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Sodium glucose co-transporter-2 inhibitors (SGLT-2i) improve cardiorenal outcomes in patients with chronic kidney disease (CKD), with and without type 2 diabetes. The molecular mechanisms underlying these pleiotropic effects remain unclear, yet it is speculated that SGLT-2i elicit a neurohormonal modulation resulting in renin-angiotensin system (RAS) activation. We hypothesized that combined SGLT-2 and angiotensin-converting enzyme inhibition (ACEi) favours RAS regulation towards the beneficial angiotensin-(1-7)-driven axis.This randomized controlled prospective study investigated the effect of 12 weeks treatment with the SGLT-2i empagliflozin on top of ACEi on the molecular RAS dynamics in 24 diabetic and 24 non-diabetic patients with CKD. Systemic RAS peptides were quantified by mass spectrometry.In patients with type 2 diabetes, combined SGLT-2i and ACEi significantly upregulated plasma renin activity [pre-treatment median and interquartile range 298.0 (43.0-672.0) pmol/L versus post-treatment 577.0 (95.0-1543.0) pmol/L; p = .037] and angiotensin I levels [pre-treatment 289.0 (42.0-668.0) pmol/L versus post-treatment 573.0 (93.0-1522.0) pmol/L; p = .037], together with a significant increase of angiotensin-(1-7) levels [pre-treatment 14.0 (2.1-19.0) pmol/L versus post-treatment 32.0 (5.7-99.0) pmol/L; p = .012]. Empagliflozin treatment resulted in a 1.5 to 2-fold increase in main RAS peptides in patients with diabetes compared with placebo. No significant effect of empagliflozin on top of ACEi on RAS peptides was found in patients with CKD without diabetes.A distinct RAS modulation by SGLT-2i occurs in diabetic kidney disease reflected by enhancement of the beneficial angiotensin-(1-7) providing a molecular background for this renoprotective therapeutic approach.

A 14‐year‐old male castrated domestic medium‐hair cat with diabetes mellitus was evaluated for vomiting, diarrhea, and anorexia. Two weeks before presentation, the cat had been diagnosed with congestive heart failure and started on furosemide. Initial diagnostic testing identified hypokalemia, systemic hypertension, and hypertrophic cardiomyopathy phenotype, and plasma aldosterone concentration was moderately increased. Abdominal ultrasound examination disclosed bilateral adrenomegaly and a right renal mass, and cytology of a needle aspirate of the mass was consistent with malignant neoplasia. The cat was treated with amlodipine and spironolactone. Because of the unusual presentation for hyperaldosteronism, a comprehensive profile of renin‐angiotensin‐aldosterone system (RAAS) peptides was performed. Results from multiple timepoints indicated persistently and markedly increased plasma renin activity and generalized RAAS upregulation. In addition to the lack of adrenal tumor, the markedly increased plasma renin activity was atypical for primary hyperaldosteronism. These clinical findings are suggestive of primary hyperreninism, a condition previously unreported in cats. The concurrent presence of a renal neoplasm suggests the possibility of a renin‐secreting tumor.

Brain renin–angiotensin system (RAS) activation is thought to mediate deoxycorticosterone acetate (DOCA)-salt hypertension, an animal model for human primary hyperaldosteronism. Here, we determined whether brainstem angiotensin II is generated from locally synthesized angiotensinogen and mediates DOCA-salt hypertension. To this end, chronic DOCA-salt-hypertensive rats were treated with liver-directed siRNA targeted to angiotensinogen, the angiotensin II type 1 receptor antagonist valsartan, or the mineralocorticoid receptor antagonist spironolactone (n = 6–8/group). We quantified circulating angiotensinogen and renin by enzyme-kinetic assay, tissue angiotensinogen by Western blotting, and angiotensin metabolites by LC-MS/MS. In rats without DOCA-salt, circulating angiotensin II was detected in all rats, whereas brainstem angiotensin II was detected in 5 out of 7 rats. DOCA-salt increased mean arterial pressure by 19 ± 1 mmHg and suppressed circulating renin and angiotensin II by >90%, while brainstem angiotensin II became undetectable in 5 out of 7 rats (

