional evidence for a tissuespecific response of both the ACE2 gene and ACE2 activity is furnished by the finding that cardiac ACE2 activity increased in the hearts of Lewis rats medicated with the combination of lisinopril and losartan [6]. Two distinct biochemical axis-pathways determine the formation of Ang II and Ang-(1-7) from Ang I. Within the first arm of the system ACE cleaves Ang II from Ang I, whereas in most conditions, neprilysin is principally involved in the formation of Ang-(1-7) from Ang I [23]. The demonstration that ACE2 converts Ang II into Ang-(17) connects downstream the two axis of the RAS system [1, 18]. ACE2 is widely localized in cardiovascular tis-
Combination
Fig. 3. Effects of the various treatments on angiotensin-converting enzyme (ACE)2 activity measures determined as the rate of angiotensin-(1-7) [Ang-(1-7)] formation from angiotensin II (Ang II) in renal membranes isolated from the kidney cortex. Values are means ± SE. ∗ P < 0.05 compared to the vehicle group.
sues mainly in the media and endothelium of large and small arterial vessels, cardiac myocytes, and the tubular epithelia of the kidneys [24]. In addition Leydig cells in the rat testis, Leydig and Sertoli cells in the human testis, and the uteroplacental complex showed positive staining for ACE2 [25, 26]. Neoexpression of ACE2 in glomerular and peritubular capillary endothelium was reported in biopsies obtained from patients with nephropathies of various origins and in transplanted kidneys [27]. Tikellis et al [28] first showed that the expression of the ACE2 mRNA was decreased in diabetic renal tubules by approximately 50% and was not influenced by treatment with ramipril. This observation agrees with our findings and those obtained by Lely et al [27] in human subjects. On the other hand, we now show that inhibition of ACE caused a 72% increase in ACE2 activity in the renal cortex. The direct assessment of the functional activity of the ACE2 gene provides a strong argument of a role for
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this enzyme in modulating intrarenal generation of Ang(1-7). Paralleling the effects of lisinopril on renal ACE2 activity, we also found that blockade of AT 1 receptors is associated with a 43% increase in renal ACE2 activity. Thus, both suppression of Ang II formation or blockade of Ang II binding to AT 1 receptors increased ACE2 activity within the renal cortex. Because the increased ACE2 activity is associated with elevations in both plasma concentrations of Ang-(1-7) and urinary excretion rates of Ang-(1-7), the data further underscore a functional role of this novel ACE homologue in the formation of Ang-(17) from Ang II. The significant correlations among renal cortex ACE2 activity and plasma and urine Ang I and Ang-(1-7) reinforced this interpretation. Additionally, Li et al [29] recently reported that ACE2 is widely localized in the tubules, glomeruli, and endothelial cells of the rat kidney; however, they demonstrate an important role of ACE2 in the formation of Ang-(1-7) from Ang I in isolated proximal tubules. Previous studies using purified preparations of ACE2 have emphasized that while Ang I and Ang II share similar Km values for the enzyme, the catalytic rate is far higher for Ang II than Ang I [1, 18, 30]. In addition, studies of Ang-(1-7) formation from Ang II in human hearts confirmed the involvement of ACE2 in the production of Ang-(1-7) [31, 32]. Because our ACE2 assay directly measured the rate of conversion of Ang II to Ang-(1-7) in isolated membranes of the renal cortex from the treated animals our results clearly implicate ACE2 in the formation of intrarenal Ang-(1-7). Combination therapy abrogated the increased renal ACE2 activity obtained in rats medicated with either agent alone. A differential effect of Ang II blockade was previously reported by us in the heart of Lewis rats after coronary artery ligation [6]. In those experiments, both losartan and the combination of lisinopril and losartan were associated with increased ACE2 activity, while lisinopril therapy reduced cardiac ACE2 activity to values found in vehicle-treated rats [6]. Therefore, in both this and in our previous study [6], the same therapeutic agents given to the same rat strain at the same doses and for the same period of time resulted in differential effects on cardiac and renal ACE2 mRNAs and activity. The differential effects on cardiac and renal ACE2 may not be attributed to hemodynamic factors since the fall in blood pressure obtained by single or combined administration of lisinopril and losartan were not different between the groups within this study or a cross-comparison with the results reported previously [6]. These findings and those reported elsewhere [22] suggest a tissue-specific regulation of the ACE2 gene expression and protein in response to maneuvers that alter either the formation of Ang II or its activity. Uncoupling between gene expression and protein is a known phenomenon as gene expression is regulated at transcription, translation, or by posttranslational modification. Furthermore, Zhong et al [33]
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showed that all-trans retinoic acid up-regulated ACE2 gene expression in the heart and kidney of spontaneously hypertensive rats, a finding that emphasizes a role of transcriptional factors in modulation of ACE2 mRNA. Further studies will be required to determine whether the absence of changes in renal cortical mRNA compared to cardiac mRNA in response to blockade of Ang II implies a differential action of these agents on transcription factors. While our studies shed no light on the signaling mechanisms that are a stimulus for ACE2 gene expression and protein activity, in both astrocytes and myocytes in culture we have preliminary evidence for a role of Ang II in reducing ACE2 mRNA while the addition of Ang-(17) into the medium blocks the inhibitory action of Ang II [Gallagher, Tallant, and Ferrario, unpublished observations]. If the in vitro data can be interpolated to the current findings, we can surmise that the combination therapy producing a marked increase in plasma Ang-(17) and suppressing Ang II formation should prevent stimulation of ACE2 activity. On the other hand, additional factors may be at play since the reduction in ACE2 activity found in animals receiving the combination therapy was not associated with a decrease in urinary excretion rates of Ang I or Ang-(1-7). These data suggest that renal excretion of Ang-(1-7), although reflecting primarily intrarenal formation of the peptide, may be under the control of other Ang-(1-7) forming enzymes, as demonstrated by us previously [11, 20]. The observation that renal neprilysin mRNA was not changed by any of the treatments alone or in combination does not necessarily exclude this Ang-(1-7)–forming enzyme from participating in the increased Ang-(1-7) found in the urine of all treatment groups since neprilysin activity was not determined in these experiments.
CONCLUSION The lessons learned from this study are (1) renal ACE2 activity increases as a result of suppression of Ang II synthesis or activity, a finding that suggests a feed forward mechanism for the components of the RAS axis regulating the production of Ang-(1-7); (2) although our experiments confirmed that ACE2 is not sensitive to blockade of ACE, our data now show that ACE inhibition is associated with increased renal ACE2 activity; (3) the absence of changes in renal AT 1 and mas receptors with the various treatment protocols suggests that the enzymes rather than the receptors affect the regulation of the system in these conditions; and (4) the experiments reported here and those described elsewhere [34], collectively, implicate increased Ang-(1-7) as a factor contributing to the renoprotective actions of blockade of Ang II with either ACE inhibitors or Ang II receptor blockers.
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ACKNOWLEDGMENTS The work described here was supported in part by grants HL-051952, HL-068258, HL-056973, HL-67363, HL-42631, and the American Heart Association, AHA-151521. Unrestricted grants from the Unifi Corporation (Greensboro, NC) and the Farley-Hudson Foundation (Jacksonville, 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 and Vascular Disease Center, Wake Forest University School of Medicine, Winston-Salem, NC 27157. E-mail: [email protected]
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