Angiotensin-converting enzyme and / or angiotensin-converting enzyme 2 as fecal biomarkers, methods and uses thereof

Abstract

The present disclosure relates to the use of angiotensin-converting enzyme and / or angiotensin-converting enzyme 2 as a fecal biomarker for the diagnosis of a disease or disorder related to the renin-angiotensin-aldosterone system, and also to the use of angiotensin-converting enzyme and angiotensin-converting enzyme 2 isoforms as fecal biomarkers for the detection of dysbiosis.

Description

[0001] D E S C R I P T I O N

[0002] ANGIOTENSIN-CONVERTING ENZYME AND / OR ANGIOTENSINCONVERTING ENZYME 2 AS FECAL BIOMARKERS, METHODS

[0003] AND USES THEREOF

[0004] TECH NICAL FIELD

[0005] The present disclosure relates to the use of angiotensin-converting enzyme and / or angiotensin-converting enzyme 2 as a fecal biomarker for a disease or disorder related to the renin-angiotensin-aldosterone system or for the detection of a dysbiosis.

[0006] BACKGROUND

[0007] The intestinal microbiota, also known as gut microbiota or gut flora, is crucial for human health in several ways. Alterations of the gut microbiota, known as dysbiosis, have been associated with several diseases and non-disease conditions. The microbiota is the core of the gut-heart axis. The composition of the microbiota is altered in cardiovascular diseases as hypertension and heart failure, and microbiota-derived metabolites as shortchain fatty acids and trimethylamine N-oxide are thought to be the key players in this interaction [1],

[0008] The renin-angiotensin-aldosterone system (RAAS) features Angiotensin (Ang) II, which causes vasoconstrictive, pro-inflammatory, and pro-fibrotic effects and is primarily generated by angiotensin-converting enzyme (ACE) [2], ACE2 metabolizes Angll into Angl-7, which exerts opposing effects [2], Thus, the balance between ACE and ACE2 determines RAAS-mediated actions. ACE2 presents only one extracellular catalytic domain, while ACE encompasses two catalytic domains (N- and C-domain): ACE C- domain mainly metabolizes Angl into Angll; the N-domain hydrolyzes Angl-7 [3],

[0009] There are local / tissue RAAS, where Ang II participates in secretion, sodium and water absorption, and smooth muscle tone. In the gut, Ang II promotes smooth muscle contraction, glucose absorption, and electrolyte balance [4], ACE contributes to peptide digestion and ACE2 mediates intestinal amino acid transport and microbiota regulation [4]. Different diseases and dietary patterns are known to affect the intestinal system, particularly the gut, leading to significant disruptions such as imbalance of the microbiota.

[0010] Diabetes Mellitus is a chronic disease characterized by hyperglycemia due to insufficient production of insulin by the pancreas or to an incapacity of the body to use it. Gastrointestinal symptoms are common, but usually neglected by both patients and clinicians. The diagnosis of those gastrointestinal complications [5] is frequently difficult and occurs as a diagnosis of exclusion since no specific test of biomarker exists. The gut microbiota is also altered in Diabetes Mellitus and these alterations might be associated with the gastrointestinal manifestations of the disease. In experimental diabetes, the intestinal expression of ACE mRNA was reported to be decreased in isolated jejunal enterocytes, while ACE2 protein and mRNA levels were found to be increased in the rat everted jejunum. Also, increasing the expression of intestinal ACE2 improves the compromised gut barrier integrity and ameliorates diabetic retinopathy in the Akita mice independently of the glycemia.

[0011] Alternative dietary patterns are being adopted particularly in Western countries due to sustainability, animal protection, but also concerns about health. The vegan diet is known to alter the gut microbiota and has been associated with both health advantages and drawbacks. There is no information on the impact of the vegan diet on intestinal RAAS.

[0012] The role for the RAAS in the pathophysiology of IBD is conflicting. Similar circulating Angl I and ACE activity levels in IBD patients and controls have been reported, as others report similar or reduced serum ACE activity between Crohn's disease (CD) and ulcerative colitis (UC) patients. Experimental studies show beneficial anti-inflammatory and clinical effects of ACE inhibition. ACE2 activity in IBD patients is reported as similar or increased, and mRNA levels are contradictory. In experimental colitis, ACE2 mRNA levels are similar or decreased in diseased animals [6], while ACE2-KO mice with experimental colitis display severe inflammation compared to controls [7], To this date, there are only two studies assessing ACE in the feces. Deddish et al. [8] have described the presence of an isoform of ACE in the ileal fluid of ostomized individuals, but failed to describe any clinical characteristics, never reporting them as patients with IBD, or any other pathology. Letizia et al. [9] assessed the fecal activity of ACE in celiac patients, but never report data on which ACE isoform they were assessing.

[0013] Ferreira-Duarte et al.

[0010] reported that ACE and ACE2 are catalytically active in the intestinal tissue and in the intestinal content. It was also noted that the activity of the intestinal content was superior to that determined in the correspondent tissue. However, no correlation was made between the activity of both enzymes and an existent pathology or dietary restriction.

[0014] Diagnosis and monitoring of Inflammatory Bowel Disease (IBD), including UC and CD, relies on subjective clinical scores and procedures like colonoscopy, which are invasive and expensive. Therefore, non-invasive biomarkers are of the utmost importance.

[0015] Serum biomarkers of disease are often non-specific and require blood collection, a simple, but many times uncomfortable, procedure. Overall, fecal biomarkers are extremely important as they present non-invasive and cost-effective alternatives to existent biomarkers of gastrointestinal diseases. Thus, fecal biomarkers are needed to support diagnosis, identify individuals at risk of developing disease states or nutrient deficiencies and to support disease and treatment monitoring, or prediction of response to treatment.

[0016] Document WO2021123456A1 discloses ACE2-Fc fusion proteins comprising the extracellular domain of human ACE2 linked to immunoglobulin Fc regions for therapeutic use against viral infections, particularly coronaviruses.

[0017] Document WO2019123456 Al describes C -terminally truncated variants of recombinant murine ACE2 proteins, designed to reduce molecular size and enable kidney glomerular filtration and tubular uptake for increased renal ACE2 activity.