The aldosterone/renin ratio (ARR) is currently considered the most reliable approach for case detection of primary aldosteronism (PA). ACE (Angiotensin-converting enzyme) inhibitors are known to raise renin and lower aldosterone levels, thereby causing false-negative ARR results. Because ACE inhibitors lower angiotensin II levels, we hypothesized that the aldosterone/equilibrium angiotensin II (eqAngII) ratio (AA2R) would remain elevated in PA. Receiver operating characteristic curve analysis involving 60 patients with PA and 40 patients without PA revealed that the AA2R was not inferior to the ARR in screening for PA. When using liquid chromatography-tandem mass spectrometry to measure plasma aldosterone concentration, the predicted optimal AA2R cutoff for PA screening was 8.3 (pmol/L)/(pmol/L). We then compared the diagnostic performance of the AA2R with the ARR among 25 patients with PA administered ramipril (5 mg/day) for 2 weeks. Compared with basally, plasma levels of equilibrium angiotensin I (eqAngI) and direct renin concentration increased significantly ( P P P

Background An angiotensin‐converting enzyme (ACE) gene polymorphism occurs in dogs; however, functional importance is not well studied. Hypothesis We hypothesized that dogs with the polymorphism would show alternative renin‐angiotensin aldosterone system (RAAS) pathway activation and classical RAAS pathway suppression before and after ACE‐inhibitor administration, as compared to dogs without the polymorphism that would show this pattern only after ACE‐inhibitor administration. Animals Twenty‐one dogs with mitral valve disease that were genotyped for the ACE gene polymorphism. Methods This retrospective study utilized stored samples from 8 ACE gene polymorphism‐negative (PN) dogs and 13 ACE gene polymorphism‐positive (PP) dogs before and after enalapril administration. Equilibrium analysis was performed to evaluate serum RAAS metabolites and enzyme activities. Results were compared before and after enalapril, and between groups. Results The classical RAAS pathway was suppressed and the alternative RAAS pathway was enhanced for both genotypes after administration of enalapril, with no differences before enalapril administration. Aldosterone breakthrough occurred in both PN (38%) and PP (54%) dogs despite angiotensin II suppression. Aldosterone was significantly higher (P = .02) in ACE gene PP dogs (median, 92.17 pM; IQR, 21.85‐184.70) compared to ACE gene PN dogs (median, 15.91 pM; IQR

Objective The branch of the renin--angiotensin system constituting angiotensin-(1-7) [Ang-(1-7)], the Ang II type 2 receptor, the Mas receptors and the Ang-(1-7)-forming enzyme ACE-2, by counteracting the Ang II type 1 receptor (AT1R)-mediated effects, are held to be cardiovascular protective in several conditions. However, whether Ang-(1-7) and ACE-2 are detectable in human adrenocortical tissues and whether they affect aldosterone and cortisol biosynthesis was unknown. Methods We measured angiotensin peptides with liquid chromatography tandem-mass spectrometry and ACE-2 mRNA with digital droplet (dd)PCR in human aldosterone-producing adenoma (APA) and APA-adjacent tissue obtained from patients with primary aldosteronism. We also investigated the effects of Ang-(1-7) and the ACE-2 activator diminazene aceturate (DIZE) on aldosterone synthase (CYP11B2) and 11β-hydroxylase (CYP11B1) gene expression, in the absence or presence of the AT1R antagonist irbesartan, or of the MasR antagonist A779. Results APA and APA-adjacent adrenocortical tissues express ACE-2 mRNA and contain detectable amounts of Ang II and Ang-(2-8), but not of Ang I, Ang-(1-5), Ang (3-8) and Ang-(1-7). Ang-(1-7) did not blunt CYP11B1 and CYP11B2 mRNA both under unstimulated and under Ang II-stimulated conditions. At supraphysiological concentrations (10-4 mol/l), Ang-(1-7) stimulated both CYP11B1 and CYP11B2 mRNA via the AT1R. The ACE-2 activator DIZE increased by 1.5-fold ACE-2 mRNA but did not blunt Ang II- upregulated CYP11B1 and CYP11B2 expression. Conclusion These results do not support the hypothesis that the ACE-2/Ang-(1-7)/MasR axis play a protective role by counteracting enhanced aldosterone secretion in humans.

