ACE and ACE2 catalytic activity in the fecal content along the gut

Angiotensin‐converting enzyme (ACE) and ACE2 are two major enzymes of the renin–angiotensin–aldosterone system (RAAS), which control the formation/degradation of angiotensin (Ang) II and Ang1‐7, regulating their opposite effects. We aimed at evaluating the catalytic activity of ACE and ACE2 in the intestinal content and corresponding intestinal tissue along the gut of Wistar Han rats.


| INTRODUC TI ON
The renin-angiotensin-aldosterone system (RAAS) is a multienzymatic cascade traditionally associated with blood pressure regulation and fluid homeostasis. 1,2 Angiotensin (Ang) II, its main effector peptide, is mostly generated by the cleavage of Ang I by angiotensin-converting enzyme (ACE), and promotes vasoconstriction, pro-inflammatory, and fibrotic effects (classic RAAS). [1][2][3] Also, AngII is metabolized by ACE2 into Ang1-7 that mediates the opposite effects (counter-regulatory RAAS). Further, AngI can also be converted to Ang1-9 by ACE2, and then to Ang1-7 by ACE. 1,2,4 ACE and ACE2 are membrane-bounded carboxypeptidases with a single transmembrane helix and an intracellular segment, but are also found in their soluble form, which results from shedding of their extracellular domain. [5][6][7] While ACE2 has only one catalytic domain, 8,9 the extracellular domain of ACE has two catalytic domains (N-and C-domains) with different specificity and affinity for different substrates: the C-domain predominantly hydrolyzes AngI into AngII, while the N-domain is responsible for the metabolism of Ang1-7 into Ang 1-5, as well as other bioactive peptides [10][11][12] (Figure 1). ACE and ACE2 are the major RAAS enzymes and ACE2/ACE balance is essential for determining RAAS activity since it reflects the activation of the classic and counter-regulatory arms and, subsequently, overall RAAS-mediated effects.
Besides its classical view as an endocrine circulating system, several RAAS have been found to exist locally in different organs and tissues, 13 with an independent regulation of the circulating RAAS. 2 For instance, RAAS enzymes and peptides have been found in the gastrointestinal (GI) tract, [13][14][15][16] where they play a fundamental role in GI physiology and pathophysiology. Intestinal AngII regulates glucose absorption, water, and electrolytes homeostasis or bicarbonate secretion. 15 Also, we and others reported that AngII is also responsible for regulating intestinal smooth muscle tone, [17][18][19] through a complex network of intestinal cells as neurons, interstitial cells of Cajal and enteric glia, thus regulating normal intestinal motility. RAAS enzymes can also show RAAS-independent effects associated with the intestinal function. ACE participates in peptide digestion 13 and ACE2 is crucial for the intestinal expression of the neutral amino acid transporter B 0 AT1, thus regulating the nutritional environment of the gut and the composition of the microbiota. 20 In fact, an interaction between the intestinal RAAS and microbiota has been suggested. 21 This interaction is bidirectional: genetic deletion of the counter-regulatory Mas receptor alters gut microbiota 22 while GI microbiota reduces ACE2 intestinal expression [23][24][25] and a bacterial aspartate beta-decarboxylase is fundamental for the endogenous synthesis of alamandine, 26 the newest RAAS peptide.
Although there is a GI RAAS and the referred evidence of an interaction with the gut microbiota, information on the presence of a functional RAAS in the stools or intestinal content remains scarce.
In fact, to our knowledge, there are only two scientific papers that assessed the presence/activity of ACE in the human ileal fluid 27 and human stools of healthy individuals, this being altered in patients with celiac disease. 28 No one has looked for fecal ACE2. So, the aim of this work was to investigate the concentration and catalytic activity of ACE and ACE2 throughout the intestinal content and excreted fecal pellets of Wistar rats, and to compare the results on the intestinal content with that of the corresponding intestinal tissue. . Enrichment material such as nesting material and paper tunnels was provided to all cages. For reduction purposes (3Rs), animals were shared with ongoing study that only included male rats.

