Angiotensin-converting enzyme 2 regulates renal atrial natriuretic peptide through angiotensin-(1–7)

Clinical Science (2012) 123, 29–37 (Printed in Great Britain) doi:10.1042/CS20110403 29

Angiotensin-converting enzyme 2 regulatesrenal atrial natriuretic peptide through

angiotensin-(1–7)

Stella BERNARDI∗†1, Wendy C. BURNS∗1, Barbara TOFFOLI‡,Raelene PICKERING∗†, Maryio SAKODA∗, Despina TSOROTES∗, Edward GRIXTI∗,Elena VELKOSKA§, Louise M. BURRELL§, Colin JOHNSTON∗, Merlin C. THOMAS∗,Bruno FABRIS‡ and Christos TIKELLIS∗∗Baker IDI Heart and Diabetes Research Institute, 75 Commercial Road, Melbourne, VIC 3004, Australia, †Department ofMorphology and Embriology, University of Ferrara, Via Fossato di Mortara 66, 44100 Ferrara, Italy, ‡Department of Medical,Surgical, Health Sciences, University of Trieste, Ospedale di Cattinara, Strada di Fiume 447, 34149 Trieste, Italy, and§Department of Medicine, University of Melbourne, Austin Health, Heidelberg, VIC 3081, Australia

A B S T R A C T

Deficiency of ACE2 (angiotensin-converting enzyme 2), which degrades Ang (angiotensin) II,promotes the development of glomerular lesions. However, the mechanisms explaining why thereduction in ACE2 is associated with the development of glomerular lesions have still to be fullyclarified. We hypothesized that ACE2 may regulate the renoprotective actions of ANP (atrialnatriuretic peptide). The aim of the present study was to investigate the effect of ACE2 deficiencyon the renal production of ANP. We evaluated molecular and structural abnormalities, as wellas the expression of ANP in the kidneys of ACE2-deficient mice and C57BL/6 mice. We alsoexposed renal tubular cells to AngII and Ang-(1–7) in the presence and absence of inhibitorsand agonists of RAS (renin–angiotensin system) signalling. ACE2 deficiency resulted in increasedoxidative stress, as well as pro-inflammatory and profibrotic changes. This was associated with adown-regulation of the gene and protein expression on the renal production of ANP. Consistentwith a role for the ACE2 pathway in modulating ANP, exposing cells to either Ang-(1–7) or ACE2or the Mas receptor agonist up-regulated ANP gene expression. This work demonstrates thatACE2 regulates renal ANP via the generation of Ang-(1–7). This is a new mechanism wherebyACE2 counterbalances the renal effects of AngII and which explains why targeting ACE2 may bea promising strategy against kidney diseases, including diabetic nephropathy.

INTRODUCTION

The RAS (renin–angiotensin system) is a group ofenzymes and peptides whose primary effector is Ang(angiotensin) II. The final effects of RAS activation

depend not only on AngII synthesis through ACE(angiotensin-converting enzyme) but also on its degrad-ation, which relies upon ACE2, a carboxypeptidase thatdegrades AngII to Ang-(1–7). Since Ang-(1–7) exertsopposing actions to those of AngII [1], ACE2 seems

Key words: angiotensin-(1–7), angiotensin-converting enzyme 2 (ACE2), atrial natriuretic peptide (ANP), diabetes, epithelial tubularcell, kidney disease.Abbreviations: ACE, angiotensin-converting enzyme; ACE2KO, ACE2-knockout; ACEi, ACE inhibitor; Ang, angiotensin; ANP,atrial natriuretic peptide; ARB, AngII receptor blocker; AT1, AngII type 1; AT2, AngII type 2; BP, blood pressure; Col1, collagen1; CTGF, connective tissue growth factor; CTL, control; ICAM-1, intercellular adhesion molecule 1; IL-1β, interleukin-1β; iNOS,inducible NO synthase; MCP-1, monocyte chemotactic protein-1; NEP, neutral endopeptidase; NPR-A, natriuretic peptide receptor-A; PAS, periodate–Schiff; RAS, renin–angiotensin system; rACE2, recombinant ACE2; RT, reverse transcription.1 These authors have equally contributed to this paper.Correspondence: Dr Stella Bernardi (email [email protected]).

C© The Authors Journal compilation C© 2012 Biochemical Society

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30 S. Bernardi and others

to counterbalance the circulating and vascular effects ofAngII by both degrading bioactive AngII and producingbioactive Ang-(1–7) [1–3].