Objective While evidence on the interface between severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection and the renin-angiotensin-aldosterone-system (RAAS) is accumulating, clinical data on RAAS peptide alteration among coronavirus disease-19 (COVID-19) patients is missing. Design and methods In this exploratory study, we prospectively included adult patients (aged ≥ 18 years) admitted between February 26 and April 30, 2020 to a tertiary care hospital in Switzerland. We assessed the association of an underlying SARS-CoV-2 infection and equilibrium serum levels of RAAS peptides in hospitalized COVID-19 patients 1:1 propensity-score matched with patients suffering from SARS-CoV-2-negative respiratory infections. Subgroup analyses involved stratification for taking RAAS inhibitors. Results COVID-19 patients had about 50% lower equilibrium serum RAAS peptide levels as compared with matched controls (angiotensin I: 31.6 vs 66.8 pmol/L, −52.7% (95%CI: −68.5% to −36.9%); angiotensin II: 37.7 vs 92.5 pmol/L, −59.2% (95%CI: −72.1% to −46.3%); angiotensin (1–5): 3.3 vs 6.6 pmol/L, −49.7% (95%CI: −59.2% to −40.2%); angiotensin (1–7): 4.8 vs 7.6 pmol/L, −64.9% (95%CI: −84.5% to −45.3%)). While the plasma renin activity was lower in COVID-19 patients (88.6 vs 207.9 pmol/L, −58.5% (95%CI: −71.4% to −45.6%)), there was no difference of angiotensin-converting enzyme (ACE) and ACE2 plasma activity between the groups. Subgroup analyses revealed a pronounced RAAS peptide profile depression in COVID-19 patients among those not on RAAS inhibitors. Conclusions As compared with SARS-CoV-2-negative patients, we found a downregulated RAAS in presence of a SARS-CoV-2 infection. Whether the lower levels of the protective angiotensin (1–5) and (1–7) are linked to adverse outcomes in COVID-19 warrants further investigation.

Rationale: Kidney homeostasis is critically determined by the coordinated activity of the renin-angiotensin system (RAS), including the balanced synthesis of its main effector peptides Ang (angiotensin) II and Ang (1–7). The condition of enzymatic overproduction of Ang II relative to Ang (1–7) is termed RAS dysregulation and leads to cellular signals, which promote hypertension and organ damage, and ultimately progressive kidney failure. ACE2 (angiotensin-converting enzyme 2) and NEP (neprilysin) induce the alternative, and potentially reno-protective axis by enhancing Ang (1–7) production. However, their individual contribution to baseline RAS balance and whether their activities change in chronic kidney disease (CKD) has not yet been elucidated. Objective: To examine whether NEP-mediated Ang (1–7) generation exceeds Ang II formation in the healthy kidney compared with diseased kidney. Methods and Results: In this exploratory study, we used liquid chromatography-tandem mass spectrometry to measure Ang II and Ang (1–7) synthesis rates of ACE, chymase and NEP, ACE2, PEP (prolyl-endopeptidase), PCP (prolyl-carboxypeptidase) in kidney biopsy homogenates in 11 healthy living kidney donors, and 12 patients with CKD. The spatial expression of RAS enzymes was determined by immunohistochemistry. Healthy kidneys showed higher NEP-mediated Ang (1–7) synthesis than Ang II formation, thus displaying a strong preference towards the reno-protective alternative RAS axis. In contrast, in CKD kidneys higher levels of Ang II were recorded, which originated from mast cell chymase activity. Conclusions: Ang (1–7) is the dominant RAS peptide in healthy human kidneys with NEP rather than ACE2 being essential for its generation. Severe RAS dysregulation is present in CKD dictated by high chymase-mediated Ang II formation. Kidney RAS enzyme analysis might lead to novel therapeutic approaches for CKD.

