DETECTION OF IMMUNOREACTIVE ALDOSTERONE
IN MURINE GASTROINTESTINAL TRACT

Aldosterone is a steroid hormone traditionally regarded as being synthetized in and secreted from zona glomerulosa of the adrenal glands. Its synthesis is stimulated mainly by angiotensin II and III, high serum potassium and low sodium levels as well as adrenocorticotrophic hormone (ACTH), although the latter is of less importance (1). In addition to these factors, plasma acidosis, atrial stretch receptors sensing a reduction in blood pressure and a lipid factor, adrenoglomerulotropin (2) can increase aldosterone secretion. Aldosterone plays a key role in water and electrolyte transport in the distal tubules and collecting ducts of the nephrons of the kidney by increasing reabsorption of sodium and water back into the bloodstream and excretion of potassium into urine. Aldosterone acting on nuclear mineralocorticoid receptors upregulates and activates the basolateral Na+/ K+ pump (Na-K-ATPase) which exchanges three sodium ions and water for two potassium ions into the tubular lumen and urine; chloride ions are reabsorbed with sodium. This exchange is ‘fine-tuning’ of total urinary water and electrolyte excretion in nephrons being about 3%. Interestingly, similar processes of sodium and water reabsorption in potassium exchange exist also in the gut, salivary and sweat glands (3). In the colon, the regulation of sodium transport by aldosterone develops very early in human infants. It has even been suggested that in preterm neonates, the colon is the major organ for sodium conservation, i.e. more important than the kidney (4). These similarities in the handling of electrolytes in the intestine and renal tubules were described already over 50 years ago (5). It has also been speculated that the effects of angiotensin II on intestinal fluid and electrolyte transport (6) are possibly mediated by aldosterone.

Close connection between the local renin-angiotensin-system (RAS) and steroid hormone (corticosterone) synthesis in the gastrointestinal tract of rodents has been reported (3, 7). Different sections of the gut behave differently; in rats, the expression of angiotensinogen mRNA has been detected in large intestine and stomach, but not in small intestine (8). In chronic renal failure, colonic angiotensin II receptors are upregulated which enhances potassium excretion (9). In animal models of inflammatory bowel disease (IBD), a dietary excess of salt exacerbates the pathology (10), but the possible role of local aldosterone synthesis has not been investigated.

Nakamura et al. (11) used a strain of mineralocorticoid receptor knock-out mice and reported that not only renal but also intestinal receptors made contributions to intestinal sodium absorption, especially in colon. This led to excessive sodium retention and the further development of hypertension. They also suggested these receptors could be attractive targets for antihypertensive drugs.

While corticosterone is formed locally in the intestine in mice and rats and released in tissue incubation (7, 12, 13), and is an intermediate product in the pathway from progesterone to aldosterone, we speculated that also aldosterone could be synthetized in the intestine. Generally, the evidence for local hormone production is based on the detection of mRNA and the enzyme protein involved in the synthesis or degradation of the hormone as well as its release in tissue incubation. These indicators may be present at quite a low level under normal conditions, but become strongly upregulated in conditions of inflammation (14, 15) or immunological stress (16) since they may also confer protection against tissue damage (17, 18).

Based on these observations, we hypothesized that aldosterone can be produced locally in the gastrointestinal tract. For the evidence, we aimed to measure aldosterone as detected by ELISA i.e. immunoreactive aldosterone, in different sections of the murine gastrointestinal tract as this has not been previously described in the literature.

MATERIAL AND METHODS

The study protocol was approved by National Animal Experimentation Committee of Finland (ESAVI/114/04.10.07/2015) according to EC Directive 86/609/EEC and Finnish Experimental Animal Act 62/2006.

Five to 10 mm long sections of different parts of gastrointestinal tract (from esophagus to distal colon) were dissected from euthanatized (isoflurane) of Balb/C male mice (age 8 weeks, weight 20 ± 2 g) (Envigo, Horst, The Netherlands). The animals were housed, two animals in a cage under standard animal laboratory conditions (12 h light and 12 dark cycle at 22 ± 2ºC, relative humidity 55% ± 15%) and fed with a 2018 Teklad Global 18% protein diet (Harlan Laboratories, Indianapolis, IN, USA) and tap water ad libitum (15).

The locations of the tissue samples are as follows: the esophagus just proximal to the stomach ‘sphincter’, the stomach sample from the fundus, the duodenal sample next from the pylorus, the jejunum, ileum and colon samples were from the middle part of the organs, and the caecum sample close to the border to the colon. The tissue samples were opened, rinsed with Krebs buffer (NaCl 119 mmol/l, NaHCO2 25 mmol/l, KCl 15 mmol/l, glucose 11 mmol/l, CaCl2 1.6 mmol/l, KH2PO4 1.2 mmol/l, MgSO4 1.2 mmol/l, pH 7.4), cleaned from internal contents and external fat and connective tissue and frozen in liquid nitrogen and kept at –20ºC until further processing. The protein content of the tissue samples was determined after homogenization for 3 × 20 s at 5500 rpm at 4ºC (Precellys 24 Cryobed Homogenizer, Bertin Technologies, Montigny le Bretonneux, France) in tissue lysis buffer (NaCl 136 mmol/l, Na2HPO4 8 mmol/l, KCl 2.7 mmol/l, KH2PO4 4.46 mmol/l, Tween 0.05%, pH 7.4) and the commercial protein assay kit (Pierce TM, BCA Protein Assay Kit, Thermo Scientific, Waltham, MA, USA). Immunoreactive aldosterone concentrations in the supernatant after centrifugation of the homogenates (15 min at 13,300 g at +4ºC) and in serum separated from blood taken by aortic puncture were determined using a commercial kit (Aldosterone ELISA Kit, #501090, Cayman Chemicals, Minnesota, MI, USA). The aldosterone concentration in the supernatant was related to the protein content of the homogenized tissue.

