Corresponding author I. Hamming: Department of Pathology and Laboratory Medicine, University Medical Center Groningen, PO Box 30.001, 9700 RB Groningen, The Netherlands. Email: email@example.com
Angiotensin-converting enzyme (ACE) 2 is thought to counterbalance ACE by breakdown of angiotensin (Ang) II and formation of Ang(1–7). Both enzymes are highly expressed in the kidney, but reports on their regulation differ. To enhance our understanding of the regulation of renal ACE and ACE2, we investigated renal ACE and ACE2 expression during conditions of physiological (low-sodium diet) and pharmacological changes (ACE inhibition) in activity of the renin–angiotensin–aldosterone system (RAAS). Healthy rats were treated with vehicle or lisinopril with either a control or a low-sodium diet, and renal ACE2, ACE and plasma angiotensins were studied. During vehicle treatment, low sodium reduced renal ACE mRNA and activity without affecting ACE2 mRNA or activity and plasma Ang(1–7) and Ang II balance. Lisinopril significantly reduced renal ACE activity without affecting renal ACE2 activity. During ACE inhibition, low sodium reduced both ACE and ACE2 mRNA without affecting ACE2 activity or further reducing ACE activity. Measurements of renal neprilysin activity revealed no significant differences between any of the treatment groups. Plasma Ang(1–7) and Ang II balance is positively shifted towards the beneficial vasopeptide Ang(1–7) by the ACE inhibitor lisinopril, especially during a low sodium intake. In conclusion, modulation of the RAAS, by low sodium intake or ACE inhibition, does not affect renal ACE2 despite major variations in renal ACE. Thus, ACE and ACE2 are differentially regulated by low sodium and ACE inhibition. Therefore, we propose that the beneficial effects of ACE inhibitors are predominantly mediated by modulation of ACE and not ACE2. Whether this also applies to renal disease conditions should be investigated in future studies.
The renin–angiotensin–aldosterone system (RAAS) has become increasingly complex since the discovery of angiotensin-converting enzyme (ACE) 2, a homologue of ACE that is assumed to be relevant to blood pressure regulation and cardiac and renal function. Angiotensin-converting enzyme 2 is thought to counterbalance the role of ACE in the RAAS by breakdown of the vasoconstrictor angiotensin (Ang) II and formation of the vasodilator Ang(1–7). It was initially hypothesized that disruption of this delicate balance could result in abnormal blood pressure control (Yagil & Yagil, 2003); ACE2 might protect against increases in blood pressure and, conversely, ACE2 deficiency might lead to hypertension.
Angiotensin-converting enzyme and ACE2 are highly expressed in the kidney. The role of ACE in the development of renal damage is generally accepted (Dzau et al. 2001). Individual differences in renal ACE activity predict the susceptibility for proteinuria-associated renal damage in experimental conditions (Huang et al. 2001; Rook et al. 2005). Furthermore, Ang II is increased in damaged tubules and is suggested to be a possible mediator of renal damage in experimental and human renal disorders (Wolf & Ritz, 2005; Ruiz-Ortega et al. 2006). Blockade of the actions of Ang II by ACE inhibitors or AT1 receptor blockers has been proven to effectively reduce blood pressure and proteinuria (The GISEN Group, 1997; Anderson et al. 1986), thereby providing renoprotection. A disrupted balance between intrarenal ACE and ACE2 with consequent low levels of Ang II and high levels of Ang(1–7) might contribute to the renoprotective mechanisms of ACE inhibitors (Campbell et al. 1991; Ferrario & Iyer, 1998; Ferrario et al. 2005b; Kocks et al. 2005).