AngII is an important contributor to the pathogenesisof diabetic and non-diabetic kidney disease [4]. BothACE and ACE2 are localized in the proximal tubulesand the glomeruli where ACE2 may exert renoprotectiveeffects [5]. Studies in ACE2-deficient mice have shownthe progressive development of glomerulosclerosis [6].Further works exploring the effects of ACE2 inhibition[7] or its genetic deletion [8] have demonstrated thatACE2 deficiency increases albuminuria as well as theassociated glomerular lesions. In animal models [5] andhuman biopsies [9] of diabetic nephropathy, there isa reduction in ACE2 glomerular expression. Tikelliset al. [10] have also demonstrated that ACE2 is decreasedin the diabetic tubule. Consistent with this, ACE2overexpression has been reported to have a renoprotectiveeffect since it prevents experimental diabetic glomerularinjury [11]. However, the mechanisms for a reduction inACE2 being associated with kidney disease, including theprogression of diabetic nephropathy, have still to be fullyclarified.

It has been recently shown that in the heart Ang-(1–7) increases ANP (atrial natriuretic peptide) [12].ANP is an endogenous antagonist to AngII [13] bothsystemically and locally. In the kidney, this peptide hasbeen shown to reduce oxidative stress [14] as well as toexert anti-proliferative [15], anti-fibrotic [16] and anti-inflammatory effects through the generation of cGMP.On the basis of these observations, we hypothesizedthat the kidney damage associated with ACE2 deficiencycould partly depend on the loss of the renoprotectiveactions of ANP. Thus the aim of the present study was toevaluate whether and how ACE2 influences renal ANP.

MATERIALS AND METHODS

Animals and experimental protocol

ACE2-deficient miceTo evaluate the effect of ACE2 on renal ANP, two murinemodels of ACE2 deficiency were studied: 34 ACE2KO(ACE2-knockout) mice (on a C57BL/6 background)and streptozotocin-induced diabetes C57BL/6 mice. Atotal of 27 C57BL/6 [CTL (control)] and 27 ACE2KOmale mice at 8 weeks of age were randomly allocatedto three groups and were followed for 2, 12 and22 weeks respectively. ACE2KO mice were providedby Professor Joseph Penninger (Institute of MolecularBiotechnology, Vienna, Austria) and were generated asdescribed previously by his group [2,4]. At the sametime, 54 male 8-week-old C57BL/6 mice were randomlyallocated to receive streptozotocin (Sigma) at a dose of55 mg/kg of body weight for the induction of diabetes

(DM, n = 27) or buffer (sodium citrate buffer, pH 4.5;CTL, n = 27) in five consecutive daily doses. DM andCTL mice were then randomized further into threegroups (n = 9 per group) and followed for 2, 12 and22 weeks respectively. The mice were housed at the animalhouse of the Baker IDI Heart and Diabetes Instituteof Melbourne and were studied according to NHMRCInstitutional Guidelines for Care and Use of LaboratoryAnimals. The mice were fed a diet containing 0.2 %sodium by weight.

Of note, our group has demonstrated previously thatthe induction of diabetes in C57BL/6 mice followingstreptozotocin injections is associated with reducedrenal ACE2 expression [17]. Specifically, both groups(ACE2KO and diabetic mice) demonstrated absentor reduced renal cortical ACE2 mRNA expressionrespectively (P < 0.001 compared with CTL) withreduced renal Ang-(1–7) concentrations in corticalhomogenates (P < 0.05 compared with CTL) [17].

The mice that had been followed for 2 weeks, whichwere overall 10 weeks old, were put in metabolic cages for24 h to collect their urine for biochemical analysis priorto killing. At the end of each study period, all the micewere killed after an intraperitoneal injection of Euthal(Delvet) at a dose of 10 mg/kg of body weight, and thekidneys were removed, weighed and snap frozen or putinto formalin for further analysis.

