Optimum AT1 receptor-neprilysin inhibition has superior cardioprotective effects compared with AT1 receptor blockade alone in hypertensive rats
2015; Elsevier BV; Volume: 88; Issue: 1 Linguagem: Inglês
10.1038/ki.2015.107
ISSN1523-1755
AutoresLodi C.W. Roksnoer, Richard van Veghel, René de Vries, Ingrid M. Garrelds, Usha M. Bhaggoe, Edith C. H. Friesema, Frank Leijten, Marko Poglitsch, Oliver Domenig, Marian C. Clahsen‐van Groningen, Ewout J. Hoorn, A.H. Jan Danser, Wendy W. Batenburg,
Tópico(s)Renin-Angiotensin System Studies
ResumoNeprilysin inhibitors prevent the breakdown of bradykinin and natriuretic peptides, promoting vasodilation and natriuresis. However, they also increase angiotensin II and endothelin-1. Here we studied the effects of a low and a high dose of the neprilysin inhibitor thiorphan on top of AT1 receptor blockade with irbesartan versus vehicle in TGR(mREN2)27 rats with high renin hypertension. Mean arterial blood pressure was unaffected by vehicle or thiorphan alone. Irbesartan lowered blood pressure, but after 7 days pressure started to increase again. Low- but not high-dose thiorphan prevented this rise. Only during exposure to low-dose thiorphan plus irbesartan did heart weight/body weight ratio, cardiac atrial natriuretic peptide expression, and myocyte size decrease significantly. Circulating endothelin-1 was not affected by low-dose thiorphan with or without irbesartan, but increased after treatment with high-dose thiorphan plus irbesartan. This endothelin-1 rise was accompanied by an increase in renal sodium–hydrogen exchanger 3 protein abundance, and an upregulation of constrictor vascular endothelin type B receptors. Consequently, the endothelin type B receptor antagonist BQ788 no longer enhanced endothelin-1-induced vasoconstriction (indicative of endothelin type B receptor–mediated vasodilation), but prevented it. Thus, optimal neprilysin inhibitor dosing reveals additional cardioprotective effects on top of AT1 receptor blockade in renin-dependent hypertension. Neprilysin inhibitors prevent the breakdown of bradykinin and natriuretic peptides, promoting vasodilation and natriuresis. However, they also increase angiotensin II and endothelin-1. Here we studied the effects of a low and a high dose of the neprilysin inhibitor thiorphan on top of AT1 receptor blockade with irbesartan versus vehicle in TGR(mREN2)27 rats with high renin hypertension. Mean arterial blood pressure was unaffected by vehicle or thiorphan alone. Irbesartan lowered blood pressure, but after 7 days pressure started to increase again. Low- but not high-dose thiorphan prevented this rise. Only during exposure to low-dose thiorphan plus irbesartan did heart weight/body weight ratio, cardiac atrial natriuretic peptide expression, and myocyte size decrease significantly. Circulating endothelin-1 was not affected by low-dose thiorphan with or without irbesartan, but increased after treatment with high-dose thiorphan plus irbesartan. This endothelin-1 rise was accompanied by an increase in renal sodium–hydrogen exchanger 3 protein abundance, and an upregulation of constrictor vascular endothelin type B receptors. Consequently, the endothelin type B receptor antagonist BQ788 no longer enhanced endothelin-1-induced vasoconstriction (indicative of endothelin type B receptor–mediated vasodilation), but prevented it. Thus, optimal neprilysin inhibitor dosing reveals additional cardioprotective effects on top of AT1 receptor blockade in renin-dependent hypertension. Renin–angiotensin system (RAS) blockers are the cornerstone in the treatment of hypertension, heart failure, and proteinuric chronic kidney disease. Yet, morbidity and mortality of these diseases remain high, despite RAS blocker treatment. Initially, it was