Several studies show that ACE2 is involved in the pathogenesis of renal damage, but its precise role is unclear. The reports on regulation of renal ACE2 in experimental models differ from each other and from those in human renal diseases. Some studies speculate that ACE and ACE2 expression may be regulated in parallel or synergistically (Tikellis et al. 2003, 2006; Wakahara et al. 2007). However, the concept of parallel regulation has been questioned by others (Ye et al. 2004; Konoshita et al. 2006; Wysocki et al. 2006). To enhance our knowledge of the regulation of renal ACE and ACE2, we investigated renal ACE and ACE2 expression during physiological (low-sodium diet) and pharmacological modulation (ACE inhibition) of RAAS activity. Therefore, we treated healthy rats with a control or a low-sodium diet in the presence or absence the ACE inhibitor lisinopril. After 3 weeks, renal ACE2, ACE, neprilysin and plasma Ang II and Ang(1–7) were measured.
Healthy male Wistar rats (250–300 g, Harlan, Zeist, The Netherlands) were randomly assigned to receive either vehicle or the ACE inhibitor lisinopril (gift from Merck, Sharp & Dohme Research Laboratories, Rahway, NJ, USA) for a period of 3 weeks. Lisinopril was dissolved in the drinking water at a dose of 75 mg l–1. This dose results in maximal reduction of proteinuria in nephrotic rats (Wapstra et al. 1996). Both vehicle and lisinopril were combined with either a control sodium diet with modestly elevated sodium (2.0% NaCl, Hope Farms, Woerden, The Netherlands; n= 10 per group) or a low-sodium diet (0.05% NaCl, Hope Farms; n= 10 per group). The experiments were in accord with the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH publication no. 85-23, revised 1996) and were approved by the local Committee for Animal Experiments.
Rats were placed in metabolic cages for collection of 24 h urine samples. Creatinine concentrations were determined by a multi-analyzer (SMA−C®; Technicon, Tarrytown, NY, USA). Systolic blood pressure was determined in conscious animals by means of the tail-cuff method (Apollo 179; IITC Life Science, Woodland Hills, CA, USA).
After 3 weeks of treatment, rats were anesthesized with 1.5% isoflurane in N2O–O2. The abdomen was opened via a mid-line incision, aorta was cannulated and blood samples were obtained. Blood samples for determination of angiotensins were drawn into cold, 1 ml tubes containing 1.8 μg K3EDTA with an ACE inhibitor cocktail comprising 0.568 μg phenantroline, 0.053 mg enalaprilat, 0.333 ml ethanol and 1.333 mg neomycin. After centrifugation at 2000 g for 10 minutes at 4°C, the plasma was snap frozen and stored at −80°C until analysis. Kidneys were harvested, snap frozen in liquid nitrogen and stored at −80°C. Hereafter the heart was taken out.
Qualitative real-time reverse transcriptase-polymerase chain reaction (qRT-PCR) for ACE and ACE2
Total renal RNA was isolated using the Nucleospin II RNA isolation kit (Macherey-nagel, GmbH & Co. KG, Düren, Germany) according to the manufacturer's protocol. Complementary DNA was synthesized using Omniscript Reverse Transcription (Qiagen, Gmbh, Hilden, Germany) with random hexamer primer (Roche, Neuilly sur Seine Cedex, France). Gene expression levels were measured by qualitative realtime RT-PCR (Applied Biosystems, Foster City, CA, USA) based on TaqMan methodology. Levels of ACE mRNA were determined using a custom-designed primer–probe set with the primers 5′-CACCG-GCAAGGTCTGCTT-3′, 5′-CTTGGCATAGTTTCGTGA-GGAA-3′ and the probe 6-FAM5′-CAACAAG-ACTGCCACCTGCTGGTCC-3′TAMRA (Applied Biosystems). Levels of ACE2 mRNA were determined using Assay-on-Demand Rn01416295_m1 (Applied Biosystems). Levels of mRNA are expressed relative to those of β2-microglobulin housekeeping gene (B2M Rn00560865_mi; Applied Biosystems).