Ang-(1–7)-infused ratsThe effect of Ang-(1–7) on renal production ofANP was studied in 16 male Sprague–Dawley rats(body weight, 200–250 g) that were implanted withan osmotic minipump (model no. 2002; Alzet). Forimplantation of the minipumps, the animals wereanaesthetized with an intraperitoneal injection of 2,2,2-tribromoethanol (Sigma) at a dose of 25 mg/100 mgof body weight. Buprenorfine (Temgesic, ReckittBenckiser) was used as analgaesic and was injectedsubcutaneously at a dose of 0.05 mg/kg of body weighton the day of the intervention and at a dose of0.025 mg/kg of body weight on the day after. Atotal of eight rats were infused with Ang-(1–7)subcutaneously (24 μg/kg of body weight per·h) andeight rats were infused with 0.9 % saline. After 10 daysof study, rats were killed by an injection of 2,2,2-tribromoethanol at a dose of 25 mg/100 g of body weightand the kidneys were removed and snap frozen [18].

Gene expression quantification byreal-time PCRFor the gene expression analysis, 3 μg of total RNAextracted from the kidneys of the 10-week-old mice wasused to synthesize cDNA with Superscript First Strandsynthesis system for RT (reverse transcription)–PCR,as described previously [19]. The renal gene expression


ACE2 and regulates ANP through Ang-(1–7) 31

of ACE2, ANP, CTGF (connective tissue growthfactor), Col (collagen) 1, Col4, fibronectin, ICAM-1(intercellular adhesion molecule 1), iNOS (inducibleNO synthase), IL-1β (interleukin-1β), IL-6 and MCP-1(monocyte chemotactic protein-1) was analysed by real-time quantitative RT–PCR using the TaqMan systembased on real-time detection of accumulated fluorescence.

Renal ANP gene expression was measured followingthe same method in RNA extracted from the kidneysof the Ang-(1–7)-infused rats and their controls.

Renal morphology and ANPimmunostainingParaffin sections (4 μm thick) were obtained from4 % (w/v) paraformaldehyde-fixed paraffin-embeddedkidneys and were stained by nitrotyrosine immuno-staining for nitrosylation of proteins by PAS (periodate–Schiff) for glomerulosclerosis and by Masson’s trichromefor renal fibrosis. To detect renal ANP both mouse andrat sections were stained with an anti-ANP antibody.In both immunostainings after neutralizing endogenousperoxidases, the sections of kidneys were incubatedwith rabbit anti-nitrotyrosine antibodies diluted 1:100(Upstate) and with rabbit anti-ANP antibodies diluted1:200 (Millipore). Biotinylated immunoglobulins (VectorLaboratories), diluted 1:500 and 1:200, were then appliedas secondary antibodies. Specific staining was detectedusing the standard avidin–biotin complex method(Vector Laboratories) and then visualized by reactionwith 3,3′-diaminobenzidine tetrahydrochloride (Sigma).Sections were then counterstained with haematoxylinand eosin. Each section was examined by lightmicroscopy (Olympus BX50WI) and digitized witha high-resolution camera (Q-Imaging Fast 1394). Theproportional areas of brown staining, bright pink stainingand blue staining were then calculated to quantifythe nitrosylation of proteins, ANP expression in thetubuli, glomerulosclerosis and fibrosis of the glomerulirespectively using an image analysis system (Image ProPlus 6.3 Software; Media-Cybernetics).

General parameters and biochemical dataIntrarenal concentrations of Ang-(1–7) were measuredon snap-frozen renal cortical samples by a commercialRIA (ProSearch International), as described previously[17]. The measurement of urinary cGMP was performedusing an EIA (enzyme immunoassay) kit (581021;Cayman Chemical), according to manufacturer’s instruc-tions. Albuminuria was measured by ELISA (BethylLaboratories), according to manufacturer’s instructions.Creatinine clearance was estimated from serum and urineconcentrations, as determined by HPLC.

In vitro experimentsThe gene expression of ANP was determined inNRK-52E cells (rat non-transformed proximal tubularepithelial cells), which express ANP according to theprevious reports [20]. NRK-52E cells were treated withrACE2 (recombinant ACE2) (50 ng/ml), Ang-(1–7) (0.1;0.5; 1; 5; 10; 25 μM; 2588; Auspep), an agonist of the Masreceptor [the Ang-(1–7) receptor] AVE-0991 (10 μM) orAngII (1 nM; 2078; Auspep) for 3 days. Untreated cellswere used as controls. Blockade of AT1 (AngII type 1)and AT2 (AngII type 2) receptors, both combined andseparately, was achieved by treating with the AT1 receptorblocker valsartan (1 μM; Novartis) and with the AT2

receptor antagonist PD123319 (1 μM; Sigma) for 30 minprior to the addition of AngII [18].