thought that this related to incomplete RAS blockade, for example, because of renin upregulation and/or non-ACE-mediated angiotensin II (Ang II) formation. However, trials evaluating dual RAS blockade to achieve near-complete suppression revealed an increased risk of adverse events (including hypotension, hyperkalemia, and acute kidney injury) without additional benefit.1.de Boer R.A. Azizi M. Danser A.H.J. et al.Dual RAAS suppression: recent developments and implications in light of the ALTITUDE study.J Renin Angiotensin Aldosterone Syst. 2012; 13: 409-412Crossref PubMed Scopus (19) Google Scholar Therefore, we need new therapeutic strategies. The natriuretic peptide system normally counterbalances the renin–angiotensin system (RAS), so that enhancing the activity of this system on top of RAS blockade might be beneficial.2.Seymour A.A. Norman J.A. Asaad M.M. et al.Antihypertensive and renal activity of SQ 28,603, an inhibitor of neutral endopeptidase.J Cardiovasc Pharmacol. 1991; 17: 296-304Crossref PubMed Scopus (33) Google Scholar Neutral endopeptidase (NEP), also known as neprilysin, has an important role in the degradation of the three currently known natriuretic peptides, that is, atrial, brain, and C-type natriuretic peptide (ANP, BNP, and CNP), with the least susceptibility to degradation for BNP.3.von Lueder T.G. Sangaralingham S.J. Wang B.H. et al.Renin–angiotensin blockade combined with natriuretic peptide system augmentation: novel therapeutic concepts to combat heart failure.Circ Heart Fail. 2013; 6: 594-605Crossref PubMed Scopus (109) Google Scholar These peptides stimulate diuresis, natriuresis, and vasodilation. Their second messenger cyclic guanosine 3′5′ monophosphate (cGMP) improves myocardial relaxation and reduces hypertrophy. Yet, NEP additionally degrades the vasodilator bradykinin, and the constrictors endothelin-1 (ET-1) and Ang II, and ET-1 rises have been observed during NEP inhibition in humans.4.Ferro C.J. Spratt J.C. Haynes W.G. Webb D.J. Inhibition of neutral endopeptidase causes vasoconstriction of human resistance vessels in vivo.Circulation. 1998; 97: 2323-2330Crossref PubMed Scopus (151) Google Scholar,5.Ando S. Rahman M.A. Butler G.C. et al.Comparison of candoxatril and atrial natriuretic factor in healthy men. Effects on hemodynamics, sympathetic activity, heart rate variability, and endothelin.Hypertension. 1995; 26: 1160-1166Crossref PubMed Google Scholar Consequently, NEP inhibition may even lead to an increase in blood pressure. Combined RAS/NEP inhibition is less likely to cause this problem. Indeed, compared with ACE inhibition alone, the combined ACE/NEP inhibitor omapatrilat showed superior effects on blood pressure, left ventricular function, and combined mortality/morbidity end points in patients with congestive heart failure.6.McClean D.R. Ikram H. Garlick A.H. et al.The clinical, cardiac, renal, arterial and neurohormonal effects of omapatrilat, a vasopeptidase inhibitor, in patients with chronic heart failure.J Am Coll Cardiol. 2000; 36: 479-486Abstract Full Text Full Text PDF PubMed Scopus (109) Google Scholar,7.Rouleau J.L. Pfeffer M.A. Stewart D.J. et al.Comparison of vasopeptidase inhibitor, omapatrilat, and lisinopril on exercise tolerance and morbidity in patients with heart failure: IMPRESS randomised trial.Lancet. 2000; 356: 615-620Abstract Full Text Full Text PDF PubMed Scopus (389) Google Scholar Unfortunately, omapatrilat often caused angioedema, which might have been expected on the basis of the elevated bradykinin levels occurring when the two major bradykinin-degrading enzymes NEP and ACE are being blocked.8.Kostis J.B. Packer M. Black H.R. et al.Omapatrilat and enalapril in patients with hypertension: the Omapatrilat Cardiovascular Treatment vs. Enalapril (OCTAVE) trial.Am J Hypertens. 