Cardiovascular and renal pathologies are frequently associated with an activated renin-angiotensin-system (RAS) and increased levels of its main effector and vasoconstrictor hormone angiotensin II (Ang II). Angiotensin-converting-enzyme-2 (ACE2) has been described as a crucial enzymatic player in shifting the RAS towards its so-called alternative vasodilative and reno-protective axis by enzymatically converting Ang II to angiotensin-(1-7) (Ang-(1-7)). Yet, the relative contribution of ACE2 to Ang-(1-7) formationin vivohas not been elucidated. Mass spectrometry based quantification of angiotensin metabolites in the kidney and plasma of ACE2 KO mice surprisingly revealed an increase in Ang-(1-7), suggesting additional pathways to be responsible for alternative RAS activationin vivo. Following assessment of angiotensin metabolism in kidney homogenates, we identified neprilysin (NEP) to be a major source of renal Ang-(1-7) in mice and humans. These findings were supported by MALDI imaging, showing NEP mediated Ang-(1-7) formation in whole kidney cryo-sections in mice. Finally, pharmacologic inhibition of NEP resulted in strongly decreased Ang-(1-7) levels in murine kidneys. This unexpected new role of NEP may have implications for the combination therapy with NEP-inhibitors and angiotensin-receptor-blockade, which has been shown being a promising therapeutic approach for heart failure therapy.

The renin-angiotensin-aldosterone system (RAAS) is a dominant player in several cardiovascular pathologies. This study investigated whether alterations induced by l-NAME, (NLG)-nitro-l-arginine methyl ester, a nitric oxide synthase inhibitor, and the protective effect of melatonin are associated with changes in the RAAS. Four groups of 3-month-old male Wistar rats (n = 10) were treated as follows for four weeks: untreated controls, rats treated with melatonin (10 mg/kg/day), rats treated with l-NAME (40 mg/kg/day), and rats treated with l-NAME + melatonin. l-NAME administration led to hypertension and left ventricular (LV) fibrosis in terms of enhancement of soluble, insoluble and total collagen concentration and content. Melatonin reduced systolic blood pressure enhancement and lowered the concentration and content of insoluble and total collagen in the LV. The serum concentration of angiotensin (Ang) 1–8 (Ang II) and its downstream metabolites were reduced in the l-NAME group and remained unaltered by melatonin. The serum aldosterone level and its ratio to Ang II (AA2-ratio) were increased in the l-NAME group without being modified by melatonin. We conclude that l-NAME-hypertension is associated with reduced level of Ang II and its downstream metabolites and increased aldosterone concentration and AA2-ratio. Melatonin exerts its protective effect in l-NAME-induced hypertension without affecting RAAS.

Ivabradine, the selective inhibitor of the If current in the sinoatrial node, exerts cardiovascular protection by its bradycardic effect and potentially pleiotropic actions. However, there is a shortage of data regarding ivabradine’s interaction with the renin-angiotensin-aldosterone system (RAAS). This study investigated whether ivabradine is able to protect a hypertensive heart in the model of L-NAME-induced hypertension and to interfere with the RAAS. Four groups (n = 10/group) of adult male Wistar rats were treated as follows for four weeks: control, ivabradine (10 mg/kg/day), L-NAME (40 mg/kg/day), and L-NAME plus ivabradine. L-NAME administration increased systolic blood pressure (SBP) and left ventricular (LV) weight, enhanced hydroxyproline concentration in the LV, and deteriorated the systolic and diastolic LV function. Ivabradine reduced heart rate (HR) and SBP, and improved the LV function. The serum concentrations of angiotensin Ang 1–8 (Ang II), Ang 1–5, Ang 1–7, Ang 1–10, Ang 2–8, and Ang 3–8 were decreased in the L-NAME group and ivabradine did not modify them. The serum concentration of aldosterone and the aldosterone/Ang II ratio were enhanced by L-NAME and ivabradine reduced these changes. We conclude that ivabradine improved the LV function of the hypertensive heart in L-NAME-induced hypertension. The protective effect of ivabradine might have been associated with the reduction of the aldosterone level.