[0018] These facts are disclosed in order to illustrate the technical problem addressed by the present disclosure.

[0019] GENERAL DESCRIPTION

[0020] The present disclosure relates to the field of diagnostic biomarkers. In particular, it pertains to the use of angiotensin-converting enzyme and / or angiotensin-converting enzyme 2 as fecal biomarkers for diagnosing diseases or disorders related to the renin- angiotensin-aldosterone system (RAAS), including inflammatory and cardiovascular diseases.

[0021] One of the aims of the present disclosure is to provide a non-invasive, reliable, and disease-specific method for diagnosing RAAS-related disorders— such as inflammatory disease of the digestive system— using easily accessible biological samples, for example fecal sample.

[0022] The present disclosure provides a non-invasive diagnostic method for assessing RAAS- related conditions by measuring the levels of ACE and / or ACE2 in a biological sample, namely in fecal samples. This approach leverages the unexpected presence and diagnostic relevance of these enzymes in biological samples, namely in human feces. It was surprisingly found that fecal concentrations of ACE and ACE2 can correlate with pathophysiological RAAS activation, particularly in inflammatory disease of the digestive system, namely inflammatory bowel disease, preferably UC or CD.

[0023] In a further embodiment, the inflammatory disease of the digestive system is an inflammatory bowel disease, preferably ulcerative colitis or Crohn's disease.

[0024] In an embodiment, the molecular weight of angiotensin-converting enzyme ranges from 50-70 KDa, preferably 70 kDa, or 50kDa. In another embodiment, the molecular weight of angiotensin-converting enzyme 2 ranges from 50-70 KDa, preferably 70 kDa, or 50kDa.

[0025] In an embodiment, the content of the biomarker is determined in a fecal sample collected from a patient to be tested, wherein in this context a patient can be a human or an animal (use in medicine or veterinary).

[0026] The present disclosure also relates to the use of angiotensin-converting enzyme and / or angiotensin-converting enzyme 2 isoforms as fecal biomarkers for the detection of dysbiosis. Preferably, to the use of angiotensin-converting enzyme or angiotensinconverting enzyme 2 isoforms as fecal biomarkers for the detection of dysbiosis.

[0027] In an embodiment, the dysbiosis results from a dietary restriction, and / or pathological condition. In a further embodiment, the pathological condition is diabetes mellitus, hypertension, heart failure, obesity, chronic kidney disease, autism spectrum disorder, toxoplasma infection, cancer, amyotrophic lateral sclerosis. Preferably, the pathological condition is diabetes mellitus, hypertension, heart failure, or obesity.

[0028] A fecal biomarker refers to a measurable substance or component found in feces that can provide information about the health or disease status of an individual. Fecal biomarkers are often used in medical research and clinical practice to diagnose or monitor conditions and can provide non-invasive means of assessing disease activity, treatment response, disease progression, or intestinal health status.

[0029] Dysbiosis refers to an imbalance in the microbial communities inhabiting a particular environment, such as the gut. In the context of human health, it often refers to an imbalance or disruption in the gut microbiota, where there is an overgrowth of harmful microorganisms or a reduction in beneficial ones. This imbalance can lead to various health issues, including digestive problems, inflammation, and susceptibility to infections. Dysbiosis can be influenced by factors such as diet, antibiotics, stress, and certain medical conditions or medicines.

[0030] Another aspect of the present disclosure relates to, a method for diagnosing in a sample a disease or disorder related to the renin-angiotensin-aldosterone system in a subject, comprising the following steps: measuring a level of angiotensin-converting enzyme (ACE) and / or angiotensin-converting enzyme 2 (ACE2) in the sample; preferably a fecal sample; comparing the measured level to a reference value; wherein a deviation from the reference value is indicative of a disease or disorder associated with RAAS.

[0031] BRIEF DESCRIPTION OF THE DRAWINGS

[0032] The following figures provide preferred embodiments for illustrating the disclosure and should not be seen as limiting the scope of invention.

[0033] Figure 1: Embodiment of clinical parameters assessed throughout the protocol, (a) Body weight change (%), (b) Food intake (g), (c) Fluid intake (mL) and (d) number of excreted fecal pellets, between control (white circles, n=10) and TNBS-induced animals (black circles, n=10). *p < 0.05 and ***p < 0.001 between groups for each day. Figure 2: Schematic representation of the colonic portions collected for different protocols. "X" marks the first 1 cm-long segment that was discarded.

[0034] Figure 3: Embodiment of results of macroscopic and microscopic evaluation, (a) macroscopic score (MaS) of control (n=10) and TNBS-induced rats (n=10); (b) Distribution of animals with mild (pointed) and moderate (striped) TNBS-induced colitis; (c) MaSpartiai of TNBS-induced animals with mild (n=4, pointed bars) and moderate (n=6, striped bars) colitis, (d, e) Representative images of the proximal colon of (d) a control animal and (e) a TNBS-induced animal; no significant histological differences are found between groups, (f-g.2) Representative images of the distal colon of (f) a control animal and (g.l and g.2) a TNBS-induced animal. Note the presence of moderate inflammatory infiltrate in the mucosa responsible for glandular dissociation (g.l), and extensive and deep ulceration with exuberant inflammatory reaction (g.2). DC - distal colon; PC - proximal colon. *p<0.05 between DC and PC with the same colitis severity degree;#p < 0.0001.

[0035] Figure 4: Representative Western Blots showing ACE and ACE2 expression in the feces of control (C) and TNBS-induced (T) rats (n=9 for each). Lung (L) and kidney (K) were used as positive controls for ACE and ACE2, respectively. One band of ~50kDa reacted with the anti-ACE antibody in control (C) and TNBS-induced (T) rats, and an additional band of ~70 kDa reacted with the anti-ACE antibody only in TNBS-induced rats (T). One band of ~70 kDa reacted with the anti-ACE2 antibody in both control (C) and TNBS-induced rats (T). MW - Molecular Weight.

[0036] Figure 5: Embodiment of results of (a) ACE and (b) ACE2 concentrations, and of (c) ACE2 / ACE concentration ratios in the distal colon (DC) and proximal colon (PC) of control (n=10) and TNBS-induced (n=10) animals, (d) ACE and (e) ACE2 concentrations, and (f) ACE2 / ACE concentration ratio in the serum of control (n=10) and TNBS-induced (n=10) animals. *p < 0.05; **p < 0.01; ***p < 0.0001.