2004; 17: 103-111Crossref PubMed Scopus (480) Google Scholar,9.Messerli F.H. Nussberger J. Vasopeptidase inhibition and angio-oedema.Lancet. 2000; 356: 608-609Abstract Full Text Full Text PDF PubMed Scopus (206) Google Scholar A better approach would therefore be to combine an Ang II type 1 (AT1) receptor blocker with an NEP inhibitor (ARNI). The first ARNI that has been tested clinically, LCZ696, consisted of the NEP inhibitor prodrug AHU377 and the AT1 receptor blocker valsartan. LCZ696 was superior to valsartan in the treatment of hypertension,10.Ruilope L.M. Dukat A. Bohm M. et al.Blood-pressure reduction with LCZ696, a novel dual-acting inhibitor of the angiotensin II receptor and neprilysin: a randomised, double-blind, placebo-controlled, active comparator study.Lancet. 2010; 375: 1255-1266Abstract Full Text Full Text PDF PubMed Scopus (449) Google Scholar and also to the ACE inhibitor enalapril in the treatment of heart failure.11.McMurray J.J. Packer M. Desai A.S. et al.Angiotensin-neprilysin inhibition versus enalapril in heart failure.N Engl J Med. 2014; 371: 993-1004Crossref PubMed Scopus (3938) Google Scholar Preclinically, the combination of valsartan and the NEP inhibitor CGS25354 was almost as effective as a dual ACE/NEP inhibitor in lowering blood pressure and improving vascular remodeling in stroke-prone spontaneously hypertensive rats.12.Pu Q. Brassard P. Javeshghani D.M. et al.Effects of combined AT1 receptor antagonist/NEP inhibitor on vascular remodeling and cardiac fibrosis in SHRSP.J Hypertens. 2008; 26: 322-333Crossref PubMed Scopus (30) Google Scholar However, it is currently unknown how much NEP inhibition on top of AT1 receptor blockade is optimal. This question is especially relevant because NEP inhibition can increase ET-1.4.Ferro C.J. Spratt J.C. Haynes W.G. Webb D.J. Inhibition of neutral endopeptidase causes vasoconstriction of human resistance vessels in vivo.Circulation. 1998; 97: 2323-2330Crossref PubMed Scopus (151) Google Scholar,5.Ando S. Rahman M.A. Butler G.C. et al.Comparison of candoxatril and atrial natriuretic factor in healthy men. Effects on hemodynamics, sympathetic activity, heart rate variability, and endothelin.Hypertension. 1995; 26: 1160-1166Crossref PubMed Google Scholar ET-1 not only is a potent vasoconstrictor but also affects kidney sodium handling. ET-1 increases the sodium-hydrogen exchanger type 3 (NHE3) in the proximal tubule and decreases the epithelial sodium channel (ENaC) in the distal tubule; both effects are mediated through the endothelin type B (ETB) receptor.13.Donowitz M. Li X. Regulatory binding partners and complexes of NHE3.Physiol Rev. 2007; 87: 825-872Crossref PubMed Scopus (157) Google Scholar, 14.Bugaj V. Pochynyuk O. Mironova E. et al.Regulation of the epithelial Na+ channel by endothelin-1 in rat collecting duct.Am J Physiol Renal Physiol. 2008; 295: F1063-F1070Crossref PubMed Scopus (85) Google Scholar, 15.Higa T. Horinouchi T. Aoyagi H. et al.Endothelin type B receptor-induced sustained Ca2+ influx involves G(q/11)/phospholipase C-independent, p38 mitogen-activated protein kinase-dependent activation of Na+/H+ exchanger.J Pharmacol Sci. 2010; 113: 276-280Crossref PubMed Scopus (7) Google Scholar In the present study, we hypothesized that too much NEP inhibition might be detrimental. We compared single NEP inhibition (thiorphan) and AT1 receptor blockade (irbesartan) versus the ARNI approach (thiorphan+irbesartan), applying both a low and a high thiorphan dose.16.Eberlin M. Muck T. Michel M.C. A comprehensive review of the pharmacodynamics, pharmacokinetics, and clinical effects of the neutral endopeptidase inhibitor racecadotril.Front Pharmacol. 