Apelin-13 in septic shock: effective in supporting hemodynamics in sheep but compromised by enzymatic breakdown in patients

Sepsis is a prevalent life-threatening condition related to a systemic infection, and with unresolved issues including refractory septic shock and organ failures. Endogenously released catecholamines are often inefficient to maintain blood pressure, and low reactivity to exogenous catecholamines with risk of sympathetic overstimulation is well documented in septic shock. In this context, apelinergics are efficient and safe inotrope and vasoregulator in rodents. However, their utility in a larger animal model as well as the limitations with regards to the enzymatic breakdown during sepsis, need to be investigated. The therapeutic potential and degradation of apelinergics in sepsis were tested experimentally and in a cohort of patients. (1) 36 sheep with or without fecal peritonitis-induced septic shock (a large animal experimental design aimed to mimic the human septic shock paradigm) were evaluated for hemodynamic and renal responsiveness to incremental doses of two dominant apelinergics: apelin-13 (APLN-13) or Elabela (ELA), and (2) 52 subjects (33 patients with sepsis/septic shock and 19 healthy volunteers) were investigated for early levels of endogenous apelinergics in the blood, the related enzymatic degradation profile, and data regarding sepsis outcome. APLN-13 was the only one apelinergic which efficiently improved hemodynamics in both healthy and septic sheep. Endogenous apelinergic levels early rose, and specific enzymatic breakdown activities potentially threatened endogenous apelin system reactivity and negatively impacted the outcome in human sepsis. Short-term exogenous APLN-13 infusion is helpful in stabilizing cardiorenal functions in ovine septic shock; however, this ability might be impaired by specific enzymatic systems triggered during the early time course of human sepsis. Strategies to improve resistance of APLN-13 to degradation and/or to overcome sepsis-induced enzymatic breakdown environment should guide future works.

Sepsis is a dysregulated host response to infection that includes a combination of heterogeneous and inflammatory events 1 . Septic shock with multiple organ failure is a major issue of sepsis resulting in elevated death rates. Associated cardiovascular dysfunction is one of the three main acute organ dysfunctions related with shortterm mortality 2 . Endogenous stress-released vasopressor catecholamines spilled into the bloodstream during the initial inflammatory storm in septic shock are ineffective. Those have reduced signaling capacity because they are oxidized by the reactive oxygen species-rich environment and associated with diminished downstream adrenergic receptor sensitivity 3,4 . Strategies aimed at improving the catecholamine-to-adrenergic receptor ratio and potentially countering this ineffectiveness with exogenous unoxidized catecholamines are often similarly inefficient, leading to sympathetic overstimulation and ultimately harmful effects [5][6][7] .
Alternative noncatecholaminergic therapies therefore represent new avenues for medical interventions in sepsis. The apelin system, mediator of cardiovascular control, is one such option, and it has been reported to have valuable cardiovascular support properties (i.e., cardiac inotropy, vasoregulation, fluid homeostasis, and renal protection) in experimental and clinical research 8 . The recognized endogenous agonists (hereinafter, "apelinergics") of the only known specific G protein-coupled receptor (GPCR) in the apelin system (named APJ; gene symbol, APLNR) are (1) apelins (APLNs), namely, the APLN-36, APLN-17, APLN-13, and APLN-12 amino acid isoforms, of which the pyroglutamate-modified form of APLN-13 (pyr-APLN-13) is the dominant form in the cardiovascular system 9 ; and (2) Elabela (ELA) (32 amino acids), which has also been described as an important regulator of cardiovascular function 10 . Infusion of these molecules has been shown to produce cardioprotective effects and to stabilize hemodynamics in rodent models of heart failure 11,12 and sepsis-induced myocardial dysfunction 13 , promoting improved survival and exhibiting a good safety profile 14 . However, reported spontaneous increases of APLN-13 levels in the blood have been modest in the acute phase of heart failure 15 as well as in cardiovascular dysfunction induced by sepsis 16 , suggesting inadequate reactivity of the apelin system or accelerated degradation/clearance of effective peptides. In sepsis, knowledge is lacking on how this apelin system is reactive; if it has therapeutic potential in larger animal models, and what is potentially affecting the homeostasis of the apelinergics in this context. Indeed, although nothing is known about the enzymatic breakdown pathways specific to ELA, apelins are recognized to be small peptides that are very susceptible to degradation and corresponding loss of function 17 . More specifically, members of the renin-angiotensin (RAS) and kallikrein-kinin (KKS) systems, i.e., angiotensinconverting enzyme type II (ACE2) 18,19 , kallikrein (KLK1) 20 , and neprilysin (NEP) 21,22 , can degrade native apelin isoforms and affect bioavailability, with varying impacts on activity. Consequently, e.g., through increased apelin levels with consecutive enhanced endogenous homeostasis and hemodynamic responsiveness, inhibition of the RAS system could lead to better shock outcomes. De facto, chronic use of a RAS inhibitor for cardiovascular protection-by patients has been reported to improve septic shock outcomes 23,24 .