[0037] Figure 6: Embodiment of results of ACE Z-FHL, ACE h-HL and ACE2 catalytic activities in the (a, b, c) colon, (d, e, f) serum, and (g, h, i) stools of control (n=10) and TNBS-induced (n=10) animals. *p < 0.05, **p < 0.01. Figure 7: Embodiment of results of ACE Z-FHL, ACE h-HL and ACE2 specific catalytic activities in the (a, b, c) colon and (d, e, f) serum of control (n=10) and TNBS-induced (n=10) animals. *p < 0.05.

[0038] Figure 8: Embodiment of results of (a) ACE Z-FHL / h-HL activity ratio, (b) ACE2 / ACE Z-FHL activity ratio and (c) ACE2 / ACE h-HL activity ratio between control (white bars, n=10) and TNBS-induced (stripped bars, n=10) animals. *p < 0.05.

[0039] DETAILED DESCRIPTION

[0040] The present disclosure relates to the use of angiotensin-converting enzyme and / or angiotensin-converting enzyme 2 as a fecal biomarker for the diagnosis of a disease or disorder related to the renin-angiotensin-aldosterone system, and also to the use of angiotensin-converting enzyme and angiotensin-converting enzyme 2 isoforms as fecal biomarkers for the detection of dysbiosis.

[0041] The in vivo experiments were approved by the national (003511 / 2018-DGAV) and local (179 / 2017-ORBEA-ICBAS-UP) competent authorities and followed the EU Directive 2010 / 63 / EU, the PREPARE guidelines for planning experiments, and the ARRIVE guidelines for reporting animal experiments.

[0042] In an embodiment, twenty male specific-pathogen free Wistar Han rats from Charles River Laboratories, France, were kept at the animal facility of ICBAS-UP. Animals were housed in pairs in individually ventilated cages (GR900 sealsafe plus; Tecniplast-ltaly) with proper bedding (Corncob ultra 12; Ultragene-Portugal), nesting and enrichment material, and ad libitum access to autoclaved tap water and laboratory rodent diet (4RF21, Mucedola S.r.l., Italy). The facilities had controlled ventilation, temperature (20- 24°C), relative humidity (40-70%), and regular light / dark cycles (12 / 12h). After a one- week acclimatization period, eleven to twelve weeks old rats, weighting 363.5±6.6 g, were blindly and randomly allocated to the control (n=10) or the TNBS (n=10) group, using the function RAND in Microsoft Excel (Microsoft®, Redmond, Washington, EUA). On day -1, rats were individually housed in clean cages and fasted for 8 hours with ad libitum access to a 5% sucrose solution. On day 0, under ad effectum anesthesia with isoflurane (Isoflo®, Esteve), animals were rectally instilled with a 21% ethanolic solution of 2,4,6-trinitrobenzene sulfonic acid (TNBS, Sigma-Aldrich Inc, St. Lois, MO, USA) (15 mg / rat; 250 pL) using a 7.6 cm ball-tipped catheter (STI 75-0285, Harvard Apparatus). Rats were then maintained in a head-down position for 60 seconds to minimize TNBS leakage. Analgesia was provided by tramadol on day 0 (Labesfal, 200 pL / kg, PO) and paracetamol (Paracetamol®, Farmoz, PO, 500 mg / kg) mixed in an aqueous solution of condensed milk (1:1:1), daily, from day 0 until the end of the protocol. To ensure intestinal motility, metoclopramide (Labesfal, 1 mg / kg, PO) was administered from day -1 to day 2 of the protocol. Controls were not manipulated.

[0043] All animals were daily monitored for body weight, food and water intake and fecal pellets excretion, and welfare was daily assessed and attributed a clinical score, as listed in Table 1. No mortality was observed.

[0044] Table 1 - Clinical score sheet to evaluate TNBS-induced colitis severity.

[0045] Body weight (g) _

[0046] No weight loss 0

[0047] Weight loss between 1 and 6% 1

[0048] Weight loss between 7 and 14% 2

[0049] Weight loss between 15 and 19% 3

[0050] Weight loss >20% _ 4

[0051] Food intake (g) _

[0052] > 18g 0

[0053] 12-18 g 1

[0054] 6-12 g 2

[0055] < 6g 3

[0056] <6g from day 5 _ 4

[0057] Water intake (g) _

[0058] Normal (20 g / day - 40 g / day) 0

[0059] Altered (< 20 g / day or > 40 g / day) _ 1

[0060] Feces (ns) _

[0061] > 40 pellets / day 0

[0062] 25-39 pellets / day and / or diarrhea 1

[0063] 16-26 pellets / day 2

[0064] < 15 pellets / day 3

[0065] < 15 pellets / day after day 5 _ 4

[0066] _ BEFORE STIMULI _ Facial expression _

[0067] Normal expression 0

[0068] Eyes half-closed 1

[0069] Facial contraction 2

[0070] Both _ 3

[0071] Resting posture Normal posture 0

[0072] Mild ventroflexion 1

[0073] Moderate ventroflexion 2

[0074] Severe Ventroflexion 3

[0075] Excessive grooming in the perineal area ( +1 )

[0076] Frequent alteration of the resting position _ ( +1 )

[0077] Piloerection _

[0078] No piloerection 0

[0079] Mild 1

[0080] Moderate 2

[0081] Severe 3

[0082] With Chromodacriorea _ ( +1 )

[0083] Moving posture _

[0084] Normal posture 0

[0085] Mild kyphosis 1

[0086] Moderate kyphosis 2

[0087] Severe kyphosis 3

[0088] Immobility _ 4

[0089] Abdominal contractions _

[0090] No Abdominal contractions 0

[0091] Twitch 1

[0092] Writhing or Mild Back Arching (< 3 s) 2

[0093] Writhing or Moderate Back Arching (> 3 s / repeated) 3

[0094] Writhing or Severe Back Arching (> 5 s / repeated) or Stagger _ 4

[0095] Breathing _

[0096] Normal 0

[0097] Irregular 1

[0098] Mild Taqui / Bradipnea 2

[0099] Persistent Taqui / Bradipnea 3

[0100] Severe Taqui or Bradipnea _ 4

[0101] _ AFTER STIMULI _

[0102] Locomotion _

[0103] Normal locomotion 0

[0104] Slightly altered locomotion 1

[0105] Highly altered locomotion 2

[0106] Unbalance 3

[0107] Immobility _ 4

[0108] Hydration _

[0109] Normal hydration 0

[0110] Mild dehydration 1

[0111] Moderate dehydration 2

[0112] Before stimuli - parameters were evaluated without manipulation of the animals; After stimuli - parameters were evaluated after lids were open and animals were left to explore their cage.