2012; 3: 93Crossref PubMed Scopus (54) Google Scholar Studies were performed in heterozygous TGR(mREN2)27 (Ren2) rats. These rats, by overexpressing the mouse Ren2 gene, display severe Ang II-dependent hypertension, myocardial hypertrophy, and vascular remodeling,17.Mullins J.J. Peters J. Ganten D. Fulminant hypertension in transgenic rats harbouring the mouse Ren-2 gene.Nature. 1990; 344: 541-544Crossref PubMed Scopus (812) Google Scholar,18.Bachmann S. Peters J. Engler E. et al.Transgenic rats carrying the mouse renin gene—morphological characterization of a low-renin hypertension model.Kidney Int. 1992; 41: 24-36Abstract Full Text PDF PubMed Scopus (162) Google Scholar and have an activated natriuretic peptide system.19.Stasch J.P. Hirth-Dietrich C. Ganten D. Wegner M. Renal and antihypertensive effects of neutral endopeptidase inhibition in transgenic rats with an extra renin gene.Am J Hypertens. 1996; 9: 795-802Crossref PubMed Scopus (20) Google Scholar In view of the potential consequences of NEP inhibition, as described above, we focused on blood pressure, natriuresis, diuresis, cardiac hypertrophy, and vascular reactivity. Additionally, to obtain a better understanding of the biochemical consequences of ARNI, we quantified its effects on the RAS, ANP, cGMP, and ET-1. Finally, we evaluated whether ARNI affects well-known Ang II and ET-1 targets such as vascular ET receptors and kidney sodium transporters, as changes therein, on top of changes in RAS, ANP, cGMP, and ET-1, may underlie the hemodynamic and renal effects of ARNI. Neither vehicle nor thiorphan alone affected mean arterial pressure (MAP; Figure 1). Irbesartan, either alone or combined with the low or high dose of thiorphan, did lower MAP compared with vehicle (Figure 1), with no effect on heart rate (363±5, 347±5, 342±3, 348±4, and 348±3 beats per minute after 3 weeks of treatment with vehicle, thiorphan, irbesartan, irbesartan+low-dose thiorphan, and irbesartan+high-dose thiorphan, respectively). The effects of irbesartan alone on MAP were significant (P<0.01) already on the first treatment day, and reached a maximum around days 1–4. Thereafter, MAP slowly started to rise again, although it was still ≈30 mm Hg below the MAP of vehicle-treated rats after 3 weeks. This rise was prevented by the low but not by the high dose of thiorphan. Urinary volume increased during vehicle treatment, while no such increase was observed during any of the drug treatments (Table 1). Changes in urinary volume paralleled those in water intake. Body weight, kidney weight and food intake were not affected by any of the treatments (Table 1).Table 1Main characteristics in Ren2 rats treated with vehicle, TH0.1, IRB, IRB+TH0.1, or IRB+TH1.0ParameterBaselineVehicleTH0.1IRBIRB+TH0.1IRB+TH1.0BW (g)—558±12553±9554±10561±21560±13HW (g)—2.2±0.062.1±0.042.0±0.081.8±0.08*2.0±0.1HW/tibia length (g/cm)—0.50±0.010.49±0.010.46±0.020.43±0.02*0.48±0.02HW/BW (g/kg)—4.0±0.13.8±0.13.6±0.23.3±0.1*3.7±0.2KW (g)—1.6±0.031.3±0.041.5±0.031.5±0.081.5±0.04KW/BW (g/kg)—2.9±0.062.9±0.042.7±0.072.7±0.12.7±0.04Water intake (ml per day)40±2.251±7.746±1.528±3.3*28±2.5*34.4±3.5Food intake (g per day)22±0.921±1.822±1.321±3.018±2.723±2.0Plasma Creatinine (nmol/ml)—42±1.833±1.740±1.937±1.843±1.6 Creatinine clearance (ml/min)—3.0±0.23.4±0.23.0±0.13.4±0.42.7±0.2 Na+ (mmol/l)—141±1.0143±1.0142±1.1142±1.3142±1.3 K+ (mmol/l)—4.7±0.34.8±0.35.3±0.45.0±0.25.7±0.2*Urine Volume (ml per day)23±235±5.0#24±2.3*14±0.9*15±0.9*17±2.3* Protein (mg per day)22±226±319±214±2*12±1*15±2* Albumin (mg per day)14±319±57.2±1*5.7±2*2.8±2*4.9±2* Aldosterone (ng per day)41±565±1327±3*14±3*11±3*,#18±6* Na+ (mmol per day)1.6±0.11.8±0.22.3±0.3#1.3±0.21.3±0.1*1.5±0.1 K+ (mmol per day)3.4±0.24.0±0.33.3±0.23.2±0.23.2±0.23.5±0.2 Endothelin-1 (pg per day)12±19.8±1.45.4±0.8*,#8.5±1.210.8±1.110.5±1.1 