Statistical analysisStatistical analysis was performed using SAS. Resultsare expressed as means +− S.E.M. One-way ANOVA wasperformed to evaluate the differences in continuousvariables among the groups. A P < 0.05 was consideredstatistically significant.

RESULTS

Molecular pathological renal changes inACE2 deficiencyPathological kidney microscopic abnormalities wereobserved in 10-weeks-old ACE2KO mice. Their kidneysshowed a significant induction of pro-oxidative, pro-inflammatory and pro-fibrotic mediators, such as iNOS,IL-6, IL-1β, ICAM-1, MCP-1, Col1, Col4, CTGF andfibronectin respectively (1.5–4.5-fold increased, P < 0.05compared with CTL) (Figure 1A). This was associa-ted with an increase in oxidative stress (P < 0.001compared with CTL), evaluated as the amount ofnitrosylated proteins in the glomeruli as well asglomerulosclerosis (P < 0.005) and glomerular fibrosis(P < 0.05 compared with CTL) (Figure 1B). In our study,these molecular renal changes were not associated withovert renal dysfunction, since 10-week-old ACE2KOmice did not show any changes in water intake, diuresis,albuminuria and creatinine clearance with respect to theirCTLs. Albuminuria was 43 +− 1.3 μg/day in ACE2KOmice and 46 +− 1.1 μg/day in CTLs, whereas creatinineclearance was 0.32 +− 0.02 ml/min in ACE2KO mice and0.26 +− 0.03 ml/min in CTLs. However, later changescannot be excluded [6,21].

ACE2 deficiency and ANPLower intrarenal concentrations of Ang-(1–7) werefound in 10-week-old ACE2KO mice (14.56 +−0.81 fmol/mg in the CTL group and 5.98 +− 0.17fmol/mg in the ACE2KO mice; P < 0.001). Genetic



Figure 1 Molecular features of kidney damage in ACE2 deficiency(A) Renal mRNA expression of iNOS, IL-6, IL-1β , ICAM-1, MCP-1, Col1, Col4, CTGF and fibronectin in the kidneys of C57BL/6 (CTL) and ACE2KO mice of 10 weeks of age.mRNA expression is reported as relative gene units; results are expressed as means +− S.E.M. ∗P < 0.05 compared with CTL; §P < 0.005 compared with CTL. (B) Glome-rular nitrotyrosine, PAS for glomerulosclerosis and Masson’s trichrome staining for fibrosis are reported as the fold induction of a percentage of the stained area; results areexpressed as means+− S.E.M. ∗P < 0.05 compared with CTL; §P < 0.001 compared with CTL. On the left representative images of nitrotyrosine immunostained sections(C and D), PAS-stained sections (E and F) and Masson’s trichrome-stained sections (G and H) of kidneys of control mice and ACE2KO (original magnification ×20).

ACE2 deficiency was also associated with a significantdown-regulation of ANP gene expression in thekidneys of ACE2KO mice at 10, 20 and 30 weeks(P < 0.05 compared with CTL) (Figure 2A). This wasconfirmed by immunostaining of kidney sections in10-week-old ACE2KO mice, which showed a significantdecrease in tubular ANP (P < 0.05 compared withCTL) (Figures 2C–2E). The urinary levels of cGMP,which is the ANP second messenger, were also reducedin the ACE2KO mice, although not significantly.In the CTL mice at 10 weeks of age, urinary cGMPlevels were 7398.5 +− 820.1 pmol/l, whereas they were6650.2 +− 741.8 pmol/ml in ACE2KO mice.

The same findings were observed in a mouse modelof acquired ACE2 deficiency, with 10-week-old diabeticmice having a reduction in renal ACE2 mRNA expressioncompared with the CTL group (P < 0.01). Accordingly,Ang-(1–7) levels were also significantly reduced indiabetic mice being 2.09 +− 0.12 fmol/mg (P < 0.001compared with CTL). As observed in ACE2KO mice, theacquired ACE2 deficiency of diabetic mice was associatedwith a significant down-regulation of ANP geneexpression in the kidney at 10, 20 and 30 weeks (P < 0.05compared with CTL) (Figure 2B). Moreover, the urinarylevels of cGMP were also significantly reduced in diabetic

mice (3802.9 +− 472.7 pmol/ml; P < 0.005 compared withCTL).