[0113] In an embodiment, body weight of TNBS-induced rats decreased until day 3, then increased, regaining their initial value by the end of the protocol (Figure 1). Food intake was similar between groups, except for days 2-3 where it was lower in TNBS-induced animals. Fluid intake was similar between groups except on days 1 and 4 where it was higher in TNBS-induced animals. Excretion of fecal pellets was lower in TNBS-induced animals than in controls during the first three days of protocol. TNBS-induced animals presented a higher welfare score than control animals throughout the protocol, except on day 6.

[0114] Sample collection and macroscopic evaluation

[0115] In an embodiment of sample collection and macroscopic evaluation, on days 7-8, rats were sacrificed by decapitation (small Decapitator, Harvard apparatus). Whole blood was collected, allowed to rest for 1 hour at room temperature, centrifuged at 670 g for 20 minutes, and the serum collected and kept at -20°C until assayed. Then, the abdomen was opened, the interior observed, and the colon excised and cleaned of fecal content using a Krebs-Henseleit solution [(in mM): 118 NaCI, 4.8 KCI, 2.5 CaCl2.2H2O, 1.2 NaH2PO4.H2O, 1.2 MgSO4.7H2O, 25 Na-HCO3, 0.02 Na2EDTA, 0.3 Ascorbic Acid and 11 glucose mono-hydrated]. Afterwards, the whole colon was longitudinally opened through the mesenteric border for mucosal macroscopic evaluation and attribution of a macroscopic score (MaS), as defined in Table 2. MaS was calculated as the mean between partial MaS (MaSpartiai) of the proximal colon (PC), a region without any visible mucosal damage, and distal colon (DC), a region with obvious mucosal damage. MaS allowed categorization of TNBS-induced experimental colitis in mild, moderate or severe

[0011] .

[0116] Table 2 - Macroscopic score (MaS).

[0117] Macroscopic Scoring Adhesions to , Mucosal

[0118] Colon thickness , „ Mucosal ulcers

[0119] Parameters adjacent organs edema / hyperemia

[0120] 0 Absent Normal Absent Absent

[0121] „ 1 Mild / focal Mild Mild Single

[0122] Score i / i i

[0123] 2 Moderate / zonal Moderate Moderate At one site

[0124] 3 Severe / diffuse Marked increased Severe At more sites

[0125] Finally, segments of 1 cm in length were collected as described in Figure 2. The four 1 cm-long portions of the PC and DC were cut in small pieces and the fragments randomly collected for the different protocols. Fecal pellets from each animal's cage were collected on the day of animal's sacrifice for the different protocols. In an embodiment, samples collected for microscopic evaluation (Figure 3) were fixed in 10% buffered formalin, embedded in paraffin wax, and cut in 3 pm full thickness sections that were stained with haematoxylin and eosin. Histological evaluation was performed in full thickness segments of PC and DC of control and TNBS-induced animals3. TNBS- induced animals presented higher MaS than controls (Figure 3a): 6 / 10 presented moderate experimental colitis and 4 / 10 presented mild colitis (Figure 3b). MaSpartiai of the DC was higher than that of the PC in TNBS-induced animals independently of the severity of the colitis (Figure 3c). MaSpartiai of the DC of rats with moderate colitis was higher than that of animals with mild colitis (Figure 3c).

[0126] In an embodiment, microscopic evaluation revealed that control animals did not exhibit histological changes in the colon (Figure 3d and 3f). Contrarily, all TNBS-induced animals displayed histological damage of different degrees of severity in DC (Figure 3g.1 and 3g.2), but not in PC (Figure 3e). In 3 animals, discrete inflammatory infiltrate and edema was observed in the lamina propria. In 4 cases, focal superficial inflammatory lesions (including ulcers and a moderate mixed inflammation in the lamina propria and submucosa, mostly consisting of lymphocytes, macrophages, and eosinophils) were found. Moreover, 3 animals exhibited extensive and deep ulceration of the intestinal wall, reaching the muscular layers.

[0127] In an embodiment, the analysis of fecal ACE and ACE2 isoforms was performed by Western blotting after sample preparation, based on the study from Morampudi et al.

[0012] , Cage fecal pellets (approximately 60 mg) were collected and homogenized in 500 pL of a U9 buffer containing: 9M Urea, 2% (m / v) CHAPS, 50 mM Tris, protease inhibitor cocktail (complete™, Mini, EDTA-free Protease Inhibitor Cocktail, 1 pill to 10 mL buffer) (all from Merck®, Darmstadt, Germany), pH 9.0, in a 2 mL round bottom tube containing 2.8 mm and 5 mm ceramic beads, using a Precellys Evolution Tissue Homogenizer (Bertin Instruments, USA). Then, they were left for 1 hour at room temperature. Afterwards, 500 pL of U1 buffer (0.9 M Urea, 2% (m / v) CHAPS, 50 mM Tris, protease inhibitor cocktail; complete™, Mini, EDTA-free Protease Inhibitor Cocktail, 1 pill to 10 mL buffer), pH 9.0 was added to the sample, vortexed gently, and centrifuged at 14500 g for 10 minutes. Supernatants were collected and total proteins were quantified following the Bradford assay