cGMP (nmol/day)50±357±869±4#60±453±658±10Abbreviations: BW, body weight; cGMP, cyclic guanosine 3′5′ monophosphate; HW, heart weight; IRB, irbesartan; IRB+TH0.1, irbesartan+thiorphan 0.1 mg/kg per day; IRB+TH1.0, irbesartan+thiorphan 1.0 mg/kg per day; KW, kidney weight; TH0.1, thiorphan 0.1 mg/kg per day.Urine was collected on days 0 and 21 and plasma was collected on day 21. Data are mean±s.e.m. of n=8. #P<0.05 vs. baseline; *P<0.05 vs. vehicle. Open table in a new tab Abbreviations: BW, body weight; cGMP, cyclic guanosine 3′5′ monophosphate; HW, heart weight; IRB, irbesartan; IRB+TH0.1, irbesartan+thiorphan 0.1 mg/kg per day; IRB+TH1.0, irbesartan+thiorphan 1.0 mg/kg per day; KW, kidney weight; TH0.1, thiorphan 0.1 mg/kg per day. Urine was collected on days 0 and 21 and plasma was collected on day 21. Data are mean±s.e.m. of n=8. #P<0.05 vs. baseline; *P<0.05 vs. vehicle. Urinary cGMP, which reflects the renal effects of ANP20.Wong K.R. Xie M.H. Shi L.B. et al.Urinary cGMP as biological marker of the renal activity of atrial natriuretic factor.Am J Physiol. 1988; 255: F1220-F1224PubMed Google Scholar and may thus serve as a marker NEP inhibition efficacy, was increased in rats treated with thiorphan alone (Table 1). Yet, no increase was observed in the plasma levels of cGMP after thiorphan (Figure 2a). Irbesartan alone did not alter cGMP excretion or plasma cGMP, but prevented the rise in urinary cGMP excretion in combination with thiorphan (Table 1), and even lowered plasma cGMP in combination with the high thiorphan dose (Figure 2a). In agreement with the above concept,20.Wong K.R. Xie M.H. Shi L.B. et al.Urinary cGMP as biological marker of the renal activity of atrial natriuretic factor.Am J Physiol. 1988; 255: F1220-F1224PubMed Google Scholar changes in plasma ANP levels paralleled those in urinary cGMP excretion (Figure 2b). The generation of ANP from proANP is accompanied by the appearance of proANP's N-terminal end, NT-proANP. Since the latter is not degraded by NEP, its levels should better reflect ANP expression during NEP inhibition than the levels of ANP. NT-proANP levels were lowest during irbesartan+low-dose thiorphan and increased versus this condition when irbesartan was combined with the high dose of thiorphan (Figure 2c). Thiorphan alone decreased urinary ET-1 excretion (Table 1), without affecting plasma ET-1 levels (Figure 2d). Irbesartan, when given on top of thiorphan, normalized urinary ET-1 excretion, and increased plasma ET-1 when combined with the high thiorphan dose only (Figure 2d). Irbesartan alone did not affect circulating or urinary ET-1. Irbesartan, with or without thiorphan, increased plasma renin two- to three-fold, although significance was reached only for the combination of irbesartan and the high thiorphan dose (Figure 2e). Thiorphan alone did not affect plasma renin. Plasma prorenin (1727±117 pmol Ang I per mlper hour in vehicle-treated rats) was unaffected by all treatments (2250±176, 1710±170, 1534±145, and 1893±129 pmol Ang I per mlper hour after treatment with thiorphan, irbesartan, irbesartan+low-dose thiorphan, and irbesartan+high-dose thiorphan, respectively). Changes in plasma renin activity, Ang I, and Ang II paralleled those in plasma renin (Figure 2f and g), and the same was true for the changes in the renal tissue levels of Ang I, Ang-(1–9), Ang-(1–7), Ang-(1–5), and Ang-(2–10) (Table 2). Renal Ang II, Ang-(2–8), and Ang-(3–8) levels were largely unaltered and/or tended to decrease in the irbesartan-treated rats. As a consequence, the renal Ang II/I ratio decreased (in agreement with the concept that, after AT1 receptor blockade, Ang II can no longer accumulate in renal tissue via AT1 receptor binding; van Esch et al.21.van Esch J.H.M. Gembardt