In vitro and in vivo effects of Ang-(1–7)and other inhibitors and agonists of RASsignallingTreatment of NRK-52E cells with Ang-(1–7) significantlyup-regulated ANP gene expression in a dose-dependentmanner until a plateau was reached (Figure 3A). Themaximum response was observed when cells were treatedwith Ang-(1–7) at a dose range between 5 and 10 μM(Figure 3A). Consistent with this effect, exposing thesecells to rACE2 (50 ng/ml) or the Mas receptor agonistAVE-0991 (10 μM) significantly increased ANP geneexpression (P < 0.05 compared with CTL) (Figure 3B).In contrast, AngII did not have any significant effect onANP gene expression (Figure 3C).

It has been shown that RAS blockade leads alsoto an increase in Ang-(1–7) levels by the degradationof an excess of AngII [2], therefore we treated ourcells with the AT1 receptor blocker valsartan (1 μM)and the AT2 receptor blocker PD123319 (1 μM) inthe presence or absence of AngII (1 nM). Tubular cellssignificantly up-regulated ANP gene expression whenthey were treated with both AT1 and AT2 receptors



Figure 2 ACE2 deficiency and ANP(A) Renal mRNA expression of ANP in the kidneys of C57BL/6 (CTL) and ACE2KO mice at 10, 20 and 30 weeks of age. mRNA expression is reported as relative geneunits; results are expressed as means +− S.E.M. ∗P < 0.05 compared with CTL. (B) Renal mRNA expression of ANP in the kidneys of C57BL/6 (CTL) and diabetic(DM) mice of 10, 20 and 30 weeks of age. mRNA expression is reported as relative gene units; results are expressed as means+− S.E.M. ∗P < 0.05 compared withCTL. (C) Tubular ANP staining is reported as a percentage of the stained area; results are expressed as means+− S.E.M. ∗P < 0.05 compared with CTL. (D and E)Representative images of ANP immunostained sections of kidneys of CTL mice and ACE2KO (original magnification ×25)

blockers (P < 0.05 compared with CTL) (Figure 3C).In these cells, ANP gene expression increased evenfurther when AngII was added to AT1 and AT2 receptorsblockers (P < 0.05 compared with CTL), which couldhave resulted from an increase in local unbound AngIIwhich then degraded to Ang-(1–7). Of note, the separateblockade of the AT1 and AT2 receptors did not affectrenal ANP expression (results not shown). The effect ofAng-(1–7) on renal ANP expression observed in vitro wasthen confirmed in vivo. Rats infused with Ang-(1–7) for10 days had a 3-fold increase in ANP expression in theirkidneys (3.18 +− 0.69-fold increase) compared with CTLs(1 +− 0.25-fold increase) (P < 0.05). This was consistentwith the significant increase in tubular ANP observedin Ang-(1–7)-infused rats (P < 0.05 compared with CTL)after immunostaining the kidney (Figures 4A–4C).

DISCUSSION

The novel finding of the present study is that, inthe kidney, unlike the heart where the synthesis ofANP is dependent on volume expansion and pressureoverload, ACE2 directly regulates renal ANP productionthrough the generation of Ang-(1–7). This is in agreementwith the previous finding that Ang-(1–7) increases

cardiac ANP secretion at high atrial pacing [12]. Inthe in vivo study, ACE2 deficiency (either genetic oracquired), which is characterized by low levels of Ang-(1–7) [17] and high levels of AngII [2], was associatedwith a significant down-regulation of renal ANP. NEP(neutral endopeptidase) is also an enzyme involved inthe metabolism of Ang-(1–7) and ANP. Although thepresent study did not examine the role of NEP in thegeneration of Ang-(1–7) and the cleavage of ANP, thefact that ACE2KO had low levels of Ang-(1–7) in theirkidneys suggests that NEP may not have a relevant rolein the local generation of Ang-(1–7) in our model [22].Thus in ACE2 deficiency ANP down-regulation could becaused either by a decrease in Ang-(1–7) or to an increasein AngII. To clarify which mechanism was responsible,we treated epithelial proximal tubular cells, which area local renal source of ANP [20], with both Ang-(1–7) and AngII. This conclusively established that theACE2 effect was related to Ang-(1–7). These resultswere then confirmed in vivo by demonstrating that Ang-(1–7) infusion significantly increased renal ANP gene andprotein expression. Consistent with these observations,it has also been shown that basal cGMP productionwas significantly increased in kidney slices from Ang-(1–7)-treated mice compared with saline-infused animals[23]. Interestingly, we also observed that blocking both