Dipeptidyl peptidase 3 (DPP3) is a zinc-dependent hydrolase involved in degrading oligopeptides with 4–12 amino acid residues. It has been associated with several pathophysiological processes, including blood pressure regulation, pain signaling, and cancer cell defense against oxidative stress. However, the physiological substrates and the cellular pathways that are potentially targeted by DPP3 to mediate these effects remain unknown. Here, we show that global DPP3 deficiency in mice (DPP3−/−) affects the renin–angiotensin system (RAS). LC–MS–based profiling of circulating angiotensin peptides revealed elevated levels of angiotensin II, III, IV, and 1–5 in DPP3−/− mice, whereas blood pressure, renin activity, and aldosterone levels remained unchanged. Activity assays using the purified enzyme confirmed that angiotensin peptides are substrates for DPP3. Aberrant angiotensin signaling was associated with substantially higher water intake and increased renal reactive oxygen species formation in the kidneys of DPP3−/− mice. The metabolic changes and altered angiotensin levels observed in male DPP3−/− mice were either absent or attenuated in female DPP3−/− mice, indicating sex-specific differences. Taken together, our observations suggest that DPP3 regulates the RAS pathway and water homeostasis by degrading circulating angiotensin peptides.

Primary aldosteronism is recognized as the most frequent cause of secondary hypertension, and its screening is expected to become a routine evaluation in most patients with hypertension. The interference of antihypertensive therapies with the aldosterone-to-renin ratio during screening process is a major confounder. Renin-angiotensin-aldosterone system Triple-A analysis is a novel liquid chromatography/tandem mass spectrometry diagnostic assay that allows simultaneous quantification of aldosterone, equilibrium Ang I (angiotensin I), and Equilibrium Ang II in a single sample of serum. We performed a comparative evaluation of the diagnostic performance of the aldosterone-to-Ang II ratio and 5 renin-based diagnostic ratios, differing in methods to determine aldosterone levels and renin activity in a cohort of 110 patients with hypertension (33 patients with confirmed primary aldosteronism and 77 with essential hypertension). All ratios showed comparable areas under the curves ranging between 0.924 and 0.970 without significant differences between each other. The evaluation of the Ang II-to-Ang I ratio revealed persistent drug intake in some patients as cause for suppressed renin-based diagnostic ratios, while aldosterone-to-Ang II ratio remained unaffected. The Youden index optimal cutoff value for the aldosterone-to-Ang II ratio was 6.6 ([pmol/L]/[pmol/L]) with a sensitivity of 90% and a specificity of 93%, proving noninferiority compared with the aldosterone-to-renin ratio while pointing to the potential for an interference-free application in patients under ACE (angiotensin-converting enzyme) inhibitor therapy. This study shows for the first time the accuracy and reliability of renin-angiotensin-aldosterone system triple-A analysis for the screening of primary aldosteronism that can be applied in clinical routine.

