Recombinant Expression and Characterization of Human and Murine ACE2: Species-Specific Activation of the Alternative Renin-Angiotensin-System

Angiotensin-converting enzyme 2 (ACE2) is a monocarboxypeptidase of the renin-angiotensin-system (RAS) which is known to cleave several substrates among vasoactive peptides. Its preferred substrate is Angiotensin II, which is tightly involved in the regulation of important physiological functions including fluid homeostasis and blood pressure. Ang 1–7, the main enzymatic product of ACE2, became increasingly important in the literature in recent years, as it was reported to counteract hypertensive and fibrotic actions of Angiotensin II via the MAS receptor. The functional connection of ACE2, Ang 1–7, and the MAS receptor is also referred to as the alternative axis of the RAS. In the present paper, we describe the recombinant expression and purification of human and murine ACE2 (rhACE2 and rmACE2). Furthermore, we determined the conversion rates of rhACE2 and rmACE2 for different natural peptide substrates in plasma samples and discovered species-specific differences in substrate specificities, probably leading to functional differences in the alternative axis of the RAS. In particular, conversion rates of Ang 1–10 to Ang 1–9 were found to be substantially different when applying rhACE2 or rmACE2 in vitro. In contrast to rhACE2, rm ACE2 is substantially less potent in transformation of Ang 1–10 to Ang 1–9

Profiling endogenous adrenal function during veno-venous ECMO support in COVID-19 ARDS: a descriptive analysis

BackgroundProlonged critical illness is often accompanied by an impairment of adrenal function, which has been frequently related to conditions complicating patient management. The presumed connection between hypoxia and the pathogenesis of this critical- illness- related corticosteroid insufficiency (CIRCI) might play an important role in patients with severe acute respiratory distress syndrome (ARDS). Since extracorporeal membrane oxygenation (ECMO) is frequently used in ARDS, but data on CIRCI during this condition are scarce, this study reports the behaviour of adrenal function parameters during oxygenation support with veno-venous (vv)ECMO in coronavirus disease 2019 (COVID-19) ARDS.MethodsA total of 11 patients undergoing vvECMO due to COVID-19 ARDS at the Medical University of Vienna, who received no concurrent corticosteroid therapy, were retrospectively included in this study. We analysed the concentrations of cortisol, aldosterone, and angiotensin (Ang) metabolites (Ang I–IV, Ang 1–7, and Ang 1–5) in serum via liquid chromatography/tandem mass spectrometry before, after 1 day, 1 week, and 2 weeks during vvECMO support and conducted correlation analyses between cortisol and parameters of disease severity.ResultsCortisol concentrations appeared to be lowest after initiation of ECMO and progressively increased throughout the study period. Higher concentrations were related to disease severity and correlated markedly with interleukin-6, procalcitonin, pH, base excess, and albumin during the first day of ECMO. Fair correlations during the first day could be observed with calcium, duration of critical illness, and ECMO gas flow. Angiotensin metabolite concentrations were available in a subset of patients and indicated a more homogenous aldosterone response to plasma renin activity after 1 week of ECMO support.ConclusionOxygenation support through vvECMO may lead to a partial recovery of adrenal function over time. In homogenous patient collectives, this novel approach might help to further determine the importance of adrenal stress response in ECMO and the influence of oxygenation support on CIRCI