Figure 3 Effects of various peptides of the RAS on ANP expression in tubular cells(A) Effects of increasing doses of Ang-(1–7) mRNA expression of ANP in proximal tubular epithelial cells is reported as relative gene units; results areexpressed as means+− S.E.M. ∗P < 0.05 compared with CTL; §P < 0.001 compared with CTL. (B) Effects of ACE2, Ang-(1–7) and the Mas agonist. mRNAexpression of ANP in proximal tubular epithelial cells is reported as relative gene units; results are expressed as means +− S.E.M. ∗P < 0.05 compared with CTL.(C) AngII blockade in presence and absence of AngII. mRNA expression of ANP in proximal tubular epithelial cells is reported as relative gene units; results areexpressed as means+− S.E.M. ∗P < 0.05 compared with CTL. val, valsartan; PD, PD123319.

Figure 4 Effect of Ang-(1–7) infusion on ANP protein expression(A) Tubular ANP staining is reported as the percentage stained area; results are expressed as means+− S.E.M. ∗P < 0.05 compared with CTL. (B, C) Representativeimages of ANP immunostained sections of kidneys of saline-infused (Control) and Ang-(1–7)-infused rats (original magnification ×25).

AT1 and AT2 receptors led to a significant up-regulationof ANP gene expression, which was even greater afterthe addition of AngII. We speculate that the additionof AngII caused a further increase in local unboundAngII, which is the substrate for Ang-(1–7), and thereforethis led to an increase in local Ang-(1–7) production.On the contrary, when tubular cells were treated eitherwith an AT1 or an AT2 receptor blocker there was

no increase in ANP gene expression, possibly becausein our model the partial blockade did not lead to asignificant increase in unbound AngII as the completeblockade did. Experimental evidence has shown that RASblockade leads to an increase in Ang-(1–7), which is animportant contributor to the protective effects of ACEis(ACE inhibitors) or ARBs (AngII receptor blockers). Inspontaneously hypertensive rats, the combination of the



ACEi lisinopril and the ARB losartan has been reportedto increase plasma levels of Ang-(1–7), which was thoughtto contribute to the anti-hypertensive effect of thesedrugs [24]. Moreover, the ARB olmesartan increased thecardiac levels of Ang-(1–7) through the overexpressionof ACE2 [25] and, in the kidney, treatment with anACEi promoted ACE2 activity, leading to an increasein Ang-(1–7) levels [26,27]. Thus we postulate that theup-regulation of ANP gene expression that we observedin vitro after blocking both AT1 and AT2 receptorscould have been due to the conversion of AngII intoAng-(1–7). This is also consistent with the report thatACEi and ARB directly stimulate ANP secretion in vivo[28].

The concept that ACE2 increases renal ANPthrough Ang-(1–7) highlights a new mechanism wherebyACE2 opposes AngII. Overall, ACE2 is believed tocounterbalance the circulating and local effects of AngIIby degrading active AngII and simultaneously producingbioactive Ang-(1–7) [1–3]. In the present study, we showthat ACE2 increases renal ANP and this might representa third mechanism whereby ACE2 counterbalancesAngII [1], since ANP is an endogenous antagonistto AngII. Systemic administration of ANP inducesnatriuresis, diuresis and a fall in BP (blood pressure)[13]. Recently, its chemical analogues have also beendemonstrated to be effective in inhibiting angiotensin-dependent hypertension and vascular damage [29]. Thediuretic and natriuretic properties of ANP are related toits ability to inhibit AngII-dependent sodium and watertransport in the proximal tubule and water transport inthe cortical collecting duct [30]. In addition, ANP inhibitsaldosterone release and antagonizes the distal tubularactions of aldosterone by binding its receptor A [31]. Italso inhibits vasopressin release and stimulates vasculardilation [32]. Because of these properties, it has beenproposed that one of the negative feedback mechanismscompensating for RAS activation is indeed the AngII-dependent stimulation of ANP release from the atriaof the heart through both direct, via AT1 receptor, andindirect actions, via atrial distension.