Rationale: Neurogenic hypertension is characterized by an increase in sympathetic activity and often resistance to drug treatments. We previously reported that it is also associated with a reduction of angiotensin-converting enzyme type 2 (ACE2) and an increase in a disintegrin and metalloprotease 17 (ADAM17) activity in experimental hypertension. In addition, while multiple cells within the central nervous system have been involved in the development of neurogenic hypertension, the contribution of ADAM17 has not been investigated. Objective: To assess the clinical relevance of this ADAM17-mediated ACE2 shedding in hypertensive patients and further identify the cell types and signaling pathways involved in this process. Methods and Results: Using a mass spectrometry-based assay, we identified ACE2 as the main enzyme converting angiotensin II into angiotensin-(1–7) in human cerebrospinal fluid. We also observed an increase in ACE2 activity in the cerebrospinal fluid of hypertensive patients, which was correlated with systolic blood pressure. Moreover, the increased level of tumor necrosis factor-α in those cerebrospinal fluid samples confirmed that ADAM17 was upregulated in the brain of hypertensive patients. To further assess the interaction between brain renin–angiotensin system and ADAM17, we generated mice lacking angiotensin II type 1 receptors specifically on neurons. Our data reveal that despite expression on astrocytes and other cells types in the brain, ADAM17 upregulation during deoxycorticosterone acetate–salt hypertension occurs selectively on neurons, and neuronal angiotensin II type 1 receptors are indispensable to this process. Mechanistically, reactive oxygen species and extracellular signal-regulated kinase were found to mediate ADAM17 activation. Conclusions: Our data demonstrate that angiotensin II type 1 receptors promote ADAM17-mediated ACE2 shedding in the brain of hypertensive patients, leading to a loss in compensatory activity during neurogenic hypertension.

Direct renin inhibition (DRI) is clinically inferior to other blockers of the renin-angiotensin system (RAS). Thus far, the underlying molecular causes of this finding remain unknown.Twenty four patients with non-diabetic chronic kidney disease (CKD) stages III-IV and albuminuria were randomized to DRI or angiotensin receptor blocker (ARB). Employing a novel mass-spectrometry method, the concentrations of renin, aldosterone and plasma angiotensin peptides [Ang I, Ang II, Ang-(1-7), Ang-(1-5), Ang-(2-8), Ang-(3-8)] were quantified before and after an 8-week treatment.While blood pressure, renal function and albuminuria decreased comparably in both groups, profound RAS component differences were observed: DRI led to a massive renin increase, while suppressing both vasoconstrictive (Ang I and Ang II) and vasodilatory RAS metabolites (Ang-(1-7) and Ang-(1-5)). In contrast, ARB led to a four-fold increase of Ang I and Ang II, while Ang-(1-7) and Ang-(1-5) increased moderately but significantly. With ARB treatment, a decreased aldosterone-to-Ang II ratio suggested efficacy in blocking AT1 receptor.DRI therapy abolishes all RAS effector peptides. ARB increases both vasoconstrictive and vasodilative angiotensins, while this is accompanied by efficient blockade of vasoconstrictive effects. These differential molecular regulations should be considered when selecting optimal antihypertensive and disease-modifying therapy in CKD patients. Key messages Direct renin inhibition leads to a complete and lasting abolition of both classical and alternative RAS components. Angiotensin receptor blockade leads to effective receptor blockade and up-regulation of alternative RAS components. Differential molecular regulations of the RAS should be considered when selecting optimal antihypertensive and disease-modifying therapy in CKD patients.

Deoxycorticosterone acetate (DOCA)-salt is suggested to increase blood pressure via selective upregulation of the brain renin-angiotensin system (RAS), while suppressing the circulating RAS. Yet, we have observed parallel downregulation of plasma and brain renin in mice. Here, we quantified brainstem angiotensinogen (AGT) and angiotensin (Ang) levels in rats exposed to DOCA (200mg; 60-day release) and 0.9% NaCl as drinking water for 7 weeks. To determine the contribution of AGT (liver vs. kidney/brain), Ang II type 1 receptors (AT1R) and blood pressure to tissue Ang II content, rats received vehicle, liver-targeted AGT siRNA (10 mg/kg fortnightly; s.c.), valsartan (31 mg/kg/day; s.c.) or spironolactone (80 mg/kg/day; s.c.) during the final 3 weeks, the latter fully normalizing blood pressure (n=3-9 per group). Plasma renin and AGT were determined by enzyme-kinetic assay, tissue AGT by western blotting, and Ang I and II by LC-MS/MS. Plasma renin, AGT, Ang I and II in rats not exposed to DOCA-salt were 24±8ng Ang I/mL per h, 731±51nM, 123±38pM and 109±43pM. AGT was present in liver, kidney and brainstem. Kidney Ang I and II were 540±135and 510±97fmol/g, while brainstem Ang I and II were undetectable in all and 50% of the rats, respectively (

