Rat descending colon absorbs fluid and Na+ against a high luminal hydraulic resistance and absorption is increased by a reduction in Na+ intake (Naftalin et al. 1999). We have now investigated whether the effects of low Na+ (LS) diet are due to increased osmotic concentration gradients, generated by Na+ and reduced solute leakage across the crypt wall.
Experiments were conducted on Sprague-Dawley rats (200-250 g) that were fed a high NaCl (HS) diet (NaCl 4500 µmol day-1) for 8-9 days and then switched to a LS diet (NaCl 35 µmol day-1) ± captopril (125 mg kg-1 day-1) for 1-9 days then were humanely killed prior to tissue isolation. Experiments were approved by the Ethical Committee in Animal Experimentation of the University of Barcelona.
[Na+] in isolated rat distal colonic mucosa from rats was determined using a modification of a dual wavelength laser scanning confocal microscopic method in which low affinity Na+-sensitive dye (Sodium Red, and BODIPY) is bound to 50 nm diameter polystyrene beads (Jayaraman et al. 2001). Crypt permeability to dextran was monitored by the rate of escape of FITC labelled dextran (10 kDa) from the crypt lumen into the pericryptal space at 37°C. These effects were correlated with plasma aldosterone and with trophic changes in crypt colonocytes, pericryptal myofibroblasts and the surrounding extracellular matrix, using immunolocalization in paraformaldehyde-fixed colon, with specific antibodies and fluorescence-labelled secondary antibodies. The [Na+] in the pericryptal sheath of HS rats was 144 ± 4 mM (n = 4; S.E.M.), and in the crypt lumen 5-10 µm distal to the opening [Na+] fell to 75-65 mM. During 9 days after switching to LS diet, pericryptal [Na+] increased, to 285 ± 7 mM (S.E.M.) (n = 5, P < 0.025 Students unpaired t test) as in murine distal colonic crypts in vivo (Thiagarajah et al. 2001), but luminal [Na+] was unaltered. Recovery of pericryptal [Na+] 3-5 days after switching to LS diet was significantly delayed in captopril-treated rats. Crypt wall permeability to dextran decreased by 4-5-fold within 3 days but was delayed by captopril. Significant captopril-sensitive increases in the pericryptal myofibroblast smooth muscle actin, OB-cadherin, collagen IV expression and also increases in ATII type I and TGF-β receptors within 3 days after switching to LS diet.
This work was supported by The Wellcome Trust