The main hypotheses of this work were that (i) exogenously infused apelinergics would ameliorate the initial hemodynamic instability in a large animal model of acute experimental septic shock and that (ii) in humans, the endogenous apelin system is reactive in early septic shock but potentially inactivated by enzymatic degradation, which can compromise its hemodynamic impact. To test these hypotheses, (1) we evaluated the hemodynamic dose-response to apelinergic infusion in a sheep model of septic shock induced by fecal peritonitis (FP), and (2) we assessed the reactivity of the endogenous apelin system and its related enzymatic degradation pathway in Apelin-13 (APLN-13) improves cardiorenal axis function in an ovine model of polymicrobial peritonitis mimicking human septic shock. Experimental design for hemodynamic monitoring in sheep with FP-induced acute septic shock is presented in Fig. 2A. Shock criteria were met 251 ± 15 min after FP induction and accompanied by several ensuing disorders (i.e., increased, lactate, troponin T (Tn T), cortisol and arginine-vasopressin (AVP) circulating levels as well as KIM-1 urinary levels or reduced ScvO2, base excess, creatinine clearance or oxygen consumption (VO 2 )) ( Table 1). The average sheep Sequential Organ Failure Assessment (SOFA) score was 10.1 ± 1.5.
Sustained translocation of gram-negative bacilli (Serratia marcescens, Klebsiella oxytoca, Citrobacter freundii, and Pseudomonas aeruginosa) was revealed by blood culture performed one and two hours after FP induction (data not shown).
The apelinergic release-degradation network is dysregulated in early sepsis/septic shock. Thirty-three out of 45 eligible patients with sepsis agreed to participate in this study (9 were excluded because informed consent was refused or not obtained, and 3 were excluded because of unavailability of the recruitment staff). Calculated and effective outcomes, types of ICU admission, sepsis categories, sources of infections, and cyto-biochemical characteristics are shown in Table 2.
The plasma stability of exogenously added APLN-13 and ELA was lower in septic plasma than in healthy plasma in vitro, suggesting enhanced degradation in the sepsis environment. The ELA half-life was shortened to a greater extent and reached more rapidly than that of APLN-13 (APLN-13, sepsis: 143 ± 20 min, healthy volunteers: 194 ± 15 min, p = 0.045; ELA, sepsis: 69 ± 12 min, healthy volunteers: 258 ± 45 min, p = 0.003) (Fig. 4A,B). Nevertheless, septic plasma exhibited higher levels of APLNs and ELA (Fig. 4C,D) and lower ACE2 activity but higher ACE1 (data not shown), NEP, and KLK1 enzymatic activities than plasma from healthy volunteers ( Fig. 4E-G). The ACE1/2 activity ratio was thus further enhanced in septic plasma (Fig. 4H) and associated with patient negative outcome (nonsurvivors, n = 10: 62.1 [6.3-77.9] vs. survivors, n = 23: 8.8 [0.7-26.9], p = 0.035). In addition, the decreased APLN-13 half-life was associated with increased NEP and KLK1 enzymatic activities (Additional file: Fig. S3A and B). Moreover, higher NEP but not ACE2 activity was associated with higher www.nature.com/scientificreports/ (A) Study Design: Sheep were first prepared as described in Fig. 1. After baseline assessment, fecal peritonitis was induced by intraperitoneal injection of a stool slurry (2 g/kg) and five criteria of shock must be met before fluid resuscitation challenge (RL 30 mL/kg for 1 h) and start of infusions of 20 min-incremental doses of APLN-13 or Elabela (ELA) vs. normal saline (NS), as described in Fig. 1  Principal component analysis (PCA) was performed on 15 intercorrelated continuous variables related to sepsis outcomes and apelin system metabolism, revealing two principal subspaces (PC1 and PC2) accounting for 19.46 and 23.94%, respectively (Fig. 4I). PCA showed a strong association among the following variables: arterial lactate levels, circulating creatinine and cortisol levels, and Acute Physiology and Chronic Health Evaluation (APACHE) II and SOFA scores, which altogether defined the PC1 axis. A second relationship among circulating apelin, ELA, and Ang1-7 levels and ACE1, KLK1, and NEP activities defined the PC2 axis. In an individual dispersion graph, sepsis nonsurvivors were widely distributed in the positive area of the PC1 and PC2 axes, suggesting that worse sepsis outcomes could have been influenced by the profile of the apelinergic release-degradation network in critically ill patients (Fig. 4J).