F. Sterner-Kock A. et al.Cardiac phenotype and angiotensin II levels in AT1a, AT1b and AT2 receptor single, double and triple knockouts.Cardiovasc Res. 2010; 86: 401-409Crossref PubMed Scopus (58) Google Scholar), the renal Ang-(1–7)/Ang II ratio increased, whereas the renal Ang-(1–7)/Ang I ratio was unchanged after irbesartan (Table 2).Table 2Renal levels of angiotensin metabolites and their ratios in kidneys of Ren2 rats treated with vehicle, TH0.1, IRB, IRB+TH0.1, or IRB+TH1.0RAS metabolite (pg/g)VehicleTH0.1IRBIRB+TH0.1IRB+TH1.0Ang-(1–10)=(Ang I)163±6979±16370±154568±66627±206Ang-(1–8)=(Ang II)388±36337±18288±65289±44328±47Ang-(1–7)20±318±248±2062±2272±33Ang-(1–5)13±49±116±523±422±7Ang-(2–8)26±318±230±1230±1032±8Ang-(3–8)5±0.35±0.15±0.85±0.46±0.6Ang-(2–10)30±1516±2.389±45123±23131±46Ang-(1–9)43±1220±338±1257±1663±12Ang-(1–7)/Ang I (× 100)18±327±416±210±313±3Ang-(1–7)/Ang II (× 100)5±15±0.514±320±5*20±6*Ang II/Ang I (× 100)437±109527±87191±6651±5*137±61*Abbreviations: Ang, angiotensin; IRB, irbesartan; IRB+TH0.1, irbesartan+thiorphan 0.1 mg/kg per day; IRB+TH1.0, irbesartan+thiorphan 1.0 mg/kg per day; TH0.1, thiorphan 0.1 mg/kg per day.Data are mean±s.e.m. of n=5–8.*P<0.05 vs. vehicle. Open table in a new tab Abbreviations: Ang, angiotensin; IRB, irbesartan; IRB+TH0.1, irbesartan+thiorphan 0.1 mg/kg per day; IRB+TH1.0, irbesartan+thiorphan 1.0 mg/kg per day; TH0.1, thiorphan 0.1 mg/kg per day. Data are mean±s.e.m. of n=5–8. *P<0.05 vs. vehicle. As expected, irbesartan decreased urinary aldosterone excretion versus baseline (Table 1), both with and without thiorphan, although significance for this effect was reached only in the low thiorphan group. This decrease in aldosterone was accompanied by a decrease in urinary Na+ excretion and a rise in plasma K+ levels (Table 1). Plasma Na+ levels and urinary K+ excretion were unchanged after irbesartan with or without thiorphan (Table 1). Of interest, thiorphan alone increased natriuresis, without altering urinary K+ or circulating Na+ and K+ (Table 1). Thiorphan alone did not lower aldosterone versus baseline (Table 1), although it did lower aldosterone excretion versus vehicle. The same was true for irbesartan under all conditions, and this is related to an unexpected rise in aldosterone excretion in the vehicle-treated group (Table 1). Vehicle treatment did not alter urinary protein and albumin excretion (Table 1). Irbesartan plus the low dose of thiorphan tended to decrease urinary albumin compared with baseline, but this was not significant. Thiorphan and irbesartan, both alone and in combination, lowered urinary albumin excretion compared with vehicle, whereas only irbesartan, independent of its combination with thiorphan, lowered urinary protein excretion (Table 1). There were no significant changes in plasma creatinine and creatinine clearance (Table 1). Irbesartan plus the high thiorphan dose increased the abundance of NHE3 more than twofold (Figure 3). In contrast, irbesartan alone or irbesartan plus the low thiorphan dose increased the abundances of α- and γ-ENaC almost twofold, but had no significant effect on NHE3. Aquaporin-2 (AQP2) increased in all rats treated with irbesartan, although this did not reach significance for the group treated with irbesartan plus the low dose of thiorphan. Urine osmolality changes paralleled the AQP2 increases (Figure 3). No differences were found in the abundances of the other kidney sodium transporters (Na-K-Cl cotransporter (NKCC2) and Na-Cl cotransporter (NCC)) or the aldosterone-sensitive proteins ubiquitin ligase NEDD4-2 (neural precursor cell expressed, developmentally downregulated protein 4-2) and SGK1 (serum- and glucocorticoid-regulated kinase 1). Focal and segmental glomerulosclerosis were only minimally present in