| Animals and sample collection
On the day of the experiment, animals were weighted and then sacrificed by decapitation. Subsequently, the abdomen was opened and the intestine was collected from the rectum until the stomach.
Then, samples from distal and proximal colon (DC and PC, respectively), cecum, and terminal ileum were collected. These portions were opened through the mesenteric border, allowing the collection of the intestinal content directly from the corresponding intestinal tissue portion, to avoid contamination. Fecal pellets from the cages were also collected. Other intestinal portions, as well as other organs, were used for other ongoing projects (3Rs-reuse).
Each intestinal portion was minced to pieces, randomized within assays, and then homogenized. Intestinal content and cage fecal pellets (CFPs) were also homogenized.
For enzyme activity assay, intestinal tissue and content, and CFPs were homogenized (200 mg tissue/feces to 1 mL of buffer) in a buffer containing: 100 mM sodium borohydride buffer, pH 7.2, 340 mM sucrose, 300 mM NaCl, and 1 mM phenylmethylsulphonyl fluoride (PMSF) inhibitor. PMSF was prepared as a concentrated

Key Points
• ACE and ACE2 activity is found in the rat intestinal content along the GI tract.
• ACE is more prevalent in the intestinal tissue than in the intestinal content.
• ACE2 is more prevalent in the intestinal content than in tissue (except the ileum).
• ACE and ACE2 are more active in intestinal content than in intestinal tissue.

| ACE and ACE2 concentration
ACE and ACE2 concentrations in the homogenate supernatants of intestinal tissue were quantified using ELISA commercial kits (cat. no. abx255667, Abbexa®, for ACE; cat. no. MBS014209, MyBioSource, for ACE2) following the manufacturer's instructions, while total proteins were quantified according to Lowry et al. 30 using bovine serum albumin as a standard. Results were expressed as mg of enzyme/mg of total proteins.

| ACE and ACE2 activity
ACE activity was assessed as previously described. 31  In the intestinal tissue, ACE activity was presented as global enzyme activity normalized by the concentration of total proteins (nmol/ min/mg of total proteins) or normalized by the concentration ACE, to get the correspondent specific activity of the enzyme (nmol/min/ ng of ACE). In the intestinal content, ACE activity was presented as global enzyme activity normalized by the concentration of total proteins (nmol/min/mg of total proteins).

| Statistical analysis
Data analysis was performed using GraphPad Prism (Graphpad Software). Data were tested for normality using the Shapiro-Wilk test (small sample size) and the evaluated parameters were found to have a non-normal distribution. Hence, we used a paired experimental Only control animals were used in this study and no criteria were set to include or exclude the animals. The experimental unit was considered the individual animal.