Discussion
The specific aim of this work was to investigate the hemodynamic impact of apelinergic infusion in a sheep model of septic shock, and to assess the reactivity of the endogenous apelin system and its related enzymatic degradation environment in patients with acute septic shock. This study is the first reporting a positive hemodynamic impact of APLN-13 infusion in a large animal model of septic shock and revealing simultaneous intricate homeostatic disturbances among the renin-angiotensin, kallikrein-kinin, and apelin systems involved in outcomes of acute human sepsis.
Given the therapeutic potential of adding exogenous apelinergics described in studies of sepsis in rodents 13,14 , the hemodynamic impact of administering incremental doses of APLN-13 or ELA was studied in healthy and septic sheep. Sheep are a significant large animal species with similar organ size to humans, and thus advantageous in modelling human sickness and to trial new therapies. A previous study described biphasic MAP and heart rate responses to a large intravenous (i.v.) bolus injection of APLN-13, showing that sheep were responsive to apelins 25 . Herein, although it was found that human APLN-13 and ELA bind ovine APJ, only APLN-13 acted www.nature.com/scientificreports/ www.nature.com/scientificreports/ as a positive inotrope upon infusion in healthy sheep, while ELA had cardiodepressant and hypotensive effects. These discrepant hemodynamic responses were unexpected based on previous studies reporting similar effects of ELA and APLN-13 in rodents 13,26 . Compared with that of APLN-13, the structure-activity relationship of ELA related to reduced APJ-dependent activation of G αi1 and recruitment of β-arrestin-2 could explain, at least in part, the different hemodynamic profiles observed in sheep. These findings will need consideration with regards to future development relating to human disease. Beyond being efficient in healthy condition, candidate apelinergics must be operating in disease. The characteristics of this ovine experimental model of sepsis are consistent with past reports in sheep 27,28 and compatible with human disease. This includes (1) compromised hemodynamics that is poorly fluid responsive; (2) an acute onset of multiple organ injury; and (3) metabolic perturbations with decreased systemic oxygen consumption. Concomitantly, circulating APLN levels but not ELA levels increased, suggesting defective paracrine release of ELA, with a contrasting decrease in APLN mRNA expression in the cardiorenal tissue axis. Of note, ELA mRNA expression was not detected in these ovine tissues, in contrast with the specific and almost exclusive renal expression of ELA reported in rats 29 . However, the plasma stability of APLN-13 and ELA was unaffected, and the low level of enzymatic degradation is probably related to the short-timeframe design of this ovine model.
According to evidence from this study and the literature, APLN-13 infusion is effective in improving cardiorenal functions without significant chronotropic changes but with a favorable cardiac energy balance and protection; and could also act as a levosimendan-like inotrope with additional anti-inflammatory; calcium sensitizing and antioxidative properties 13,30 . In contrast, while ELA showed interesting and promising hemodynamic effects with improved fluid homeostasis in a model of peritonitis in rats 13 , no beneficial effect was observed in the present setting. This highlights that the apelin system is complex and how these findings cannot be easily applied across species.
