The distal colon has a key function in NaCl and water absorption. It has been demonstrated that distal colon absorptive function depends both on crypt luminal cells and on the myofibroblast cells of the surrounding pericryptal sheath (1). This layer of myofibroblasts generates collagen IV, and is held together predominantly by intercellular adhesion molecules (2), which link with the cytoskeletal smooth-muscle actin (α-SMA) (3) via adherens junctions in the cell membrane (4). Colonic Na+ absorption is influenced by a low-sodium (LS) diet following activation of the renin-angiotensin-aldosterone system by increasing renin secretion. This in turn activates a process that generates angiotensin II, which stimulates aldosterone production and secretion within the adrenal cortex (5).
Aldosterone stimulates sodium absorption by distal colon and renal distal tubule by enhancing expression of the epithelial Na+ channel in apical membranes and increasing the Na+/K+ ATPase activity in the basolateral membrane (6). Another consequence is that the crypt wall permeability to both Na+ and dextran (10 kDa) is decreased, as determined by monitoring the rates of equilibration of fluorescein-labelled dextran across the crypt wall using confocal microscopy of tissue in vitro taken from adrenalectomised rats pretreated with aldosterone via an osmotic minipump (7). Events related to the renin-angiotensin-aldosterone system in the distal colon also stimulate myofibroblast growth (8) in the pericryptal sheath, as determined by increased expression of smooth-muscle actin in these cells. Animals fed an LS diet accumulate high sodium concentrations in the pericryptal space, whereas animals fed a high-sodium diet do not accumulate hypertonic Na+ in the pericryptal sheath. The presence of this stable hypertonic solution surrounding the crypts provides a large driving force for water absorption across the crypt wall (9), and is used to create a suction tension within the crypt lumen that is used to dehydrate faeces in the colonic lumen.
Because an LS diet increases plasma levels of both aldosterone and angiotensin II, it is difficult to discriminate between the effects of each hormone in colonic permeability and in the growth of pericryptal myofibroblasts, so their specific role or the possibility of a synergic action has until now been unclear. Both aldosterone and angiotensin II have been shown to have roles in fibrosis in other tissues, such as the heart and the kidney. Mineralocorticoid receptors have been detected in cardiac myocytes and endothelial cells (10). The Randomized Aldactone Evaluation Study (11), which evaluated the benefits of spironolactone therapy on congestive heart failure, showed that aldosterone-receptor blockade decreased morbidity and mortality associated with excessive intracardiac myofibroblast stimulation; however, angiotensin-converting enzyme (ACE) activity (12,13) and angiotensin II type 1 receptors have been observed at sites of fibrosis (14). In cardiac myofibroblasts angiotensin II has trophic effects, increasing α-SMA expression after 2 days and increasing collagen synthesis after 14 days of infusion (15,16). Given that ACE inhibitors do not inhibit aldosterone synthesis completely, prolonged exposure or increased sensitivity to aldosterone results in cardiac remodelling and eventual failure (17).
ROLES OF ALDOSTERONE AND ANGIOTENSIN II
Postadrenalectomy osmotic minipumps were implanted to Sprague-Dawley rats to maintain plasma concentrations of both hormones (18,19). The actions of aldosterone and angiotensin II were followed during the 3 days after changing from high-sodium (150 mmol/L NaCl in drinking water) to an LS diet (150 μmol/L NaCl). To study the transport and permeability properties of the crypt wall, the following variables were observed: Na+ accumulation in the pericryptal space; dextran permeability across the crypt wall; the electrical variables transmural potential difference, short-circuit current, and transepithelial electrical resistance (TER) in the presence or absence of amiloride; and expression of epithelial Na+ channel in colonocytes. Myofibroblast growth was studied by the tissue growth factor β receptor and angiotensin II type 1 receptor expression, E- and OB-cadherin expression, ACE activity, α-SMA and claudin IV expression, and collagen IV production. To corroborate that each hormone was active, separate groups of animals received the ACE inhibitor captopril, the angiotensin II receptor type I inhibitor losartan, or the aldosterone antagonist spironolactone.
Our results showed that aldosterone alone reproduced all of the typical effects of an LS diet in the distal colon, without angiotensin II present and independent of salt intake. Aldosterone infusion decreased crypt wall permeability to dextran (Fig. 1), increased Na+ accumulation in the pericryptal sheath (Fig. 2), and raised the expression of epithelial Na+ channel in colonocytes (Fig. 3). Increases in potential difference, short-circuit current, and TER were observed with aldosterone perfusion (18). The aldosterone-sensitive decrease in crypt-wall permeability to dextran correlates with the aldosterone-dependent increase in TER, and these changes relate to the increased Na+ accumulation in the pericryptal space because Na+ accumulates in part because its leakage across the crypt wall is diminished. In contrast, angiotensin II perfusion had no effect on these electrical variables and increased dextran permeability across the crypt wall (18). The angiotensin II–dependent rise in dextran permeability may result from angiotensin II–dependent activation of adrenomedullin, which in turn is known to activate matrix metalloproteinases, thereby causing breakdown of the extracellular matrix (20) and hence increased leakage of dextran across the crypt wall and a reduced TER.
Increased pericryptal sheath growth, relating to pericryptal sheath myofibroblasts, was observed within 3 days of instituting an LS diet. We studied the separate roles of aldosterone and angiotensin II on the myofibroblast growth; expression of α-SMA, a myofibroblast specific protein; collagen IV, an exclusive product of myofibroblasts; and claudin IV, a junctional protein that participates in epithelial barrier function and regulates paracellular permeability (21). The trophic effects of aldosterone were demonstrated in the adrenalectomized animals with aldosterone infusion, given that it increased α-SMA (Fig. 3) and claudin IV expression and collagen IV production to the same extent as an LS diet, in which both aldosterone and angiotensin II are present (19). Angiotensin II perfusion has no positive trophic effect on the extracellular matrix.
It also has been reported in cardiac muscle that aldosterone alone can stimulate the growth of myofibroblasts (22). In the absence of raised aldosterone, angiotensin II inhibits collagen synthesis and this increases the view that angiotensin II could activate adrenomedullin, which in turn activates collagenases in the extracellular matrix (23), as has been demonstrated.
It is evident from these studies that aldosterone is the primary effector of both colonic absorptive function and of the trophic response of pericryptal myofibroblasts to LS. Angiotensin II has no direct effect on these functions. Aldosterone induces not only the proteins that control transcellular Na+ transport but also the junctional proteins necessary to maintain vectorial transport across epithelia, without which neither net fluid nor electrolyte flow can exist. The aldosterone effect on myofibroblasts may result from a direct genomic response on adhesion molecule expression between adjacent colonocytes and between the colonocytes and the myofibroblasts adhering to the basal membranes of the colonocytes. Alternatively, the myofibroblast growth may be an indirect response related to stretch, resulting from enhanced fluid absorption distending the pericryptal space between the myofibroblasts and basal membranes of the colonocytes and the lateral intercellular spaces, as has been observed with myofibroblasts in culture (24). Although our results unequivocally show that aldosterone is the key hormone in colonic permeability and the growth of pericryptal myofibroblasts, further work is required to establish possible synergisms between aldosterone and other factors implicated in sodium and water homeostasis, such as vasopressin.
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