the kidneys of the Ren2 rats, and the same was true for tubular injury (Supplementary Figure S1 online). No significant effects of the various drug combinations on these parameters were seen. Download .doc (2.29 MB) Help with doc files Supplementary Information In vehicle-treated Ren2 rats, acetylcholine (ACh) fully relaxed preconstricted mesenteric arteries. This relaxation is due to NO generation by NO synthase and intermediate+small conductance Ca2+-activated K+ channel activation by endothelium-derived hyperpolarizing factor(s) (EDHF). Indeed, the NO synthase inhibitor L-NAME, but not combined intermediate+small conductance Ca2+-activated K+ channel inhibition with TRAM34+apamin, partially blocked this effect, whereas all inhibitors together fully blocked the effect of ACh (Figure 4a). Results in all thiorphan-treated groups were identical to those in vehicle-treated rats, whereas only in the rats treated with irbesartan alone, TRAM34+apamin significantly shifted the ACh concentration–response curve (CRC) to the right and decreased Emax compared with vehicle-treated rats (pEC50 (the negative logarithm of the half-maximal effective concentration) 7.6±0.2 vs. 8.4±0.2 and Emax 73±8.0% vs. 91±1.4%, respectively; P<0.05 for both). Thus, irbesartan upregulated the EDHF component of the ACh–induced relaxation, and thiorphan reversed this effect. The endothelium-independent vasodilator S-nitroso-N-penicillamine, like ACh, fully relaxed preconstricted mesenteric arteries obtained from vehicle-treated rats, and this effect was unchanged in all treatment groups (Supplementary Figure S2 online). ET-1 potently constricted mesenteric arteries of vehicle-treated Ren2 rats (pEC50 8.4±0.2; Figure 4b), and the endothelin type A (ETA) receptor antagonist BQ123 (pEC50 7.8±0.1) prevented this effect. Similar results were obtained after all drug treatments. Simultaneously, the ETB receptor antagonist BQ788 marginally shifted the ET-1 CRC to the left in all treatment groups, except in the rats exposed to irbesartan plus the high dose of thiorphan, where the Emax after BQ788 was significantly lower than in the vehicle-treated group (142±8.4% vs. 202±18.4%, respectively; P<0.05). These data suggest that NEP inhibition, at its highest dose, abolished the vasodilatory effect of the ETB receptor. Thiorphan alone, but none of the other treatments, upregulated vascular ETA receptor mRNA expression (Figure 5a). A similar upregulation was observed for the vascular ETB receptor mRNA expression in all thiorphan-exposed rats, although this was significant only in the rats receiving irbesartan plus the highest thiorphan dose (Figure 5b). Combined with the absence of ETB receptor-induced vasodilation in this treatment group, these data suggest that the upregulation concerns constrictor ETB receptors. Only Ren2 rats treated with irbesartan plus the low thiorphan dose displayed a reduced heart weight (HW), HW/body weight ratio, and HW/tibia length ratio (Table 1). Cardiac ANP expression and cardiomyocyte size paralleled these changes (Figure 5c and e, see Supplementary Figure S3 online for representative images), whereas cardiac β-myosin heavy chain (β-MHC) expression was unaltered (Figure 5d). Unexpectedly, irbesartan increased the degree of cardiac fibrosis, whereas cardiac fibrosis in all other treatment groups was unaltered (Figure 5f, see Supplementary Figure S3 online for representative images). HW correlated positively with MAP (Supplementary Figure S4 online). This study shows that NEP inhibition has favorable effects on top of AT1 receptor blockade, but that this approach has a limit, that is, these effects disappear when increasing the degree of NEP inhibition. Indeed, a low dose of the NEP inhibitor thiorphan (0.1 mg/kg per day) potentiated