[0013] , using bovine serum albumin (BSA) as a standard. Equal amounts of fecal proteins (25 pg) were boiled at 95°C for 10 minutes in a 6X sample buffer (0.35 M Tris-HCI pH 6.8, 4% (m / v) SDS, 30% (v / v) glycerol, 9.3% (v / v) DTT, 0.01% (v / v) bromophenol blue, 5% (v / v) mercaptoethanol), and then loaded in an 8% (v / v) SDS- PAGE 1 mm gel. Lung and kidney (20 pg of total proteins) were used as positive controls for ACE and ACE2, respectively. Gels were run for 30 minutes at 40 V, 350 mA maximum, followed by 2 hours at 120 V, 350 mA maximum. Then, proteins were electrotransferred to nitrocellulose membranes for 20 minutes at 1.3 A and 25 V maximum, using a transfer buffer containing 48 mM Tris and 52 mM Glycine. Membranes were then incubated 1 hour in a blocking solution with 5% (m / v) BSA in TBS-T buffer (IX Tris-Buffered Saline containing 0.05% (v / v) Tween-20, pH = 7.4) at room temperature, and then probed overnight at 4°C with primary antibodies: rabbit polyclonal anti-ACE (1:500, ab28311, Abeam, Cambridge, UK) and rabbit polyclonal anti-ACE2 (1:500, abl08252, Abeam, Cambridge, UK). After, membranes were incubated with a secondary antibody goat antirabbit IgG conjugated to horseradish peroxidase (1:10000 for ACE and 1:5000 for ACE2, sc2004, Santa Cruz Biotechnology, Dallas, Texas, USA). Proteins were detected with Radiance ECL detection kit (Azure Biosystems, Dublin, California, USA), following manufacturer's instructions, using ChemiDoc Imaging Systems (BioRad, Hercules, California, USA).

[0128] In another embodiment, the concentration of both enzymes was quantified in the serum and in the supernatants of the colon homogenates using ELISA commercial kits (Cat. No.: abx255667, Abbexa®, Cambrige, UK, for ACE; Cat. No.: MBS014209, MyBioSource, San Diego, CA, for ACE2) following manufacturer's instructions, while total proteins were quantified according to Bradford et al.

[0013] , using BSA as a standard. Results were expressed as ng / mLforthe serum, and ng of enzyme / mg of total proteins for the colonic portions.

[0129] In yet another embodiment, ACE and ACE2 activities were assessed as previously described

[0010] , ACE activity was performed by a fluorimetric assay using Hippuryl-His- Leu (h-HL) and Z-Phe-His-Leu (Z-FHL) as substrates, as it has been described that the Z- FHL / h-HL ratio is characteristic for each type of ACE: ACE (both domains): Z-FHL / h-HL ratio = 1; ACE N-domain: Z-FHL / h-HL ratio = 4.5; and ACE C-domain: Z-FHL / h-HL ratio = 0.7. ACE2 activity was performed by a fluorometric kinetic assay using Mca-APK(Dnp) (Cat. No.: BML-P163-0001, Enzo Life Sciences, Inc., NY, USA) as a substrate in the absence or presence of the selective ACE2 inhibitor DX600 (Abeam pic, Cambrige, UK). Total proteins were quantified according to Bradford et al.

[0013] , using BSA as a standard. Results are presented as global enzyme activity (nmol / min / mg of total proteins for colonic portions and feces, or nmol / min / mL for serum), or normalized by the concentration of ACE or ACE2, to get the correspondent specific activity of the enzyme (nmol / min / ng of ACE orACE2) in the colonic portions and serum.

[0130] In an embodiment of results, western Blot analysis regarding ACE showed a band of ~50 kDa in the feces of control and TNBS-induced animals, while in TNBS-induced animals, a band of ~70 kDa was also present (Figure 4). Considering ACE2, a band of ~70 kDa was found present in the feces of control and TNBS-induced rats (Figure 4).

[0131] In an embodiment of results, ACE concentration was increased in the DC of TNBS- induced animals compared to the PC (mainly due to animals induced with moderate colitis (Table 3)), while in control animals there were no differences between intestinal regions (Figure 5a). No differences were found between groups in either intestinal region (Figure 5a). ACE2 concentration was decreased in the PC of TNBS-induced animals compared to that of control rats (Figure 5b) but no differences were found between intestinal regions within the same experimental group (Figure 5b). In the PC, ACE2 / ACE concentration ratio was >1 in controls (ACE2 more prevalent than ACE), but <1 in TNBS- induced animals (ACE more prevalent than ACE2) (Figure 5c). In the DC, ACE2 / ACE concentration ratio was <1 in both experimental groups (ACE more prevalent than ACE2) and lower in the TNBS-induced animals than in controls (Figure 5c). ACE2 / ACE concentration ratio was decreased in the DC compared to the PC in both experimental groups (Figure 5c). In the serum, ACE concentration was similar between experimental groups (Figure 5d) and ACE2 concentration was lower in TNBS-induced animals than in controls (Figure 5e). ACE2 / ACE concentration ratio was <1 in both experimental groups and lower in the TNBS-induced animals (mainly due to animals with moderate colitis, Table 3, reinforcing the prevalence of ACE over ACE2 (Figure 5f).

[0132] Table 3 - ACE and ACE2 concentration in the PC, DC and serum of controls and animals with mild and moderate TNBS-induced colitis. Results are expressed and median [interquartile range]. Control Mild TNBS Moderate TNBS

[0133] ACE concentration (ng / mg of total proteins)

[0134] Proximal Colon 7.7(5.8-15.0] 5.2[2.2-8.1] 6.6]3.5-9.2]*

[0135] Distal Colon 18.2(11.5-39.1] 15.8(7.2-21.1] 40.8(29.2-46.0]

[0136] Serum 20.4(9.0-22.2] 17.8(12.7-25.2] 16.7(1.4-32.0]

[0137] ACE2 concentration (ng / mg of total proteins)

[0138] Proximal Colon 9.5(6.4-14.7] 5.4[1.8-6.0]* 4.9[3.6-9.0]

[0139] Distal Colon 7.6[6.5-9.6] 5.7[1.3-6.9] 7.1(5.8-10.5]

[0140] Serum 1.2[1.5-0.6] 0.5[0.4-0.6]* 0.0[0.0-0.0]*

[0141] ACE2 / ACE concentration ratio

[0142] Proximal Colon 1.3[0.7-2.1] 0.7[0.4-1.7] 0.8[0.5-1.8]