Physiological regulation of fluid homeostasis is also a critical event in hemodynamic stability and is a matter of balance between the apelin and vasopressin systems 31 . A direct counteracting effect of APLN on the antidiuretic activity of AVP can be observed in collecting ducts, modulating the docking of aquaporin-2 (AQP-2) and thus enhancing urinary output 32 . Herein, increased diuresis and improved creatinine clearance were also observed with APLN-13 infusion in septic sheep, confirming the reported effects regarding sepsis-induced reversal of blood AVP elevation, improved fluid clearance, and a negative fluid balance in rodents 13 . Overall, the APLN-13-driven beneficial effects on kidney function in sepsis seem to be closely related to its physiological interaction with the vasopressin system. This specific network is thus an important link that should be considered in future studies because fluid homeostasis and kidney function are both key issues in acute sepsis.
Whether the observed responsiveness of the apelin system in human sepsis and septic shock is appropriate is, however, unclear. Previously, we were the first to report elevated APLN-13 blood concentrations in septic shock patients, as well as in critically ill nonseptic patients 16 . In this study, considering all isoforms and degradation moieties, the increase in APLN-13 blood concentrations was higher than that previously reported, averaging more than threefold the concentrations documented in healthy volunteers, which rivals the fold increase in catecholamine levels in a similar context [33][34][35] . Notably, quantified ELA blood concentrations showed a smaller 1.5-fold increase in septic patients. For unclear reasons, one-fourth and one-fifth of the septic patients in this cohort remained "unreactive", with normal ranges of circulating APLNs and ELA, respectively. This could mean that patients should be screened to select those most likely to benefit from an apelinergic therapy.
A comprehensive analysis of apelinergic blood levels requires examination of the breakdown environment. Activated RAS and KKS proteases have been associated with both accelerated protein/peptide degradation and microvascular dysfunction with multiple organ failure in sepsis and septic shock 36,37 . While the process of ELA enzymatic degradation remains essentially unknown, biologically less active or fully inactive APLN moieties can be generated by RAS-and KKS-dependent proteolysis through ACE2, NEP and KLK1 hydrolysis 20 . Indeed, exogenous APLN-13 or ELA incubated with septic plasma displayed a further shortened half-life ex vivo. Prehospital use of ACE1 inhibitors has been linked to less need for vasopressor administration and better outcomes in septic shock-may be through higher levels of APLN-13 before the onset of sepsis 38,39 . However, ACE1 blockade may be associated with sepsis-induced endothelial dysfunction and the resulting AngII deficiency related to refractory shock and impaired outcomes 40 .
Under such conditions, an alternative bypass toward a nonclassical RAS pathway producing Ang1-7 under predominantly NEP-driven activity has been suggested 41 . A bloodstream phenotype of RAS activation (i.e., high ACE1/ACE2 ratio and NEP activity), along with Ang1-7 release, was found in human sepsis. Moreover, KKS has been described as a specific node linking the inflammatory and coagulation responses to microbial infection and thus contributing to the pathogenesis of sepsis 42 . The biological efficiency of the apelin system is therefore compromised under this environmental pressure, given the very short half-lives of endogenous APLNs 43 . Accordingly, a PCA of this cohort revealed that nonsurvivors were widely located within a component corresponding to enhanced apelinergic release-degradation system activity. Based on these results, we hypothesize that endogenous apelinergic system instability related to sepsis-induced dysregulation of KKS and RAS could influence the natural history and outcome of this disease.
Several limitations of this work must be mentioned. First, the large animal model selected was ovine, which was found to have defective kidney ELA expression, and reduced signaling efficacy/potency upon human ELA binding to sheep APJ was observed. Septic shock was induced by peritonitis (which was not the primary cause of sepsis in the human cohort), and the experimental period was not long enough to trigger enzymatic degradation systems. A long-term animal model of sepsis including more hemodynamic support, antibiotic therapy, and outcome assessment would offer additional distinctive advantages with more accurate and extended clinical readouts. Additionally, the human data were observational, were derived from a relatively small sample size, and were imperfectly matched for sex, and need further confirmation in a larger cohort.