New Findings What is the central question of this study? Is autonomic dysregulation in a mouse model of muscular dystrophy dependent on left ventricular systolic dysfunction and/or activation of the renin–angiotensin system (RAS) and does it predict development of dilated cardiomyopathy (DCM)? What is the main finding and its importance? The results demonstrate that autonomic dysregulation precedes and predicts left ventricular dysfunction and DCM in sarcoglycan-δ-deficient (Sgcd−/−) mice. The autonomic dysregulation is prevented by treatment of young Sgcd−/− mice with the angiotensin II type 1 receptor blocker losartan. Measurements of RAS activation and autonomic dysregulation may predict risk of DCM, and therapies targeting the RAS and autonomic dysregulation at a young age may slow disease progression in patients. Sarcoglycan mutations cause muscular dystrophy. Patients with muscular dystrophy develop autonomic dysregulation and dilated cardiomyopathy (DCM), but the temporal relationship and mechanism of autonomic dysregulation are not well understood. We hypothesized that activation of the renin–angiotensin system (RAS) causes autonomic dysregulation prior to development of DCM in sarcoglycan-δ-deficient (Sgcd−/−) mice and that the severity of autonomic dysfunction at a young age predicts the severity of DCM at older ages. At 10–12 weeks of age, when left ventricular function assessed by echocardiography remained normal, Sgcd−/− mice exhibited decreases in arterial pressure, locomotor activity, baroreflex sensitivity and cardiovagal tone and increased sympathetic tone compared with age-matched C57BL/6 control mice (P

Objective: We aimed at assessing the molecular adaptation of the renin-angiotensin system (RAS) after successful kidney transplantation (KTX). Materials and methods: In this prospective, exploratory study we analyzed 12 hemodialysis (HD) patients, who received a KTX and had excellent graft function six to 12 months thereafter. The concentrations of plasma Angiotensin (Ang) peptides (Ang I, Ang II, Ang-(1–7), Ang-(1–5), Ang-(2–8), Ang-(3–8)) were simultaneously quantified with a novel mass spectrometry-based method. Further, renin and aldosterone concentrations were determined by standard immunoassays. Results: Ang values showed a strong inter-individual variability among HD patients. Yet, despite a continued broad dispersion of Ang values after KTX, a substantial improvement of the renin/Ang II correlation was observed in patients without RAS blockade or on angiotensin receptor blocker (HD: renin/Ang II R2 = 0.660, KTX: renin/Ang II R2 = 0.918). Ang-(1–7) representing the alternative RAS axis was only marginally detectable both on HD and after KTX. Conclusions: Following KTX, renin-dependent Ang II formation adapts in non-ACE inhibitor-treated patients. Thus, a largely normal RAS regulation is reconstituted after successful KTX. However, individual Ang concentration variations and a lack of potentially beneficial alternative peptides after KTX call for individualized treatment. The long-term post-transplant RAS regulation remains to be determined.

Because of the presence of the blood–brain barrier, brain renin–angiotensin system activity should depend on local (pro)renin synthesis. Indeed, an intracellular form of renin has been described in the brain, but whether it displays angiotensin (Ang) I–generating activity (AGA) is unknown. Here, we quantified brain (pro)renin, before and after buffer perfusion of the brain, in wild-type mice, renin knockout mice, deoxycorticosterone acetate salt–treated mice, and Ang II–infused mice. Brain regions were homogenized and incubated with excess angiotensinogen to detect AGA, before and after prorenin activation, using a renin inhibitor to correct for nonrenin-mediated AGA. Renin-dependent AGA was readily detectable in brain regions, the highest AGA being present in brain stem (>thalamus=cerebellum=striatum=midbrain>hippocampus=cortex). Brain AGA increased marginally after prorenin activation, suggesting that brain prorenin is low. Buffer perfusion reduced AGA in all brain areas by >60%. Plasma renin (per mL) was 40× to 800× higher than brain renin (per gram). Renin was undetectable in plasma and brain of renin knockout mice. Deoxycorticosterone acetate salt and Ang II suppressed plasma renin and brain renin in parallel, without upregulating brain prorenin. Finally, Ang I was undetectable in brains of spontaneously hypertensive rats, while their brain/plasma Ang II concentration ratio decreased by 80% after Ang II type 1 receptor blockade. In conclusion, brain renin levels (per gram) correspond with the amount of renin present in 1 to 20 μL of plasma. Brain renin disappears after buffer perfusion and varies in association with plasma renin. This indicates that brain renin represents trapped plasma renin. Brain Ang II represents Ang II taken up from blood rather than locally synthesized Ang II.