Statistical analysis

One-way analysis of variance followed by Dunnett’s test in SPSS Statistics version 26 (IBM, Armonk, NY, USA) was used to compare the concentration of immunoreactive aldosterone in caecum to the other sections of the gastrointestinal tract.

RESULTS

After homogenization and centrifugation, the clear supernatants of the tissue samples from different parts of the gastrointestinal tract displayed immunoreactivity in the assay’s range of 15.6 – 2000 pg/ml (standard curve) and sensitivity 30 pg/ml expressed in the kit’s instructions (Cayman). The only exception was esophagus where only two out of six samples showed values over the lowest standard 15.6 pg/ml. When the tissue concentrations were related to the tissue protein content (as the indicator of the amount of tissue which produced aldosterone), stomach and colon showed the lowest values being 12.2 ± 2.8 pg/mg protein (mean ± SEM) and 15.5 ± 1.7 pg/mg protein, respectively (Fig. 1). In the different sections of the small intestine rather similar concentrations were detected i.e. in duodenum (33.5 ± 4.9 pg/mg protein), jejunum (45.4 ± 12.2 pg/mg protein) and ileum (33.1 ± 8.8 pg/mg protein). The highest values 80.1 ± 11.1 pg/mg protein were found in caecum (P < 0.05 versus jejunum, and P < 0.01 or P < 0.001 versus the other regions). The mean serum concentration was 286.6 ± 37.8 pg/ml. To confirm the immunoassay findings by mass-spectrometry, we sent some samples to a central hospital laboratory. Unfortunately, the mass-spectrometric procedure available for us had been applied for the measurement of aldosterone from urine and saliva being therefore of too low sensitivity (1 nMol/l i.e. 360 pg/ml) for our tissue supernatants.

Fig. 1. Immunoreactive aldosterone concentrations (pg/mg protein) in different sections of mouse gastrointestinal tract. Median of individual values (n = 4 – 6). Measurements from tissue homogenates with ELISA (Cayman), protein determination by Pierce TM, BCA Protein assay (Thermo Scientific). The immunoreactive aldosterone between groups was compared by using ANOVA followed by Dunnett’s test comparing caecum to the other sections (*P < 0.05; **P < 0.01; ***P < 0.001).

DISCUSSION

We and others have described local corticosteroid synthesis in the intestinal epithelium of mice and rats in healthy tissue and in experimental inflammation (7, 12-18). Corticosterone synthesis seems to be associated with the local activity of the renin-angiotensin-system (RAS) (7, 14, 15), with angiotensin II being a physiological stimulant, as for aldosterone in adrenal glands.

Here our aim was to test whether another steroid hormone, aldosterone, can be found locally in the murine gastrointestinal tract similarly to many other tissues (3) including vascular smooth muscle cells (19) and ovaries (20). An immunological ELISA method, which is specific for aldosterone but with moderate cross-reactivity with the urinary metabolite, tetrahydro-aldosterone (Cayman) was used. Therefore, we propose that the term ‘immunoreactive aldosterone’ should be at present used for the compound analyzed. It was detected in whole tissue sections throughout the intestine, less in the proximal (upper) part of the tract, increasing towards caecum. For comparison, the serum concentrations in the present study gave concentrations which are in the range of those previously reported in mice and rats (21-23) and in humans (Cayman). Therefore, we consider that the commercial ELISA method, used also in some of the studies referred here, was a reliable way to measure aldosterone levels also from clear Krebs buffer supernatant after the tissue homogenization. Nonetheless, one should be careful in interpreting the measurement using only immunological methods (24). Because in biological tissue samples, the measured concentrations vary in addition due to biological variation and methodological differences. Therefore, we were only interested to examine whether aldosterone as a whole could be detected in the murine gastrointestinal tract and if so, in which parts would the concentrations be highest. It was interesting that it was caecum which showed the highest levels; this tissue is known to be the location in rodents where there is extensive water and electrolyte exchange.