[0143] Distal Colon 0.4[0.3-0.7] 0.3[0.1-0.4] 0.2[0.1-0.3]

[0144] Serum 0.1[0.0-0.3] 0.0[0.0-0.1] 0.0[0.0-0.0]*

[0145] In an embodiment of results of ACE activity, it was increased in the DC of TNBS-induced rats compared to that of the correspondent PC (mainly due to animals with moderate colitis (Table 4, Figure 6a and 6b), as well as in controls. No differences were found between experimental groups, except for the ACE activity (Z-FHL) of the PC in TNBS- induced animals that was lower than that of the controls (Figure 6a and 6b). Also, no differences were found in ACE2 activity between experimental groups nor between colonic regions (Figure 6c). In the serum, ACE and ACE2 activities, were similar between control and TNBS-induced animals (Figure 6d, 6e and 6f, respectively). In the feces, ACE activity was similar between control and TNBS-induced animals (Figure 6g and 6h), but ACE2 activity was increased in the stools of TNBS-induced rats compared to that of controls (Figure 6i).

[0146] Table 4 - ACE and ACE2 activities in the PC, DC serum and feces of controls and animals with mild and moderate TNBS-induced colitis. Results are expressed and median[interquartile range].

[0147] Control Mild TNBS Moderate TNBS

[0148] ACE activity (h-HL) (nmol / min / mg of total proteins)

[0149] Proximal Colon 12.9(8.7-19.1] 11.1(2.1-22.9] 7.4(2.7-12.9]*

[0150] Distal Colon 68.8(41.4-92.2] 36.1(15.7-70.0] 103.9(8.4-147.2] Serum 30.6(23.9-39.7] 81.2(29.8-183.6] 32.8(0.8-172.4]

[0151] Feces 15.6(9.8-18.1] 15.6(12.9-22.6] 24.4(11.1-29.5]

[0152] ACE activity (Z-FHL) (nmol / min / mg of total proteins)

[0153] Proximal Colon 45.7(32.8-59.0] 19.3(4.6-27.2]* 19.5(6.7-37.3]*,#

[0154] Distal Colon 228.7(112.8-301.4] 124.4(44.7-232.6] 275.0(181.1-396.1]

[0155] Serum 342.3(275.8-423.1] 444.6(267.6-835.3] 468.1(181.9-616.5]

[0156] Feces 38.6(18.3-50.5] 36.3(23.0-40.9] 55.0(43.6-90.6]

[0157] ACE2 activity (nmol / min / mg of total proteins)

[0158] Proximal Colon 71.3(28.7-130.4] 92.3(5.7-187.2] 17.5(0.0-95.0]

[0159] Distal Colon 56.5(28.9-125.2] 41.0(7.9-318.7] 56.5(28.9-125.2]

[0160] Serum 9.1(1.9-19.4] 19.9(1.5-56.3] 25.7(3.9-41.4]

[0161] Feces 19.4(12.5-34.2] 41.3(31.1-83.5] 51.4(21.9-73.4]

[0162] ACE Z-FHL / h-HL activity ratio

[0163] Proximal Colon 3.5[2.9-4.0] 2.0(1.1-32.2] 2.2[1.7-3.8]

[0164] Distal Colon 3.5[2.9-4.0] 3.4[2.7-3.7] 2.5[2.0-3.2]*

[0165] Serum 11.7(9.8-14.2] 5.4(3.9-13.0] 6.7(3.5-249.4]

[0166] Feces 2.1[1.7-2.3] 2.3[1.5-3.2] 2.6[1.8-3.2]

[0167] ACE2 / ACE h-HL activity ratio

[0168] Proximal Colon 6.4[2.9-8.8] 6.3(0.2-20.7] 1.8[0.0-7.3]

[0169] Distal Colon 1.3[0.8-1.6] 1.9(0.2-63] 0.9[0.0-2.4]

[0170] Serum 0.4[0.0-0.5] 0.1[0.0-1.4] 0.2(0.0-17.8]

[0171] Feces 1.6[0.8-3.1] 2.9[1.6-5.7] 2.9[0.8-4.6]

[0172] ACE2 / ACE Z-FHL activity ratio

[0173] Proximal Colon 1.7[0.7-2.4] 4.5(0.2-10.2] 0.9[0.0-3.5]

[0174] Distal Colon 03(0.2-0.5] 0.5[0.1-1.8] 0.4[0.0-1.0]

[0175] Serum 0.0[0.0-0.0] 0.0[0.0-0.2] 0.0[0.0-0.4]

[0176] Feces 0.8[0.4-1.7] 1.5[0.7-2.4] 1.0[0.4-1.4]

[0177] Regarding ACE (Z-FHL and h-HL) specific catalytic activities, meaning the absolute activity normalized to the concentration of ACE, they were increased in the DC of control and TNBS-induced rats, compared to the correspondent PC (Figure 7a and 7b). No differences were found in the specific catalytic activity of ACE2 between groups nor between colonic regions (Figure 7c), even when analyzing by disease severity (Table 5). Also, serum ACE (Z-FHL and h-HL) specific catalytic activities, as well as ACE2 specific catalytic activity, were similar between experimental groups (Figure 7d, 7e and 7f). Table 5 - ACE and ACE2 specific activities in the PC, DC and serum of controls and animals with mild and moderate TNBS-induced colitis. Results are expressed and medianfinterquartile range].

[0178] Control Mild TNBS Moderate TNBS

[0179] ACE specific activity (h-HL) (nmol / min / ng of ACE)

[0180] Proximal Colon 1.6[1.0-2.5] 1.4[0.3-7.6] 1.4[0.3-2.7]

[0181] Distal Colon 2.8[1.6-6.0] 3.2[1.4-4.1] 3.3[1.6-4.0]

[0182] Serum 1.7(1.1-11.6] 3.8(1.6-13.0] 3.7(0.0-13.1]

[0183] ACE activity (Z-FHL) (nmol / min / ng of ACE)

[0184] Proximal Colon 4.6[3.5-9.7] 2.4[0.9-8.7] 2.8(0.9-11.1]

[0185] Distal Colon 9.7(5.9-21.2] 9.6(4.3-14.0] 6.9(4.6-11.9]

[0186] Serum 20.8(16.4-70.7] 18.3(17.1-60.46] 27.0(18.7-67.2]

[0187] ACE2 activity (nmol / min / ng of ACE2)

[0188] Proximal Colon 4.8{3.9-10.1] 15.1(1.0-35.1] 4.7(0.0-19.0]

[0189] Distal Colon 10.5(3.9-13.2] 38.6(5.3-72.0] 7.6(0.0-42.4]

[0190] Serum 7.3(1.6-18.8] 39.6(3.0-87.1] Not enough data

[0191] ACE Z-FHL / h-HL activity ratios were >1 for all matrices (Figure 7a) and was lower in the PC (p=0.0524) and the DC of TNBS-induced rats compared to that of controls (Figure 8a).