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) formation in vivo has 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 activation in 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.

Background: Primary aldosteronism (PA) is a widely under-diagnosed, potentially curable and specifically treatable cause of hypertension. PA screening involves measuring the aldosterone-to-renin-ratio (ARR), but false negative results can occur in the setting of medications, which block the renin-angiotensin system (RAS). Withdrawing RAS blockers from patients with resistant hypertension is not without cardiovascular risk. A novel diagnostic approach, the aldosterone-to-angiotensin-II-ratio (AA2-Ratio), has the potential for less drug interference and improved reliability in PA screening and confirmation of diagnosis. Methods: Serum samples from 80 patients undergoing PA confirmation testing were analyzed. Sampling was performed in a recumbent (7 a.m.) and in an upright (10 a.m.) position before and after 4 days of oral administration of fludrocortisone and salt loading. The concentrations of renin, aldosterone and equilibrium Angiotensin-II were determined and ARR and AA2-Ratios were calculated. The interference of ACE-inhibition with the AA2-Ratio was investigated in healthy volunteers receiving 10mg enalapril daily for 8 days. Results: Renin concentration was undetectable in more than 40% of samples, while equilibrium Angiotensin-II was measurable in 98% of all 320 samples analyzed. Angiotensin-II levels were significantly higher in upright collected samples compared to samples collected in a recumbent position. Comparison of the ARR with the AA2-Ratio revealed a significantly larger diagnostic window for the AA2-Ratio. While the ARR was significantly suppressed by ACE-inhibitor treatment, the AA2-Ratio remained unaffected by ACE-inhibition. Conclusion: The AA2-Ratio may be superior to the ARR in PA screening among hypertensive patients. Equilibrium Angiotensin-II levels show expected responses to posture and appear to outperform renin concentration as a marker for RAS activation in terms of sensitivity, giving a measurable readout even in clinical states characterized by markedly suppressed RAS activity. The stability of the AA2-Ratio in the presence of ACE-inhibition points to a potential use of the AA2-Ratio PA screening in hypertensive patients without ACE-inhibitor discontinuation.

Background Blockade of the renin-angiotensin system (RAS) exerts beneficial effects in patients with mild-to-moderate chronic kidney disease, yet evidence suggesting a similar benefit in haemodialysis (HD) patients is not available. Furthermore, knowledge of the effects of RAS blockade on systemic RAS components in HD patients is limited. Analysis of the quantity and dynamics of all known peripheral constituents of the RAS may yield important pathomechanistic information of a widespread therapeutic measure in HD patients. Methods Fifty-two HD patients from the following groups were analysed cross-sectionally: patients without RAS blockade (n = 16), angiotensin-converting enzyme inhibitor (ACEi) users (n = 8), angiotensin receptor blocker (ARB) users (n = 11), patients on ACEi plus ARB (dual blockade, n = 8) and anephric patients (n = 9). Ten healthy volunteers served as controls. Angiotensin metabolites were quantified by mass spectrometry. Results In general, HD patients showed a broad variability of RAS activity. Patients without RAS blockade displayed angiotensin metabolite patterns similar to healthy controls. ACEi therapy increased plasma Ang 1-10 and Ang 1-7 concentrations, whereas ARB treatment increased both Ang 1-8 and Ang 1-5, while suppressing Ang 1-7 to minimal levels. Dual RAS blockade resulted in high levels of Ang 1-10 and suppressed levels of other angiotensins. Anephric patients were completely devoid of detectable levels of circulating angiotensins. Conclusion In HD patients, the activity status of the systemic RAS is highly distorted with the emergence of crucial angiotensin metabolites upon distinct RAS blockade. The characterization of molecular RAS patterns associated with specific RAS interfering therapies may help to individualize future clinical studies and therapies.