the blood pressure-lowering effects of irbesartan in Ren2 rats, and this was accompanied by a significant reduction in cardiac hypertrophy, cardiomyocyte size, cardiac ANP expression, and vascular EDHF release. Increasing the thiorphan dose 10-fold abolished these effects. This most likely relates to the rise in circulating ET-1 with subsequent effects on kidney NHE3 abundance and vascular ETB receptor expression that occurred at such high doses, in agreement with previous literature.22.Kobayashi T. Miyauchi T. Sakai S. et al.Expression of endothelin-1, ETA and ETB receptors, and ECE and distribution of endothelin-1 in failing rat heart.Am J Physiol. 1999; 276: H1197-H1206PubMed Google Scholar,23.Peng Y. Amemiya M. Yang X. et al.ET(B) receptor activation causes exocytic insertion of NHE3 in OKP cells.Am J Physiol Renal Physiol. 2001; 280: F34-F42PubMed Google ScholarFigure 6 summarizes these observations. Thiorphan, when given alone at a dose of 0.1 mg/kg per day, did not affect blood pressure or cardiac hypertrophy. This is in full agreement with previous studies showing no such effects of NEP inhibition in human hypertension.24.Campbell D.J. Vasopeptidase inhibition: a double-edged sword?.Hypertension. 2003; 41: 383-389Crossref PubMed Scopus (115) Google Scholar Nevertheless, at this dose, thiorphan did increase circulating ANP, urinary cGMP, and natriuresis, without affecting circulating cGMP. These data confirm that urinary cGMP is largely of renal cellular origin, and correlates with ANP-induced natriuresis.20.Wong K.R. Xie M.H. Shi L.B. et al.Urinary cGMP as biological marker of the renal activity of atrial natriuretic factor.Am J Physiol. 1988; 255: F1220-F1224PubMed Google Scholar The latter effects of thiorphan were not seen on top of irbesartan, most likely because irbesartan, via its blood pressure-lowering effects, reduced atrial stretch, and thus diminished ANP release. Indeed, in combination with the high thiorphan dose, this even resulted in a significant decrease in circulating cGMP. Given the physiological antagonism between cGMP and ET-1, for example, with regard to cardiac hypertrophy,25.Irvine J.C. Ganthavee V. Love J.E. et al.The soluble guanylyl cyclase activator bay 58-2667 selectively limits cardiomyocyte hypertrophy.PLoS One. 2012; 7: e44481Crossref PubMed Scopus (45) Google Scholar this phenomenon may underlie the absence of favorable cardiac effects when combining irbesartan with the high thiorphan dose. Clinical trials evaluating the effects of the ARNI approach in heart failure (making use of LCZ696) observed a reduction in the plasma levels of NT-proBNP and a rise in BNP, despite the fact that both peptides are generated in equal amounts from proBNP.26.Packer M. McMurray J.J. Desai A.S. et al.Angiotensin receptor neprilysin inhibition compared with enalapril on the risk of clinical progression in surviving patients with heart failure.Circulation. 2015; 131: 54-61Crossref PubMed Scopus (487) Google Scholar The authors speculated that the decrease in NT-proBNP (which is not degraded by neprilysin) reflects reduced cardiac wall stress, whereas the rise in BNP reflects NEP inhibition. In the present study, we focused on ANP, given its greater susceptibility to degradation by NEP as compared with BNP.3.von Lueder T.G. Sangaralingham S.J. Wang B.H. et al.Renin–angiotensin blockade combined with natriuretic peptide system augmentation: novel therapeutic concepts to combat heart failure.Circ Heart Fail. 2013; 6: 594-605Crossref PubMed Scopus (109) Google Scholar In agreement with the above concept, plasma NT-proANP levels better reflected cardiac ANP expression than plasma ANP levels (see Figures 2c and 5c): rats receiving the high
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