| RE SULTS
3.1 | ACE2 and ACE are present and active along the rat gut ACE2 concentration was similar across the intestinal regions studied ( Figure 2A) but ACE concentration was higher in the DC than in the ileum and cecum ( Figure 2B). In line with this, the ACE2/ACE concentration ratio was lower in the DC than in the ileum and cecum ( Figure 2C); this ratio was lower than 1 in the DC, around 1 in the PC, but higher than 1 in the cecum and ileum ( Figure 2C), showing that in the ileum ACE2 concentration prevails over ACE but this is attenuated along the gut so that in the DC ACE concentration prevails over ACE2.
ACE2 activity ( Figure 3A) and ACE N-domain activity ( Figure 3E) were similar among the intestinal regions studied, and the same was observed for ACE2 and ACE N-domain specific activities ( Figure 3B,F). As for the ACE C-domain activity, the ileum showed increased global enzyme activity than the cecum and increased specific activity than the DC ( Figure 3C,D).
The ratio between the activities of the N-and C-domains of ACE was higher than 1 and similar between all intestinal tissue samples ( Figure 4A), suggesting that the intestinal tissue ACE activity occurs more at the N-domain than at the C-domain. Also, the ACE2/ACE C-domain activities ratio was over 1 in all samples but similar among them, except for an increase in cecum over DC that was not observed when specific activities were taken ( Figure 4B,C). Moreover, there was no difference in the ACE2/ACE N-domain activities ratios between intestinal regions ( Figure 4D,E), although these ratios were always lower than 1 for all regions except the cecum, where they were higher than 1 ( Figure 4D) or just around 1 ( Figure 4E). These results suggest that overall, in the intestinal tissue ACE N-domain is more active than ACE2 which is more active than the ACE C-domain.
3.2 | ACE2 and ACE are active in the rat intestinal content along the gut ACE2 activity was similar among the intestinal content of the different regions studied, or the CFPs ( Figure 5A). Regarding ACE activity, the catalytic activity in the ileal content was higher than that of DC for both catalytic domains ( Figure 5B,C), and also higher than that of CFPs for the N-domain ( Figure 5C).
The ratio between the activity of the ACE N-and C-domains was higher than 1 in all fecal samples and did not differ along the intestine ( Figure 6A), again pointing to a higher activity of the N-over the C-domain of ACE. The ratios between the activity of ACE2 and that of either the N-or C-domains of ACE did not statistically differ between fecal samples ( Figure 6B,C). However, the ACE2/ACE Cdomain activity ratio was higher than 1 in all fecal samples except the ileal content ( Figure 6B), and the ACE2/ACE N-domain activity ratio was higher than 1 only in the DC and CFP ( Figure 6D). These results suggest that in the intestinal content (similar to that observed in the intestinal tissue), ACE N-domain is more active than ACE2 which is more active than the ACE C-domain.  (Table 1). Regarding ACE activity, both C-domain and Ndomain activities were higher in the intestinal content than the corresponding intestinal segment for all intestinal regions (Table 1).

| ACE2 and ACE activities are different between the intestinal content and anatomically corresponding intestinal tissue
Regarding activity ratios, no differences were found in the ACE-N-domain/C-domain activity ratio, neither in ACE2/ACE-C-domain or ACE2/ACE-N-domain activity ratios between intestinal content and corresponding intestinal tissue ( Table 2).