Background Angiotensin-converting enzyme (ACE) inhibitors (ACEis) are beneficial in patients with heart failure, yet their role after heart transplantation (HTx) remains ambiguous. Particularly, the effects of ACEis on plasma and cardiac metabolites of the "classical" and "alternative" renin-angiotensin system (RAS) in HTx patients are unknown. Methods This cross-sectional study used a novel mass spectrometry–based approach to analyze plasma and tissue RAS regulation in homogenates of heart biopsy specimens from 10 stable HTx patients without RAS blockade and in 15 patients with ACEi therapy. Angiotensin (Ang) levels in plasma and Ang formation rates in biopsy tissue homogenates were measured. Results Plasma Ang II formation is exclusively ACE dependent, whereas cardiac Ang II formation is primarily chymase dependent in HTx patients. ACEi therapy substantially increased plasma Ang-(1–7), the key effector of the alternative RAS, leaving plasma Ang II largely intact. Importantly, neprilysin and prolyl-carboxypeptidase but not angiotensin converting enzyme 2 are essential for cardiac tissue Ang-(1–7) formation. Conclusion ACE is the key enzyme for the generation of plasma Ang II, whereas chymase is responsible for cardiac tissue production of Ang II. Furthermore, our findings reveal that neprilysin and prolyl-carboxypeptidase are the essential cardiac enzymes for the alternative RAS after HTx. These novel insights into the versatile regulation of the RAS in HTx patients might affect future therapeutic avenues, such as chymase and neprilysin inhibition, beyond classical Ang II blockade.

Primary aldosteronism (PA) is severe form of hypertension characterized by a strongly increased aldosterone secretion mediated by adenomas or other forms of adrenal hyper-activity. Once detected, PA can be usually cured by either surgical intervention or by appropriate pharmacologic treatments. This is also reflected in clinical guidelines of Endocrine Societies in Europe and the US, suggesting extensive PA screening activities among resistant hypertensive patients. The incidence of PA among hypertensive patients varies strongly between different studies, which is in part caused by the complex state-of-the-art testing procedure that unfortunately is far away from being a versatile PA screening tool. Despite strong limitations regarding selectivity, sensitivity and the interference with multiple anti-hypertensive drugs, the aldosterone-renin-ratio (ARR) is widely used for PA case detection. However, there is still a strong demand for accurate and reliable and patient friendly PA case detection. The use of novel and more accurate technologies for quantification of aldosterone and renin activity might help to improve the power of the ARR as a diagnostic tool for PA. However, there is a big need for a versatile PA screening assay that doesn’t interfere with anti-hypertensive treatments and therefore allows the clear identification of PA patients without complex corrections and adaptions being necessary and without increasing the patient’s cardiovascular risk in the course of the diagnostic process. The Aldosterone-to-Angiotensin-II-Ratio (AA2-Ratio) is a novel mass-spectrometry based high-throughput test for PA that combines the plasma levels of aldosterone and physiologically active angiotensin II into a diagnostic ratio. The test performance is superior to the ARR in terms of the diagnostic window and method accuracy. The AA2-Ratio does not interfere with standard anti-hypertensive drugs including ACE inhibitors. First data obtained in a proof-of-concept study investigating PA positive and negative patients proved the AA2-Ratio to be a powerful and cost-effective diagnostic tool for the diagnosis of PA in clinical practice.