Biochemistry for angiotensins, ACE and ACE2 activity, and neprilysin activity
Plasma levels of Ang(1–7) and Ang II were measured after SepPak (Waters, Zellik, Belgium) extraction of plasma samples and HPLC seperation (Admiraal et al. 1990). Radioimmunoassays of dried collected fractions were used for quantification of angiotensins using specific antibodies. The antibody for Ang(1–7), raised in New Zealand White rabbits, had cross-reactivities with Ang I and Ang II of 1.1 and 1.7% respectively, and <0.1 and 1.7% with Ang(1–4) and Ang(2–8), the angiotensins eluting nearest to Ang(1–7). Detection limits were 0.5–1.0 fmol per tube. Renal ACE and ACE2 activity were determined as described previously (Hirsch et al. 1991; Riviere et al. 2005). For ACE activity, renal cortex tissue was homogenized in a 50 mm K2PO4 buffer at pH 7.5. Subsequently, 100 μl of the diluted sample was pipetted into a 0.5 m K2PO4 buffer. Then, the substrate (100 μl of 12.5 mm Hip-His-Leu; Sigma-Aldrich, Zwijndrecht, Netherlands), which is cleaved by ACE, was added. This was incubated at 37°C for exactly 15 min. At this amount, the substrate is present in excess and is thus not rate limiting for the reaction. The conversion of the substrate was stopped by adding 1.45 ml of 280 mm sodium hydroxide. Then, 100 μl of 1% phtaldialdehyde, which adheres to the formed bipeptide His−Leu, was added. The amount of tagged His−Leu was determined fluorimetrically at 364 nm excitation and 486 nm emission wavelength. This yields a measure of the amount of His−Leu generated in the sample. In blank samples, sodium hydroxide was added to prevent conversion. The substrate was added after the incubation period. The coefficient of variation was 6% for these measurements of ACE activity using this method.
Measurement of ACE2 activity was performed with Ang II as a substrate in the presence of protease inhibitors (perindoprilat, amastatin and bestatin; 10, 100 and 100 μmol l–1, respectively). The specificity of the reaction was confirmed by control incubation in the presence of a specific ACE2 inhibitor, DX600 (R&D systems, Minneapolis, MN, USA). After incubation, conversion of Ang II to Ang(1–7) was measured quantitatively by reverse-phase HPLC.
For assay of neprilysin activity, tissue was homogenised in Radioimmunoprecipitation assay (RIPA) lysis buffer [50 mm Tris pH 8.0, 150 mm NaCl, 1% Nonidet-P40 (NP-40), 0.5% deoxycholate, 0.02% sodium azide, 0.1% sodium dodecyl sulphate, 4% (v/v) EDTA-free protease inhibitor cocktail (Roche, France), 0.2% (v/v) benzonase nuclease (Novagen, Madison, WI, USA)]. Bicinchoninic assay was used to determine protein concentration of samples (Smith et al. 1985). Neprilysin activity assays were carried out with 1 μg of sample in 100 mm Tris-HCl, pH 8.0 assay buffer. The assays used the synthetic peptide substrate Suc-AAF-AMC (Bachem International, Weil am Rhein, Germany) at a final concentration of 25 μm with 2 microunits (i.u.) of leucine aminopeptidase. The assay was monitored by measuring the increase in fluorescence (excitation at 380 nm; emission at 460 nm) upon substrate hydrolysis using a Wallac Victor2 fluorescence plate reader (Turku, Finland). Specific neprilysin activity was determined using 10 μm thiorphan (a neprilysin-specific inhibitor; Marleau et al. 1990) pre-incubated with the samples for 20 min before the addition of substrate. Initial velocities were determined from the linear rate of fluorescence increase. The reaction product was quantified using standard solutions of Suc-AAF-AMC.
Differences between groups were analysed with a non-parametric Kruskal–Wallis test followed by a Mann–Whitney U test with correction for multiple comparisons. Statistical significance was accepted at P < 0.05.