[0192] Regarding ACE2 / ACE activity (Z-FHL) ratio, it was >1 in the PC of both experimental groups, <1 in the DC and serum of both experimental groups, and =1 in the feces of both experimental groups (Figure 8b). As for ACE2 / ACE activity (h-HL) ratio, it was >1 in the PC and feces of both experimental groups, <1 in the serum of both experimental groups, and =1 in the DC of both experimental groups (Figure 8c). No differences in ACE2 / ACE activity (Z-FHL) ratio and ACE2 / ACE activity (h-HL) ratio between control and TNBS- induced animals were found (Figure 8b and 8c).

[0193] Surprisingly, this work shows the presence of catalytically active ACE and ACE2 in the feces of animals with TNBS-induced colitis and the presence of an ACE isoform of 70 kDa only in TNBS-induced rats. Additionally, it is shown that ACE concentration is higher than that of ACE2 in the serum and in the inflamed region of the colon; the ACE N-domain seems to play an important role regulating colonic lesion; and fecal ACE2 activity is higher in TNBS-induced animals than in controls.

[0194] It is herein disclosed the presence of a ~70 kDa isoform of ACE in the feces of TNBS- induced rats, which is not present in the feces of controls, and a ~50 kDa ACE isoform in the feces of both groups. The presence of the ACE 70kDa band seems relevant in intestinal inflammation. ACE isoforms of these molecular weight have not been reported in the literature. The ACE isoforms probably result from shedding of transmembrane ACE or deglycosylation.

[0195] Regarding ACE2, it was identified a catalytically active ACE2 in the feces of rats with experimental colitis and an ACE2 isoform of ~70 kDa. Different ACE2 isoforms with molecular mass ranging 60-130 kDa have been described, with reports of deglycosylated ACE2 isoforms of 70-80 kDa in vitro and of 60-70 kDa in cultured mesangial cells and urine and kidney lysates align with our findings.

[0196] The concentration and activity of ACE and ACE2 was quantified, further analyzing several ratios that give a better insight on the balance between classic and the counter- regulatory arms of intestinal RAAS. The results show that ACE concentration prevails over ACE2 in the serum and the DC, and particularly in TNBS-induced animals, suggesting a role in TNBS-induced intestinal inflammation, as ACE generates pro-inflammatory Angll, while ACE2 degrades it. Moreover, the lower ACE Z-FHL / h-HL activity ratio observed in TNBS-induced animals vs controls anticipates decreased hydrolysis of beneficial peptides as Angl-7 and N-acetyl-seryl-aspartyl-lysyl-proline peptide (AcSDKP), which are metabolized by ACE N-domain.

[0197] ACE2 was found in the feces of control and TNBS-induced animals, with increased activity in the latter. This suggests increased ACE2 shedding from the inflamed colon epithelial cells, a mechanism already described for ACE. I ntriguingly, if shedding is the primary source of both enzymes in feces due to intestinal epithelial turnover, one would expect increased fecal activity for both ACE and ACE2.

[0198] In conclusion, ACE and ACE2 isoforms are present in the feces of control and TNBS- induced animals, with a ~70 kDa ACE isoform being only present in TNBS-induced experimental colitis. The activity of ACE2 was increased in the feces of TNBS-induced animals as compared to control.

[0199] In an embodiment, the experimental unit was considered the individual animal. Data statistical analysis was performed using GraphPad Prism 9 (Graphpad Software, San Diego, CA). Data was tested for normality using the Shapiro-Wilktest (small sample size). Regarding ACE and ACE2 concentrations in the colon, it was used the Wilcoxon matched- pairs test to compare values between intestinal regions of control animals, and the paired t-test to compare values between intestinal regions of TNBS-induced rats. The unpaired t-test and the Mann-Whitney U test were used to compare ACE and ACE2 concentrations, respectively, between experimental groups. Regarding ACE and ACE2 activities, it was used Wilcoxon matched-pairs test to compare values between intestinal regions for the same experimental group and the Mann-Whitney U test for comparisons between groups. Regarding the feces, we used the unpaired t-test for comparisons of ACE and ACE2 activities between groups.

[0200] In another embodiment, ACE and ACE2 activity was also assessed in individual following an omnivorous (OMNI) or vegan (VEG) dietary pattern for at least 1 year. The correlation analysis suggested that blood pressure might be regulated not only by classic mechanisms including the systemic RAAS, but also by modulation of a fecal RAAS or, at least, by the fecal activity of its major enzymes or products. The study showed a negative correlation between ACE2 activity and systolic blood pressure (SBP) in the VEG group and a negative correlation between ACE Z-FHL activity and diastolic blood pressure (DBP) in the OMNI group and when all participants were considered. In the OMNI group, the correlation was stronger than when all participants were considered. In VEG individuals, those with higher activity of fecal ECA2 may have lower levels of fecal angiotensin II (due to increased catabolism), which may decrease the absorption of sodium and thus be associated with lower SBP.

[0201] In another embodiment, studies with fecal pellets from germ-free, ACE and ACE2 knockout (KO) mice, and from the corresponding controls, showed that fecal ACE and ACE2 activity may stem from a combination of intestinal shedding and microbiota activity, either by producing ACEs' inhibitors or degrading them, or by producing ACEs- like enzymes. Thus, it is surprisingly shown that dysregulations in the gut microbiota (dysbiosis) can be detected by analyzing the activity of ACE and / or ACE2 enzymes in the feces of a subject.