Conclusion
The apelin system is complex, with confirmed variability in cardiovascular effects across species 44 . Clearly efficient in experimental settings, this system is reactive in real-life human disease but highly susceptible to increased specific enzymatic degradation activities. From this standpoint, apelin system disturbance could influence the severity of sepsis and septic shock, opening doors for future explorative studies. The APLN-13/APJ axis thus represents a novel candidate pathway to manage hemodynamics in septic shock and could be an alternative to inotropic drugs but preventing specific enzymatic degradation or designing apelinergics resistant to degradation should be explored.  45 . Animals were housed for at least 48 h before the experiment in dedicated accommodations and bedding. The day before surgery, autologous stool was retrieved for treatment of the sheep selected for fecal peritonitis induction and mixed overnight in 0.9% normal saline (NS) and 5% D-glucose at 37 °C. The animals were fasted overnight. On the morning of the day of surgery, sheep received premedication to ease animal handling and intubation (i.e., intramuscular injections of 0.1 mg/kg atropine and 10 mg/kg ketamine) before routing to the operating room for insertion of an external jugular vein catheter and administration of a 500 mL bolus of Ringer's lactate (RL). Sheep were then continuously infused with RL (3 mL/kg/h) and 5% D-glucose (2 mL/kg/h). Tracheal intubation (7)(8) was performed in a caddy under isoflurane (1-2%) mask inhalation and by an anterior approach. Sheep were then placed in the supine position on heating pads before ventilator connection (Aestiva 15, Datex-Ohmeda, USA). The initial parameters (Vt: tidal volume, 200-400 mL; P insp : total inspiratory pressure, + 12-20 cm H 2 O; RR: respiratory rate, 12/min) were adjusted to obtain an end-tidal CO 2 of 35-50 mmHg (Capnomac Ultima, Datex-Ohmeda USA), and the FiO 2 was adjusted for sublingual SpO 2 ≥ 92%. A Levin catheter was inserted into the ovine rumen for continuous removal of excessive secretions. General anesthesia and animal comfort were ensured with a combination of titrated isoflurane (0.5%-1.5%, Baxter) and continuous i.v. infusion of ketamine (2 mg/kg/h) and rocuronium (0.2 mg/kg/h) to achieve a Sedation-Agitation Scale (SAS) score of 1. Once mechanical ventilation/oxygenation was established, a surgical team performed sequential catheter insertions: (1) on the right: (i) arterial femoral access (Pulsiocath 4Fr thermodilution catheter, 16  Baseline hemodynamic parameters and vital sign measurements (HR: heart rate, MAP: mean arterial pressure, SVR: systemic vascular resistance, CVP: central venous pressure, PAOP: pulmonary artery occlusion pressure and wedge pressure), dP/dt max and min, LV end-systolic pressure (LVESP), CO, stroke volume (SV), GEDV, and extravascular lung water (EVLW) were then recorded. Blood temperature, baseline central venous O 2 saturation (ScvO 2 ), and arterial blood gas (RapidLab® 348, Siemens Healthcare Diagnostics, Montreal, Qc, Canada) and lactate levels (Lactate Plus, Nova Biomedical, Waltham, MA, USA) were also monitored. A slightly modified sheep SOFA 46 score was calculated after reached shock criteria defining the "shock" time point and before euthanasia named the "final" time points, defined as a MAP below 65 mmHg for level 1 of the cardiovascular system assessment and assuming a constant level 3 of the central nervous system (Glasgow score 6-9) at "shock". Additional blood sample were collected at the "shock" and "final" endpoints for the biomolecular measurements. All blood test and rumen secretion removal volumes were estimated and systematically balanced by compensatory NS infusion.

Methods
After 2 g/kg autologous stool slurry was injected into both intra-peritoneal (i.p.) tubes, RL and 5% D-glucose infusions were stopped, and all the above parameters were monitored every 30 min. Serial blood cultures (Signal Blood Culture System, Thermo Scientific™ BC0100M, USA) were performed to assess whether the septic shock criteria were met in the sheep and to document and specify active bacterial translocation. All five criteria for experimental septic shock had to be met before implementing the next interventional steps. These criteria, measured from baseline, included (1) a decrease in MAP to < 60 mmHg or ≥ 40%, (2) a decrease in CO to < 3.5 L/min/m 2 or ≥ 40%, (3) less than 1.5 mL/kg/h urine output, (4) ScvO 2 below 70%, and (5) arterial lactate content above 2 mmol/L or a threefold increase. At this time point, hemodynamic parameters were recorded, and blood tests were performed.