A considerable difference in sodium status was obtained by feeding the different sodium diets, as reflected by 24 h urinary sodium excretion, which was significantly lower in rats on a low-sodium diet during vehicle treatment as well as during ACE inhibition (Table 1). Three weeks administration of lisinopril reduced blood pressure significantly on both diets, although blood pressure reduction by lisinopril was more pronounced during a low sodium intake (Table 1). Body weight was reduced by the combination of lisinopril and a low-sodium diet.
Table 1. . Characteristics of healthy Wistar rats after 3 weeks of vehicle treatment or ACE inhibition combined with either a control or low dietary sodium intake
Data represent the median (interquartile range) per group (n= 9 per group). *P < 0.05 for low versus control sodium; †P < 0.05 for ACE inhibition versus vehicle as appropriate.
Sodium excretion (mg (24 h)–1)
Body weight (g)
Renal ACE2, ACE and neprilysin
Renal ACE2 and ACE are differentially regulated by a low-sodium diet and by lisinopril as shown in Figs 1 and 2. During vehicle treatment, a low-sodium diet reduced renal ACE mRNA and activity without affecting ACE2 mRNA or activity. Lisinopril significantly reduced renal ACE activity without affecting renal ACE2 activity. Sodium restriction in combination with lisinopril reduced renal ACE and ACE2 mRNA levels. However, renal ACE and ACE2 activity were unaffected by the addition of a low-sodium diet to lisinopril. There was no significant effect of ACE inhibition or salt depletion on neprilysin activity (Fig. 3).
Angiotensin levels of the different study groups are shown in Fig. 4. A low-sodium diet did not affect plasma Ang(1–7), and lisinopril increased plasma Ang(1–7) concentrations approximately three times compared with vehicle. The addition of low-sodium to lisinopril did not change plasma Ang(1–7). Low sodium or lisinopril alone did not affect plasma Ang II, but the combination decreased plasma Ang II significantly. The resulting balance between Ang(1–7) and Ang II, calculated as the ratio of Ang(1–7) to Ang II, was increased by lisinopril. The shift towards Ang(1–7) was most pronounced during a low sodium intake.
The major finding of the present study is that renal ACE2 and ACE are differentially regulated during physiological and pharmacological modulation of RAAS activity, i.e. low sodium intake or ACE inhibition, in healthy rats. Both a low-sodium diet and ACE inhibition significantly reduced renal ACE activity without affecting ACE2 activity. Sodium restriction potentiated the effect of ACE inhibition, as reflected by a larger reduction of blood pressure, which coincided with a reduction of renal ACE2 and ACE at the transcriptional level only. Although ACE2 activity was not affected by the interventions, the plasma balance between Ang(1–7) and Ang II was shifted towards the beneficial vasopeptide Ang(1–7) by ACE inhibition, especially during a low sodium intake.
The RAAS is delicately balanced by a two-arm cascade: the ACE–Ang II–AT1 activation pathway and the ACE2–Ang(1–7)–Mas activation pathway. The latter is implicated in what is considered the beneficial arm, associated with reduction of blood pressure and antifibrotic mechanisms, which oppose the effects of the detrimental arm, the ACE–Ang II–AT1 pathway, associated with fibrosis, inflammation and hypertension. Renal ACE activity is associated with proteinuria, glomerulosclerosis and interstitial fibrosis (Largo et al. 1999; Bos et al. 2003; Rook et al. 2005), via enhanced generation of Ang II, and ACE2 is thought to balance these detrimental effects of ACE via cleavage of Ang II and formation of the antiproliferative and vasodilatory peptide Ang(1–7).
There are contradictory reports on the effects of dietary sodium modification per se on renal ACE activity (Fox et al. 1992; Ingert et al. 2002). In our study, a reduction in renal ACE during low sodium intake, in the absence of changes in renal ACE2, suggests a beneficial role for a low-sodium diet in preventing renal damage. This is in line with recent findings by our group showing that low-sodium diet reduces nephrotic range proteinuria in renal patients (Vogt et al. 2008) Moreover, these data may explain why a high sodium intake is associated with increased urinary albumin excretion and hyperfiltration, especially in obese subjects (Verhave et al. 2004; Krikken et al. 2007).