Aldosterone, the principal and most powerful mineralocorticoid, is synthetized in the adrenal glands from progesterone, with deoxycorticosterone and corticosterone being the intermediate steroids. Even though these intermediates can also stimulate mineralocorticoid receptors, aldosterone is the most potent and important regulator of water and sodium reabsorption by exchanging it with potassium in the distal and collecting tubules in the kidney (25). It should be remembered that the intestine is another important electrolyte and water balancing organ in humans, since each day, it encounters nine liters of fluid from digestive secretions and ingested water with only about 200 ml being excreted with the feces (26). While the kidneys regulate sodium excretion, it is evident that also the large intestine included caecum participates its reabsorption and therefore both organs are important in the regulation of blood pressure (11). Over 50 years ago, Shields and coworkers (5) showed in vivo in the dog and humans that “aldosterone seems to influence the intestinal handling of electrolytes in a manner which, in several aspects resembles its effect upon renal tubules”. These findings can be regarded as a good basis for searching for a local aldosterone synthesis in the intestine i.e. in a similar manner to that demonstrated for corticosterone (brain, heart, salivary glands and intestine) (12). In searching for the enzymes responsible for the synthesis of steroids, e.g. aldosterone synthase (CYP11b2) or for glucocorticoid synthesis (CYP11b1), outside of the adrenal glands, only low expression levels have been found in healthy tissues (3, 27), but these levels are upregulated in inflammation (7) or immunological stress (16) as well as in situations of tissue repair (17, 18). Our preliminary findings on release of aldosterone in in vitro incubation of murine caecum and on expression of immunoreactive aldosterone synthase protein (CYP11b2) (Pang et al., unpublished data) give further evidence for our hypothesis that this hormone could be synthetized in intestine.

Mineralocorticoid receptors have been described in the distal colon of rat and reported to participate in active sodium and potassium transport (28). Nonetheless, these receptor subtypes are different in kidney and intestine; in the renal tubular epithelium, they are the ENaC subtype gamma, whereas in the intestinal epithelium, they consist of the beta and gamma subtypes (11). In renal distal nephron epithelial cells, aldosterone receptors showed biphasic responses. A low concentration of aldosterone (K1/2 = 0.5 nM) evoked a transient effect, but a high concentration (K1/2 90 nM) induced a long-lasting response in a collecting duct cell line (25). Because the serum levels of aldosterone in our present study (286.6 pg/ml i.e. 0.79 nM) and in earlier reports (0.56 nM (21); 1.1 nM (22); 0.67 nM (23)) have been clearly lower than those needed to exert this long-lasting effect, it is tempting to hypothesize that a local synthesis of the hormone is needed to induce long-lasting stimulation of aldosterone receptors, also in the intestine. Sztechman et al. (29) classified mineralocorticoid receptors (MRs) in normal and pathophysiological processes of cardiovascular system into classical genomic MRs and non-genomic MRs. The former ones are less selective to aldosterone. Their activation induces slowly developing (in an hour) and long lasting (for hours) effects. The non-genomic MRs induce rapid cellular responses within seconds and minutes. Interestingly, angiotensin II, regarded as the physiological stimulant of aldosterone secretion in adrenal glands does not influence sodium homeostasis in the colon. In a previous study, prolonged aldosterone infusion stimulated sodium reabsorption both in proximal and distal colon, but potassium excretion only in the distal colon in adrenalectomized rats (30) indicating different sensitivity of the aldosterone receptors in different parts of large intestine. Blockades of aldosterone receptors and the enzyme responsible for its synthesis (aldosterone synthase, CYP11b2) have been considered as potential targets to treat cardiovascular diseases (11, 29, 31) and recently also skin diseases while in the skin local renin-angiotensin-aldosterone system is fully expressed (32). However, this has not yet evaluated from the point of view of their effects in the gut e.g. in inflammatory bowel diseases and infectious diarrhea, in which electrolyte and water balance is disturbed.

We have applied caution in interpreting our findings because an immunological assay was used. However, there are few known cross-reactivities with other steroids except with the metabolite of aldosterone. Thereafter, the highest value, 0.1%, seems to be for corticosterone (Cayman); this could not possibly interfere with our aldosterone assay because in our previous study conducted under similar conditions, the corticosterone levels in the tissue samples were about 2000 pg/ml (7) giving a maximum cross-reaction concentration of 2 pg/ml. Similarly, if we estimate that in a tissue specimen weighing approximately 100 mg, only a few microliters of blood would remain after rinsing with buffer and further diluted in homogenization the adrenal gland derived aldosterone in blood could make only a very insignificant contribution to the final immunoreaction.

In conclusion, this is the first time that aldosterone probably synthetized locally has been detected in murine intestinal tissue, the highest levels in caecum. This finding is supported by various observations in the literature describing the presence of different components of the renin-angiotensin-aldosterone-system in the gastrointestinal tract. It can be suggested that in addition to kidney, large intestine, especially caecum in rodents has a physiological role in regulating electrolyte and water balance in the body. Nonetheless because an immunoassay was used, instead of calling the compound simply as aldosterone, we prefer to be cautious and use the term ‘immunoreactive aldosterone’.

Acknowledgements: Grants: Finska Läkaresällskapet, Einar och Karin Stroems Stiftelse, Finland (HV); Juhani Aho Medical Foundation, Finland (HL); Orion Research Foundation, Finland and Finnish Medical Foundation (LV).

We are grateful to Hanne Salmenkari, PhD, for tissue samples and fruitful discussions and Ewen McDonald, PhD, for checking the grammar and style.

Conflict of interest: None declared.

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