Beyond the systemic regulation of BP, ANP alsohas local ‘protective’ anti-proliferative and anti-fibroticeffects [16,33] in different organs, including the kidney.In particular, among the autocrine and/or paracrineactions [20] that ANP exerts in the kidney, this peptidehas been shown to reduce oxidative stress [14] aswell as to exert anti-proliferative [15], anti-fibrotic [16]and anti-inflammatory effects through the generationof membrane-bound cGMP. Of note, ANP generatescGMP through the activation of a membrane-boundguanylate cyclase, whereas the soluble form of thisenzyme is stimulated by NO [34]; since NO productionis notably impaired in diabetes [35], this could accountfor the difference that we observed between urinarycGMP in ACE2KO and diabetic mice. Experimental

evidence demonstrates that ANP inhibits cell growth ofvascular smooth muscle cells, fibroblasts and mesangialcells [15,31,36], where the ANP/NPR-A (natriureticpeptide receptor-A)/cGMP system would inhibit theproliferative effects of AngII and or ET-1 (endothelin-1)[37]. Moreover, overexpression of BNP (brain natriureticpeptide) in mice attenuated renal injury in a model of renalablation, glomerulonephritis and diabetic nephropathy[38–40]. This suggests that ANP exerts a paracrine directprotective effect within the kidney and it may protectagainst the progression of renal disease. Given all theseeffects of ANP, the finding that ACE2 deficiency reducesrenal ANP could explain why ACE2-deficient mice,including diabetic mice, develop spontaneous kidneyabnormalities. Notably, it has been demonstrated thatthe deletion of ACE2 leads to the development of AngII-dependent glomerular injury, which can be prevented bytreatment with the ARB irbesartan [6]. Similar to whatwas reported in the study by Oudit et al. [6], we found thatby 10 weeks of age ACE2-deficient mice have a significantup-regulation of genes involved in the generation ofoxidative stress and in inflammatory processes, as wellas in fibrogenesis. In addition, ACE2-deficient micedisplay increased glomerular nitrosylation of proteins,which is a marker of oxidative stress, glomerulosclerosisand glomerular fibrosis. Thus all of these abnormalitiescould result from a local increase in AngII and a localdecrease in both Ang-(1–7) and ANP. This is consistentwith the report by Nishikimi et al. [16] showing alink between decreased ANP and increased profibroticgene expression in the kidneys, where exogenous ANPadministration inhibited renal fibrosis. To what extentthe decrease in either Ang-(1–7) or ANP may contributeseparately to the kidney phenotype of ACE2KO miceis difficult to assess given that Ang-(1–7) seems tostimulate ANP production and that there seems to bealso a synergistic effect between both peptides [12]. Forexample, Shah et al. [12] have demonstrated that theblockage of NPR-A in the heart diminished the anti-hypertrophic effect of Ang-(1–7). Interestingly, given therenoprotective paracrine effects of ANP [14–16,37],the finding that ACE2 increases ANP in vitro couldalso explain why overexpression of ACE2 in diabeticmice results in amelioration of their glomerular injury[11]. Thus we speculate that the benefit of reintroducingACE2 in diabetic mice might partly be due to an expectedincrease in renal ANP levels.

In conclusion, the results of the present studydemonstrate that ACE2 is very important for the renalproduction of ANP through the generation of Ang-(1–7). This is a third and a new mechanism whereby ACE2may oppose AngII. Moreover, in ACE2-deficient states,such as diabetes, the loss of the renoprotective effectsof ANP could worsen the effects of increased levels ofAngII, contributing to the development and progressionof kidney disease, including diabetic nephropathy.



AUTHOR CONTRIBUTION

Stella Bernardi designed and performed the experimentalwork, analysed the data, and wrote, edited and revisedthe paper prior to submission. Wendy Burns and BarbaraToffoli performed the experimental work and analysedthe data. Raelene Pickering, Despina Tsorotes, EdwardGrixti, Mariyo Sakoda and Elena Velkoska assisted in theexperimental work. Louise Burrell designed the work.Colin Johnston and Merlin Thomas designed thework and edited the paper prior to submission. BrunoFabris designed the work, analysed the data, and wrote,edited and revised the paper prior to submission. ChristosTikellis designed and performed the experimental work,and edited the paper prior to submission.

FUNDING

This work was supported by the National HealthResearch Medical Council of Australia and the Ministerodell’Istruzione, dell’Universita e della Ricerca [grantnumber PRIN2006]. S.B. was supported by fundingfrom the Societa’ Italiana dell’Ipertensione Arteriosa(SIIA).

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Received 11 August 2011/24 January 2012; accepted 30 January 2012Published as Immediate Publication 30 January 2012, doi:10.1042/CS20110403


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Angiotensin-converting enzyme 2 regulates renal atrial natriuretic peptide through angiotensin-(1–7)