| DISCUSS ION
Our study has some groundbreaking findings that will enhance   ACE2 was evenly distributed and active from the terminal ileum to the DC. These data are in accordance with data from other studies reporting that ACE2 is expressed throughout the small and large intestine, 9,[33][34][35][36][37] although some studies have reported that ACE2 expression is higher in the ileum than in the colon. [34][35][36][37] Intestinal ACE2 actively removes C-terminal single neutral amino acid from nutrient peptides, which are then uptaken by the neutral amino acid transporter B 0 AT1. 38,39 Interestingly, it has been reported that ACE2 expression is required for the expression, proper trafficking, and functioning of B 0 AT1, 39 which is abundantly expressed in the ileum. 40 Accordingly, in ACE2 knockout mice, tryptophan uptake was impaired, leading to compromised antimicrobial peptide formation and altered gut microbiota composition. 20,41 In our study, ACE concentration was higher in the DC than in the terminal ileum and cecum, although its C-domain was more catalytically active in the ileum than in the DC (with no difference for the N-domain). ACE was also reported to be expressed in the small and large intestine. 13,15,16 But, Erickson et al. have reported that within the small intestine, ACE mRNA and protein, as well as enzymatic activity, are higher in the proximal to middle portions, decreasing toward the distal end, 42 which was the only region of the small intestine that we studied.
Despite this observation, we are, to our knowledge, the first group to report differences in ACE concentration between small and large intestinal portions. ACE may also have a role in digesting dietary proteins. Yoshioka et al. described that ACE, present in the intestinal brush-border membrane, can cleave C-terminal residues from peptides at the same hydrolysis rate than other intestinal brush-border membrane peptidases like aminopeptidase IV and aminopeptidase N. 13,43 Interestingly, ACE and ACE2 seem to be more abundant in the intestine than in other tissues and fluids. 35,44 In order to understand the relation between the fecal RAAS and the intestinal RAAS, we compared ACE and ACE2 activities between the intestinal content and the anatomically corresponding intestinal tissue. Curiously, we observed that both ACE (N-and C-domains) and ACE2 activities are higher or similar (never lower) in the intestinal content than in the corresponding intestinal tissue. The presence of catalytically active ACE and ACE2 in the intestinal content could simply result from enzyme shedding from the intestinal epithelial membrane. However, if this should be the only origin of enzyme activity in the intestinal content, and considering that our study used healthy rats, one would expect that the enzyme activity in the intestinal content would be similar or lower than that of the intestinal membrane.
Hence, we speculate that the intestinal microbiota can be a source of enzyme activity in the intestinal content. New studies are ongoing in order to test this hypothesis but, at present, the possibility is already thrilling since it would unravel the existence of a microbiota RAAS. It has been suggested that the RAAS and the microbiota interact. 21   mouse intestinal microbiota. 22 Hence, we hypothesize that the differences found in our study might reflect the different composition of the microbiota between the intestinal regions. 45 The balance of the RAAS activity relies considerably on the balance between the activities of its classic and counter-regulatory arms is more active than ACE2 which is more active than the ACE C-domain.
There is: (1) higher activity of the ACE N-domain over the C-domain, (2) higher activity of ACE2 over that of the ACE C-domain, but (3) lower activity of ACE2 over that of the ACE N-domain. ACE C-domain activity mainly determines the formation of AngII 11,12 (Figure 1), which is catabolized into Ang1-7 by ACE2 1,2,4 (Figure 1), and Ang1-7 is hydrolyzed into inactive peptides by the ACE N-domain 11,12,46,47 (Figure 1). If we reason on this RAAS picture and our ratio data, the higher ACE2/ ACE C-domain activity ratio possibly results in more Ang1-7 being formed when compared to AngII. This would also explain the higher ACE N-/C-domain activity, which would contribute to regulate the increased formation of Ang1-7 by increasing its hydrolysis and decreasing the formation of AngII, which would promptly generate more Ang1-7 through the high ACE2 activity. Finally, the lower ACE2/ACE N-domain activity ratio observed in the more proximal intestinal regions would also contribute to regulate Ang1-7 concentration by increasing its hydrolysis and decreasing its formation. Although these interpretations are speculative, our study clearly shows a dynamic balance between ACE and ACE2 not only in the intestinal tissue but also, and innovatively, in the intestinal content.
Current knowledge of the RAAS prompts us to speculate on the pathophysiological relevance of our data. The imbalance between the abundance and/or activity of gut ACE and ACE2 may have an impact on: (1) smooth muscle tone and electrolyte homeostasis, (2) intestinal inflammation, (3) gut dysbiosis, (4) dysregulation of tryptophan metabolism, (5) dysregulation of metabolic pathways (e.g., glucose absorption), and (6) susceptibility to intestinal infections and associated intestinal manifestations. 15,21,48,49 In conclusion, we report for the first time the presence of enzymatically active ACE and ACE2 along the intestinal content of the rat, with some regional differences and independence from the corresponding tissue. This work sheds new light on the RAAS, with the hint of the existence of a fecal RAAS. Quantification of other RAAS components, namely AngII and Ang1-7, in future studies will refine the present data.
Our data open a totally unexplored line of research that might be of translational relevance in the field of fecal biomarkers of intestinal diseases, and others associated with the microbiota, as well as for the use of genetically modified probiotics (that would secrete ACE inhibitors or Ang1-7) in the treatment of inflammatory GI or cardiovascular diseases.

AUTH O R CO NTR I B UTI O N S
Mariana Ferreira-Duarte designed and performed the research, ana-  TA B L E 2 Ratios between angiotensinconverting enzyme (ACE) N-domain/Cdomain activity, ACE2/ACE C-domain activity, and ACE2/ACE-N-domain activity in the intestinal content and the corresponding intestinal tissue (n = 8).