A 30 mL/kg i.v. bolus of balanced crystalloid RL for resuscitation was infused for 1 h (as recommended by the 2018 Surviving Sepsis Campaign guidelines) 47 before restarting the initial infusion, RL (3 mL/kg/h) and 5% D-glucose (2 mL/kg/h). Sheep were randomly and blindly assigned to an incremental five-step dose-response infusion procedure (20 min) using e-syringes containing a solution of the apelinergic APLN-13 or ELA in NS (d1: 0.025, d2: 0.25, d3: 2.5, d4: 6.25, d5: 12.5 nmol/kg/h), or NS alone. Oxygen consumption (VO 2 ) was calculated as VO 2 = CO x (CaO 2 -CvO 2 ) × 10 with Ca or vO 2 = (Hb × 1.34 x SaO 2 ) + (PaO 2 × 0.003) at the baseline, shock, and fluid resuscitation time points and for every assessed dose of tested article until the endpoint. Hemodynamics, arterial blood gases and lactate content and necessary blood or clinical parameters for SOFA score calculation were recorded at all dose steps. At the end of the agonist dose-response trial Sheep were euthanized (i.v. injection of 90 mg/kg phenobarbital) before sampling lung, heart, and kidney tissues. , and creatinine clearance was calculated as previously described 48 . Enzymatic degradation activities in plasma were evaluated at the experimental "final" endpoint, as detailed in the Common Methods section.
Apelinergic system expression and pharmacology. Quantitative reverse-transcription polymerase chain reaction. RNA was extracted from heart and kidney tissues with a RNeasy Mini Kit (Qiagen, Germany). iScript Reverse Transcription Supermix (Bio-Rad) was used to prepare cDNA from 1 μg of total RNA in 20 μL. Sheep APJ plasmid cloning. The sequence of the apelin receptor was synthetized with gBlocks Gene Fragments (Integrated DNA Technologies, USA) and subcloned into pEYFPC1 (a modified vector in which we replaced the YFP sequence with that for two HA tags) with an NEBuilder HiFi DNA Assembly Cloning Kit (New England Biolabs, MA, USA). The Ovis aries APJ sequence was obtained from the RefSeq database (XM_027979777.1).

Patient study design.
General blood cyto-biochemical measurements. Human arterial blood was collected in EDTA-containing tubes, and a small portion was dedicated to whole blood cell and polymorphonuclear (PMN) cell counting (10 9 /L) with band-cell (%) determination using a DxH 900 hematology analyzer de Beckman Coulter. The remaining sample was used for PMN isolation using Polymorphprep® (Axis-Shield Diagnostics Ltd., Dundee, Scotland) density centrifugation according to the manufacturer's instructions. Isolated PMN cells were suspended in RPMI medium at a concentration of 1 × 10 6 cells/mL, washed in PBS, and incubated for 30 min on ice with the fluorochrome-conjugated monoclonal antibodies anti-CD64-APC/Cy7 (clone 10.1) and anti-CD66b-PerCP/ Cy5.5 (clone G10F5) (BioLegend, San Diego, CA, USA). The cells were then fixed in 2% paraformaldehyde (BD Cytofix™, BD Pharmingen, USA) for 10 min at room temperature. Isotype controls were used to quantify the nonspecific background signal. Cells were washed twice in PBS prior to measurement with a FACSCanto instrument. PMN purity was first assessed using expression of the marker CD66b higher than 98%, and PMN CD64 expression expressed as the mean fluorescence intensity (MFI). Blood levels of pentraxin 3 (PTX3) were measured using high-sensitivity cytokine magnetic bead assays (Mil-liplex® MAP Multiplex Assays, EMD Millipore, Billerica, MA, USA) according to the manufacturer's instructions. Data were analyzed using Multiplex Assay Analysis Software (EMD Millipore) and are expressed in ng/mL.
The angiotensin 1-7 (Ang1-7) plasma concentration was quantified with a commercially available ELISA kit and performed according to the manufacturer instructions (Abbexa; abx251960; Houston, TX, USA; range of 15.625-1,000 and a sensitivity of 9.38 pg/mL).
Arterial lactate (mmol/L) was assessed in septic patients as described previously 52 . Human blood cortisol content was measured with the ADVIA Centaur system (Siemens Medical Solutions Diagnostics, Tarrytown, NY, USA) with a sensitivity of 5.5-2,069 nmol/L and a range of 85-618 nmol/L.