Widespread application of the plasma aldosterone/renin ratio (ARR) as a screening test has led to the recognition that primary aldosteronism (PA) is the most common specifically treatable and potentially curable form of hypertension, accounting for 5–10% of patients. Maximal detection requires accurate diagnostic approaches and awareness and control of factors that confound results, including most antihypertensives, posture, time of day, dietary salt, and plasma potassium. Recent studies have revealed potential for false positives in patients on beta-adrenoceptor blockers, and, when direct renin concentration (but not plasma renin activity) is used to measure renin, in women during the luteal phase of the menstrual cycle or receiving estrogen-containing contraceptives or hormonal replacement therapy. In addition to verapamil slow release, hydralazine and prazosin, moxonidine has minimal effects on the ARR and can be used to control hypertension during work-up. Fludrocortisone suppression testing, while probably the most reliable means of definitively confirming or excluding PA, is time consuming and expensive, requiring a five day inpatient stay. A novel approach, upright (seated) saline infusion suppression testing (SST), has shown excellent reliability with much greater sensitivity than conventional recumbent SST in a recent pilot study, and requires only a day visit. Accurate measurement of aldosterone is essential for each step of PA workup: introduction of new, highly reliable high-throughput mass spectrometric methods into clinical practice has represented a major advance. In response to concerns raised about accuracy of renin assays, new mass spectrometric methods for measuring angiotensin II are currently being assessed in the clinical setting.

Angiotensin-converting enzyme inhibitors (ACEis) are beneficial in patients with chronic kidney disease (CKD). Yet, their clinical effects after kidney transplantation (KTx) remain ambiguous and local renin-angiotensin system (RAS) regulation including the ‘classical’ and ‘alternative’ RAS has not been studied so far. Here, we investigated both systemic and kidney allograft-specific intrarenal RAS using tandem mass-spectrometry in KTx recipients with or without established ACEi therapy (n = 48). Transplant patients were grouped into early (12 years). Patients on ACEi displayed lower angiotensin (Ang) II plasma levels (P < 0.01) and higher levels of Ang I (P < 0.05) and Ang-(1–7) (P < 0.05) compared to those without ACEi independent of graft vintage. Substantial intrarenal Ang II synthesis was observed regardless of ACEi therapy. Further, we detected maximal allograft Ang II synthesis in the late transplant vintage group (P < 0.005) likely as a consequence of increased allograft chymase activity (P < 0.005). Finally, we could identify neprilysin (NEP) as the central enzyme of ‘alternative RAS’ metabolism in kidney allografts. In summary, a progressive increase of chymase-dependent Ang II synthesis reveals a transplant-specific distortion of RAS regulation after KTx with considerable pathogenic and therapeutic implications.

Combined inhibition of NEP (neutral endopeptidase) and ACE (angiotensin-converting enzyme), without unwanted effects, remains an attractive therapeutic strategy in cardiovascular medicine. Omapatrilat, a dual NEP inhibitor–ACE inhibitor, was a promising antihypertensive drug but failed in trials due to angioedema, an effect possibly caused by inhibition of both the N- and C-domains of ACE. Here, we aimed to determine whether lisinopril-tryptophan (lisW-S), a C-domain specific ACE inhibitor that preserves the N-domain catalytic activity, together with sacubitril (NEP inhibitor), differentially influences cardiovascular function and vascular permeability in hypertension compared with omapatrilat and lisinopril+sacubitril which inhibits both the ACE C- and N-domains. Ang II (angiotensin II)–dependent hypertensive mice (transgenic mice expressing active human renin in the liver [also known as LinA3]) received vehicle, sacubitril, lisW-S, lisinopril, lisinopril+sacubitril, or lisW-S+sacubitril for 4 weeks. Systolic blood pressure was increased in LinA3 mice, along with cardiac hypertrophy/dysfunction, impaired endothelium-dependent vasorelaxation, hypercontractile responses, vascular remodeling, and renal inflammation. LisW-S+sacubitril, lisinopril+sacubitril, and omapatrilat reduced systolic blood pressure and normalized cardiovascular remodeling and vascular hypercontractile responses in LinA3 mice. Although lisinopril+sacubitril and omapatrilat improved Ach-induced vasorelaxation, lisW-S+sacubitril had no effect. Endothelial permeability (Evans Blue assessment) was increased in omapatrilat but not in LisW-S+sacubitril–treated mice. In conclusion, lisW-S combined with sacubitril reduced systolic blood pressure and improved cardiac dysfunction in LinA3 mice, similar to omapatrilat but without effects on endothelium-dependent vasorelaxation. Moreover, increased vascular leakage (plasma extravasation) induced by omapatrilat was not evident in mice treated with lisW-S+sacubitril. Targeting ACE C-domain and NEP as a combination therapy may be as effective as omapatrilat in lowering systolic blood pressure, but without inducing vascular permeability and endothelial injury.