Defining the role of the tissue-specific Renin-Angiotensin-System

Das Renin-Angiotensin-System (RAS) ist ein Peptid-Hormon-System, welches eine zentrale Rolle in der Salz-, Flüssigkeits- und Blutdruckregulation einnimmt. Ein dereguliertes RAS, gekennzeichnet durch eine Verschiebung der klassischen und alternativen RAS-Achse, wird mit Bluthochdruck, Herz- und Nierenversagen, Diabetes und sogar Krebs in Verbindung gebracht, und stellt daher ein attraktives Ziel für diverse Therapeutika dar. Eines der größten Probleme im Bereich der angewandten RAS-Forschung ist die extreme Varianz der publizierten RAS-Peptide-Konzentration, wodurch der biochemische Hintergrund des Systems verschleiert wird. Der Mangel an reproduzierbaren und genauen Methoden für die Bestimmung der Effektorpeptide und Enzyme des RAS erschwert deren Untersuchung und kann als das bislang größtes Hindernis in der RAS-Forschung betrachtet werden. Der Eckpfeiler dieser Dissertation war die Entwicklung einer neuen Methode, mit welcher das RAS auf Peptid- und Enzymebene in Geweben untersucht werden kann. Die neu zu entwickelnde Methode basiert auf der bereits bewährten LC-MS/MS Methode für die Plasma-Angiotensin-Messung und wurde im Mensch und Nager angewandt. Die zentrale Studie untersuchte das alternative RAS in der Niere und konnte zeigen, dass dessen Aktivierung nicht über ACE2, wie allgemein angenommen, sondern über NEP geschieht. Dies könnte großen Einfluss auf das neu zugelassene Herzmedikament LCZ696 haben, welches unter anderem NEP-Aktivität blockiert. Da herzinsuffiziente Patienten oftmals an Nierenerkrankungen leiden, könnten die therapeutischen Effekte im Herzen durch Nebenwirkungen in renal beeinträchtigten Patienten zunichte gemacht werden. Weiters konnten gewebsspezifische Unterschiede in humanen Nieren- und Herzbiopsien gezeigt werden. Während in der Niere das klassische RAS durch ACE gesteuert wird, zeigte sich in Herzbiopsien, dass dies durch Chymase geschieht, wodurch das RAS im Herzen insensitiv gegenüber den klinisch verwendeten ACE Inhibitoren ist. Im Gegensatz dazu konnte aber gezeigt werden, dass ACE Inhibitoren das alternative RAS besser aktivieren als Angiotensin-Rezeptor-Blocker, was möglicherweise Unterschiede beim Behandlungserfolg der Medikamente erklärt. Neben dem wertvollen Tool für weitere Forschungen liefert diese Dissertation auch neue Erkenntnisse über das gewebsspezifische RAS und seine medikamentöse Modulierung.The Renin-Angiotensin-System (RAS) is a proteolytic peptide cascade, which is ubiquitously present and primarily responsible for salt retention as well as involved in the regulation of blood pressure and fluid homeostasis. It has been suggested that a dysregulated RAS, indicated by an imbalance of the classical and alternative RAS axis, could be an essential pathogenic factor in various disease states including arterial hypertension, heart and kidney failure, diabetes and even cancer, making the system an attractive target for therapeutic intervention. One of the major issues in the fields of basic and clinical RAS research is the wide variance of reported levels of effector peptides, so-called angiotensins, leading to contradictory results and an incompletely understood molecular background of the RAS. The lack of reliable and reproducible methods to accurately quantify RAS enzymes and peptides makes the dissection of RAS in alternative and classical axis impossible and could be considered as one of the central drawbacks in RAS research in general. The cornerstone of this PhD thesis was the development of more accurate and state-of-the-art methods for RAS analysis of angiotensins and enzymes in tissue samples on the basis of the approved LC-MS/MS assay for plasma angiotensin measurements. The novel assays were employed in clinical and rodent tissue samples. The central study of the thesis revealed that NEP, but not ACE2, as previously assumed, activates the alternative RAS in the kidney. This could be highly relevant in regard of the novel heart failure drug LCZ696 which inhibits NEP activity. The LCZ696-mediated breakdown of the alternative RAS and its beneficial effects in the kidney might cause harmful effects, especially in heart failure patients suffering from renal comorbidities. Enzymatic tissue-specificities could also be observed in human renal and and cardiac biopsies. While the classical RAS axis is driven by ACE in the kidney, cardiac classical RAS activation is maintained by chymase, making the RAS in the heart insensitive to clinically used ACE inhibitors. In contrast, molecular investigation of RAS metabolism could visualize specific alternative RAS modulation by clinically used drugs, showing superiority of ACE inhibitors over angiotensin receptor blockers, which could explain the known differences in treatment outcomes. Apart from the valuable tool for further studies, this thesis also deepens our knowledge about the tissue specificity of the RAS and its potential therapeutic modulation.submitted by Oliver Domenig, BSc MScZusammenfassung in deutscher SpracheAbweichender Titel laut Übersetzung der Verfasserin/des VerfassersMedizinische Univesität Wien, Dissertation, 2016OeB