Apelinergic enzymatic degradation activities in plasma. Neprilysin (NEP). As previously described 53 , 20 µL of plasma, 10 µL of substrate (5 mmol/L glutaryl-Ala-Ala-Phe-AMC; Peptides International, Louisville, KY, USA) and 50 µL of assay buffer (0.1 mol/L Tris-HCl, pH 7.6) were incubated at 37 °C for 30 min. The reaction was stopped by adding 10 µL of the NEP inhibitor phosphoramidon (0.1 mmol/L; Sigma, Oakville, ON, Canada) and incubating the samples on ice. Background controls were processed in the same manner except that phosphoramidon was added before the incubation at 37 °C. In the next step, the samples were incubated at 37 °C for 30 min with 10 µL of aminopeptidase M (500 mg/L, EMD Millipore, Etobicoke, ON, Canada) and 5 mmol/L EDTA. The reaction products were diluted in 3 mL of assay buffer, and the fluorescence was measured using a Tecan Infinite M1000 plate reader at an excitation wavelength of 360 nm and an emission wavelength of 440 nm. NEP activity was calculated from the difference between the sample (S) and control (C) values with the equation (S-C)/194 53 .
Angiotensin converting enzyme 2 (ACE2). The assay to measure ACE2 activity was performed as previously described (10); briefly, 2 µL of plasma was incubated with 100 µl of buffer (100 mM Tris-HCl, 600 mM NaCl, 0.5 mM ZnCl 2 , pH 7.5) and 2 µl of 1 mM quenched fluorescent substrate (Mca-Ala-Pro-Lys (Dnp)-OH; Enzo Life Sciences, Farmingdale, NY, USA) at 37 °C for 16 h. Fluorescence was measured at 405 nm with excitation at 320 nm. The results were expressed as relative fluorescence units (RFU)/µL of plasma/h. Angiotensin converting enzyme 1(ACE1). The assay to measure ACE1 activity was performed as previously described 54 ; briefly, 0.83 µL of plasma was incubated at 37 °C for 25 min with 73 µL of assay buffer (0.5 M borate, 15.63 mM ZnCl 2 , 5.45 M N-hippuryl-His-Leu; Sigma). Fluorescence was measured at an excitation wavelength of 355 nm and an emission wavelength of 535 nm. The results are expressed as RFU/µL of plasma.
Kallikrein (KLK1). The plasma KLK1 activity was quantified with a commercially available fluorescence assay kit and performed according to the manufacturer instructions (Anaspec; AS-72255; Fremont, CA, USA). Fluorescence was measured at an excitation wavelength of 490 nm and an emission wavelength of 520 nm. Results were expressed as relative fluorescent units/minute. Common methods. Apelinergic concentrations in plasma. Plasma was obtained from EDTA-containing blood samples collected from included patients and sheep by centrifuging at 1,600 × g for 10 min at 4 °C. Additional extraction on a C18-E SEP column (Phenomenex, Torrance, CA, USA) with overnight freeze-drying was mandatory for samples used for ELA measurements. APLN-36, APLN-17, APLN-16, APLN-13, and APLN-12 plasma concentrations, as well as those of the corresponding shorter degradation products, were measured with a specific commercially available ELISA kit (LifeSpan BioSciences; LS-F25717; WA, USA; detection range: 31.25-2,000 pg/mL; sensitivity: less than 18.75 pg/mL). ELA was quantified using a commercially available ELISA kit (Peninsula Laboratories International; S-1508; CA, USA) with a measurement range of 0-100 ng/mL. Aperlinergic stability in plasma. As previously described 43 , 27 μL of plasma mixed with 6 μL of a 1 mM aqueous solution of APLN-13 or ELA were incubated at 37 °C. Proteolytic degradation was stopped by adding 140 μL of 50% acetonitrile, 50% ethanol, and 0.25 mM N, N-dimethylbenzamide at 0, 0.5, 1, 2, 4, or 6 h. The reaction mixture was filtered by centrifugation at 2,000 rpm with Impact Protein Precipitation Filter plates (Phenomenex, CA, USA). Next, the reaction mixture was diluted with 80 μL of water and analyzed overnight with an Acquity