[0202] In an embodiment, fecal pellets from germ-free, ACE and ACE2 knockout (KO) mice, and from the corresponding controls were collected for enzymes' activities fluorimetric assays. ACE activity was assessed using Hippuryl-His-Leu and Z-Phe-His-Leu as substrates. ACE2 activity was assessed using Mca-APK(Dnp) as a substrate, in the presence and absence of the selective ACE2 inhibitor MLN -4760. ACE and ACE2 activities were increased in the feces of germ-free animals compared to that of controls. ACE2 activity was abolished in the feces of ACE2-KO mice, while fecal ACE activity remained similar compared to that of controls. In the feces of ACE-KO mice, ACE activity was decreased, but not abolished, compared to that of controls, while ACE2 activity remained similar to that of controls. In ACE C- and N-domain KO, ACE activity was not different from that of controls. Inhibition with captopril completely abolished fecal ACE activity using Hippuryl-His-Leu, but not Z-Phe-His-Leu, in those animals.

[0203] The term "comprising" whenever used in this document is intended to indicate the presence of stated features, integers, steps, components, but not to preclude the presence or addition of one or more other features, integers, steps, components or groups thereof.

[0204] The terms "determining," "measuring," "evaluating," "assessing," "assaying," and "analyzing" are often used interchangeably herein to refer to forms of measurement. The terms include determining if an element is present or not (for example, detection). These terms can include quantitative, qualitative or quantitative and qualitative determinations. Assessing can be relative or absolute. "Detecting the presence of" can include determining the amount of something present in addition to determining whether it is present or absent depending on the context.

[0205] The disclosure should not be seen in any way restricted to the embodiments described and a person with ordinary skill in the art will foresee many possibilities to modifications thereof. The above-described embodiments are combinable. The following dependent claims further set out particular embodiments of the disclosure.

[0206] References:

[0207] 1. Bui TVA. et al.The Gut-Heart Axis: Updated Review for The Roles of Microbiome in Cardiovascular Health. Korean Circ J. 2023 Aug;53(8):499-518.

[0208] 2. Crowley SD, Coffman TM. Recent advances involving the renin-angiotensin system. Exp Cell Res. 2012;318:1049-56.

[0209] 3. lawata MG, B. H. Ectodomain Shedding of ACE and ACE2 as Regulators of Their Protein Functions. Current Enzyme Inhibition. 2011;7:42-55.

[0210] 4. Lo SW, Segal JP, Lubel JS, Garg M. What do we know about the renin angiotensin system and inflammatory bowel disease? Expert Opin Ther Targets. 2022 Qct;26(10):897-909.

[0211] 5. Krishnan B, Babu S, Walker J, Walker AB, Pappachan JM. Gastrointestinal complications of diabetes mellitus. World J Diabetes. 2013 Jun 15;4(3):51-63.

[0212] 6. Burgueno JF, Reich A, Hazime Het al. Expression of SARS-CoV-2 Entry Molecules ACE2 and TMPRSS2 in the Gut of Patients With IBD. Inflamm Bowel Dis. 2020;26:797-808.

[0213] 7. Hashimoto T, Perlot T, Rehman Aet al. ACE2 links amino acid malnutrition to microbial ecology and intestinal inflammation. Nature. 2012;487:477-81.

[0214] 8. Deddish PAW, J.; Michel B.; Morris, P. W.; Davidson N. O.; Skidgel, R. A.; Erdos, E. G. Naturally occurring active N-domain of human angiotensin l-converting enzyme. Proceedings of the National Academy of Sciences. 1994;91:7807-11.

[0215] 9. Letizia CP, A.; De Ciocchis, A.; Di Giovambattistas, F.; Greco, M.; Cerci, S.; Torsoli,A.; Scavo, D. Angiotensin-converting enzyme activity in stools of healthy subjects and patients with celiac disease. Digestive Diseases and Sciences. 1996;41:2268-71.

[0216] 10. Ferreira-Duarte M, Oliveira LCG, Quintas Cet al. ACE and ACE2 catalytic activity in the fecal content along the gut. Neurogastroenterol Motil. 2023:el4598. 11. Ferreira-Duarte M, Rodrigues-Pinto T, Menezes-Pinto Det al. 2,4,6- trinitrobenzenesulfonic acid-induced colitis in Rattus norgevicus: a categorization proposal. Exp Anim. 2021;70:245-56.

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Claims

C L A I M S1. Use of angiotensin-converting enzyme and / or angiotensin-converting enzyme 2 as a fecal biomarker for the diagnosis of a disease or disorder related to the renin- angiotensin-aldosterone system.

2. Use according to the previous claim wherein the disease or disorder related to renin-angiotensin-aldosterone system is an inflammatory disease, or a cardiovascular disease.

3. Use according to any of the previous claims wherein the inflammatory disease is an inflammatory disease of the digestive system.

4. Use according to the previous claim wherein the inflammatory disease of the digestive system is an inflammatory bowel disease, preferably ulcerative colitis or Crohn's disease.

5. Use according to any of the previous claims wherein the molecular weight of angiotensin-converting enzyme ranges from 50-70 Kda; preferably 70 kDa, or 50 kDa.

6. Use according to any of the previous claims wherein the molecular weight of angiotensin-converting enzyme 2 ranges from 50-70 Kda; preferably 70 kDa, or 50 kDa.

7. Use according to any of the previous claims wherein the content of the biomarker is determined in a fecal sample collected from a patient to be tested.

8. Use of angiotensin-converting enzyme and angiotensin-converting enzyme 2 isoforms as fecal biomarkers for the detection of dysbiosis.

9. Use according to the previous claim wherein the dysbiosis results from a dietary restriction, and / or pathological condition.

10. Use according to the previous claim wherein the pathological condition is diabetes mellitus, hypertension, heart failure, obesity, chronic kidney disease, autism spectrum disorder, toxoplasma infection, amyotrophic lateral sclerosis, or cancer.

11. A method for diagnosing in a sample a disease or disorder related to the renin- angiotensin-aldosterone system in a subject, comprising the following steps measuring a level of angiotensin-converting enzyme (ACE) and / or angiotensinconverting enzyme 2 (ACE2) in the sample; preferably a fecal sample.

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