Suspected primary hyperreninism in a cat with malignant renal sarcoma and global renin-angiotensin-aldosterone system upregulation

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.This is the peer-reviewed version of the following article: Evans, Jeremy, Jessica Ward, Oliver Domenig, Jonathan P. Mochel, and Kate Creevy. "Suspected primary hyperreninism in a cat with malignant renal sarcoma and global renin‐angiotensin‐aldosterone system upregulation." Journal of Veterinary Internal Medicine (2021). DOI: 10.1111/jvim.16329. Attribution 4.0 International (CC BY 4.0). Copyright 2021 The Authors. Posted with permission

Comprehensive characterization of the effect of mineralocorticoid receptor antagonism with spironolactone on the renin-angiotensin-aldosterone system in healthy dogs

Objective – To characterize the dose-exposure-response effect of spironolactone on biomarkers of the classical and alternative arms of the renin-angiotensin-aldosterone system (RAAS) in healthy dogs. Animals – Ten healthy purpose-bred Beagle dogs. Procedures – Study dogs were randomly allocated to 2 spironolactone dosing groups (2 mg/kg PO q24hr, 4 mg/kg PO q24hr). The dogs received 7-day courses of spironolactone followed by a 14-day washout period in a crossover (AB/BA) design. Angiotensin peptides and aldosterone were measured in serum using equilibrium analysis, and plasma canrenone and 7-α-thiomethyl spironolactone (TMS) were quantified via liquid chromatography-mass spectrometry/mass spectroscopy (LC-MS/MS). Study results were compared before and after dosing and between groups. Results – Following spironolactone treatment, dogs had a significant increase in serum aldosterone concentration (P = 0.07), with no statistical differences between dosing groups. Significant increases in angiotensin II (P = 0.09), angiotensin I (P = 0.08), angiotensin 1–5 (P = 0.08), and a surrogate marker for plasma renin activity (P = 0.06) were detected compared to baseline following spironolactone treatment during the second treatment period only. Overall, changes from baseline did not significantly differ between spironolactone dosages. RAAS analytes were weakly correlated (R < 0.4) with spironolactone dosage and plasma canrenone or plasma TMS. There were no adverse clinical or biochemical effects seen at any spironolactone dosage during treatment. Conclusions – Treatment with spironolactone increased serum aldosterone concentration in healthy dogs and impacted other biomarkers of the classical and alternative arms of the RAAS. There was no difference in effect on the RAAS between 2 and 4 mg/kg/day dosing. Dosage of 4 mg/kg/day was safe and well-tolerated in healthy dogs.This is a manuscript of an article published as Masters, Allison K., Jessica L. Ward, Emilie Guillot, Oliver Domenig, Lingnan Yuan, and Jonathan P. Mochel. "Comprehensive characterization of the effect of mineralocorticoid receptor antagonism with spironolactone on the renin-angiotensin-aldosterone system in healthy dogs." (2023). DOI: 10.21203/rs.3.rs-2917558/v1. Copyright 2023 The Authors. Attribution 4.0 International (CC BY 4.0). Posted with permission

Molecular remodeling of the renin-angiotensin system after kidney transplantation

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-(17), Ang-(15), Ang-(28), Ang-(38)) 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-(17) 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.(VLID)456375