Lisinopril decreased renal ACE activity, as previously described (Ferrario et al. 2005b) without affecting renal ACE2, suggesting that the beneficial effects of ACE inhibition on proteinuria and blood pressure can be ascribed to modulation of ACE and not ACE2. Ferrario et al. (2005b) reported that ACE inhibition, angiotensin receptor blockade, or the combination of both did not affect renal ACE2 mRNA in healthy Lewis rats; however, cardiac ACE2 activity was increased by both lisinopril and losartan. In rats with streptozotocin-induced diabetes mellitus, ACE2 mRNA levels were low in the diabetic kidney and were unaffected by ramipril, while renal ACE2 protein was markedly increased (Tikellis et al. 2003). Unfortunately, ACE2 activity was not measured in the study by Tikellis et al. (2003). The discrepancies between these studies and ours may be related to differences in species, timing and dosage of ACE inhibitor, sodium status or disease model. This is further supported by a study which showed that during ACE inhibition and AT1 receptor blockade, renal ACE2 mRNA was not affected in normotensive conditions but increased in hypertensive rats (Jessup et al. 2006).
Sodium restriction potentiated the effect of ACE inhibition, as reflected by a larger reduction in blood pressure, which coincided with reduced renal ACE and ACE2 mRNA. Surprisingly, renal ACE2 activity was unaffected by the combination of low sodium and ACE inhibition, nor was ACE activity further decreased. Several studies show discrepancies between renal mRNA and activity of ACE and ACE2 (Ferrario et al. 2005b; Wysocki et al. 2006), which suggests post-transcriptional regulation or uncoupling between gene expression and protein. The changes in mRNA are difficult to interpret because enzymatic activity eventually determines the concentrations of Ang II and Ang(1–7). During ACE inhibition with low sodium intake, plasma Ang II was decreased. Several experimental and human studies show that chronic ACE inhibition does not suppress the formation of Ang II, at least not during normal and high sodium intake (Luque et al. 1996; Mento & Wilkes, 1987; Kocks et al. 2005). Our group recently showed that, in healthy human subjects, ACE inhibition is able to reduce Ang II levels during a low sodium intake (Kocks et al. 2005), which is in accord with our present findings in animals.
How can increased plasma Ang(1–7) be explained in the absence of changes in ACE2 activity? A reduction in ACE, although depleting Ang II as a substrate for ACE2 to be subsequently converted to Ang(1–7), will increase the overall level of Ang I. Angiotensin(1–7) can then be generated through pathways that bypass the formation Ang II, for instance by the action of tissue-specific endopeptidases, such as neprilysin. The measurement of the renal activity of neprilysin, an Ang(1–7)-forming neutral endopeptidase, however, did not reveal significant differences between the various treatment groups, although the low-salt groups had numerically lower values. Since ACE is an important enzyme involved in the inactivation of Ang(1–7) (Chappell et al. 1998; Allred et al. 2000), and lisinopril decreases renal ACE activity by approximately 80%, breakdown of Ang(1–7) might be attenuated as well (Yamada et al. 1998). Also, plasma Ang(1–7) levels might be modulated by ACE2 activity in organs other than the kidney. This concept is supported by studies that show differential effects of RAAS blockade on cardiac and renal ACE2 (Ferrario et al. 2005a,b), suggesting tissue-specific regulation of ACE2 (Gembardt et al. 2005; Igase et al. 2005).
In conclusion, manipulation of the RAAS in healthy rats, by a low sodium intake or ACE inhibition, does not result in changes in renal ACE2 despite major variations in renal ACE. Therefore, we propose that renal ACE and ACE2 are differentially regulated by low sodium and ACE inhibition. This may also apply to renal disease conditions and, accordingly, the renoprotective effects of ACE inhibition should be investigated in further studies.